[go: up one dir, main page]

WO2004085457A2 - 17beta-hydroxysteroid dehydrogenase inhibitors - Google Patents

17beta-hydroxysteroid dehydrogenase inhibitors Download PDF

Info

Publication number
WO2004085457A2
WO2004085457A2 PCT/GB2004/001234 GB2004001234W WO2004085457A2 WO 2004085457 A2 WO2004085457 A2 WO 2004085457A2 GB 2004001234 W GB2004001234 W GB 2004001234W WO 2004085457 A2 WO2004085457 A2 WO 2004085457A2
Authority
WO
WIPO (PCT)
Prior art keywords
group
alkyl
substituted
compound
amide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2004/001234
Other languages
French (fr)
Other versions
WO2004085457A3 (en
Inventor
Nigel Vicker
Harshani Rithma Ruchiranani Lawrence
Gillian Margaret Allan
Christian Bubert
Delphine Sophie Marion Fischer
Alan Purohit
Michael John Reed
Barry Victor Lloyd Potter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sterix Ltd
Original Assignee
Sterix Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sterix Ltd filed Critical Sterix Ltd
Priority to JP2006505995A priority Critical patent/JP5000294B2/en
Priority to CA2519956A priority patent/CA2519956C/en
Priority to MXPA05010340A priority patent/MXPA05010340A/en
Priority to BRPI0408697-0A priority patent/BRPI0408697A/en
Priority to AU2004224061A priority patent/AU2004224061B2/en
Priority to EP04722316A priority patent/EP1631579A2/en
Publication of WO2004085457A2 publication Critical patent/WO2004085457A2/en
Publication of WO2004085457A3 publication Critical patent/WO2004085457A3/en
Priority to US11/234,868 priority patent/US8003783B2/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/06—Antiabortive agents; Labour repressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/30—Oestrogens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring

Definitions

  • the present invention relates to a compound.
  • the present invention provides compounds capable of inhibiting 17 ⁇ -hydroxysteroid dehydrogenase (17 ⁇ - HSD).
  • the aim in the treatment is no longer to cure but to reach a palliative control. This is the case when metastases of the tumour have reached locations such as bones, skin, lymph, node or brain.
  • the treatment varies depending on the hormonal status of the patient (whether it is a pre- or post-menopausal woman to be treated) and depending on the type of tumour. Certain tumours have indeed been proven to rely on estrogens for their growth and development, leading to what is called a Hormone Dependent Breast Cancer (HDBC, see 1-1). While non HDBC are treated with chemotherapy, where the aim is to kill differentially tumour cells using a combination of cytotoxic agents, 5 HDBC are expected to respond to endocrine therapy.
  • HDBC Hormone Dependent Breast Cancer
  • hER human Oestrogen Receptor
  • ER+ oestrogen-receptor- positive
  • ER- oestrogen-receptor-negative
  • Anti-oestrogens as antagonists of the oestrogen receptor, have been one of the first treatment considered for HDBC. Their action rely on their ability to bind competitively to the specific receptor protein hER, thus preventing access of endogenous estrogens to their specific binding site. Consequently, the natural hormone is unable to maintain tumour growth.
  • tamoxifen (below) is the most widely used because of the very low toxicity profile of the molecule. Despite its non-steroidal skeleton, tamoxifen possesses a mixed agonist-antagonist activity that limits its therapeutic potential. 9 In addition, some for of drug resistance has been reported in patients after long-term tamoxifen treatment. 10
  • Novel pure anti-oestrogenic drugs such as ICI 164384 (below) have since been discovered but the loss of potency compared with that of tamoxifen suggested the need to design more highly potent targets. 11
  • the use of therapeutic agents that inhibit one or several enzyme of the steroid biosynthesis pathway represents another important strategy to control of the development of oestrogen-dependent tumours.
  • the enzyme aromatase which converts androgenic C19 steroids to estrogenic C18 steroids, has been the prime target for reducing oestrogen levels.
  • This enzyme complex which contains a cytochrome P450 haemoprotein, catalyses the aromatisation of the androgen A-ring with the subsequent loss of the C19 methyl group to yield estrogens.
  • Aminoglutethimide (below) was the first aromatase inhibitor used for the treatment of breast cancer. It however showed a number of undesirable side effects given its wide spectrum of inhibitory effects on other P450-dependant enzymes, and attempts to improve on the original structure have led to a number of non-steroidal compounds entering clinical trials. 15 The last generation developed compounds such as letrozole, which combine high potency and high selectivity for the enzyme, and are also better tolerated.
  • Generation I aminoglutethimide, AG; generation III, letrozole.
  • aromatase inhibitors are reserved as second line treatment for advanced HDBC patients whose diseases are no longer controlled by tamoxifen.
  • tamoxifen because of the extreme good toxicity profile of some of the latest aromatase inhibitors, recent clinical trials have been conducted to assess their suitability as first line treatment for HDBC.
  • estrogens are synthesised from the highly available precursor oestrone-sulphate, via two enzymes (scheme below): oestrone sulphatase (STS) which hydrolyses oestrone-sulphate into oestrone, and 17 ⁇ -hydroxysteroid dehydrogenase (17 ⁇ -HSD) which reduces oestrone into oestradiol.
  • STS oestrone sulphatase
  • 17 ⁇ -HSD 17 ⁇ -hydroxysteroid dehydrogenase
  • AR aromatase
  • ST steroid sulfotransferase
  • STS steroid sulphatase
  • 17 ⁇ -HSD 17 ⁇ -hydroxysteroid dehydrogenase
  • 3 ⁇ -IS 3 ⁇ -hydroxysteroid dehydrogenase ⁇ 5 , ⁇ 4 -isomerase
  • ER oestrogen receptor
  • EMATE was shown to inhibit steroid sulphatase activity in a time- and concentration-dependent manner 17 and was active in vivo on oral administration. 18 It was however revealed to be highly estrogenic which raised the need to design STS inhibitors devoid of agonist activity on hER.
  • steroid sulphatase inhibitors are sulphamate esters, such as N,N-dimethyl oestrone-3-sulphamate and, preferably, oestrone-3-sulphamate (EMATE). It is known that EMATE is a potent E1-STS inhibitor as it displays more than 0 99% inhibition of E1-STS activity in intact MCF-7 cells at 0.1 mM. EMATE also inhibits the E1-STS enzyme in a time- and concentration-dependent manner, indicating that it
  • EMATE acts as an active site-directed inactivator.
  • DHA-STS dehydroepiandrosterone sulphatase
  • EMATE is also active in vivo as almost complete inhibition of rat liver E1-STS (99%) and DHA-STS (99%) activities resulted when it is administered either orally or subcutaneously.
  • EMATE has been shown to have a memory enhancing 0 effect in rats. Studies in mice have suggested an association between DHA-STS activity and the regulation of part of the immune response. It is thought that this may also occur in humans.
  • the bridging O-atom of the sulphamate moiety in EMATE is important for inhibitory activity. Thus, when the 3-O-atom is replaced by other heteroatoms as in oestrone-3-N-sulphamate and oestrone-3-S-sulphamate, these analogues are weaker 5 non-time-dependent inactivators.
  • 17 ⁇ -HSD which catalyses the final step in estrogens and androgens biosynthesis, also appeared as a target for oestrogen deprivation strategies.
  • This enzyme is responsible for the interconversion of the oxidised form (less active) and the reduced form (more active) of steroids. Its activity directly supports the growth and development of oestrogen dependent tumours since it preferably reduces oestrone into estradiol 25 and in a minor extend, via the conversion of the androgen DHEA into androstenediol (Adiol), which has recently been proven to have estrogenic properties and to be able to bind to the oestrogen receptor ' . 26
  • 17 ⁇ -HSD belongs to a family of isoenzymes, 11 of which have been so far identified and cloned. 27 Each type has a selective substrate affinity and directional activity which means that selectivity of drug action has to be achieved. 17 ⁇ -HSD type 1 is the isotype that catalyses the interconversion of oestrone and oestradiol.
  • 17 ⁇ -HSD inhibitors Unlike STS inhibitors, only few 17 ⁇ -HSD inhibitors have been reported. Most of the steroidal inhibitors for 17 ⁇ -HSD type 1 have in common a D-ring modified structure. Oestradiol derivatives which contain a side-chain with a good leaving group at the 16 ⁇ - position have been shown to be a potent class of inhibitors. In particular, 16 ⁇ - (bromoalkyl)-estradiol 28 where the side-chains exhibit high reactivity towards nucleophilic amino-acids residues in the active site of the enzyme were found to be promising irreversible inhibitors.
  • Analogues containing short bromoalkyl moieties at position 16 exhibited the highest activity with 16 -(Bromopropyl)-oestradiol, followed by 16 ⁇ - (Bromobutyl)-oestradiol, the most potent of the series (3 and 4). They, however, turned out to be pure agonists of the oestrogen receptor.
  • 17 ⁇ -HSD type 1 inhibitors 16 ⁇ -(bromopropyl)-oestradiol, 3; 16 ⁇ -(bromopropyl)-oestradiol, 4 and a flavone derivative, apigenin.
  • Flavonoids which are structurally similar to estrogens, are able to bind to the oestrogen receptor with estrogenic or anti-estrogenic activities. 30 Their action on aromatase activity is well documented and in recent studies, they were found to reduce the conversion of oestrone into oestradiol catalysed by 17 ⁇ -HSD type 1. 31 Flavone derivatives, such as apigenin emerged from a SAR study as a promising compounds with some inhibitory activity on 1 ⁇ -HSD type 1 without being estrogenic at the inhibitory concentration. 32
  • DH Steroid dehydrogenases
  • E2HSD oestradiol 17 ⁇ -hydroxysteroid dehydrogenases
  • E2HSD Type I reduces oestrone (E1) to the biologically active oestrogen, oestradiol (E2), while E2HSD Type II inactivates E2 by catalysing its oxidation to E1.
  • E1 oestrone
  • E2HSD Type II inactivates E2 by catalysing its oxidation to E1.
  • the present invention provides novel compounds which are capable of acting as effective 17 ⁇ -hydroxysteroid dehydrogenase (17 ⁇ -HSD) inhibitors.
  • the present invention identifies that the compounds of the present application are effective 17 ⁇ - hydroxysteroid dehydrogenase (17 ⁇ -HSD) inhibitors.
  • Figures 1 shows some of the enzymes involved in the in situ synthesis of oestrone from oestrone sulphate, and oestradiol.
  • STS denotes Oestrone Sulphatase
  • E2DH Type I denotes Oestradiol 17 ⁇ -hydroxysteroid dehydrogenase Type I or Oestradiol 17 ⁇ -hydroxysteroid dehydrogenase Type 1, 3, 5 and/or 7
  • E2DH Type II denotes Oestradiol 17 ⁇ -hydroxysteroid dehydrogenase Type II or Oestradiol 17 ⁇ -hydroxysteroid dehydrogenase Type 2 and/or 8.
  • the present invention therefore seeks to overcome one or more of the problems associated with the prior art methods of treating breast and endometrial cancers.
  • the present invention provides a use of a compound for the preparation of a medicament that can or affect, such as substantially inhibit, the steroid dehydrogenase pathway - which pathway converts oestrone to and from oestradiol.
  • This aspect of the present invention is advantageous because by the administration of one type of compound it is possible to block the synthesis of oestradiol from oestrone.
  • the present invention provides compounds that have considerable therapeutic advantages, particularly for treating breast and endometrial cancers.
  • the compounds of the present invention may comprise other substituents. These other substituents may, for example, further increase the activity of the compounds of the present invention and/or increase stability (ex vivo and/or in vivo).
  • DETAILED ASPECTS OF THE PRESENT INVENTION may, for example, further increase the activity of the compounds of the present invention and/or increase stability (ex vivo and/or in vivo).
  • R 2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group;
  • R 3 is selected from
  • R 1 is a selected from
  • alkylaryl group wherein the aryl group is substituted by other than a methyl group
  • alkenylaryl group v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group
  • R 2 is selected from groups capable of forming a hydrogen bond , a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and (III) R 3 is selected from
  • R is a selected from (i) an alkyloxyalkyl group
  • alkenylaryl group wherein the aryl group is substituted (v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group
  • the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
  • R 2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group;
  • R 3 is selected from
  • a pharmaceutical composition comprising a compound having Formula I
  • the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
  • R 2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group;
  • R 1 is a selected from
  • a compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 17 ⁇ -hydroxysteroid dehydrogenase (17 ⁇ -HSD) levels wherein the compound is of Formula I Formula
  • R 1 is a selected from
  • alkenylaryl group wherein the aryl group is substituted (v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group
  • (x) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH 2 - or - CH 2 CH 2 -, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group, (xi) -CHO so that Ri together with R 3 provide the enol tautomer
  • R 2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and ,
  • alkylaryl group wherein the aryl group is substituted by other than a C1-10 group
  • alkenylaryl group wherein the aryl group is substituted
  • the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
  • R 2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group;
  • a method of inhibiting 17 ⁇ -hydroxysteroid dehydrogenase (17 ⁇ -HSD) activity in a subject in need of same comprising administering a compound a compound having Formula
  • R 1 is a selected from
  • alkyiheteroaryl group wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group
  • One key advantage of the present invention is that the compounds of the present invention can act as 17 ⁇ -HSD inhibitors.
  • Another advantage of the compounds of the present invention is that they may be potent in vivo.
  • non-oestrogenic compounds means exhibiting no or substantially no oestrogenic activity.
  • Another advantage is that some of the compounds may not be capable of being metabolised to compounds which display or induce hormonal activity.
  • Some of the compounds of the present invention are also advantageous in that they may be orally active.
  • Some of the compounds of the present invention may useful for the treatment of cancer, such as breast cancer, as well as (or in the alternative) non-malignant conditions, such as the prevention of auto-immune diseases, particularly when pharmaceuticals may need to be administered from an early age.
  • cancer such as breast cancer
  • non-malignant conditions such as the prevention of auto-immune diseases, particularly when pharmaceuticals may need to be administered from an early age.
  • some of the compounds of the present invention are also believed to have therapeutic uses other than for the treatment of endocrine-dependent cancers, such as the treatment of autoimmune diseases.
  • capable of forming a hydrogen bond means a group having a region of negative charge capable of forming one part of a hydrogen bond.
  • hydrocarbyl group means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo, alkoxy, nitro, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. A non- limiting example of a hydrocarbyl group is an acyl group.
  • a typical hydrocarbyl group is a hydrocarbon group.
  • hydrocarbon means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may be linear, branched or cyclic, or an aryl group.
  • the term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
  • the hydrocarbyl group is selected from optionally substituted alkyl group, optionally substituted haloalkyl group, aryl group, alkylaryl group, alkylarylakyl group, and an alkene group.
  • the hydrocarbyl group is an optionally substituted alkyl group.
  • the hydrocarbyl group is selected from Cj-C 10 alkyl group, such as C
  • Typical alkyl groups include G, alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, C 7 alkyl, and C 8 alkyl.
  • the hydrocarbyl group is selected from C.
  • Typical haloalkyl groups include C, haloalkyl, C 2 haloalkyl, C 3 haloalkyl, C 4 haloalkyl, C 5 haloalkyl, C 7 haloalkyl, C 8 haloalkyl, C, bromoalkyl, C 2 bromoalkyl, C 3 bromoalkyl, C 4 bromoalkyl, C 5 bromoalkyl, C 7 bromoalkyl, and C 8 bromoalkyl.
  • the hydrocarbyl group is selected from aryl groups, alkylaryl groups, alkylarylakyl groups, -(CH 2 ) 1-10 -aryl, -(CH 2 ) 1-10 -Ph, (CH 2 ) 1-10 -Ph- C 1-10 alkyl, -(CH ⁇ -Ph, (CH 2 ) 1-5 -Ph-C 1-5 alkyl, -(CH 2 ) 1-3 -PhBOOK (CH 2 ) 1-3 -Ph-C 1-3 alkyl, -CH 2 - Ph, and -CH 2 -Ph-C(CH 3 ) 3 .
  • the aryl group or one or more of the aryl groups may contain a hetero atom.
  • the aryl group or one or more of the aryl groups may be carbocyclic or more may heterocyclic.
  • Typical hetero atoms include O, N and S, in particular N.
  • the hydrocarbyl group is selected from -(CH 2 ) ⁇ . 10 -cycloalkyl, -(CH 2 ) 1-10 -C 3-10 cycloalkyl, ⁇ (CH 2 ) 1-7 -C 3-7 cycloalkyl, -(CH 2 ) 1-5 -C 3 . 5 cycloalkyl, - (CH 2 ) -3 -C 3-5 cycloalkyl, and -CH 2 - C 3 cycloalkyl.
  • the hydrocarbyl group is an alkene group.
  • Typical alkene groups include Cj-C ⁇ 0 alkene group, C
  • bio-isostere is when any one or more of rings A, B, C and D is a heterocyclic ring and/or when any one or more of rings A, B, C and D has been substituted and/or when any one or more of rings A, B, C and D has been modified; but wherein the bio-isostere has steroidal properties.
  • the ring system of the present invention is analogous to a steroidal ring structure and may be a bio-isostere of a steroidal ring structure.
  • the structure of a present polycyclic structure can be presented as:
  • each ring A', B', and C independently represents a heterocyclic ring or a non- heterocyclic ring, and wherein each ring may be independently substituted or unsubstituted, saturated or unsaturated.
  • any one or more of rings A', B', C and D' may be independently substituted with suitable groups - such as an alkyl group, an aryl group, a hydroxy group, a halo group, a hydrocarbyl group, an oxyhydrocarbyl group etc.
  • At least one of A', B', and C may be a heterocyclic group (a heterocycle) or a non- heterocyclic group.
  • At least one of A', B', C and D' may be a saturated ring structure or an unsaturated ring structure (such as an aryl group).
  • At least one of A', B', C and D' is an aryl ring.
  • the compound will contain, inclusive of all substituents, no more than 50 about carbon atoms, more usually no more than about 30 to 40 carbon atoms.
  • D' is a five or six membered ring.
  • Preferred steroidal nuclei rings A'-D' on which the compounds of the present invention may be based include rings A-D of:
  • oestrones and substituted oestrones viz: oestrone 16 ⁇ -OH-oestrone
  • dehydroepiandrosterones and substituted dehvdroepiandrosterones. viz: dehydroepiandrosterones 16 ⁇ -OH-dehydroepiandrosterone
  • the compound is of Formula II
  • the compound is of Formula III Formula III
  • the compound is of Formula IV
  • R1 of formula I is an alkyloxyalkyl group.
  • the alkyloxyalkyl group is a -alkyl-O-alkyl group.
  • Each alkyl of the group is preferably independently has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
  • Each alkyl of the group may be independently branched or straight chain. Preferably one or each alkyl is straight chain
  • Particulariy preferred alkyloxyalkyl groups are -EtOEt, -EtOMe, -MeOEt, and -MeOMe.
  • R 1 may be a nitrile group or comprise a nitrile group.
  • Nitrile group is understood to mean a -CsN group.
  • R of formula I is a nitrile group.
  • R 1 is a nitrile group
  • R 2 is -OH
  • R 1 of formula I is a nitrile group and R 2 is -OH.
  • R1 of formula I is an alkylaryl group.
  • alkylaryl group it is meant a group denoted by -alkyl-aryl.
  • the alkyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
  • the alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
  • the aryl group typically has a six membered ring
  • the aryl group is substituted.
  • the aryl group of the alkylaryl group is substituted by a group selected from N,N-dialkyl, alkoxy, nitro (-NO 2 " ), nitrile, and a ⁇ aalkyl groups.
  • N,N-dialkyl is -N(C1-10 alkyl) 2 , -N(C1-5 alkyl) 2 , or -N(C1-3 alkyl) 2 .
  • NMe 2 is particularly preferred.
  • the alkoxy group is C1-10 alkoxy, C1-5 alkoxy, and C1-3 alkoxy. Particularly preferred is methoxy.
  • the alkylaryl group is -CH 2 -Ph.
  • R1 of formula I is an alkenylaryl group.
  • alkylaryl group it is meant a group denoted by -alkenyl-aryl.
  • the aryl group of the alkenylaryl group is substituted.
  • the alkenyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
  • the alkenyl group may be branched or straight chain. Preferably the alkenyl group is straight chained.
  • the aryl group typically has a six membered ring
  • the aryl group is substituted.
  • the aryl group of the alkylaryl group is substituted by a group selected from N,N-dialkyl, alkoxy, nitro (-NO 2 " ), nitrile, and azaalkyl groups.
  • N,N-dialkyl is -N(C1-10 alkyl) 2 , -N(C1-5 alkyl) 2 , or -N(C1-3 Particularly preferred is NMe 2 .
  • the alkoxy group is C1-10 alkoxy, C1-5 alkoxy, and C1-3 alkoxy. Particularly preferred is methoxy.
  • R1 of formula I is an alkyiheteroaryl group.
  • the alkyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
  • the alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
  • the aryl group typically has a six membered ring
  • the heteroarylgroup contain carbon and hetero atoms.
  • Typical hetero atoms include O, N and S, in particular N.
  • the aryl group may be substituted or unsubstituted.
  • the aryl group is substituted.
  • the aryl group of the alkylaryl group is substituted by a group selected from N,N-dialkyl, alkoxy, nitro (-NO 2 " ), nitrile, and azaalkyl groups.
  • the N,N-dialkyl is -N(C1-10 alkyl) 2 , -N(C1-5 alkyl) 2 , or -N(C1-3 alkyl) 2 .
  • NMe 2 Particularly preferred is NMe 2 .
  • the alkoxy group is C1-10 alkoxy, C1-5 alkoxy, and C1-3 alkoxy. Particularly preferred is methoxy.
  • alkyiheteroaryl group is selected from
  • R1 of formula I is an alkenylheteroaryl group. In one preferred aspect, R1 of formula I is an alkenylheteroaryl group, wherein the aryl group is substituted. Preferably R1 of formula I is an alkenylheteroaryl group wherein the aryl group is unsubstituted.
  • R1 of formula I is an alkyiheteroaryl group.
  • the alkenyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
  • the alkenyl group may be branched or straight chain. Preferably the alkenyl group is straight chained.
  • the aryl group typically has a six membered ring
  • the heteroarylgroup contain carbon and hetero atoms.
  • Typical hetero atoms include O, N and S, in particular N.
  • the aryl group may be substituted or unsubstituted.
  • the aryl group is substituted.
  • the aryl group of the alkylaryl group is substituted by a group selected from N,N-dialkyl, alkoxy, nitro (-NO 2 ), nitrile, and azaalkyl groups.
  • the N,N-dialkyl is -N(C1-10 alkyl) 2 , -N(C1-5 alkyl) or -N(C1-3 alkyl) 2 .
  • NMe 2 Particularly preferred is NMe 2 .
  • the alkoxy group is C1-10 alkoxy, C1-5 alkoxy, and C1-3 alkoxy. Particularly preferred is methoxy.
  • alkenylheteroaryl group is selected from
  • the alkyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
  • the alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
  • composition comprising a compound having Formula V
  • a method of inhibiting 7 ⁇ -hydroxysteroid dehydrogenase (17 ⁇ -HSD) activity in a subject in need of same comprising administering a compound a compound having Formula V
  • R 1 of formula I is a carboxylic acid ester.
  • R 1 of formula I is a carboxylic acid ester
  • R 3 O and/or the ester is selected from -CO 2 X, -CH 2 CO 2 X, and -CH 2 CH 2 CO 2 X, wherein X is a hydrocarbyl group.
  • the carboxylic acid ester is selected from -CO 2 X, -CH 2 CO 2 X, and -CH 2 CH 2 CO 2 X, wherein X is a hydrocarbyl group.
  • R 1 of formula I is a carboxylic acid ester
  • R 3 O and the ester is selected from -CO 2 X, -CH 2 CO 2 X, and -CH 2 CH 2 CO 2 X, wherein X is a hydrocarbyl group.
  • X is a hydrocarbon group. More preferably X is an alkyl group such as an alkyl group preferably having from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1, 2 or 3 carbons.
  • the alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
  • R1 of formula I is -CO 2 H or alkyl-CO 2 H group
  • the alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
  • the alkyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
  • R1 of formula I is a branched alkenyl group.
  • the branched alkenyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2, 3 or 4 carbons.
  • Alkyl-Alcohol Group Or Alkenyl-Alcohol Group
  • R1 of formula I is an alkyl-alcohol group or alkenyl-alcohol group.
  • the alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
  • alkyl-alcohol group or alkenyl-alcohol group it is meant a group of the formula C x H 2x-2n-y (OH)y wherein x is an integer, y is an integer and n is the degree of unsaturation.
  • x is from 1 to 20, preferably from 1 to 10, preferably from 1 to 5, preferably 1 , 2, 3 or 4.
  • y is 1 , 2 or 3.
  • n is 0, 1 , 2 or 3.
  • alkenyl-alcohol has the following structural formula:
  • the alkenyl alcohol is substituted.
  • a substituent replaces H a and/or H b in the following structural formula:
  • Suitable substituents include ester groups, haloalkyl groups, aryl groups such as heteroaryl groups, and alkyl groups.
  • Preferred substituted alkenyl-alcohol groups include
  • X is a hydrocarbyl group, preferably a hydrocarbon, more preferably an alkyl group, preferably having from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1, 2, or 3 carbons.
  • X is an ethyl group.
  • R1 of formula I is an amide or alkylamide group.
  • R1 of formula I is an amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH 2 - or -CH 2 CH 2 -, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
  • R1 of formula I is an amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH 2 - or -CH 2 CH 2 -, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
  • Preferred amide groups include NHCO-C 1-10 alkyl, CONH C 1-10 alkyl, NHCO(CH 2 ) 1-10 CH 3 , CONH(CH 2 ) 1-10 CH 3 , NHCO(CH 2 ) 3 . 7 CH 3 , CONH(CH 2 ) 3-7 CH 3 , NHCO(CH 2 ) 6 CH 3 , CONH(CH 2 ) 6 CH 3 .
  • the amide or alkylamide is an alkylamide.
  • Preferred alkylamide groups include C 1-10 alkyl-NHCO-C 1-10 alkyl, Cnoalkyl-CO H-C,. 10 alkyl, C 1-10 alkyl-NHCO(CH 2 ) 1-10 CH 3 , C 1-10 alkyl-CONH(CH 2 ) 1-10 CH 3 , C 1-10 alkyl- NHCO(CH 2 ) 3-7 CH 3 , C 1-10 alkyl-CONH(CH 2 ) 3-7 CH 3 , C 1-10 alkyl-NHCO(CH 2 ) 6 CH 3 , C- M oalkyl- CONH(CH 2 ) 6 CH 3 .
  • substituents of the di-substituted amide together form a cyclic structure.
  • substituents of the di-substituted amide together form an aryl ring.
  • the substituents of the di-substituted amide together form a heterocyclic ring.
  • R1 of formula I is together with R 3 provide the enol tautomer
  • R 1 of formula I together with R 3 form a pyrazole.
  • the pyrazole is substituted.
  • the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl group, branched alkyl group, and an amide.
  • R 1 of formula I together with R 3 form a pyrazole, wherein the pyrazole is substituted with one or more (i) an alkyloxyalkyl group, (ii) a nitrile group, (iii) alkylaryl group, (iv) alkenylaryl group, (v) alkyiheteroaryl group, (vi) alkenylheteroaryl group, (viii) carboxylic acid ester, (ix) CO 2 H or alkyl-CO 2 H group, (x) branched alkenyl, (xi) alkyl-alcohol group or alkenyl-alcohol group, (xii) amide or alkylamide, as described herein.
  • R 1 and R 3 form a pyrazole
  • the 2 position of the ring system is substituted with a group selected from -OH and -O- hydrocarbyl, preferably selected from -OH and -O-alkyl.
  • the alkyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2, 3 or 4 carbons.
  • the 2 position of the ring system it is meant the position on the steriodal ring system which is labelled '2' on the formula below:
  • R and R 3 when R and R 3 form a pyrazole the pyrazole is substituted.
  • the addition of substituents to the pyrazole ring may remove unsaturation.
  • substituted pyrazole rings examples include:
  • R 1 of formula I together with R 3 forms a heteroaryl ring
  • the aryl group typically has a six membered ring
  • the heteroarylgroup contains carbon and hetero atoms.
  • Typical hetero atoms include O, N and S, in particular N.
  • the aryl group may be substituted or unsubstituted. Preferably the aryl group is unsubstituted.
  • R 2 is selected from groups capable of forming a hydrogen bond , a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group.
  • R 2 is selected from groups capable of forming a hydrogen bond and a sulphamate group.
  • the sulphamate group is of the formula wherein R 9 and R 10 are independently selected from H, alkyl, cycloalkyl, alkenyl and aryl, or combinations thereof, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
  • At least one of R 9 and R 10 is H.
  • R 9 and R 10 are both H
  • the group capable of forming a hydrogen bond are selected from -OH
  • the compound of the present invention may have substituents other than those of the ring systems show herein.
  • the ring systems herein are given as general formulae and should be interpreted as such.
  • the absence of any specifically shown substituents on a given ring member indicates that the ring member may substituted with any moiety of which H is only one example.
  • the ring system may contain one or more degrees of unsaturation, for example is some aspects one or more rings of the ring system is aromatic.
  • the ring system may be carbocyclic or may contain one or more hetero atoms.
  • the compound of the invention in particular the ring system compound of the invention of the present invention may contain substituents other than those show herein.
  • substituents may be one or more of: one or more sulphamate group(s), one or more phosphonate group(s), one or more thiophosphonate group(s), one or more sulphonate group(s), one or more sulphonamide group(s), one or more halo groups, one or more O groups, one or more hydroxy groups, one or more amino groups, one or more sulphur containing group(s), one or more hydrocarbyl group(s) - such as an oxyhydrocarbyl group.
  • the ring system A'B'C'D' of the present compounds may contain a variety of non-interfering substituents.
  • the ring system A'B'C'D' may contain one or more hydroxy, alkyl especially lower (C.,-C 6 ) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n- butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C,-C 6 ) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
  • the ring system is substituted with a oxyhydrocarbyl group. More preferably the A' ring of the ring system is substituted with a oxyhydrocarbyl group.
  • At least the 2 position of the A' ring of the ring system is substituted with a oxyhydrocarbyl group.
  • oxyhydrocarbyl group as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the oxyhydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen.
  • the oxyhydrocarbyl group is a oxyhydrocarbon group.
  • oxyhydrocarbon means any one of an alkoxy group, ah oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group.
  • the term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
  • the oxyhydrocarbyl group is of the formula G ⁇ O (such as a C 1-3 O).
  • the A' ring of the ring system is substituted with an alkoxy group.
  • At least the 2 position of the A' ring of the ring system is substituted with an alkoxy group.
  • the alkoxy group is methoxy
  • the ring system is substituted with a hydrocarbylsulphanyl group. More preferably the A' ring of the ring system is substituted with a hydrocarbylsulphanyl group.
  • hydrocarbylsulphanyl means a group that comprises at least hydrocarbyl group (as herein defined) and sulphur, preferably -S-hydrocarbyl, more preferably -S- hydrocarbon. That sulphur group may be optionally oxidised.
  • At least the 2 position of the A' ring of the ring system is substituted with a hydrocarbylsulphanyl group.
  • the hydrocarbylsulphanyl group is -S-C 1-10 alkyl, more preferably -S-C,. 5 alkyl, more preferably -S-C 1-3 alkyl, more preferably -S-CH 2 CH 2 CH 3 , -S-CH 2 CH 3 or -SCH 3
  • At least the A' ring of the ring system is substituted with an hydrocarbyl group.
  • At least the 2 position of the A' ring of the ring system is substituted with an alkyl group.
  • the alkyl group is ethyl.
  • the compound comprises at least two or more of sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group or a sulphonamide group.
  • the compound comprises at least two sulphamate groups. .
  • the compound comprises at least two sulphamate groups, wherein said sulphamate groups are not on the same ring.
  • the A' ring of the ring system comprises at least one sulphamate group and wherein the D' ring of the ring system comprises at least one sulphamate group.
  • the A' ring preferably contain one or more of an alkoxy substituent and an alkyl substituent.
  • the compound has one of the following formulae Formula VI
  • R 1 is an alkoxy group or an alkyl group
  • R 1 is an alkyl group.
  • R 1 is a C,-C 10 alkyl group, preferably a C C 6 alkyl group, preferably a C
  • R 1 is -CH 3 or -CH 2 CH 3 .
  • R 1 is an alkoxy group.
  • R 11 is methoxy.
  • a method comprising (a) performing a steroid dehydrogenase assay with one or more candidate compounds having the formula as defined herein; (b) determining whether one or more of said candidate compounds is/are capable of modulating steroid dehydrogenase activity; and (c) selecting one or more of said candidate compounds that is/are capable of modulating steroid dehydrogenase activity.
  • a method comprising (a) performing a steroid dehydrogenase assay with one or more candidate compounds having the formula as defined herein; (b) determining whether one or more of said candidate compounds is/are capable of inhibiting steroid dehydrogenase activity; and (c) selecting one or more of said candidate compounds that is/are capable of inhibiting steroid dehydrogenase activity.
  • the method may also include the step of modifying the identified candidate compound (such as by chemical and/or enzymatic techniques) and the optional additional step of testing that modified compound for steroid dehydrogenase inhibition effects (which may be to see if the effect is greater or different).
  • the method may also include the step of determining the structure (such as by use of crystallographic techniques) of the identified candidate compound and then performirtg computer modelling studies - such as to further increase its steroid dehydrogenase inhibitory action.
  • the present invention also encompasses a computer having a dataset (such as the crystallographic co-ordinates) for said identified candidate compound.
  • the present invention also encompasses that identified candidate compound when presented on a computer screen for the analysis thereof - such as protein binding studies.
  • a compound according to the present invention for use in medicine.
  • a pharmaceutical composition comprising the compound according to the present invention optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • a compound according to the present invention in the manufacture of a medicament for use in the therapy of a condition or disease associated with steroid dehydrogenase.
  • a compound according to the present invention in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse steroid dehydrogenase levels.
  • the compounds have no, or a minimal, oestrogenic effect.
  • the compounds have an oestrogenic effect.
  • the compounds have a reversible action.
  • the compounds have an irreversible action.
  • the compounds of the present invention are useful for the treatment of breast cancer.
  • the compounds of the present invention may be in the form of a salt.
  • the present invention also covers novel intermediates that are useful to prepare the compounds of the present invention.
  • the present invention covers novel alcohol precursors for the compounds.
  • the present invention covers bis protected precursors for the compounds. Examples of each of these precursors are presented herein.
  • the present invention also encompasses a process comprising each or both of those precursors for the synthesis of the compounds of the present invention.
  • present compounds may also inhibit the activity of steroid dehydrogenase (HSD).
  • HSD steroid dehydrogenase
  • steroid dehydrogenase or HSD it is meant 17 ⁇ hydroxy steroid dehydrogenase.
  • the 17 ⁇ hydroxy steroid dehydrogenase is EC 1.1.1.62
  • the HSD is of Type 1 , 3, 5 and/or 7.
  • the HSD converts oestrone (ketone) to oestradiol (hydroxy).
  • the HSD is of Type 2 and/or 8.
  • the HSD converts oestradiol (hydroxy) to oestrone (ketone).
  • the steroid dehydrogenase is steroid dehydrogenase Type I.
  • the steroid dehydrogenase is steroid dehydrogenase Type II
  • the HSD is of Type 1 , 3, 5 and/or 7.
  • the HSD converts oestrone (ketone) to oestradiol (hydroxy).
  • the HSD is of Type 2 and/or 8.
  • the HSD converts 'oestradiol (hydroxy) to oestrone (ketone).
  • Steroid dehydrogenase or "DH" for short may be classified as consisting of two types - Type I and Type II.
  • the two types of enzyme such as oestradiol 17 ⁇ -hydroxysteroid dehydrogenases (E2HSD), have pivotal roles in regulating the availability of ligands to interact with the oestrogen receptor.
  • E2HSD oestradiol 17 ⁇ -hydroxysteroid dehydrogenases
  • Type I reduces oestrone (E1) to the biologically active oestrogen, oestradiol (E2) while E2HSD Type II inactivates E2 by catalysing its oxidation to E1.
  • inhibitor includes reduce and/or eliminate and/or mask and/or prevent the detrimental action of DH.
  • the compound of the present invention is capable of acting as an DH inhibitor.
  • inhibitor as used herein with respect to the compound of the present invention means a compound that can inhibit DH activity - such as reduce and/or eliminate and/or mask and/or prevent the detrimental action of DH.
  • the DH inhibitor may act as an antagonist.
  • the compound of the present invention may have other beneficial properties in addition to or in the alternative to its ability to inhibit DH activity.
  • the compounds defined herein may also inhibit steroid sulphatase.
  • Steroid sulphatase which is sometimes referred to as steroid sulphatase or steryl sulphatase or "STS" for short - hydrolyses several sulphated steroids, such as oestrone sulphate, dehydroepiandrosterone sulphate and cholesterol sulphate.
  • STS has been allocated the enzyme number EC 3.1.6.2.
  • STS is an enzyme that has been implicated in a number of disease conditions.
  • STS has also been implicated in other disease conditions.
  • Le Roy et al (Behav Genet 1999 Mar;29(2):131-6) have determined that there may be a genetic correlation between steroid sulphatase concentration and initiation of attack behaviour in mice. The authors conclude that sulphatation of steroids may be the prime mover of a complex network, including genes shown to be implicated in aggression by mutagenesis.
  • inhibit includes reduce and/or eliminate and/or mask and/or, prevent the detrimental action of STS.
  • the compound of the present invention is capable of acting as an STS inhibitor.
  • inhibitor as used herein with respect to the compound of the present invention means a compound that can inhibit STS activity - such as reduce and/or eliminate and/or mask and/or prevent the detrimental action of STS.
  • the STS inhibitor may act as an antagonist.
  • the compound is further characterised by the feature that if the sulphamate group were to be substituted by a sulphate group to form a sulphate derivative, then the sulphate derivative would be hydrolysable by an enzyme having steroid sulphatase (E.G. 3.1.6.2) activity - i.e. when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37°C.
  • E.G. 3.1.6.2 an enzyme having steroid sulphatase
  • sulphamate group of the compound were to be replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity and would yield a Km value of less than 200 mmolar, preferably less 0 than 150 mmolar, preferably less than 100 mmolar, preferably less than 75 mmolar, preferably less than 50 mmolar, when incubated with steroid sulphatase EC 3.1.6.2 at
  • sulphamate group of the compound were to be 5 replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable » by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity and would yield a Km value of less than 200 ⁇ molar, preferably less than 150 ⁇ molar, preferably less than 100 ⁇ molar, preferably less than 75 ⁇ molar, preferably less than 50 ⁇ molar, when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 0 37°C.
  • E.C. 3.1.6.2 an enzyme having steroid sulphatase
  • the compound of the present invention is not hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity.
  • the compound of the present invention has at least about a 100 fold selectivity to a desired target (e.g. STS), preferably at least about a 150 fold selectivity to the desired target, preferably at least about a 200 fold selectivity to the desired target, preferably at least about a 250 fold selectivity to the desired target, preferably at least about a 300 fold selectivity to the desired target, preferably at least 0 about a 350 fold selectivity to the desired target.
  • a desired target e.g. STS
  • the compound of the present invention may have other beneficial properties in addition to or in the alternative to its ability to inhibit HSD activity.
  • the ring X has a sulphamate group as a substituent.
  • sulphamate as used herein includes an ester of sulphamic acid, or an ester of an N- substituted derivative of sulphamic acid, or a salt thereof.
  • R 1 is a sulphamate group then the compound of the present invention is referred to as a sulphamate compound.
  • the sulphamate group has the formula:
  • R 9 and R 10 are independently selected from H, alkyl, cycloalkyl, alkenyl and aryl, or combinations thereof, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
  • the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms.
  • R 9 and/or R 0 alkyl
  • the preferred values are those where R 9 and R 10 are each independently selected from lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc.
  • R 4 and R 5 may both be methyl.
  • R 9 and/or R 10 is aryl
  • typical values are phenyl and tolyl (PhCH 3 ; o).
  • R 9 and R 10 represent cycloalkyl
  • typical values are cyclopropyl, cyclopentyl, cyclohexyl etc.
  • R 9 and R 10 typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. to provide a 5 membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
  • alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question.
  • substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
  • the sulphamate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
  • there may be two sulphamates i.e. bis-sulphamate compounds. If these compounds are based on a steroidal nucleus, preferably the second (or at least one of the additional) sulphamate group is located at position 17 of the steroidal nucleus. These groups need not be the same.
  • At least one of R 9 and R 10 is H.
  • each of R 9 and R 10 is H.
  • R 1 is a phosphonate group then the compound of the present invention is referred to as a phosphonate compound.
  • the phosphonate group has the formula:
  • R 12 is H, alkyl, cycloalkyl, alkenyl or aryl, or combinations thereof, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
  • the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms.
  • R 2 is alkyl
  • R 12 may be a lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. By way of example, R 12 may be methyl.
  • R 6 is aryl
  • typical values are phenyl and tolyl (PhCH 3 ;o).
  • R 6 represents cycloalkyl
  • typical values are cyclopropyl, cyclopentyl, cyclohexyl etc.
  • R 12 may even comprise an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. to provide a 5 membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
  • alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question.
  • Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
  • the phosphonate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
  • there may be two phosphonates i.e. bis-phosphonate compounds. If these compounds are based on a steroidal nucleus, preferably the second (or at least one of the additional) phosphonate group is located at position 17 of the steroidal nucleus. These groups need not be the same.
  • R 2 is a thiophosphonate group then the compound of the present invention is referred to as a thiophosphonate compound.
  • the thiophosphonate group has the formula:
  • R 13 is H, alkyl, cycloalkyl, alkenyl or aryl, or combinations thereof, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
  • the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms.
  • R 7 When R 7 is alkyl, R 7 may be a lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. By way of example, R 7 may be methyl.
  • R 13 is aryl, typical values are phenyl and tolyl (PhCH 3 ;o).
  • R 13 represents cycloalkyl
  • typical values are cyclopropyl, cyclopentyl, cyclohexyl etc.
  • R 13 may even comprise an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. to provide a 5 membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
  • alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question.
  • Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
  • the thiophosphonate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
  • there may be two thiophosphonates i.e. bis-thiophosphonate compounds. If these compounds are based on a steroidal nucleus, preferably the second (or at least one of the additional) thiophosphonate group is located at position 17 of the steroidal nucleus. These groups need not be the same.
  • R 2 is a sulphonate group then the compound of the present invention is referred to as a sulphonate compound.
  • the sulphonate group has the formula:
  • R 14 is H, alkyl, cycloalkyl, alkenyl or aryl, or combinations thereof, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
  • the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms.
  • R 14 When R 14 is alkyl, R 14 may be a lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. By way of example, R 14 may be methyl.
  • R 14 is aryl, typical values are phenyl and tolyl (PhCH 3 ;o).
  • R 14 represents cycloalkyl
  • typical values are cyclopropyl, cyclopentyl, cyclohexyl etc.
  • R 14 may even comprise an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. to provide a 5 membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
  • alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question.
  • substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
  • the sulphonate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
  • there may be two sulphonates i.e. bis- sulphonate compounds. If these compounds are bdsed on a steroidal nucleus, preferably the second (or at least one of the additional) sulphonate group is located at position 17 of the steroidal nucleus. These groups need not be the same.
  • a sulphonate as herein defined or a phosphonate as herein defined or a thiophosphonate as herein defined or a sulphamate as herein defined there may be present one of a sulphonate as herein defined or a phosphonate as herein defined or a thiophosphonate as herein defined or a sulphamate as herein defined; and another of a sulphonate as herein defined or a phosphonate as herein defined or a thiophosphonate as herein defined or a sulphamate as herein defined.
  • the compound of the present invention may comprise one sulphamate group and one phosphonate group.
  • these compounds of the present invention are based on a steroidal nucleus, preferably the other of said groups is located at position 17 of the steroidal nucleus.
  • Steroid sulphatase activity is measured in vitro using intact MCF-7 human breast cancer cells. This hormone dependent cell line is widely used to study the control of human breast cancer cell growth. It possesses significant steroid sulphatase activity (Maclndoe et al. Endocrinology, 123, 1281-1287 (1988); Purohit & Reed, Int. J. Cancer, 50, 901- 905 (1992)) and is available in the U.S.A. from the American Type Culture Collection (ATCC) and in the U.K. (e.g. from The Imperial Cancer Research Fund).
  • ATCC American Type Culture Collection
  • MEM Minimal Essential Medium
  • HEPES Flow Laboratories, Irvine, Scotland
  • 5% foetal bovine serum 20 mM HEPES
  • 2 mM glutamine 2 mM glutamine
  • non- essential amino acids 0.075% sodium bicarbonate.
  • Up to 30 replicate 25 cm2 tissue culture flasks are seeded with approximately 1 x 10 5 cells/flask using the above medium. Cells are grown to 80% confluency and the medium is changed every third day.
  • Intact monolayers 'of MCF-7 cells in triplicate 25 cm 2 tissue culture flasks are washed with Earle's Balanced Salt Solution (EBSS from ICN Flow, High Wycombe, U.K.) and incubated for 3-4 hours at 37°C with 5 pmol (7 x 10 5 dpm) [6,7-3H]oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass.., U.S.A.) in serum-free MEM (2.5 ml) together with oestrone-3-sulphamate (11 concentrations: 0; 1fM; 0.01pM; 0.1pM; 1pM; 0.01nM; 0.1nM; 1nM; O.OImM; 0.1mM; 1mM).
  • EBSS Earle's Balanced Salt Solution
  • the mass of oestrone-3-sulphate hydrolysed was calculated from the 3H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of [14C] oestrone added) and the specific activity of the substrate.
  • Each batch of experiments includes incubations of microsomes prepared from a sulphatase-positive human placenta (positive control) and flasks without cells (to assess apparent non- enzymatic hydrolysis of the substrate). The number of cell nuclei per flask is determined using a Coulter Counter after treating the cell monolayers with Zaponin. One flask in each batch is used to assess cell membrane status and viability using the Trypan Blue exclusion method (Phillips, H.J.
  • Sulphatase-positive human placenta from normal term pregnancies are thoroughly minced with scissors and washed once with cold phosphate buffer (pH 7.4, 50 mM) then re-suspended in cold phosphate buffer (5 ml/g tissue). Homogenisation is accomplished with an Ultra-Turrax homogeniser, using three 10 second bursts separated by 2 minute cooling periods in ice. Nuclei and cell debris are removed by centrifuging (4°C) at 2000g for 30 minutes and portions (2 ml) of the supernatant are stored at 20°C. The protein concentration of the supematants is determined by the method of Bradford (Anal. Biochem., 72, 248-254 (1976)).
  • Incubations (1 ml) are carried out using a protein concentration of 100 mg/ml, substrate concentration of 20 mM [6,7-3H]oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass., U.S.A.) and an incubation time of 20 minutes at 37°C. If necessary eight concentrations of compounds are employed: 0 (i.e. control); O.O ⁇ mM; 0.1 mM; 0.2mM; 0.4mM; 0.6mM; 0.8mM; I.OmM.
  • the mass of oestrone-3- sulphate hydrolysed is calculated from the 3H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of [14C]oestrone added) and the specific activity of the substrate.
  • the compounds of the present invention may be studied using an animal model, in particular in ovariectomised rats.
  • an animal model in particular in ovariectomised rats.
  • compounds which are oestrogenic stimulate uterine growth in this model compounds which are oestrogenic stimulate uterine growth.
  • the compound (10 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol).
  • a further group received the compound EMATE subcutaneously in an amount of 10 ⁇ g/day for five days.
  • samples of liver tissue were obtained and oestrone sulphatase activity assayed using 3H oestrone sulphate as the substrate as previously described (see PCT/GB95/02638).
  • the compounds of the present invention may be studied using an animal model, in particular in ovariectomised rats.
  • an animal model in particular in ovariectomised rats.
  • compounds which are oestrogenic stimulate uterine growth.
  • the compound (10 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol).
  • a further group received the estrogenic compound EMATE subcutaneously in an amount of 10 ⁇ g/day for five days.
  • uteri were obtained and weighed with the results being expressed as uterine weight/whole body weight x 100.
  • the ability of compounds to inhibit oestrone sulphatase activity can also be assessed using amino acid sequences or nucleotide sequences encoding STS, or active fragments, derivatives, homologues or variants thereof in, for example, high-through put screens.
  • any one or more of appropriate targets - such as an amino acid sequence and/or nucleotide sequence - may be used for identifying an agent capable of modulating STS in any of a variety of drug screening techniques.
  • the target employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of target activity or the formation of binding complexes between the target and the agent being tested may be measured.
  • the assay of the present invention may be a screen, whereby a number of agents are tested.
  • the assay method of the present invention is a high through put screen.
  • Techniques for drug screening may be based on the method described in Geysen, European Patent Application 84/03564, published on September 13, 1984.
  • large numbers of different small peptide test compounds are synthesised on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with a suitable target or fragment thereof and washed. Bound entities are then detected - such as by appropriately adapting methods well known in the art.
  • a purified target can also be coated directly onto plates for use in a drug screening techniques.
  • non-neutralising antibodies can be used to capture the peptide and immobilise it on a solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a target specifically compete with a test compound for binding to a target.
  • HTS high throughput screening
  • the present invention relates to a method of identifying agents that selectively modulate STS, which compounds have the formula (la).
  • reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • reporter molecules i ⁇ clude but are not limited to ( ⁇ -galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, (- glucuronidase, exo-glucanase and glucoamylase.
  • radiolabelled or fluorescent tag-labelled nucleotides can be incorporated into nascent transcripts which are then identified when bound to oligonucleotide probes.
  • reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3817837; US-A-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 and US-A-4366241.
  • host cell in relation to the present invention includes any cell that could comprise the target for the agent of the present invention.
  • a further embodiment of the present invention provides host cells transformed or transfected with a polynucleotide that is or expresses the target of the present invention.
  • a polynucleotide that is or expresses the target of the present invention.
  • said polynucleotide is carried in a vector for the replication and expression of polynucleotides that are to be the target or are to express the target.
  • the cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
  • the gram negative bacterium E. coli is widely used as a host for heterologous gene expression.
  • large amounts of heterologous protein tend to accumulate inside the cell.
  • Subsequent purification of the desired protein from the bulk of E.coli intracellular proteins can sometimes be difficult.
  • bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium.
  • Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas.
  • eukaryotic hosts such as yeasts or other fungi may be preferred.
  • yeast cells are preferred over fungal cells because they are easier to manipulate.
  • some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.
  • suitable expression hosts within the scope of the present invention are fungi such as Aspergillus species (such as those described in EP-A-0184438 and EP-A- 0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; and yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species.
  • typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var.
  • suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational ' modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
  • post-translational ' modifications e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation
  • organism in relation to the present invention includes any organism that could comprise the target according to the present invention and/or products obtained therefrom. Examples of organisms may include a fungus, yeast or a plant.
  • transgenic organism in relation to the present invention includes any organism that comprises the target according to the present invention and/or products obtained.
  • the host organism can be a prokaryotic or a eukaryotic organism.
  • suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.
  • nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
  • the transgenic organism can be a yeast.
  • yeast have also been widely used as a vehicle for heterologous gene expression.
  • the species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression.
  • Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey ef al (1987, Yeast Biotechnology, D R Berry ef al, eds, pp 401-429, Allen and Unwin, London) and by King ef al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).
  • Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
  • yeast vectors include integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
  • expression constructs are prepared by inserting the nucleotide sequence into a construct designed for expression in yeast.
  • constructs used for heterologous expression have been developed.
  • the constructs contain a promoter active in yeast fused to the nucleotide sequence, usually a promoter of yeast origin, such as the GAL1 promoter, is used.
  • a promoter of yeast origin such as the GAL1 promoter
  • a signal sequence of yeast origin such as the sequence encoding the SUC2 signal peptide, is used.
  • a terminator active in yeast ends the expression system.
  • transgenic Saccharomyces can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
  • the transformed yeast cells are selected using various selective markers.
  • markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, e.g. G418.
  • Another host organism is a plant.
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • Several techniques exist for inserting the genetic information the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system.
  • a review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.
  • the present invention also provides a method of transforming a host cell with a nucleotide sequence that is to be the target or is to express the target.
  • Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for the expression of the encoded protein.
  • the protein produced by a recombinant cell may be displayed on the surface of the cell.
  • expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).
  • the present invention also encompasses the use of variants, homologue and derivatives thereof.
  • the term “homology” can be equated with “identity”.
  • an homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be' calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the nucleotide sequence for use as the target or for expressing the target can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate and express the nucleotide sequence in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • the protein produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
  • the coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • the target amino acid sequence may be produced as a fusion protein, for example to aid in extraction and purification.
  • fusion protein partners include glutathione-S- transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and (-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the target.
  • the fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention.
  • the fusion protein may be a non-naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system.
  • the antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
  • the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence for example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric'substance expressing a heterologous epitope that is recognised by a commercially available antibody.
  • the compounds of the present invention may be used as therapeutic agents - i.e. in therapy applications.
  • the term "therapy” includes curative effects, alleviation effects, and prophylactic effects.
  • the therapy may be on humans or animals, preferably female animals.
  • the present invention provides a pharmaceutical composition, which comprises a compound according to the present invention and optionally a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Preservatives may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the formulation may be designed to be delivered by both routes.
  • the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
  • compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously.
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • the compound of the present invention may be used in combination with one or more other active agents, such as one or more other pharmaceutically active agents.
  • the compounds of the present invention may be used in combination with other STS inhibitors and/or other inhibitors such as an aromatase inhibitor (such as for example, 4hydr ⁇ xyandrostenedione (4-OHA)) and/or steroids - such as the naturally occurring sterneursteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and/or other structurally similar organic compounds.
  • an aromatase inhibitor such as for example, 4hydr ⁇ xyandrostenedione (4-OHA)
  • steroids - such as the naturally occurring sterneurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and/or other structurally similar organic compounds.
  • DHEAS dehydroepiandrosterone sulfate
  • PS pregnenolone sulfate
  • the compound of the present invention may be used in combination with a biological response modifier.
  • the term biological response modifier includes cytokines, immune modulators, growth factors, haematopoiesis regulating factors, colony stimulating factors, chemotactic, haemolytic and thrombolytic factors, cell surface receptors, ligands, leukocyte adhesion molecules, monoclonal antibodies, preventative and therapeutic vaccines, hormones, extracellular matrix components, fibronectin, etc.
  • the biological response modifier is a cytokine.
  • cytokines include: interleukins (IL) - such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-
  • TNF Tumour Necrosis Factor
  • TNF Tumour Necrosis Factor
  • the cytokine is tumour necrosis factor (TNF).
  • TNF may be any type of TNF - such as TNF- ⁇ , TNF- ⁇ , including derivatives or mixtures thereof. More preferably the cytokine is TNF- ⁇ . Teachings on TNF may be found in the art - such as WO-A-98/08870 and WO-A-98/13348.
  • a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.
  • the dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
  • compositions of the present invention may be administered by direct injection.
  • the composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
  • the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • the agents of the present invention may be administered in accordance with a regimen of 1 to 4 times per day, preferably once or twice per day.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing, therapy.
  • administered also includes delivery by techniques such as lipid mediated transfection, liposomes, immuholiposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
  • routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
  • administered includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
  • the STS inhibitors of the present invention can be formulated in any suitable manner utilising conventional pharmaceutical formulating techniques and pharmaceutical carriers, adjuvants, excipients, diluents etc. and usually for parenteral administration.
  • Approximate effective dose rates may be in the range from 1 to 1000 mg/day, such as from 10 to 900 mg/day or even from 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of average (70Kg) bodyweight. More usual dosage rates for the preferred and more active compounds will be in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day, most preferably from 200 to 250 mg/day.
  • the compounds may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days.
  • oral administration they may be formulated in tablets, capsules, solution or suspension containing from 100 to 500 mg of compound per unit dose.
  • the compounds will be formulated for parenteral administration in a suitable parenterally administrable carrier and providing single daily dosage rates in the range 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250 mg.
  • Such effective daily doses will, however, vary depending on inherent activity of the active ingredient and on the bodyweight of the patient, such variations being within the skill and judgement of the physician.
  • the compounds of the present invention may be useful in the method of treatment of a cell cycling disorder.
  • Yeast cells can divide every 120 min., and the first divisions of fertilised eggs in the embryonic cells of sea urchins and insects take only 1530 min. because one large pre-existing cell is subdivided. However, most growing plant and animal cells take 10-20 hours to double in number, and some duplicate at a much slower rate. Many cells in adults, such as nerve cells and striated muscle cells, do not divide at all; others, like the fibroblasts that assist in healing wounds, grow on demand but are otherwise quiescent.
  • FACS fluorescence-activated cell sorter
  • the stages of mitosis and cytokinesis in an animal cell are as follows
  • each visible chromosome structure is composed of two chromatids held together at their centromeres. Each chromatid contains one of the two newly replicated daughter DNA molecules.
  • the microtubular spindle begins to radiate from the regions just adjacent to the centrioles, which are moving closer to their poles. Some spindle fibres reach from pole to pole; most go to chromatids and attach at kinetochores.
  • Metaphase The chromosomes move toward the equator of the cell, where they become aligned in the equatorial plane. The sister chromatids have not yet separated.
  • cell cycling is an extremely important cell process. Deviations from normal cell cycling can result in a number of medical disorders. Increased and/or unrestricted cell cycling may result in cancer. Reduced cell cycling may result in degenerative conditions. Use of the compound of the present invention may provide a means to treat such disorders and conditions.
  • the compound of the present invention may be suitable for use in the treatment of cell cycling disorders such as cancers, including hormone dependent and hormone independent cancers.
  • the compound of the present invention may be suitable for the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer, pancreatic cancer etc. and other solid tumours.
  • cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer, pancreatic cancer etc. and other solid tumours.
  • cell cycling is inhibited and/or prevented and/or arrested, preferably wherein cell cycling is prevented and/or arrested.
  • cell cycling may be inhibited and/or prevented and/or arrested in the G 2 /M phase.
  • cell cycling may be irreversibly prevented and/or inhibited and/or arrested, preferably wherein cell cycling is irreversibly prevented and/or arrested.
  • irreversibly prevented and/or inhibited and/or arrested it is meant after application of a compound of the present invention, on removal of the compound the effects of the compound, namely prevention and/or inhibition and/or arrest of cell cycling, are still observable. More particularly by the term “irreversibly prevented and/or inhibited and/or arrested” it is meant that when assayed in accordance with the cell cycling assay protocol presented herein, cells treated with a compound of interest show less growth after Stage 2 of the protocol I than control cells. Details on this protocol are presented below.
  • the present 'invention provides compounds which: cause inhibition of growth of oestrogen receptor positive (ER+) and ER negative (ER-) breast cancer cells in vitro by preventing and/or inhibiting and/or arresting cell cycling; and/or cause regression of nitroso-methyl urea (NMU)-induced mammary tumours in intact animals (i.e. not ovariectomised), and/or prevent and/or inhibit and/or arrest cell cycling in cancer cells; and/or act in vivo by preventing and/or inhibiting and/or arresting cell cycling and/or act as a cell cycling agonist.
  • NMU nitroso-methyl urea
  • MCF-7 breast cancer cells are seeded into multi-well culture plates at a density of 105 cells/well. Cells were allowed to attach and grown until about 30% confluent when they are treated as follows:
  • Steroids were extracted from the aqueous medium with diethyl ether
  • the marked regions were cut out and placed in scintillation vials containing methanol
  • the compounds of the present invention may be useful in the treatment of a cell cycling disorder.
  • a particular cell cycling disorder is cancer.
  • Cancer remains a major cause of mortality in most Western countries.
  • Cancer therapies developed so far have included blocking the action or synthesis of hormones to inhibit the growth of hormone-dependent tumours.
  • more aggressive chemotherapy is currently employed for the treatment of hormone-independent tumours.
  • oestrogens undergo a number of hydroxylation and conjugation reactions after their synthesis. Until recently it was thought that such reactions were part of a metabolic process that ultimately rendered oestrogens water soluble and enhanced their elimination from the body. It is now evident that some hydroxy metabolites (e.g. 2- hydroxy and 16alpha-hydroxy) and conjugates (e.g. oestrone sulphate, E1S) are important in determining some of the complex actions that oestrogens have in the body.
  • hydroxy metabolites e.g. 2- hydroxy and 16alpha-hydroxy
  • conjugates e.g. oestrone sulphate, E1S
  • 2- and 16-hydroxylated oestrogens in relation to conditions that alter the risk of breast cancer. There is now evidence that factors which increase 2-hydroxylase activity are associated with a reduced cancer risk, while those increasing 16alpha-hydroxylation may enhance the risk of breast cancer. Further interest in the biological role of oestrogen metabolites has been stimulated by the growing body of evidence that 2-methoxyoestradiol is an endogenous metabolite with anti-mitotic properties.
  • 2-MeOE2 is formed from 2-hydroxy oestradiol (2-OHE2) by catechol oestrogen methyl transferase, an enzyme that is widely distributed throughout the body.
  • 2-MeOE2 inhibits the growth of tumours arising from the subcutaneous injection of Meth A sarcoma, B16 melanoma or MDA-MB-435 oestrogen receptor negative (ER-) breast cancer cells. It also inhibits endothelial cell proliferation and migration, and in vitro angiogenesis. It was suggested that the ability of 2-MeOE2 to inhibit tumour growth in vivo may be due to its ability to inhibit tumour- induced angiogenesis rather than direct inhibition of the proliferation of tumour cells.
  • 2-MeOE2 exerts its potent anti-mitogenic and anti-angiogenic effects. There is evidence that at high concentrations it can inhibit microtubule polymerisation and act as a weak inhibitor of colchicine binding to tubulin. Recently, however, at concentrations that block mitosis, tubulin filaments in cells were not found to be depolymerised but to have an identical morphology to that seen after taxol treatment. It is possible, therefore, that like taxol, a drug that is used for breast and ovarian breast cancer therapy, 2-MeOE2 acts by stabilising microtubule dynamics.
  • 2-MeOE2 While the identification of 2-MeOE2 as a new therapy for cancer represents an important advance, the bioavailability of orally administered oestrogens is poor. Furthermore, they can undergo extensive metabolism during their first pass through the liver. As part of a research programme to develop a steroid sulphatase inhibitor for breast cancer therapy, oestrone-3-O-sulphamate (EMATE) was identified as a potent active site-directed inhibitor. Unexpectedly, EMATE proved to possess potent oestrogenic properties with its oral uterotrophic activity in rats being a 100-times higher than that of oestradiol.
  • EMATE oestrone-3-O-sulphamate
  • rbcs red blood cells
  • the compound of the present invention provides a means for the treatment of cancers and, especially, breast cancer.
  • the compound of the present invention may be useful in the blocking the growth of cancers including leukaemias and solid tumours such as breast, endometrium, prostate, ovary and pancreatic tumours.
  • the compounds of the present invention may be useful in the control of oestrogen levels in the body - in particular in females.
  • some of the compounds may be useful as providing a means of fertility control - such as an oral contraceptive tablet, pill, solution or lozenge.
  • the compound could be in the form of an implant or as a patch.
  • the compounds of the present invention may be useful in treating hormonal conditions associated with oestrogen.
  • the compound of the present invention may be useful in treating hormonal conditions in addition to those associated with oestrogen.
  • the compound of the present invention may also be capable of affecting hormonal activity and may also be capable of affecting an immune response.
  • STS inhibitors may be useful in the enhancing the memory function of patients suffering from illnesses such as amnesia, head injuries, Alzheimer's disease, epileptic dementia, presenile dementia, post traumatic dementia, senile dementia, vascular dementia and post-stroke dementia or individuals otherwise seeking memory enhancement.
  • inflammatory conditions such as conditions associated with any one or more of: autoimmunity, including for example, rheumatoid arthritis, type I and II diabetes, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, thyroiditis, vasculitis, ulcerative colitis and Crohn's disease, skin disorders e.g. psoriasis and contact dermatitis; graft versus host disease; eczema; asthma and organ rejection following transplantation.
  • autoimmunity including for example, rheumatoid arthritis, type I and II diabetes, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, thyroiditis, vasculitis, ulcerative colitis and Crohn's disease, skin disorders e.g. psoriasis and contact dermatitis; graft versus host disease; eczema; asthma and organ rejection following transplantation.
  • STS inhibitors may prevent the normal physiological effect of DHEA or related steroids on immune and/or inflammatory responses.
  • the compounds of the present invention may be useful in the manufacture of a medicament for revealing an endogenous glucocorticoid-like effect.
  • the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-99/52890 - viz:
  • the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-98/05635.
  • cancer inflammation or inflammatory disease
  • dermatological disorders fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis
  • the compound or composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/07859.
  • cytokine and cell proliferation/differentiation activity e.g. for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumour immunity
  • regulation of haematopoiesis e.g. treatment of myeloid or lymphoid diseases
  • promoting growth of bone, cartilage, tendon, ligament and nerve tissue e.g.
  • follicle-stimulating hormone for healing wounds, treatment of burns, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic/chemokinetic activity (e.g. for mobilising specific cell types to sites of injury or infection); haemostatic and thrombolytic activity (e.g. for treating haemophilia and stroke); antiinflammatory activity (for treating e.g. septic shock or Crohn's disease); as antimicrobials; modulators of e.g. metabolism or behaviour; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine.
  • composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/09985.
  • macrophage inhibitory and/or T cell inhibitory activity and thus, anti-inflammatory activity i.e.
  • inhibitory effects against a cellular and/or humoral immune response including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-
  • retinitis or cystoid macular oedema retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus .disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferate vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g.
  • monocyte or leukocyte proliferative diseases e.g. leukaemia
  • monocytes or lymphocytes for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.
  • the sulphamate compounds of the present invention may be prepared by reacting an appropriate alcohol with a suitable chloride.
  • the sulphamate compounds of the present invention may be prepared by reacting an appropriate alcohol with a suitable sulfamoyl chloride, of the formula R 9 R 10 NSO 2 CI.
  • the alcohol is derivatised, as appropriate, prior to reaction with the sulfamoyl chloride.
  • functional groups in the alcohol may be protected in known manner and the protecting group or groups removed at the end of the reaction.
  • the sulphamate compounds are prepared according to the teachings of Page ef al (1990 Tetrahedron 46; 2059-2068).
  • the phosphonate compounds may be prepared by suitably combining the teachings of Page et al (1990 Tetrahedron 46; 2059-2068) and PCT/GB92/01586.
  • the sulphonate compounds may be prepared by suitably adapting the teachings of Page ef a/ (1990 Tetrahedron 46; 2059-2068) and PCT/GB92/01586.
  • the thiophosphonate compounds may be prepared by suitably adapting the teachings of Page ef al (1990 Tetrahedron 46; 2059-2068) and PCT/GB91/00270.
  • the present invention provides compounds for use as steroid dehydrogenase inhibitors, and pharmaceutical compositions for the same.
  • the starting 16-methylene estradiol was dissolved in a volume of THF and the same volume of ethanol added to the solution. The solution was then degassed by bubbling nitrogen through for 40min before Pd/C (5%wt., catalytic) was added and hydrogen gas (balioon) was passed over the reaction. The reaction was stirred under hydrogen at room temperature overnight before being filtered through celite.
  • the bis-acetate protected starting material (0.0137g) was dissolved in ethanol (1cm 3 ): To this was added a solution of KOH (0.004g) in ethanol/water (2cm 3 of 1/1) and the mixture was shaken then left at room temperature for 30min. TLC at this stage showed no starting material remaining therefore the mixture was acidified with glacial acetic acid, concentrated in vacuo and water added. The solution was left standing overnight during which time a pale orange precipitate formed. This powder was collected by filtration. Yield 46%.
  • GMA03028 (3-Benzyloxy-2-ethyl-13-methyl-17-oxo-6,7,8,9,11,12,13,14,15,17- ecahydro- cycl ⁇ p ⁇ n£a[ajphenanthren-16-ylid ⁇ n ⁇ )-hydr ⁇ xy-acetie acid ethyl ester
  • the loaded resin (0.03 g) was placed in a fritted syringe and washed with TFA/DCM (20 cm 3 of 1/1, added portion-wise with 5 min between washings) followed by DCM. The washings were combined and concentrated in vacuo. The resin loading from the weight of the crude was calculated to be 0.8 mmol/g.
  • the resin was collected in a fritted syringe and washed with DMF, DCM and MeOH (x3) and again with DCM (x3) before cleavage of the product using TFA/DCM (1/1).
  • GRHA03036 2-Bensylo ⁇ y-7-(2-cyano-@thyl)-3-ethyl-4b,5,6,6a,7,10,10a,10b,11,12-d ⁇ cahydro-7,8- diazapentaleno[2,1-a]phenanthrene-9-carboxylic acid ethyl ester
  • the reaction was stirred at 0°C for 1 h and allowed to react at room temperature for a
  • Stepl Loading ofJAC01041 on oxime resin.
  • oxime resin loading of 1.06 mmol/g resin
  • 251 mg of JAC01041 were added followed by the addition of 41 ⁇ L DIC and 358 mg HOBT.
  • the mixture was shaken for 48h at room temperature under inert atmosphere and the resin was filtered, washed with DCM, DMF, MeOH (3 cycles) and MeOH (5 times) and dried in vacuo.
  • Step2 Amidification. 200 mg of oxime resin loaded with JAC01041 (maximum loading of 1.06 mmol/g resin) were swollen in 4 mL dry DCM and 47 ⁇ L (0.53 mmol) furfurylamine were added to the suspension. The mixture was shacked at 40°C under nitrogen atmosphere for 24h. After filtration, the resin was washed with DCM (5 times) and the filtrated was evaporated in vacuo.
  • Step 2 General procedure for Amidification.
  • the active ester JAC01177 (0.17 mmol) was solubilized in 2mL dry acetonitrile and the amine (2 eq) was added to the mixture. The solution was stirred overnight at room temperature and the solvents were evaporated in vacuo. The residue was purified by column chromatography using flash column ISOLUTE SI 5g and an elution gradient Hexane / Ethyl Acetate.
  • Step 3 General procedure for debenzylation: The benzylated amide was solubilized in THF (3 mL). A catalytic amount of 5% Pd/C was added and the reaction mixture was stirred at room temperature under H 2 for 24 h. Pd/C was filtered over paper and the filtrat was evaporated in vacuo. The final product was precipitated in a mixture Ethyl Acetate / Hexane.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Neurology (AREA)
  • Diabetes (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Neurosurgery (AREA)
  • Biomedical Technology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Endocrinology (AREA)
  • Oncology (AREA)
  • Rheumatology (AREA)
  • Pulmonology (AREA)
  • Dermatology (AREA)
  • Hematology (AREA)
  • Reproductive Health (AREA)
  • Virology (AREA)
  • Urology & Nephrology (AREA)
  • Communicable Diseases (AREA)
  • Psychology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Emergency Medicine (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Vascular Medicine (AREA)
  • Pain & Pain Management (AREA)
  • Molecular Biology (AREA)

Abstract

There is provided a compound of Formula (I) wherein (I) R1 is a selected from (i) an alkyloxyalkyl group (ii) a nitrile group, and wherein R2 is capable of forming a hydrogen bond (iii) alkylaryl group, wherein the aryl group is substituted by other than a C1-10 group (iv) alkenylaryl group wherein the aryl group is substituted (v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group (vi) alkenylheteroaryl group, (vii) =N-O-alkyl or =N-O-H group (viii) branched alkenyl (ix) alkyl-alcohol group (x) amide or alkylamide wherein (a) the alkyl of the alkylamide is - CH2- or -CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of a I kyl heterocycle group, al ke nyl heterocycle group, alkylheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group, (xi) -CHO so that R, together with R3 provide the enol tautomer (a); OR R1 together with R3 form (xii) a pyrazole wherein (a) R4 is =N-0-alkyl or =N-0-H group, (b) the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl. group, branched alkyl group, and an amide and/or (c) the 2 position is substituted with a group selected from -OH and -0-hydrocarbyl (xiii) a heteroaryl ring to provide a compound of the formula (b); (II) R2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and (III) R3 is selected from -OH, =O, or a -C(=O)- mimetic.

Description

COMPOUND
FIELD OF INVENTION
The present invention relates to a compound. In particular the present invention provides compounds capable of inhibiting 17β-hydroxysteroid dehydrogenase (17β- HSD).
BACKGROUND TO THE INVENTION
Breast cancer is a devastating disease which remains to be a major cause of death for women in most Western countries. It is estimated to affect approximately 1 million women per year across the globe.1
Britain has one of the highest mortality rate for breast cancer in the world with over 35,000 women diagnosed each year accounting for nearly one in five of all cancer cases. It is estimated that 1 in 10 women living to the age of 85 in Britain will develop breast cancer during the course of her life. Although modern methods of treatment as well as an earlier detection of the disease have greatly improved survival rates, breast cancer remains the leading cause of death for women aged between 35-54.2
All women are at risk of breast cancer although a number of risk factors have been identified, most of them being related to women's hormonal and reproductive history as well as their family background of the disease. Women at higher risk are generally those with a strong family history of the disease, early onset of menarche, late onset of menopause or a first full-term pregnancy after the age of 30.2
In the earliest stages of a breast cancer, surgery appears to be the treatment of choice. In most of the cases, breast conserving surgical techniques, such as local incision of lump(s) in the breast(s), are involved rather than mastectomy. To prevent any recurrence of the disease, radiotherapy is often prescribed, particularly if breast conserving techniques have been involved.3 It is also used to reduce large tumours to an operable size so that conservational surgery can be carried out.4
For advanced breast cancers, when the tumour has spread or recurred, the aim in the treatment is no longer to cure but to reach a palliative control. This is the case when metastases of the tumour have reached locations such as bones, skin, lymph, node or brain. The treatment varies depending on the hormonal status of the patient (whether it is a pre- or post-menopausal woman to be treated) and depending on the type of tumour. Certain tumours have indeed been proven to rely on estrogens for their growth and development, leading to what is called a Hormone Dependent Breast Cancer (HDBC, see 1-1). While non HDBC are treated with chemotherapy, where the aim is to kill differentially tumour cells using a combination of cytotoxic agents,5 HDBC are expected to respond to endocrine therapy.
The concept of hormone dependent tumours appeared in the early 1960s, when the model of estrogens action was first introduced.6 In order for estrogens to regulate cell growth and function in humans, a specific protein, called the human Oestrogen Receptor (hER), must be present.7 This protein, localised in the nucleus, interacts with estrogens resulting in the formation of a binding complex. This acts as a transcription factor by activating production of m-RNA from specific genes, one or more of which are probably essential for efficient tumour cell growth.
Patients with a measurable level of receptor protein are classified as oestrogen-receptor- positive (ER+) with opposition to oestrogen-receptor-negative (ER-). About 50% of pre- menopausal women and 75% of post-menopausal women fall into the ER+ group8 where the development of breast cancers can be directly linked to the presence of estrogens. Endocrine therapy, where the use of drugs results in a deprivation of estrogenic stimulation to cells, has proven to be an effective approach to the treatment of HDBC. Originally, two classes of drugs, responding to different strategies, were developed: anti- oestrogens and aromatase inhibitors.
Anti-oestrogens, as antagonists of the oestrogen receptor, have been one of the first treatment considered for HDBC. Their action rely on their ability to bind competitively to the specific receptor protein hER, thus preventing access of endogenous estrogens to their specific binding site. Consequently, the natural hormone is unable to maintain tumour growth.
Of the anti-oestrogens commonly used in breast cancer therapy, tamoxifen (below) is the most widely used because of the very low toxicity profile of the molecule. Despite its non-steroidal skeleton, tamoxifen possesses a mixed agonist-antagonist activity that limits its therapeutic potential.9 In addition, some for of drug resistance has been reported in patients after long-term tamoxifen treatment.10
Novel pure anti-oestrogenic drugs, such as ICI 164384 (below), have since been discovered but the loss of potency compared with that of tamoxifen suggested the need to design more highly potent targets.11
Figure imgf000005_0001
Tamoxifen ICI 164384
For some years now, a new type of anti-oestrogen has emerged, combining oestrogen agonism on target tissues such as bone or liver and antagonism and/or minimal agonism in reproductive tissues such as breasts or uterus.12 These compounds, designed as Selective Oestrogen Receptor Modulators (SERMs), are not only potentially effective in reducing a patient's risk of breast carcinoma but they have also been shown to increase bone mineral density and prevent osteoporosis in post-menopausal women. Raloxifen is the first of this class of compounds to be used clinically.13 More SERMs are currently in clinical trials and these molecules might one day replace tamoxifen as the first line treatment for women with HDBC.
The use of therapeutic agents that inhibit one or several enzyme of the steroid biosynthesis pathway represents another important strategy to control of the development of oestrogen-dependent tumours.14 The enzyme aromatase, which converts androgenic C19 steroids to estrogenic C18 steroids, has been the prime target for reducing oestrogen levels. This enzyme complex, which contains a cytochrome P450 haemoprotein, catalyses the aromatisation of the androgen A-ring with the subsequent loss of the C19 methyl group to yield estrogens.
Aminoglutethimide (below) was the first aromatase inhibitor used for the treatment of breast cancer. It however showed a number of undesirable side effects given its wide spectrum of inhibitory effects on other P450-dependant enzymes, and attempts to improve on the original structure have led to a number of non-steroidal compounds entering clinical trials.15 The last generation developed compounds such as letrozole, which combine high potency and high selectivity for the enzyme, and are also better tolerated.
Figure imgf000006_0001
AG Letrozole
Structure of different types of aromatase inhibitors. Generation I : aminoglutethimide, AG; generation III, letrozole.
Traditionally, aromatase inhibitors are reserved as second line treatment for advanced HDBC patients whose diseases are no longer controlled by tamoxifen. However, because of the extreme good toxicity profile of some of the latest aromatase inhibitors, recent clinical trials have been conducted to assess their suitability as first line treatment for HDBC.
Strong evidence has emerged over the past decade, both biochemically and clinically, that the sole inhibition of the enzyme aromatase cannot afford an effective reduction of estrogenic stimulation to HDBC, the reason being that other pathways are involved in oestrogen biosynthesis. The sulphatase pathway is now considered to be the major route for breast tumour oestrogen synthesis since sulphatase activity was found to provide 10 fold more oestrone than the aromatase activity.16
In the sulphatase pathway, estrogens are synthesised from the highly available precursor oestrone-sulphate, via two enzymes (scheme below): oestrone sulphatase (STS) which hydrolyses oestrone-sulphate into oestrone, and 17β-hydroxysteroid dehydrogenase (17β-HSD) which reduces oestrone into oestradiol. These two enzymes represent the latest targets for oestrogen deprivation strategies. ER
Estradiol Adiol
Figure imgf000007_0001
DHEA-sulfate
Figure imgf000007_0002
Origin of estrogens in normal and tumoral breast cells. AR, aromatase; ST: steroid sulfotransferase; STS, steroid sulphatase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; 3β-IS, 3β-hydroxysteroid dehydrogenase Δ5,Δ4-isomerase; ER, oestrogen receptor.
Several potent inhibitors have been identified for oestrone sulphatase. They all share the common structural feature of an aromatic ring bearing a substituent that mimics the phenolic A-ring of the enzyme substrate, oestrone-sulphate. On the development of steroidal inhibitors, a wide variety of chemical groups have been introduced at C3, of which the 3-O-sulfamate was found to be the most potent for the oestrone molecule. The resulting compound, estrone-3-O-sulfamate (below) led to the identification of the aryl-O-sulphamate structure as an active pharmacophore required for potent inhibition of STS. EMATE was shown to inhibit steroid sulphatase activity in a time- and concentration-dependent manner17 and was active in vivo on oral administration.18 It was however revealed to be highly estrogenic which raised the need to design STS inhibitors devoid of agonist activity on hER.
To avoid the problems linked to an active steroid nucleus, non steroid-based inhibitors have been synthesised. Coumarin sulphamate such as 4-methylcoumarin-7-O- sulfamate (COUMATE, below), where the active pharmacophore is conserved, have been among the first inhibitors of that type to be identified.19 Although COUMATE is less potent than EMATE, it has the advantage of being non estrogenic.20 Some tricyclic coumarin-based sulphamates have also been developed and turned out to be much more potent than COUMATE, while retaining its non estrogenic characteristic.21 667COUMATE, which is some 3 times more potent than EMATE in vitro is now in pre- clinical development for clinical trials.22
Figure imgf000008_0001
EMATE COUMATE 667COUMATE .
Structures of the steroid sulphatase inhibitors EMATE, COUMATE and 667COUMATE.
5 PCT/GB92/01587 teaches novel steroid sulphatase inhibitors and pharmaceutical compositions containing them for use in the treatment of oestrone dependent tumours, especially breast cancer. These steroid sulphatase inhibitors are sulphamate esters, such as N,N-dimethyl oestrone-3-sulphamate and, preferably, oestrone-3-sulphamate (EMATE). It is known that EMATE is a potent E1-STS inhibitor as it displays more than 0 99% inhibition of E1-STS activity in intact MCF-7 cells at 0.1 mM. EMATE also inhibits the E1-STS enzyme in a time- and concentration-dependent manner, indicating that it
, , acts as an active site-directed inactivator. Although EMATE was originally designed for the inhibition of E1-STS, it also inhibits dehydroepiandrosterone sulphatase (DHA-STS), which is an enzyme that is believed to have a pivotal role in regulating the biosynthesis 5 of the oestrogenic steroid androstenediol. Also, there is now evidence to suggest that androstenediol may be of even greater importance as a promoter of breast tumour growth. EMATE is also active in vivo as almost complete inhibition of rat liver E1-STS (99%) and DHA-STS (99%) activities resulted when it is administered either orally or subcutaneously. In addition, EMATE has been shown to have a memory enhancing 0 effect in rats. Studies in mice have suggested an association between DHA-STS activity and the regulation of part of the immune response. It is thought that this may also occur in humans. The bridging O-atom of the sulphamate moiety in EMATE is important for inhibitory activity. Thus, when the 3-O-atom is replaced by other heteroatoms as in oestrone-3-N-sulphamate and oestrone-3-S-sulphamate, these analogues are weaker 5 non-time-dependent inactivators.
Although optimal potency for inhibition of E1-STS may have been attained in EMATE, it is possible that oestrone may be released during sulphatase inhibition and that EMATE and its oestradiol congener may possess oestrogenic activity. 0
17β-HSD, which catalyses the final step in estrogens and androgens biosynthesis, also appeared as a target for oestrogen deprivation strategies. This enzyme is responsible for the interconversion of the oxidised form (less active) and the reduced form (more active) of steroids. Its activity directly supports the growth and development of oestrogen dependent tumours since it preferably reduces oestrone into estradiol25 and in a minor extend, via the conversion of the androgen DHEA into androstenediol (Adiol), which has recently been proven to have estrogenic properties and to be able to bind to the oestrogen receptor'.26
17β-HSD belongs to a family of isoenzymes, 11 of which have been so far identified and cloned.27 Each type has a selective substrate affinity and directional activity which means that selectivity of drug action has to be achieved. 17β-HSD type 1 is the isotype that catalyses the interconversion of oestrone and oestradiol.
Unlike STS inhibitors, only few 17β-HSD inhibitors have been reported. Most of the steroidal inhibitors for 17β-HSD type 1 have in common a D-ring modified structure. Oestradiol derivatives which contain a side-chain with a good leaving group at the 16α- position have been shown to be a potent class of inhibitors. In particular, 16α- (bromoalkyl)-estradiol28 where the side-chains exhibit high reactivity towards nucleophilic amino-acids residues in the active site of the enzyme were found to be promising irreversible inhibitors. Analogues containing short bromoalkyl moieties at position 16 exhibited the highest activity with 16 -(Bromopropyl)-oestradiol, followed by 16α- (Bromobutyl)-oestradiol, the most potent of the series (3 and 4). They, however, turned out to be pure agonists of the oestrogen receptor.
Figure imgf000009_0001
3 R - (CH2)3Br Apigenin
4 R = (CH2)4Br
17β-HSD type 1 inhibitors: 16α-(bromopropyl)-oestradiol, 3; 16α-(bromopropyl)-oestradiol, 4 and a flavone derivative, apigenin.
In an attempt to eliminate the intrinsic oestrogen icity of potent inhibitors and possibly at the same time engineer anti-oestrogenic properties into the molecule, several 16α- (broadly)-oestradiol derivatives bearing the C7α-alkylamide side chain of the known anti- oestrogen ICI 164384 were synthesised.29 However, rather poor inhibition of 17β-HSD type 1 was obtained, with estrogenic and anti-oestrogenic properties not completely abolished or introduced respectively.
In parallel, non-steroidal inhibitors of 17β-HSD type 1 have been designed. Flavonoids, which are structurally similar to estrogens, are able to bind to the oestrogen receptor with estrogenic or anti-estrogenic activities.30 Their action on aromatase activity is well documented and in recent studies, they were found to reduce the conversion of oestrone into oestradiol catalysed by 17β-HSD type 1.31 Flavone derivatives, such as apigenin emerged from a SAR study as a promising compounds with some inhibitory activity on 1 β-HSD type 1 without being estrogenic at the inhibitory concentration.32
Ahmed et al (Biochem Biophys Res Commun 1999 Jan 27;254(3):811-5) report on a structure-activity relationship study of steroidal and nonsteroidal inhibitors of STS.
Steroid dehydrogenases (DH) such as oestradiol 17β-hydroxysteroid dehydrogenases (E2HSD) have pivotal roles in regulating the availability of ligands to interact with the oestrogen receptor. E2HSD Type I reduces oestrone (E1) to the biologically active oestrogen, oestradiol (E2), while E2HSD Type II inactivates E2 by catalysing its oxidation to E1. Thus the identification of compounds having DH inhibitory activity, in particular, inhibitors of E2HSD Type I, could be of therapeutic value in inhibiting the formation of E2.
SUMMARY ASPECTS OF THE PRESENT INVENTION
The present invention provides novel compounds which are capable of acting as effective 17β-hydroxysteroid dehydrogenase (17β-HSD) inhibitors. The present invention identifies that the compounds of the present application are effective 17β- hydroxysteroid dehydrogenase (17β-HSD) inhibitors.
Figures 1 shows some of the enzymes involved in the in situ synthesis of oestrone from oestrone sulphate, and oestradiol. "STS" denotes Oestrone Sulphatase, "E2DH Type I" denotes Oestradiol 17β-hydroxysteroid dehydrogenase Type I or Oestradiol 17β-hydroxysteroid dehydrogenase Type 1, 3, 5 and/or 7 and "E2DH Type II" denotes Oestradiol 17β-hydroxysteroid dehydrogenase Type II or Oestradiol 17β-hydroxysteroid dehydrogenase Type 2 and/or 8.
As can be seen, two enzymes that are involved in the peripheral synthesis of oestrogens are the enzyme Oestradiol 17β-hydroxysteroid dehydrogenase and the enzyme oestrone sulphatase.
In situ synthesis of oestrogen is thought to make an important contribution to the high levels of oestrogens in tumours and therefore specific inhibitors of oestrogen biosynthesis are of potential value for the treatment of endocrine-dependent tumours.
Moreover, even though oestrogen formation in malignant breast and endometrial tissues via the sulphatase pathway makes a major contribution to the high concentration of oestrogens, there are still other enzymatic pathways that contribute to in vivo synthesis of oestrogen. !
Thus, there is an urgent need to develop new therapies for the treatment of these cancers.
The present invention therefore seeks to overcome one or more of the problems associated with the prior art methods of treating breast and endometrial cancers.
In one aspect, therefore, the present invention provides a use of a compound for the preparation of a medicament that can or affect, such as substantially inhibit, the steroid dehydrogenase pathway - which pathway converts oestrone to and from oestradiol.
This aspect of the present invention is advantageous because by the administration of one type of compound it is possible to block the synthesis of oestradiol from oestrone. Hence, the present invention provides compounds that have considerable therapeutic advantages, particularly for treating breast and endometrial cancers.
The compounds of the present invention may comprise other substituents. These other substituents may, for example, further increase the activity of the compounds of the present invention and/or increase stability (ex vivo and/or in vivo). DETAILED ASPECTS OF THE PRESENT INVENTION
According to one aspect of the present invention, there is provided a compound having Formula I
Formula I
Figure imgf000012_0001
wherein (I) R1 is a selected from
(i) an alkyloxyalkyl group
(ii) a nitrile group, and wherein R2 is capable of forming a hydrogen bond (iii) alkylaryl group, wherein the aryl group is substituted by other than a C1-10 group
(iv) alkenylaryl group wherein the aryl group is substituted
(v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group (vi) alkenylheteroaryl group
(vii) =N-O-alkyl or =N-O-H group
(viii) branched alkenyl
(ix) alkyl-alcohol group
(x) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or - CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
(xi) -CHO so that R., together with R3 provide the enol tautomer
Figure imgf000012_0002
OR R., together with R3 form
(xii) a pyrazole wherein (a) R4 is =N-O-alkyl or =N-O-H group, (b) the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl group, branched alkyl group, and an amide and/or (c) the 2 position is substituted with a group selected from -OH and -O-hydrocarbyl (xiii) a heteroaryl ring to provide a compound of the formula
Figure imgf000013_0001
(II) R2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and
(III) R3 is selected from
-OH, =O, or a -C(=O)- mimetic
According to one aspect of the present invention, there is provided use of a compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with 17β-hydroxysteroid dehydrogenase (17β-HSD), wherein the compound is of Formula I
Formula I
Figure imgf000013_0002
wherein
(I) R1 is a selected from
(i) an alkyloxyalkyl group
(ii) a nitrile group
(iii) alkylaryl group, wherein the aryl group is substituted by other than a methyl group iv) alkenylaryl group v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group
) alkenylheteroaryl group i) =N-O-alkyl or =N-O-H group ii) carboxylic acid esters wherein (a) R3 is =O and/or (b) the ester is selected rom -CO2X, -CH2CO2X, and -CH2CH2CO2X, wherein X is a hydrocarbyl group ix) CO2H or alkyl-CO2H group, wherein alkyl is CH2 or CH2CH2 group x) branched alkenyl xi) alkyl-alcohol group or alkenyl-alcohol group xii) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or - CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group, (xiv) -CHO so that R^ together with R3 provide the enol tautomer
Figure imgf000014_0001
OR R., together with R3 form
(xii) a pyrazole wherein (a) R4 is =N-O-alkyl or =N-O-H group, (b) the pyrazole is substituted and/or (c) the 2 position is substituted with a group selected from -OH and -O-hydrocarbyl (xiii) a heteroaryl ring to provide a compound of the formula
Figure imgf000014_0002
(II) R2 is selected from groups capable of forming a hydrogen bond , a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and (III) R3 is selected from
-OH, =O, or a -C(=O)- mimetic
According to one aspect of the present invention, there is provided use of a compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with 17β-hydroxysteroid dehydrogenase (17β-HSD), wherein the compound is of Formula I
Formula I
Figure imgf000015_0001
wherein
(I) R is a selected from (i) an alkyloxyalkyl group
(ii) a nitrite group, and wherein R2 is capable of forming a hydrogen bond
(iii) alkylaryl group, wherein the aryl group is substituted by other than a C1-10 group
(iv) alkenylaryl group wherein the aryl group is substituted (v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group
(vi) alkenylheteroaryl group
(vii) =N-O-alkyl or =N-O-H group
(viii) branched alkenyl (ix) alkyl-alcohol group
(x) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or -
CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
(xi) -CHO so that R1 together with R3 provide the enol tautomer
Figure imgf000016_0001
OR R., together with R3 form
(xii) a pyrazole wherein (a) R4 is =N-O-alkyl or =N-O-H group, (b) the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl group, branched alkyl group, and an amide and/or (c) the 2 position is substituted with a group selected from -OH and -O-hydrocarbyl (xiii) a heteroaryl ring to provide a compound of the formula
Figure imgf000016_0002
(II) R2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and
(III) R3 is selected from
-OH, =O, or a -C(=O)- mimetic
According to one aspect of the present invention, there is provided a pharmaceutical composition comprising a compound having Formula I
Formula I
Figure imgf000016_0003
wherein (I) R1 is a selected from
(i) an alkyloxyalkyl group (ii) a nitrile group, and wherein R2 is capable of forming a hydrogen bond
(iii) alkylaryl group, wherein the aryl group is substituted by other than a C1-10 group
(iv) alkenylaryl group wherein the aryl group is substituted
(v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group
(vi) alkenylheteroaryl group
(vii) =N-O-aikyl or =N-O-H group
(viii) branched alkenyl
(ix) alkyl-alcohol group
(x) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or -
CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
(xi) -CHO so that ^ together with R3 provide the enol tautomer
Figure imgf000017_0001
OR R., together with R3 form
(xii) a pyrazole wherein (a) R4 is =N-O-alkyl or =N-O-H group, (b) the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl group, branched alkyl group, and an amide and/or (c) the 2 position is substituted with a group selected from -OH and -O-hydrocarbyl (xiii) a heteroaryl ring to provide a compound of the formula
Figure imgf000017_0002
(II) R2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and
(Ill) R3 is selected from
-OH, =O, or a -C(=O)- mimetic optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
According to one aspect of the present invention, there is provided a compound having Formula I
Formula I
Figure imgf000018_0001
wherein
(I) R1 is a selected from
(i) an alkyloxyalkyl group
(ii) a nitrile group, and wherein R2is capable of forming a hydrogen bond (iii) alkylaryl group, wherein the aryl group is substituted by other than a C1-10 group
(iv) alkenylaryl group wherein the aryl group is substituted
(v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group (vi) alkenylheteroaryl group
(vii) =N-O-alkyl or =N-O-H group
(viii) branched alkenyl
(ix) alkyl-alcohol group
(x) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or - CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
(xi) -CHO so that R1 together with R3 provide the enol tautomer
Figure imgf000019_0001
OR R., together with R3 form
(xii) a pyrazole wherein (a) RA is =N-O-alkyl or =N-O-H group, (b) the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl group, branched alkyl group, and an amide and/or (c) the 2 position is substituted with a group selected from -OH and -O-hydrocarbyl (xiii), a heteroaryl ring to provide a compound of the formula
Figure imgf000019_0002
(II) R2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and (III) R3 is selected from -OH, =O, or a -C(=O)- mimetic for use in medicine.
According to one aspect of the present invention, there is provided use of a compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 17β-hydroxysteroid dehydrogenase (17β-HSD) levels, wherein the compound is of Formula I Formula
Figure imgf000020_0001
wherein
(I) R1 is a selected from
( ) an alkyloxyalkyl group i) a nitrile group, and wherein R2 is capable of forming a hydrogen bond ii) alkylaryl group, wherein the aryl group is substituted by other than a C1-10 group
(iv) alkenylaryl group wherein the aryl group is substituted (v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group (vi) alkenylheteroaryl group (vii) =N-O-alkyl or =N-O-H group (viii) branched alkenyl (ix) alkyl-alcohol group
(x) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or - CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group, (xi) -CHO so that Ri together with R3 provide the enol tautomer
Figure imgf000020_0002
OR Ri together with R3 form
(xii) a pyrazole wherein (a) R4 is =N-O-alkyl or =N-O-H group, (b) the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl group, branched alkyl group, and an amide and/or (c) the 2 position is substituted with a group selected from -OH and -O-hydrocarbyl (xiii) a heteroaryl ring to provide a compound of the formula
Figure imgf000021_0001
(II) R2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and ,
(III) R3 is selected from -OH, =O, or a -C(=O)- mimetic
According to one aspect of the present invention, there is provided use of a compound having Formula I
Formula I
Figure imgf000021_0002
wherein (I) R1 is a selected from
(i) an alkyloxyalkyl group
(ii) a nitrile group, and wherein R2is capable of forming a hydrogen bond
(iii) alkylaryl group, wherein the aryl group is substituted by other than a C1-10 group (iv) alkenylaryl group wherein the aryl group is substituted
(v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group
(vi) alkenylheteroaryl group
(vii) =N-O-alkyl or =N-O-H group (viii) branched alkenyl (ix) alkyl-alcohol group
(x) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or -
CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
(xi) -CHO so that R1 together with R3 provide the enol tautomer
Figure imgf000022_0001
OR RT together with R3 form
(xii) a pyrazole wherein (a) R4 is =N-O-alkyl or =N-O-H group, (b) the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl group, branched alkyl group, and an amide and/or (c) the 2 position is substituted with a group selected from -OH and -O-hydrocarbyl (xiii) a heteroaryl ring to provide a compound of the formula
Figure imgf000022_0002
(II) R2 is selected from groups capable of forming a hydrogen bond, a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and
(III) R3 is selected from -OH, =O, or a -C(=O)- mimetic in the manufacture of a pharmaceutical for inhibiting 17β-hydroxysteroid dehydrogenase (17β-HSD) activity.
According to one aspect of the present invention, there is provided a method of inhibiting 17β-hydroxysteroid dehydrogenase (17β-HSD) activity in a subject in need of same, the method comprising administering a compound a compound having Formula
Formula I
Figure imgf000023_0001
wherein
(I) R1 is a selected from
(i) an alkyloxyalkyl group (ii) a nitrile group
(iii) alkylaryl group, wherein the aryl group is substituted by other than a methyl group
(iv) alkenylaryl group
(v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group (vi) alkenylheteroaryl group (vii) =N-O-alkyl or =N-O-H group
(viii) carboxylic acid esters wherein (a) R3 is =O and/or (b) the ester is selected from -CO2X, -CH2CO2X, and -CH2CH2CO2X, wherein X is a hydrocarbyl group (ix) CO2H or alkyl-CO2H group, wherein alkyl is CH2 or CH2CH2 group (x) branched alkenyl
(xi) alkyl-alcohol group or alkenyl-alcohol group
(xii) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or - CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group, (xiv) -CHO so that R1 together with R3 provide the enol tautomer
Figure imgf000023_0002
OR RT together with R3 form (xii) a pyrazole wherein (a) R4 is =N-O-alkyl or =N-O-H group, (b) the pyrazole is substituted and/or (c) the 2 position is substituted with a group selected from -OH and -O-hydrocarbyl (xiii) a heteroaryl ring to provide a compound of the formula
Figure imgf000024_0001
(II) R2 is selected from groups capable of forming a hydrogen bond , a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and (III) R3 is selected from -OH, =O, or a -C(=O)- mimetic
SOME ADVANTAGES
One key advantage of the present invention is that the compounds of the present invention can act as 17β-HSD inhibitors.
Another advantage of the compounds of the present invention is that they may be potent in vivo.
Some of the compounds of the present invention may be non-oestrogenic compounds. Here, the term "non-oestrogenic" means exhibiting no or substantially no oestrogenic activity.
Another advantage is that some of the compounds may not be capable of being metabolised to compounds which display or induce hormonal activity.
Some of the compounds of the present invention are also advantageous in that they may be orally active.
Some of the compounds of the present invention may useful for the treatment of cancer, such as breast cancer, as well as (or in the alternative) non-malignant conditions, such as the prevention of auto-immune diseases, particularly when pharmaceuticals may need to be administered from an early age.
Thus, some of the compounds of the present invention are also believed to have therapeutic uses other than for the treatment of endocrine-dependent cancers, such as the treatment of autoimmune diseases.
For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.
FURTHER/PREFERABLE ASPECTS
The term "capable of forming a hydrogen bond" as used herein means a group having a region of negative charge capable of forming one part of a hydrogen bond.
The term "hydrocarbyl group" as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo, alkoxy, nitro, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. A non- limiting example of a hydrocarbyl group is an acyl group.
A typical hydrocarbyl group is a hydrocarbon group. Here the term "hydrocarbon" means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch. In some aspects of the present invention, the hydrocarbyl group is selected from optionally substituted alkyl group, optionally substituted haloalkyl group, aryl group, alkylaryl group, alkylarylakyl group, and an alkene group.
In some aspects of the present invention, the hydrocarbyl group is an optionally substituted alkyl group.
In some aspects of the present invention, the hydrocarbyl group is selected from Cj-C10 alkyl group, such as C|-G6 alkyl group, and C C3 alkyl group. Typical alkyl groups include G, alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C7 alkyl, and C8 alkyl.
In some aspects of the present invention, the hydrocarbyl group is selected from C.|-C-10 haloalkyl group, 0,-Cg haloalkyl group, C,-C3 haloalkyl group, C,-C10 bromoalkyl group, C^Ce bromoalkyl group, and 0,-03 bromoalkyl group. Typical haloalkyl groups include C, haloalkyl, C2 haloalkyl, C3 haloalkyl, C4 haloalkyl, C5 haloalkyl, C7 haloalkyl, C8 haloalkyl, C, bromoalkyl, C2 bromoalkyl, C3 bromoalkyl, C4 bromoalkyl, C5 bromoalkyl, C7 bromoalkyl, and C8 bromoalkyl.
In some aspects of the present invention, the hydrocarbyl group is selected from aryl groups, alkylaryl groups, alkylarylakyl groups, -(CH2)1-10-aryl, -(CH2)1-10-Ph, (CH2)1-10-Ph- C1-10 alkyl, -(CH^-Ph, (CH2)1-5-Ph-C1-5 alkyl, -(CH2)1-3-Ph„ (CH2)1-3-Ph-C1-3 alkyl, -CH2- Ph, and -CH2-Ph-C(CH3)3.
When the hydrocarbyl group is or contains an aryl group, the aryl group or one or more of the aryl groups may contain a hetero atom. Thus the aryl group or one or more of the aryl groups may be carbocyclic or more may heterocyclic. Typical hetero atoms include O, N and S, in particular N.
In some aspects of the present invention, the hydrocarbyl group is selected from -(CH2)ι. 10-cycloalkyl, -(CH2)1-10-C3-10cycloalkyl, ~(CH2)1-7-C3-7cycloalkyl, -(CH2)1-5-C3.5cycloalkyl, - (CH2) -3-C3-5cycloalkyl, and -CH2- C3cycloalkyl.
In some aspects of the present invention, the hydrocarbyl group is an alkene group.
Typical alkene groups include Cj-Cι0 alkene group, C|-C6 alkene group, C C3 alkene group, such as C,, C2, C3, C4, C5, C6, or C7 alkene group. In a preferred aspect the alkene group contains 1 , 2 or 3 C=C bonds. In a preferred aspect the alkene group contains 1 C=C bond. In some preferred aspect at least one C=C bond or the only C=C bond is to the terminal C of the alkene chain, that is the bond is at the distal end of the chain to the ring system.
RING SYSTEM
It will be evident to one skilled in the art that the present compounds contain a steroidal ring structure or are based thereon/derived therefrom. As it is well known in the art, a classical steroidal ring structure has the generic formula of:
Figure imgf000027_0001
In the above formula, the rings have been labelled in the conventional manner.
An example of a bio-isostere is when any one or more of rings A, B, C and D is a heterocyclic ring and/or when any one or more of rings A, B, C and D has been substituted and/or when any one or more of rings A, B, C and D has been modified; but wherein the bio-isostere has steroidal properties.
In this regard, the ring system of the present invention is analogous to a steroidal ring structure and may be a bio-isostere of a steroidal ring structure.
The structure of a present polycyclic structure can be presented as:
C
A' B'
wherein each ring A', B', and C independently represents a heterocyclic ring or a non- heterocyclic ring, and wherein each ring may be independently substituted or unsubstituted, saturated or unsaturated.
By way of example, any one or more of rings A', B', C and D' may be independently substituted with suitable groups - such as an alkyl group, an aryl group, a hydroxy group, a halo group, a hydrocarbyl group, an oxyhydrocarbyl group etc.
At least one of A', B', and C may be a heterocyclic group (a heterocycle) or a non- heterocyclic group.
At least one of A', B', C and D' may be a saturated ring structure or an unsaturated ring structure (such as an aryl group).
Preferably, at least one of A', B', C and D' is an aryl ring.
Preferably the compound will contain, inclusive of all substituents, no more than 50 about carbon atoms, more usually no more than about 30 to 40 carbon atoms.
An example of D' is a five or six membered ring.
Preferred steroidal nuclei rings A'-D' on which the compounds of the present invention may be based include rings A-D of:
oestrones and substituted oestrones, viz: oestrone 16β-OH-oestrone
4-OH-oestrone 17-deoxyoestrone
6 -OH-oestrone 2-OH-oestrone
7α-OH-oestrone 2-MeO-oestrone
16α-OH-oestrone oestrone
oestradiols and substituted oestradiols, viz:
4-OH-17β-oestradiol 16β-OH-17β-oestradiol
6α-OH-17β-oestradiol 17α-oestradiol
7α-OH-17β-oestradiol 17β-oestradiol
4-OH-17α-oestradiol 17α-ethinyl-17β-oestradiol
6α-OH-17α-oestradiol 17β-ethinyl-17α-oestradiol
7α-OH-17α-oestradiol 17-deoxyoestradiol
16α-OH-17 -oestradiol 2-OH-17α-oestradiol
16α-OH-17β-oestradiol 2-OH-17β-oestradiol
16β-OH-17α-oestradiol 2-MeO-17α-oestradiol 2-MeO-17β-oestradiol
oestriols and substituted oestriols. viz: oestriol 17-deoxyoestriol
4-OH-oestriol 2-OH-oestriol
6α-OH-oestriol 2-MeO-oestriol
7α-OH-oestriol
dehydroepiandrosterones and substituted dehvdroepiandrosterones. viz: dehydroepiandrosterones 16α-OH-dehydroepiandrosterone
6α-OH-dehydroepiandrosterone 16β-OH-dehydroepiandrosterone
7α-OH-dehydroepiandrosterone 5-androstenediol
In one preferred aspect of the present invention the compound is of Formula II
Formula II
Figure imgf000029_0001
In one preferred aspect of the present invention the compound is of Formula III Formula III
Figure imgf000029_0002
In one preferred aspect of the present invention the compound is of Formula IV
Formula IV
Figure imgf000029_0003
RI
Alkyloxyalkyl Group
In one preferred aspect of the present invention R1 of formula I is an alkyloxyalkyl group.
The alkyloxyalkyl group is a -alkyl-O-alkyl group.
Each alkyl of the group is preferably independently has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
Each alkyl of the group may be independently branched or straight chain. Preferably one or each alkyl is straight chain
Particulariy preferred alkyloxyalkyl groups are -EtOEt, -EtOMe, -MeOEt, and -MeOMe.
Nitrile Group
In one preferred aspect of the present invention R1 may be a nitrile group or comprise a nitrile group. Nitrile group is understood to mean a -CsN group.
In one preferred aspect of the present invention R of formula I is a nitrile group.
In one preferred aspect when R1 is a nitrile group, R2 is -OH.
In one preferred aspect R1 of formula I is a nitrile group and R2is -OH.
Alkylaryl Group
In one preferred aspect of the present invention R1 of formula I is an alkylaryl group.
It will be understood that by alkylaryl group it is meant a group denoted by -alkyl-aryl.
The alkyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons. The alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
The aryl group typically has a six membered ring
The aryl group is substituted. In preferred aspects the aryl group of the alkylaryl group is substituted by a group selected from N,N-dialkyl, alkoxy, nitro (-NO2 "), nitrile, and a∑aalkyl groups.
Preferably the N,N-dialkyl is -N(C1-10 alkyl)2, -N(C1-5 alkyl)2, or -N(C1-3 alkyl)2. Particularly preferred is NMe2.
Preferably the alkoxy group is C1-10 alkoxy, C1-5 alkoxy, and C1-3 alkoxy. Particularly preferred is methoxy.
In a highly preferred aspect the alkylaryl group is -CH2-Ph.
Alkenylaryl Group
In one preferred aspect of the present invention R1 of formula I is an alkenylaryl group.
It will be understood that by alkylaryl group it is meant a group denoted by -alkenyl-aryl.
In one preferred aspect of the present invention, in the present use or method the aryl group of the alkenylaryl group is substituted.
The alkenyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
The alkenyl group may be branched or straight chain. Preferably the alkenyl group is straight chained.
The aryl group typically has a six membered ring The aryl group is substituted. In preferred aspects the aryl group of the alkylaryl group is substituted by a group selected from N,N-dialkyl, alkoxy, nitro (-NO2 "), nitrile, and azaalkyl groups.
Preferably the N,N-dialkyl is -N(C1-10 alkyl)2, -N(C1-5 alkyl)2, or -N(C1-3 Particularly preferred is NMe2.
Preferably the alkoxy group is C1-10 alkoxy, C1-5 alkoxy, and C1-3 alkoxy. Particularly preferred is methoxy.
In a highly preferred aspect the alkenylaryl group is =CH-Ph.
Alkyiheteroaryl Group
In one preferred aspect of the present invention R1 of formula I is an alkyiheteroaryl group.
The alkyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
The alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
The aryl group typically has a six membered ring
The heteroarylgroup contain carbon and hetero atoms. Typical hetero atoms include O, N and S, in particular N.
The aryl group may be substituted or unsubstituted. Preferably the aryl group is substituted. In preferred aspects the aryl group of the alkylaryl group is substituted by a group selected from N,N-dialkyl, alkoxy, nitro (-NO2 "), nitrile, and azaalkyl groups.
Preferably the N,N-dialkyl is -N(C1-10 alkyl)2, -N(C1-5 alkyl)2, or -N(C1-3 alkyl)2. Particularly preferred is NMe2. Preferably the alkoxy group is C1-10 alkoxy, C1-5 alkoxy, and C1-3 alkoxy. Particularly preferred is methoxy.
In a highly preferred aspect the alkyiheteroaryl group is selected from
Figure imgf000033_0001
Alkenylheteroaryl Group
In one preferred aspect of the present invention R1 of formula I is an alkenylheteroaryl group. In one preferred aspect, R1 of formula I is an alkenylheteroaryl group, wherein the aryl group is substituted. Preferably R1 of formula I is an alkenylheteroaryl group wherein the aryl group is unsubstituted.
In one preferred aspect of the present invention R1 of formula I is an alkyiheteroaryl group.
The alkenyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
The alkenyl group may be branched or straight chain. Preferably the alkenyl group is straight chained.
The aryl group typically has a six membered ring
The heteroarylgroup contain carbon and hetero atoms. Typical hetero atoms include O, N and S, in particular N.
The aryl group may be substituted or unsubstituted. Preferably the aryl group is substituted. In preferred aspects the aryl group of the alkylaryl group is substituted by a group selected from N,N-dialkyl, alkoxy, nitro (-NO2 ), nitrile, and azaalkyl groups.
Preferably the N,N-dialkyl is -N(C1-10 alkyl)2, -N(C1-5 alkyl) or -N(C1-3 alkyl)2. Particularly preferred is NMe2. Preferably the alkoxy group is C1-10 alkoxy, C1-5 alkoxy, and C1-3 alkoxy. Particularly preferred is methoxy.
In a highly preferred aspect the alkenylheteroaryl group is selected from
Figure imgf000034_0001
Oxime Group (=N-O-Alkyl or =N-O-H Group)
In one preferred aspect of the present invention R1 of formula I is an oxime group, namely =N-O-alkyl or =N-O-H group.
Preferably the oxime group is a =N-O-alkyl group
In one preferred aspect when R1 is selected from =N-O-alkyl and =N-O-H groups and R3 is a =N-O-alkyl group or =N-O-H group.
The alkyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons.
The alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
We have also surprisingly found that when R3 is a =N-O-alkyl group or =N-O-H group, it is not essential for R1 to be a =N-O-alkyl or =N-O-H group if R4 is a =N-O-alkyl or =N-O- H group. We have found that when R3 and R4 are independently selected from =N-O- alkyl and =N-O-H groups, a 17β-HSD inhibitor may be provided. Thus a further aspect the present invention provides
a compound having Formula V Formula V
Figure imgf000035_0001
wherein R3 and R4 are independently selected from =N-O-alkyl or =N-O-H group
use of a compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with 17β-hydroxysteroid dehydrogenase (17β- HSD), wherein the compound has Formula V
Formula V
Figure imgf000035_0002
wherein R3 and R4 are independently selected from =N-O-alkyl or =N-O-H group
a pharmaceutical composition comprising a compound having Formula V
Formula V
Figure imgf000035_0003
wherein R3 and R4 are independently selected from =N-O-alkyl or =N-O-H group
a compound having Formula V
Formula V
Figure imgf000035_0004
wherein R3 and R4 are independently selected from =N-O-alkyl or =N-O-H group for use in medicine.
use of a compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 17β-hydroxysteroid dehydrogenase (17β-HSD) levels, wherein the compound is of Formula V
Formula V
Figure imgf000036_0001
wherein R3 and R4 are independently selected from =N-O-alkyl or =N-O-H group
use of a compound having Formula V
Formula V
Figure imgf000036_0002
wherein R3 and R4 are independently selected from =N-O-alkyl or =N-O-H group in the manufacture of a pharmaceutical for inhibiting 17β-hydroxysteroid dehydrogenase (17β-HSD) activity.
a method of inhibiting 7β-hydroxysteroid dehydrogenase (17β-HSD) activity in a subject in need of same, the method comprising administering a compound a compound having Formula V
Formula V
Figure imgf000036_0003
wherein R3 and R4 are independently selected from =N-O-alkyl or =N-O-H group Carboxylic Acid Esters
In one preferred aspect of the present invention R1 of formula I is a carboxylic acid ester.
In some aspects, when R1 of formula I is a carboxylic acid ester, R3 is =O and/or the ester is selected from -CO2X, -CH2CO2X, and -CH2CH2CO2X, wherein X is a hydrocarbyl group.
In one preferred aspect of the present invention, in the present use or method the carboxylic acid ester is selected from -CO2X, -CH2CO2X, and -CH2CH2CO2X, wherein X is a hydrocarbyl group.
In some aspects, When R1 of formula I is a carboxylic acid ester, R3 is =O and the ester is selected from -CO2X, -CH2CO2X, and -CH2CH2CO2X, wherein X is a hydrocarbyl group.
Preferably X is a hydrocarbon group. More preferably X is an alkyl group such as an alkyl group preferably having from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1, 2 or 3 carbons.
The alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
CO H Or Alkyl-CO H Group
In one preferred aspect of the present invention R1 of formula I is -CO2H or alkyl-CO2H group
The alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
The alkyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2 or 3 carbons. Branched Alkenyl
In one preferred aspect of the present invention R1 of formula I is a branched alkenyl group.
The branched alkenyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2, 3 or 4 carbons.
Alkyl-Alcohol Group Or Alkenyl-Alcohol Group
In one preferred aspect of the present invention R1 of formula I is an alkyl-alcohol group or alkenyl-alcohol group.
The alkyl group may be branched or straight chain. Preferably the alkyl group is straight chained.
By alkyl-alcohol group or alkenyl-alcohol group it is meant a group of the formula CxH2x-2n-y(OH)y wherein x is an integer, y is an integer and n is the degree of unsaturation.
Typically x is from 1 to 20, preferably from 1 to 10, preferably from 1 to 5, preferably 1 , 2, 3 or 4. Typically y is 1 , 2 or 3. Typically n is 0, 1 , 2 or 3.
Preferably the alkenyl-alcohol has the following structural formula:
Figure imgf000038_0001
In one preferred aspect the alkenyl alcohol is substituted. In this aspect preferably a substituent replaces Ha and/or Hb in the following structural formula:
Figure imgf000038_0002
Suitable substituents include ester groups, haloalkyl groups, aryl groups such as heteroaryl groups, and alkyl groups.
Preferred substituted alkenyl-alcohol groups include
Figure imgf000039_0001
wherein X is a hydrocarbyl group, preferably a hydrocarbon, more preferably an alkyl group, preferably having from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1, 2, or 3 carbons. In a highly preferred aspect X is an ethyl group.
Amide or Alkylamide
In one preferred aspect of the present invention R1 of formula I is an amide or alkylamide group.
In one aspect, R1 of formula I is an amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or -CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
Preferably, in one aspect, R1 of formula I is an amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or -CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
In one preferred aspect the amide is of the formula -C(=O)NR5R6 or -N(CO-R7)R8, wherein R5, R6, R7 and R8 are independently selected from H and hydrocarbyl groups. Preferred amide groups include NHCO-C1-10alkyl, CONH C1-10alkyl, NHCO(CH2)1-10CH3, CONH(CH2)1-10CH3, NHCO(CH2)3.7CH3, CONH(CH2)3-7CH3, NHCO(CH2)6CH3, CONH(CH2)6CH3.
In one preferred aspect the amide or alkylamide is an alkylamide.
Preferred alkylamide groups include C1-10alkyl-NHCO-C1-10alkyl, Cnoalkyl-CO H-C,. 10alkyl, C1-10alkyl-NHCO(CH2)1-10CH3, C1-10alkyl-CONH(CH2)1-10CH3, C1-10alkyl- NHCO(CH2)3-7CH3, C1-10alkyl-CONH(CH2)3-7CH3, C1-10alkyl-NHCO(CH2)6CH3, C-Moalkyl- CONH(CH2)6CH3.
In one preferred aspect the substituents of the di-substituted amide together form a cyclic structure. '
In one preferred aspect the substituents of the di-substituted amide together form an aryl ring.
In one preferred aspect the substituents of the di-substituted amide together form a heterocyclic ring.
-CHO (Enol Tautomer)
In one preferred aspect of the present invention R1 of formula I is together with R3 provide the enol tautomer
Figure imgf000040_0001
Ri together with R3 ' Pyrazole
In one preferred aspect of the present invention R1 of formula I together with R3 form a pyrazole. In one preferred aspect of the present invention, in the present use or method the pyrazole is substituted. Preferably the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl group, branched alkyl group, and an amide.
In one preferred aspect of the present invention R1 of formula I together with R3 form a pyrazole, wherein the pyrazole is substituted with one or more (i) an alkyloxyalkyl group, (ii) a nitrile group, (iii) alkylaryl group, (iv) alkenylaryl group, (v) alkyiheteroaryl group, (vi) alkenylheteroaryl group, (viii) carboxylic acid ester, (ix) CO2H or alkyl-CO2H group, (x) branched alkenyl, (xi) alkyl-alcohol group or alkenyl-alcohol group, (xii) amide or alkylamide, as described herein.
In one preferred aspect of the present invention, when R1 and R3 form a pyrazole, the 2 position of the ring system is substituted with a group selected from -OH and -O- hydrocarbyl, preferably selected from -OH and -O-alkyl.The alkyl group preferably has from 1 to 20 carbons, preferably from 1 to 10 carbons, preferably from 1 to 5 carbons, preferably 1 , 2, 3 or 4 carbons.
By the term "the 2 position of the ring system" it is meant the position on the steriodal ring system which is labelled '2' on the formula below:
Figure imgf000041_0001
In one preferred aspect of the present invention, when R and R3 form a pyrazole the pyrazole is substituted. In this aspect, the addition of substituents to the pyrazole ring may remove unsaturation.
Examples of substituted pyrazole rings include:
Figure imgf000041_0002
Figure imgf000042_0001
R± together with R3' Heteroaryl Ring
In one preferred aspect of the present invention R1 of formula I together with R3 forms a heteroaryl ring
The aryl group typically has a six membered ring
The heteroarylgroup contains carbon and hetero atoms. Typical hetero atoms include O, N and S, in particular N.
The aryl group may be substituted or unsubstituted. Preferably the aryl group is unsubstituted.
R2
R2 is selected from groups capable of forming a hydrogen bond , a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group.
Preferably R2 is selected from groups capable of forming a hydrogen bond and a sulphamate group.
Preferably the sulphamate group is of the formula
Figure imgf000043_0001
wherein R9 and R10 are independently selected from H, alkyl, cycloalkyl, alkenyl and aryl, or combinations thereof, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
In one preferable aspect at least one of R9 and R10 is H.
In one preferable aspect R9 and R10 are both H
Preferably the group capable of forming a hydrogen bond are selected from -OH
R2 is selected from -OH, =O, or a -C(=O) mimetic.
The term "-C(=O) mimetic" would be understood to one skilled in the art
Preferably the -C(=O) mimetic is selected from -CN, =N-O-alkyl group, =N-O-H group, pyridine, pyrimidine, and groups of the formula
Figure imgf000043_0002
In one preferred aspect R3 is selected from -OH, and =O.
In one preferred aspect R3 is =O.
SUBSTITUENTS
The compound of the present invention may have substituents other than those of the ring systems show herein. Furthermore the ring systems herein are given as general formulae and should be interpreted as such. The absence of any specifically shown substituents on a given ring member indicates that the ring member may substituted with any moiety of which H is only one example. The ring system may contain one or more degrees of unsaturation, for example is some aspects one or more rings of the ring system is aromatic. The ring system may be carbocyclic or may contain one or more hetero atoms.
The compound of the invention, in particular the ring system compound of the invention of the present invention may contain substituents other than those show herein. By way of example, these other substituents may be one or more of: one or more sulphamate group(s), one or more phosphonate group(s), one or more thiophosphonate group(s), one or more sulphonate group(s), one or more sulphonamide group(s), one or more halo groups, one or more O groups, one or more hydroxy groups, one or more amino groups, one or more sulphur containing group(s), one or more hydrocarbyl group(s) - such as an oxyhydrocarbyl group.
In general terms the ring system A'B'C'D' of the present compounds may contain a variety of non-interfering substituents. In particular, the ring system A'B'C'D' may contain one or more hydroxy, alkyl especially lower (C.,-C6) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n- butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C,-C6) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
For some compounds of the present invention, it is preferred that the ring system is substituted with a oxyhydrocarbyl group. More preferably the A' ring of the ring system is substituted with a oxyhydrocarbyl group.
For some compounds of the present invention, it is highly preferred that at least the 2 position of the A' ring of the ring system is substituted with a oxyhydrocarbyl group.
The term "oxyhydrocarbyl" group as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the oxyhydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen.
In one embodiment of the present invention, the oxyhydrocarbyl group is a oxyhydrocarbon group.
Here the term "oxyhydrocarbon" means any one of an alkoxy group, ah oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
Typically, the oxyhydrocarbyl group is of the formula G^O (such as a C1-3O).
For some compounds of the present invention, it is highly preferred that the A' ring of the ring system is substituted with an alkoxy group.
For some compounds of the present invention, it is highly preferred that at least the 2 position of the A' ring of the ring system is substituted with an alkoxy group.
Preferably the alkoxy group is methoxy.
For some compounds of the present invention, it is preferred that the ring system is substituted with a hydrocarbylsulphanyl group. More preferably the A' ring of the ring system is substituted with a hydrocarbylsulphanyl group. The term "hydrocarbylsulphanyl" means a group that comprises at least hydrocarbyl group (as herein defined) and sulphur, preferably -S-hydrocarbyl, more preferably -S- hydrocarbon. That sulphur group may be optionally oxidised.
For some compounds of the present invention, it is highly preferred that at least the 2 position of the A' ring of the ring system is substituted with a hydrocarbylsulphanyl group. Preferably the hydrocarbylsulphanyl group is -S-C1-10 alkyl, more preferably -S-C,.5 alkyl, more preferably -S-C1-3 alkyl, more preferably -S-CH2CH2CH3, -S-CH2CH3 or -SCH3
For some compounds of the present invention, it is highly preferred that at least the A' ring of the ring system is substituted with an hydrocarbyl group.
For some compounds of the present invention, it is highly preferred that at least the 2 position of the A' ring of the ring system is substituted with an alkyl group.
Preferably the alkyl group is ethyl.
For some compounds of the present invention, it is highly preferred that the compound comprises at least two or more of sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group or a sulphonamide group.
For some compounds of the present invention, it is highly preferred that the compound comprises at least two sulphamate groups. .
For some compounds of the present invention, it is highly preferred that the compound comprises at least two sulphamate groups, wherein said sulphamate groups are not on the same ring.
For some compounds of the present invention, it is highly preferred that the A' ring of the ring system comprises at least one sulphamate group and wherein the D' ring of the ring system comprises at least one sulphamate group.
In some aspects of the present invention, preferably the A' ring contain one or more of an alkoxy substituent and an alkyl substituent. Thus according to further aspects of the present invention, the compound has one of the following formulae Formula VI
Figure imgf000046_0001
Formula VII
Formula VIII
Formula IX
Figure imgf000047_0001
wherein R 1 is an alkoxy group or an alkyl group
Preferably R1 is an alkyl group. Preferably R1 is a C,-C10 alkyl group, preferably a C C6 alkyl group, preferably a C|-C3 alkyl group. Preferably R1 is -CH3 or -CH2CH3.
In another preferred aspect, R 1 is an alkoxy group. Preferably R11 is methoxy.
FURTHER ASPECTS
According to a further aspect of the present invention there is provided a method comprising (a) performing a steroid dehydrogenase assay with one or more candidate compounds having the formula as defined herein; (b) determining whether one or more of said candidate compounds is/are capable of modulating steroid dehydrogenase activity; and (c) selecting one or more of said candidate compounds that is/are capable of modulating steroid dehydrogenase activity.
According to a further aspect of the present invention there is provided a method comprising (a) performing a steroid dehydrogenase assay with one or more candidate compounds having the formula as defined herein; (b) determining whether one or more of said candidate compounds is/are capable of inhibiting steroid dehydrogenase activity; and (c) selecting one or more of said candidate compounds that is/are capable of inhibiting steroid dehydrogenase activity.
In any one of the methods of the present invention, one or more additional steps may be present. For example, the method may also include the step of modifying the identified candidate compound (such as by chemical and/or enzymatic techniques) and the optional additional step of testing that modified compound for steroid dehydrogenase inhibition effects (which may be to see if the effect is greater or different). By way of further example, the method may also include the step of determining the structure (such as by use of crystallographic techniques) of the identified candidate compound and then performirtg computer modelling studies - such as to further increase its steroid dehydrogenase inhibitory action. Thus, the present invention also encompasses a computer having a dataset (such as the crystallographic co-ordinates) for said identified candidate compound. The present invention also encompasses that identified candidate compound when presented on a computer screen for the analysis thereof - such as protein binding studies.
According to one aspect of the present invention, there is provided a compound identified by the method of the present invention.
According to one aspect of the present invention, there is provided a compound according to the present invention for use in medicine.
According to one aspect of the present invention, there is provided a pharmaceutical composition comprising the compound according to the present invention optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
According to one aspect of the present invention, there is provided the use of a compound according to the present invention in the manufacture of a medicament for use in the therapy of a condition or disease associated with steroid dehydrogenase.
According to one aspect of the present invention, there is provided the use of a compound according to the present invention in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse steroid dehydrogenase levels.
For some applications, preferably the compounds have no, or a minimal, oestrogenic effect.
For some applications, preferably the compounds have an oestrogenic effect.
For some applications, preferably the compounds have a reversible action.
For some applications, preferably the compounds have an irreversible action. •
In one embodiment, the compounds of the present invention are useful for the treatment of breast cancer.
The compounds of the present invention may be in the form of a salt.
The present invention also covers novel intermediates that are useful to prepare the compounds of the present invention. For example, the present invention covers novel alcohol precursors for the compounds. By way of further example, the present invention covers bis protected precursors for the compounds. Examples of each of these precursors are presented herein. The present invention also encompasses a process comprising each or both of those precursors for the synthesis of the compounds of the present invention.
We have also identified that in some aspects of the present invention the present compounds may also inhibit the activity of steroid dehydrogenase (HSD).
By steroid dehydrogenase or HSD it is meant 17β hydroxy steroid dehydrogenase. In one aspect the 17β hydroxy steroid dehydrogenase is EC 1.1.1.62
Preferably the HSD is of Type 1 , 3, 5 and/or 7. Preferably the HSD converts oestrone (ketone) to oestradiol (hydroxy).
Preferably the HSD is of Type 2 and/or 8. Preferably the HSD converts oestradiol (hydroxy) to oestrone (ketone). In some aspects of the present invention, it is preferred that the steroid dehydrogenase is steroid dehydrogenase Type I.
In some aspects of the present invention, it is preferred that the steroid dehydrogenase is steroid dehydrogenase Type II
Preferably the HSD is of Type 1 , 3, 5 and/or 7. Preferably the HSD converts oestrone (ketone) to oestradiol (hydroxy).
Preferably the HSD is of Type 2 and/or 8. Preferably the HSD converts 'oestradiol (hydroxy) to oestrone (ketone).
STEROID DEHYDROGENASE
Steroid dehydrogenase or "DH" for short may be classified as consisting of two types - Type I and Type II. The two types of enzyme, such as oestradiol 17β-hydroxysteroid dehydrogenases (E2HSD), have pivotal roles in regulating the availability of ligands to interact with the oestrogen receptor. Type I reduces oestrone (E1) to the biologically active oestrogen, oestradiol (E2) while E2HSD Type II inactivates E2 by catalysing its oxidation to E1.
DH INHIBITION
It is believed that some disease conditions associated with DH activity are due to conversion of a nonactive, oestrone to an active, oestradiol. In disease conditions associated with DH activity, it would be desirable to inhibit DH activity.
Here, the term "inhibit" includes reduce and/or eliminate and/or mask and/or prevent the detrimental action of DH.
DH INHIBITOR
In accordance with the present invention, the compound of the present invention is capable of acting as an DH inhibitor.
Here, the term "inhibitor" as used herein with respect to the compound of the present invention means a compound that can inhibit DH activity - such as reduce and/or eliminate and/or mask and/or prevent the detrimental action of DH. The DH inhibitor may act as an antagonist.
The ability of compounds to inhibit steroid dehydrogenase activity can be assessed using either T47D breast cancer cells in which E2HSD Type I activity is abundant or MDA-MB-231 cells for Type II inhibitor studies. In both cell lines formation of products is linear with respect to time and cell numbers. Details on a suitable Assay Protocol are presented in the Examples section.
It is to be noted that the compound of the present invention may have other beneficial properties in addition to or in the alternative to its ability to inhibit DH activity.
STEROID SULPHATASE
In some aspects the compounds defined herein may also inhibit steroid sulphatase.
Steroid sulphatase - which is sometimes referred to as steroid sulphatase or steryl sulphatase or "STS" for short - hydrolyses several sulphated steroids, such as oestrone sulphate, dehydroepiandrosterone sulphate and cholesterol sulphate. STS has been allocated the enzyme number EC 3.1.6.2.
STS has been cloned and expressed. For example see Stein ef al (J. Biol. Chem. 264:13865-13872 (1989)) and Yen et al (Cell 49:443-454(1987)).
STS is an enzyme that has been implicated in a number of disease conditions.
By way of example, workers have found that a total deficiency in. STS produces ichthyosis. According to some workers, STS deficiency is fairly prevalent in Japan. The same workers (Sakura ef al, J Inherit Metab Dis 1997 Nov;20(6):807-10) have also reported that allergic diseases - such as bronchial asthma, allergic rhinitis, or atopic dermatitis - may be associated with a steroid sulphatase deficiency.
In addition to disease states being brought on through a total lack of STS activity, an increased level of STS activity may also bring about disease conditions. By way of example, and as indicated above, there is strong evidence to support a role of STS in breast cancer growth and metastasis.
STS has also been implicated in other disease conditions. By way of example, Le Roy et al (Behav Genet 1999 Mar;29(2):131-6) have determined that there may be a genetic correlation between steroid sulphatase concentration and initiation of attack behaviour in mice. The authors conclude that sulphatation of steroids may be the prime mover of a complex network, including genes shown to be implicated in aggression by mutagenesis.
STS INHIBITION
It is believed that some disease conditions associated with STS activity are due to conversion of a nonactive, sulphated oestrone to an active, nonsulphated oestrone. In disease conditions' associated with STS activity, it would be desirable to inhibit STS activity.
Here, the term "inhibit" includes reduce and/or eliminate and/or mask and/or, prevent the detrimental action of STS.
STS INHIBITOR
In accordance with the present invention, the compound of the present invention is capable of acting as an STS inhibitor.
Here, the term "inhibitor" as used herein with respect to the compound of the present invention means a compound that can inhibit STS activity - such as reduce and/or eliminate and/or mask and/or prevent the detrimental action of STS. The STS inhibitor may act as an antagonist.
The ability of compounds to inhibit oestrone sulphatase activity can be assessed using either intact MCF-7 breast cancer cells or placental microsomes. In addition, an animal model may be used. Details on suitable Assay Protocols are presented in following sections. It is to be noted that other assays could be used to determine STS activity and thus STS inhibition. For example, reference may also be made to the teachings of WO- A-99/50453.
Preferably, for some applications, the compound is further characterised by the feature that if the sulphamate group were to be substituted by a sulphate group to form a sulphate derivative, then the sulphate derivative would be hydrolysable by an enzyme having steroid sulphatase (E.G. 3.1.6.2) activity - i.e. when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37°C.
5
In one preferred embodiment, if the sulphamate group of the compound were to be replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity and would yield a Km value of less than 200 mmolar, preferably less 0 than 150 mmolar, preferably less than 100 mmolar, preferably less than 75 mmolar, preferably less than 50 mmolar, when incubated with steroid sulphatase EC 3.1.6.2 at
■ pH 7.4 and 37°C.
In one preferred embodiment, if the sulphamate group of the compound were to be 5 replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable » by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity and would yield a Km value of less than 200 μmolar, preferably less than 150 μmolar, preferably less than 100 μmolar, preferably less than 75 μmolar, preferably less than 50 μmolar, when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 0 37°C.
In a preferred embodiment, the compound of the present invention is not hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity.
5 For some applications, preferably the compound of the present invention has at least about a 100 fold selectivity to a desired target (e.g. STS), preferably at least about a 150 fold selectivity to the desired target, preferably at least about a 200 fold selectivity to the desired target, preferably at least about a 250 fold selectivity to the desired target, preferably at least about a 300 fold selectivity to the desired target, preferably at least 0 about a 350 fold selectivity to the desired target.
It is to be noted that the compound of the present invention may have other beneficial properties in addition to or in the alternative to its ability to inhibit HSD activity. SULPHAMATE GROUP
In one embodiment, the ring X has a sulphamate group as a substituent. The term "sulphamate" as used herein includes an ester of sulphamic acid, or an ester of an N- substituted derivative of sulphamic acid, or a salt thereof.
If R1 is a sulphamate group then the compound of the present invention is referred to as a sulphamate compound.
Typically, the sulphamate group has the formula:
(R9)( R10)N-S(O)(O)-O-
wherein preferably R9 and R10 are independently selected from H, alkyl, cycloalkyl, alkenyl and aryl, or combinations thereof, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R9 and/or R 0 is alkyl, the preferred values are those where R9 and R10 are each independently selected from lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. R4 and R5 may both be methyl. When R9 and/or R10 is aryl, typical values are phenyl and tolyl (PhCH3; o). Where R9 and R10 represent cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R9 and R10 typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. to provide a 5 membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl. In some embodiments, the sulphamate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
In some embodiments, there may be more than one sulphamate group. By way of example, there may be two sulphamates (i.e. bis-sulphamate compounds). If these compounds are based on a steroidal nucleus, preferably the second (or at least one of the additional) sulphamate group is located at position 17 of the steroidal nucleus. These groups need not be the same.
In some preferred embodiments, at least one of R9 and R10 is H.
In some further preferred embodiments, each of R9 and R10 is H.
PHOSPHONATE GROUP
If R1 is a phosphonate group then the compound of the present invention is referred to as a phosphonate compound.
Typically, the phosphonate group has the formula:
(R12)-P(O)(OH)-O-
wherein preferably R12 is H, alkyl, cycloalkyl, alkenyl or aryl, or combinations thereof, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R 2 is alkyl, R12 may be a lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. By way of example, R12 may be methyl. When R6 is aryl, typical values are phenyl and tolyl (PhCH3;o). Where R6 represents cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. R12 may even comprise an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. to provide a 5 membered heterocycle, e.g. morpholino, pyrrolidino or piperidino. Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
In some embodiments^ the phosphonate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
In some embodiments, there may be more than one phosphonate group. By way of example, there may be two phosphonates (i.e. bis-phosphonate compounds). If these compounds are based on a steroidal nucleus, preferably the second (or at least one of the additional) phosphonate group is located at position 17 of the steroidal nucleus. These groups need not be the same.
THIOPHOSPHONATE GROUP
If R2 is a thiophosphonate group then the compound of the present invention is referred to as a thiophosphonate compound.
Typically, the thiophosphonate group has the formula:
(R13)-P(S)(OH)-O-
wherein preferably R13 is H, alkyl, cycloalkyl, alkenyl or aryl, or combinations thereof, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R7 is alkyl, R7 may be a lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. By way of example, R7 may be methyl. When R13 is aryl, typical values are phenyl and tolyl (PhCH3;o). Where R13 represents cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. R13 may even comprise an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. to provide a 5 membered heterocycle, e.g. morpholino, pyrrolidino or piperidino. Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
In some embodiments, the thiophosphonate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
In some embodiments, there may be more than one thiophosphonate group. 'By way of example, there may be two thiophosphonates (i.e. bis-thiophosphonate compounds). If these compounds are based on a steroidal nucleus, preferably the second (or at least one of the additional) thiophosphonate group is located at position 17 of the steroidal nucleus. These groups need not be the same.
SULPHONATE GROUP
If R2 is a sulphonate group then the compound of the present invention is referred to as a sulphonate compound.
Typically, the sulphonate group has the formula:
(R 4)-S(O)(O)-O-
wherein preferably R14 is H, alkyl, cycloalkyl, alkenyl or aryl, or combinations thereof, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R14 is alkyl, R14 may be a lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. By way of example, R14 may be methyl. When R14 is aryl, typical values are phenyl and tolyl (PhCH3;o). Where R14 represents cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. R14 may even comprise an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. to provide a 5 membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
In some embodiments, the sulphonate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
In some embodiments, there may be more than one sulphonate group. By way of example, there may be two sulphonates (i.e. bis- sulphonate compounds). If these compounds are bdsed on a steroidal nucleus, preferably the second (or at least one of the additional) sulphonate group is located at position 17 of the steroidal nucleus. These groups need not be the same.
COMBINATION OF SULPHONATE/PHOSPHONATE/THIOPHOSPHONATE/SULPHAMATE
For some compounds of the present invention there may be present one of a sulphonate as herein defined or a phosphonate as herein defined or a thiophosphonate as herein defined or a sulphamate as herein defined; and another of a sulphonate as herein defined or a phosphonate as herein defined or a thiophosphonate as herein defined or a sulphamate as herein defined. By way of example, the compound of the present invention may comprise one sulphamate group and one phosphonate group.
If these compounds of the present invention are based on a steroidal nucleus, preferably the other of said groups is located at position 17 of the steroidal nucleus.
ASSAY FOR DETERMINING STS ACTIVITY USING CANCER CELLS
(PROTOCOL 1)
Inhibition of Steroid Sulphatase Activity in MCF-7 cells
Steroid sulphatase activity is measured in vitro using intact MCF-7 human breast cancer cells. This hormone dependent cell line is widely used to study the control of human breast cancer cell growth. It possesses significant steroid sulphatase activity (Maclndoe et al. Endocrinology, 123, 1281-1287 (1988); Purohit & Reed, Int. J. Cancer, 50, 901- 905 (1992)) and is available in the U.S.A. from the American Type Culture Collection (ATCC) and in the U.K. (e.g. from The Imperial Cancer Research Fund).
Cells are maintained in Minimal Essential Medium (MEM) (Flow Laboratories, Irvine, Scotland) containing 20 mM HEPES, 5% foetal bovine serum, 2 mM glutamine, non- essential amino acids and 0.075% sodium bicarbonate. Up to 30 replicate 25 cm2 tissue culture flasks are seeded with approximately 1 x 105 cells/flask using the above medium. Cells are grown to 80% confluency and the medium is changed every third day.
Intact monolayers 'of MCF-7 cells in triplicate 25 cm2 tissue culture flasks are washed with Earle's Balanced Salt Solution (EBSS from ICN Flow, High Wycombe, U.K.) and incubated for 3-4 hours at 37°C with 5 pmol (7 x 105 dpm) [6,7-3H]oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass.., U.S.A.) in serum-free MEM (2.5 ml) together with oestrone-3-sulphamate (11 concentrations: 0; 1fM; 0.01pM; 0.1pM; 1pM; 0.01nM; 0.1nM; 1nM; O.OImM; 0.1mM; 1mM). After incubation each flask is cooled and the medium (1 ml) is pipetted into separate tubes containing [14C]oestrone (7 x 103 dpm) (specific activity 97 Ci/mmol from Amersham International Radiochemical Centre, Amersham, U.K.). The mixture is shaken thoroughly for 30 seconds with toluene (5 ml). Experiments have shown that >90% [14C] oestrone and <0.1% [3H]oestrone-3-sulphate is removed from the aqueous phase by this treatment. A portion (2 ml) of the organic phase is removed, evaporated and the 3H and 14C content of the residue determined by scintillation spectrometry. The mass of oestrone-3-sulphate hydrolysed was calculated from the 3H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of [14C] oestrone added) and the specific activity of the substrate. Each batch of experiments includes incubations of microsomes prepared from a sulphatase-positive human placenta (positive control) and flasks without cells (to assess apparent non- enzymatic hydrolysis of the substrate). The number of cell nuclei per flask is determined using a Coulter Counter after treating the cell monolayers with Zaponin. One flask in each batch is used to assess cell membrane status and viability using the Trypan Blue exclusion method (Phillips, H.J. (1973) In: Tissue culture and applications, [eds: Kruse, D.F. & Patterson, M.K.]; pp. 406-408; Academic Press, New York). Results for steroid sulphatase activity are expressed as the mean ± 1 S.D. of the total product (oestrone + oestradiol) formed during the incubation period (20 hours) calculated for 106 cells and, for values showing statistical significance, as a percentage reduction (inhibition) over incubations containing no oestrone-3-sulphamate. Unpaired Student's t-test was used to test the statistical significance of results.
ASSAY FOR DETERMINING STS ACTIVITY USING PLACENTAL MICROSOMES
(PROTOCOL 2)
Inhibition of Steroid Sulphatase Activity in Placental Microsomes
Sulphatase-positive human placenta from normal term pregnancies are thoroughly minced with scissors and washed once with cold phosphate buffer (pH 7.4, 50 mM) then re-suspended in cold phosphate buffer (5 ml/g tissue). Homogenisation is accomplished with an Ultra-Turrax homogeniser, using three 10 second bursts separated by 2 minute cooling periods in ice. Nuclei and cell debris are removed by centrifuging (4°C) at 2000g for 30 minutes and portions (2 ml) of the supernatant are stored at 20°C. The protein concentration of the supematants is determined by the method of Bradford (Anal. Biochem., 72, 248-254 (1976)).
Incubations (1 ml) are carried out using a protein concentration of 100 mg/ml, substrate concentration of 20 mM [6,7-3H]oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass., U.S.A.) and an incubation time of 20 minutes at 37°C. If necessary eight concentrations of compounds are employed: 0 (i.e. control); O.OδmM; 0.1 mM; 0.2mM; 0.4mM; 0.6mM; 0.8mM; I.OmM. After incubation each sample is cooled and the medium (1 ml) was pipetted into separate tubes containing [14C]oestrone (7 x 103 dpm) (specific activity 97 Ci/mmol from Amersham International Radiochemical Centre, Amersham, U.K.). The mixture is shaken thoroughly for 30 seconds with toluene (5 ml). Experiments have shown that >90% [14C]oestrone and <0.1% [3H]oestrone-3-sulphate is removed from the aqueous phase by this treatment. A portion (2 ml) of the organic phase was removed, evaporated and the 3H and 14C content of the residue determined by scintillation spectrometry. The mass of oestrone-3- sulphate hydrolysed is calculated from the 3H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of [14C]oestrone added) and the specific activity of the substrate. ANIMAL ASSAY MODEL FOR DETERMINING STS ACTIVITY
(PROTOCOL 3)
Inhibition of oestrone sulphatase activity in vivo
The compounds of the present invention may be studied using an animal model, in particular in ovariectomised rats. In this model compounds which are oestrogenic stimulate uterine growth.
The compound (10 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol). A further group received the compound EMATE subcutaneously in an amount of 10μg/day for five days. At the end of the study samples of liver tissue were obtained and oestrone sulphatase activity assayed using 3H oestrone sulphate as the substrate as previously described (see PCT/GB95/02638).
ANIMAL ASSAY MODEL FOR DETERMINING OESTROGENIC ACTIVITY
(PROTOCOL 4)
Lack of in vivo oestrogenicity
The compounds of the present invention may be studied using an animal model, in particular in ovariectomised rats. In this model, compounds which are oestrogenic stimulate uterine growth.
The compound (10 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol). A further group received the estrogenic compound EMATE subcutaneously in an amount of 10μg/day for five days. At the end of the study uteri were obtained and weighed with the results being expressed as uterine weight/whole body weight x 100.
Compounds having no significant effect on uterine growth are not oestrogenic. BIOTECHNOLOGICAL ASSAYS FOR DETERMINING STS ACTIVITY
(PROTOCOL 5)
The ability of compounds to inhibit oestrone sulphatase activity can also be assessed using amino acid sequences or nucleotide sequences encoding STS, or active fragments, derivatives, homologues or variants thereof in, for example, high-through put screens.
Any one or more of appropriate targets - such as an amino acid sequence and/or nucleotide sequence - may be used for identifying an agent capable of modulating STS in any of a variety of drug screening techniques. The target employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of target activity or the formation of binding complexes between the target and the agent being tested may be measured.
The assay of the present invention may be a screen, whereby a number of agents are tested. In one aspect, the assay method of the present invention is a high through put screen.
Techniques for drug screening may be based on the method described in Geysen, European Patent Application 84/03564, published on September 13, 1984. In summary, large numbers of different small peptide test compounds are synthesised on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with a suitable target or fragment thereof and washed. Bound entities are then detected - such as by appropriately adapting methods well known in the art. A purified target can also be coated directly onto plates for use in a drug screening techniques. Alternatively, non-neutralising antibodies can be used to capture the peptide and immobilise it on a solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a target specifically compete with a test compound for binding to a target.
Another technique for screening provides for high throughput screening (HTS) of agents having suitable binding affinity to the substances and is based upon the method described in detail in WO 84/03564.
It is expected that the assay methods of the present invention will be suitable for both small and large-scale screening of test compounds as well as in quantitative assays.
In one preferred aspect, the present invention relates to a method of identifying agents that selectively modulate STS, which compounds have the formula (la).
REPORTERS
A wide variety of reporters may be used in the assay methods (as well as screens) of the present invention with preferred reporters providing conveniently detectable signals (e.g. by spectroscopy). By way of example, a reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties.
Other protocols include enzyme-linked immunosorbent assay, (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilising monoclonal antibodies reactive to two non- interfering epitopes may even be used. These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 15 8:121 1).
Examples of reporter molecules iηclude but are not limited to (β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, (- glucuronidase, exo-glucanase and glucoamylase. Alternatively, radiolabelled or fluorescent tag-labelled nucleotides can be incorporated into nascent transcripts which are then identified when bound to oligonucleotide probes.
By way of further examples, a number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for assay procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3817837; US-A-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 and US-A-4366241. HOST CELLS
The term "host cell" - in relation to the present invention includes any cell that could comprise the target for the agent of the present invention.
Thus, a further embodiment of the present invention provides host cells transformed or transfected with a polynucleotide that is or expresses the target of the present invention. Preferably said polynucleotide is carried in a vector for the replication and expression of polynucleotides that are to be the target or are to express the target. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
The gram negative bacterium E. coli is widely used as a host for heterologous gene expression. However, large amounts of heterologous protein tend to accumulate inside the cell. Subsequent purification of the desired protein from the bulk of E.coli intracellular proteins can sometimes be difficult.
In contrast to E.coli, bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas.
Depending on the nature of the polynucleotide encoding the polypeptide of the present invention, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. In general, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.
Examples of suitable expression hosts within the scope of the present invention are fungi such as Aspergillus species (such as those described in EP-A-0184438 and EP-A- 0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; and yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species. By way of example, typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var. awamori, Aspergillus aculeatis, Aspergillus nidulans, Aspergillus orvzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae.
The use of suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational ' modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
ORGANISM
The term "organism" in relation to the present invention includes any organism that could comprise the target according to the present invention and/or products obtained therefrom. Examples of organisms may include a fungus, yeast or a plant.
The term "transgenic organism" in relation to the present invention includes any organism that comprises the target according to the present invention and/or products obtained.
TRANSFORMATION OF HOST CELLS/HOST ORGANISMS
As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.
If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey ef al (1987, Yeast Biotechnology, D R Berry ef al, eds, pp 401-429, Allen and Unwin, London) and by King ef al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).
For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting the nucleotide sequence into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed. The constructs contain a promoter active in yeast fused to the nucleotide sequence, usually a promoter of yeast origin, such as the GAL1 promoter, is used. Usually a signal sequence of yeast origin, such as the sequence encoding the SUC2 signal peptide, is used. A terminator active in yeast ends the expression system.
For the transformation of yeast several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
The transformed yeast cells are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, e.g. G418.
Another host organism is a plant. The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material. Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.
Thus, the present invention also provides a method of transforming a host cell with a nucleotide sequence that is to be the target or is to express the target.. Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for the expression of the encoded protein. The protein produced by a recombinant cell may be displayed on the surface of the cell. If desired, and as will be understood by those of skill in the art, expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).
VARIANTS/HOMOLOGUES/DERIVATIVES
In addition to the specific amino acid sequences and nucleotide sequences mentioned herein, the present invention also encompasses the use of variants, homologue and derivatives thereof. Here, the term "homology" can be equated with "identity".
In the present context, an homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
% homology may be' calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
A further useful reference is that found in FEMS Microbiol Lett 1999 May 15;174(2):247- 50 (and a published erratum appears in FEMS Microbiol Lett 1999 Aug 1;177(1):187-8).
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Figure imgf000070_0001
EXPRESSION VECTORS
The nucleotide sequence for use as the target or for expressing the target can be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
The protein produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
FUSION PROTEINS
The target amino acid sequence may be produced as a fusion protein, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S- transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and (-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the target.
The fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention. In this embodiment, the fusion protein may be a non-naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. The antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
In another embodiment of the invention, the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric'substance expressing a heterologous epitope that is recognised by a commercially available antibody.
THERAPY
The compounds of the present invention may be used as therapeutic agents - i.e. in therapy applications.
The term "therapy" includes curative effects, alleviation effects, and prophylactic effects.
The therapy may be on humans or animals, preferably female animals.
PHARMACEUTICAL COMPOSITIONS
In one aspect, the present invention provides a pharmaceutical composition, which comprises a compound according to the present invention and optionally a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).
The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Preservatives, stabilisers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.
Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner. COMBINATION PHARMACEUTICAL
The compound of the present invention may be used in combination with one or more other active agents, such as one or more other pharmaceutically active agents.
By way of example, the compounds of the present invention may be used in combination with other STS inhibitors and/or other inhibitors such as an aromatase inhibitor (such as for example, 4hydrόxyandrostenedione (4-OHA)) and/or steroids - such as the naturally occurring sterneurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and/or other structurally similar organic compounds. Exaples of other STS inhibitors may be found in the above references. By way of example, STS inhibitors for use in the present invention include EMATE, and either or both of the 2-ethyl and 2- methoxy 17-deoxy compounds that are analogous to compound 5 presented herein.
In addition, or in the alternative, the compound of the present invention may be used in combination with a biological response modifier.
The term biological response modifier ("BRM") includes cytokines, immune modulators, growth factors, haematopoiesis regulating factors, colony stimulating factors, chemotactic, haemolytic and thrombolytic factors, cell surface receptors, ligands, leukocyte adhesion molecules, monoclonal antibodies, preventative and therapeutic vaccines, hormones, extracellular matrix components, fibronectin, etc. For some applications, preferably, the biological response modifier is a cytokine. Examples of cytokines include: interleukins (IL) - such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-
9, IL-10, IL-11 , IL-12, IL-19; Tumour Necrosis Factor (TNF) - such as TNF-α; Interferon alpha, beta and gamma; TGF-β. For some applications, preferably the cytokine is tumour necrosis factor (TNF). For some applications, the TNF may be any type of TNF - such as TNF-α, TNF-β, including derivatives or mixtures thereof. More preferably the cytokine is TNF-α. Teachings on TNF may be found in the art - such as WO-A-98/08870 and WO-A-98/13348.
ADMINISTRATION
Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
The compositions of the present invention may be administered by direct injection. The composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration. Depending upon the need, the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
By way of further example, the agents of the present invention may be administered in accordance with a regimen of 1 to 4 times per day, preferably once or twice per day. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing, therapy.
Aside from the typical modes of delivery - indicated above - the term "administered" also includes delivery by techniques such as lipid mediated transfection, liposomes, immuholiposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof. The routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
The term "administered" includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
Thus, for pharmaceutical administration, the STS inhibitors of the present invention can be formulated in any suitable manner utilising conventional pharmaceutical formulating techniques and pharmaceutical carriers, adjuvants, excipients, diluents etc. and usually for parenteral administration. Approximate effective dose rates may be in the range from 1 to 1000 mg/day, such as from 10 to 900 mg/day or even from 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of average (70Kg) bodyweight. More usual dosage rates for the preferred and more active compounds will be in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day, most preferably from 200 to 250 mg/day. They may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days. For oral administration they may be formulated in tablets, capsules, solution or suspension containing from 100 to 500 mg of compound per unit dose. Alternatively and preferably the compounds will be formulated for parenteral administration in a suitable parenterally administrable carrier and providing single daily dosage rates in the range 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250 mg. Such effective daily doses will, however, vary depending on inherent activity of the active ingredient and on the bodyweight of the patient, such variations being within the skill and judgement of the physician.
CELL CYCLING
The compounds of the present invention may be useful in the method of treatment of a cell cycling disorder.
As discussed in "Molecular Cell Biology" 3rd, Ed. Lodish et al. pages 177-181 different eukaryotic cells can grow and divide at quite different rates. Yeast cells, for example, can divide every 120 min., and the first divisions of fertilised eggs in the embryonic cells of sea urchins and insects take only 1530 min. because one large pre-existing cell is subdivided. However, most growing plant and animal cells take 10-20 hours to double in number, and some duplicate at a much slower rate. Many cells in adults, such as nerve cells and striated muscle cells, do not divide at all; others, like the fibroblasts that assist in healing wounds, grow on demand but are otherwise quiescent.
Still, every eukaryotic cell that divides must be ready to donate equal genetic material to two daughter cells. DNA synthesis in eukaryotes does not occur throughout the cell division cycle but is restricted to a part of it before cell division.
The relationship between eukaryotic DNA synthesis and cell division has been thoroughly analysed in cultures of mammalian cells that were all capable of growth and division. In contrast to bacteria, it was found, eukaryotic cells spend only a part of their time in DNA synthesis, and it is completed hours before cell division (mitosis). Thus a gap of time occurs after DNA synthesis and before cell division; another gap was found to occur after division and before the next round of DNA synthesis. This analysis led to the conclusion that the eukaryotic cell cycle consists of an M (mitotic) phase, a G-, phase (the first gap), the S (DNA synthesis) phase, a G2 phase (the second gap), and back to M. The phases between mitoses (G-j, S, and G2) are known collectively as the interphase.
Many nondividing cells in tissues (for example, all quiescent fibroblasts) suspend the cycle after mitosis and just prior to DNA synthesis; such "resting" cells are said to have exited from the cell cycle and to be in the G0 state.
It is possible to identify cells when they are in one of the three interphase stages of the cell cycle, by using a fluorescence-activated cell sorter (FACS) to measure their relative DNA content: a cell that is in G., (before DNA synthesis) has a defined amount x of DNA; during S (DNA replication), it has between x and 2x; and when in G2 (or M), it has 2x of DNA.
The stages of mitosis and cytokinesis in an animal cell are as follows
(a) Interphase. The G2 stage of interphase immediately precedes the beginning of mitosis. Chromosomal DNA has been replicated and bound to protein during the S phase, but chromosomes are not yet seen as distinct structures. The nucleolus is the only nuclear substructure that is visible under light microscope. In a diploid cell before DNA replication there are two morphologic chromosomes of each type, and the cell is said to be 2n. In G2, after DNA replication, the cell is 4n. There are four copies of each chromosomal DNA. Since the sister chromosomes have not yet separated from each other, they are called sister chromatids.
b) Early prophase. Centrioles, each with a newly formed daughter centriole, begin moving toward opposite poles of the cell; the chromosomes can be seen as long threads. The nuclear membrane begins to disaggregate into small vesicles.
(c) Middle and late prophase. Chromosome condensation is completed; each visible chromosome structure is composed of two chromatids held together at their centromeres. Each chromatid contains one of the two newly replicated daughter DNA molecules. The microtubular spindle begins to radiate from the regions just adjacent to the centrioles, which are moving closer to their poles. Some spindle fibres reach from pole to pole; most go to chromatids and attach at kinetochores. (d) Metaphase. The chromosomes move toward the equator of the cell, where they become aligned in the equatorial plane. The sister chromatids have not yet separated.
(e) Anaphase. The two sister chromatids separate into independent chromosomes. Each contains a centromere that is linked by a spindle fibre to one pole, to which it moves. Thus one copy of each chromosome is donated to each daughter cell. Simultaneously, the cell elongates, as do the pole-to-pole spindles. Cytokinesis begins as the cleavage furrow starts to form.
(f) Telophase. New membranes form around the daughter nuclei; the chromosomes uncoil and become less distinct, the nucleolus becomes visible again, and the nuclear membrane forms around each daughter nucleus. Cytokinesis is nearly complete, and the spindle disappears as the microtubules and other fibres depolymerise. Throughout mitosis the "daughter" centriole at each pole grows until if is full-length. At telophase. the duplication of each of the original centrioles is completed, and new daughter centrioles will be generated during the next interphase.
(g) Interphase. Upon the completion of cytokinesis, the cell enters the G-, phase of the cell cycle and proceeds again around the cycle.
It will be appreciated that cell cycling is an extremely important cell process. Deviations from normal cell cycling can result in a number of medical disorders. Increased and/or unrestricted cell cycling may result in cancer. Reduced cell cycling may result in degenerative conditions. Use of the compound of the present invention may provide a means to treat such disorders and conditions.
Thus, the compound of the present invention may be suitable for use in the treatment of cell cycling disorders such as cancers, including hormone dependent and hormone independent cancers.
In addition, the compound of the present invention may be suitable for the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer, pancreatic cancer etc. and other solid tumours.
For some applications, cell cycling is inhibited and/or prevented and/or arrested, preferably wherein cell cycling is prevented and/or arrested. In one aspect cell cycling may be inhibited and/or prevented and/or arrested in the G2/M phase. In one aspect cell cycling may be irreversibly prevented and/or inhibited and/or arrested, preferably wherein cell cycling is irreversibly prevented and/or arrested.
By the term "irreversibly prevented and/or inhibited and/or arrested" it is meant after application of a compound of the present invention, on removal of the compound the effects of the compound, namely prevention and/or inhibition and/or arrest of cell cycling, are still observable. More particularly by the term "irreversibly prevented and/or inhibited and/or arrested" it is meant that when assayed in accordance with the cell cycling assay protocol presented herein, cells treated with a compound of interest show less growth after Stage 2 of the protocol I than control cells. Details on this protocol are presented below.
Thus, the present 'invention provides compounds which: cause inhibition of growth of oestrogen receptor positive (ER+) and ER negative (ER-) breast cancer cells in vitro by preventing and/or inhibiting and/or arresting cell cycling; and/or cause regression of nitroso-methyl urea (NMU)-induced mammary tumours in intact animals (i.e. not ovariectomised), and/or prevent and/or inhibit and/or arrest cell cycling in cancer cells; and/or act in vivo by preventing and/or inhibiting and/or arresting cell cycling and/or act as a cell cycling agonist.
CELL CYCLING ASSAY (PROTOCOL 6)
Procedure Stage 1
MCF-7 breast cancer cells are seeded into multi-well culture plates at a density of 105 cells/well. Cells were allowed to attach and grown until about 30% confluent when they are treated as follows:
Control - no treatment
Compound of Interest (COI) 20μM
Cells are grown for 6 days in growth medium containing the COI with changes of medium/COI every 3 days. At the end of this period cell numbers were counted using a Coulter cell counter. Stage 2
After treatment of cells for a 6-day period with the COI cells are re-seeded at a density of 104 cells/well. No further treatments are added. Cells are allowed to continue to grow for a further 6 days in the presence of growth medium. At the end of this period cell numbers are again counted.
ASSAY FOR DETERMINING DH ACTIVITY USING CANCER CELLS
(PROTOCOL 7)
Conversion of oestrone to oestradiol (E1 -> E2, E2DH Type I) and oestradiol to oestrone (E2 -> E1, E2DH Type II) was measured in intact cell monolayers of T47D and MDA- MB-231 breast cancer cells respectively. Cells were cultured in flasks until they were 80-90%) confluent. 3H-E1 or 3H-E2 (6pmol, -90 Ci/mmol) were added to each flask in the absence (control) or presence of various test compounds (10μM) in 2.5ml of medium. Substrate was also added to flasks without cells and incubated in parallel (blanks).
After incubation with T47D cells for 30min or MDA cells for 3h at 37°C, 2ml of the medium was added to test tubes containing 14C-E2 or 14C-E1 (~5000 cpm) and 50μg E2 or E1 respectively. Steroids were extracted from the aqueous medium with diethyl ether
(4ml). The ether phase was decanted into separate tubes after freezing the aqueous phase in solid carbon dioxide-methanol mixture. The ether was evaporated to dryness under a stream of air at 40°C. The residue was dissolved in a small volume of diethyl ether and applied to TLC plates containing a fluorescent indicator. E1 and E2 were separated by TLC using DCM-Ethyl acetate (4:1 v/v). The position of the product from each incubation flask was marked on the TLC plate after visualisation under UV light.
The marked regions were cut out and placed in scintillation vials containing methanol
(0.5ml) to elute the product. The amount of 3H-product formed and 14C-E1 or 14C-E2 recovered were calculated after scintillation spectrometry. The amount of product formed was corrected for procedural losses and for the number of cells in each flask.
CANCER
As indicated, the compounds of the present invention may be useful in the treatment of a cell cycling disorder. A particular cell cycling disorder is cancer. Cancer remains a major cause of mortality in most Western countries. Cancer therapies developed so far have included blocking the action or synthesis of hormones to inhibit the growth of hormone-dependent tumours. However, more aggressive chemotherapy is currently employed for the treatment of hormone-independent tumours.
Hence, the development of a pharmaceutical for anti-cancer treatment of hormone dependent and/or hormone independent tumours, yet lacking some or all of the side- effects associated with chemotherapy, would represent a major therapeutic advance.
It is known that oestrogens undergo a number of hydroxylation and conjugation reactions after their synthesis. Until recently it was thought that such reactions were part of a metabolic process that ultimately rendered oestrogens water soluble and enhanced their elimination from the body. It is now evident that some hydroxy metabolites (e.g. 2- hydroxy and 16alpha-hydroxy) and conjugates (e.g. oestrone sulphate, E1S) are important in determining some of the complex actions that oestrogens have in the body.
Workers have investigated the formation of 2- and 16-hydroxylated oestrogens in relation to conditions that alter the risk of breast cancer. There is now evidence that factors which increase 2-hydroxylase activity are associated with a reduced cancer risk, while those increasing 16alpha-hydroxylation may enhance the risk of breast cancer. Further interest in the biological role of oestrogen metabolites has been stimulated by the growing body of evidence that 2-methoxyoestradiol is an endogenous metabolite with anti-mitotic properties. 2-MeOE2 is formed from 2-hydroxy oestradiol (2-OHE2) by catechol oestrogen methyl transferase, an enzyme that is widely distributed throughout the body.
Workers have shown that in vivo 2-MeOE2 inhibits the growth of tumours arising from the subcutaneous injection of Meth A sarcoma, B16 melanoma or MDA-MB-435 oestrogen receptor negative (ER-) breast cancer cells. It also inhibits endothelial cell proliferation and migration, and in vitro angiogenesis. It was suggested that the ability of 2-MeOE2 to inhibit tumour growth in vivo may be due to its ability to inhibit tumour- induced angiogenesis rather than direct inhibition of the proliferation of tumour cells.
The mechanism by which 2-MeOE2 exerts its potent anti-mitogenic and anti-angiogenic effects is still being elucidated. There is evidence that at high concentrations it can inhibit microtubule polymerisation and act as a weak inhibitor of colchicine binding to tubulin. Recently, however, at concentrations that block mitosis, tubulin filaments in cells were not found to be depolymerised but to have an identical morphology to that seen after taxol treatment. It is possible, therefore, that like taxol, a drug that is used for breast and ovarian breast cancer therapy, 2-MeOE2 acts by stabilising microtubule dynamics.
While the identification of 2-MeOE2 as a new therapy for cancer represents an important advance, the bioavailability of orally administered oestrogens is poor. Furthermore, they can undergo extensive metabolism during their first pass through the liver. As part of a research programme to develop a steroid sulphatase inhibitor for breast cancer therapy, oestrone-3-O-sulphamate (EMATE) was identified as a potent active site-directed inhibitor. Unexpectedly, EMATE proved to possess potent oestrogenic properties with its oral uterotrophic activity in rats being a 100-times higher than that of oestradiol. Its enhanced oestrogenicity is thought to result from its absorption by red blood cells (rbcs) which protects it from inactivation during its passage through the liver and which act as a reservoir for its slow release for a prolonged period of time. A number of A-ring modified analogues were synthesised and tested, including 2-methoxyoestrone-3~O-sulphamate. While this compound was equipotent with EMATE as a steroid sulphatase inhibitor, it was devoid of oestrogenicity.
We believe that the compound of the present invention provides a means for the treatment of cancers and, especially, breast cancer.
In addition or in the alternative the compound of the present invention may be useful in the blocking the growth of cancers including leukaemias and solid tumours such as breast, endometrium, prostate, ovary and pancreatic tumours.
THERAPY CONCERNING OESTROGEN
We believe that some of the compounds of the present invention may be useful in the control of oestrogen levels in the body - in particular in females. Thus, some of the compounds may be useful as providing a means of fertility control - such as an oral contraceptive tablet, pill, solution or lozenge. Alternatively, the compound could be in the form of an implant or as a patch. Thus, the compounds of the present invention may be useful in treating hormonal conditions associated with oestrogen.
In addition or in the alternative the compound of the present invention may be useful in treating hormonal conditions in addition to those associated with oestrogen. Hence, the compound of the present invention may also be capable of affecting hormonal activity and may also be capable of affecting an immune response.
NEURODEGENERATIVE DISEASES
We believe that some of the compounds of the present invention may be useful in the treatment of neurodenerative diseases, and similar conditions.
By way of example, it is believed that STS inhibitors may be useful in the enhancing the memory function of patients suffering from illnesses such as amnesia, head injuries, Alzheimer's disease, epileptic dementia, presenile dementia, post traumatic dementia, senile dementia, vascular dementia and post-stroke dementia or individuals otherwise seeking memory enhancement.
TH1
We believe that some of the compounds of the present invention may be useful in TH1 implications.
By way of example, it is believed that the presence of STS inhibitors within the macrophage or other antigen presenting cells may lead to a decreased ability of sensitised T cells to mount a TH1 (high lL-2, IFNy low lL-4) response. The normal regulatory influence of other steroids such as glucocorticoids would therefore predominate.
INFLAMATORY CONDITIONS
We believe that some of the compounds of the present invention may be useful in treating inflammatory conditions - such as conditions associated with any one or more of: autoimmunity, including for example, rheumatoid arthritis, type I and II diabetes, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, thyroiditis, vasculitis, ulcerative colitis and Crohn's disease, skin disorders e.g. psoriasis and contact dermatitis; graft versus host disease; eczema; asthma and organ rejection following transplantation.
By way of example, it is believed that STS inhibitors may prevent the normal physiological effect of DHEA or related steroids on immune and/or inflammatory responses.
The compounds of the present invention may be useful in the manufacture of a medicament for revealing an endogenous glucocorticoid-like effect.
OTHER THERAPIES
It is also to be understood that the compound/composition of the present invention may have other important medical implications.
For example, the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-99/52890 - viz:
In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-98/05635. For ease of reference, part of that list is now provided: cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulceration, retinopathy and surgical wound healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosclerosis.
In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/07859. For ease of reference, part of that list is now provided: cytokine and cell proliferation/differentiation activity; immunosuppressant or immunostimulant activity (e.g. for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumour immunity); regulation of haematopoiesis, e.g. treatment of myeloid or lymphoid diseases; promoting growth of bone, cartilage, tendon, ligament and nerve tissue, e.g. for healing wounds, treatment of burns, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic/chemokinetic activity (e.g. for mobilising specific cell types to sites of injury or infection); haemostatic and thrombolytic activity (e.g. for treating haemophilia and stroke); antiinflammatory activity (for treating e.g. septic shock or Crohn's disease); as antimicrobials; modulators of e.g. metabolism or behaviour; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine.
In addition, or in the alternative, the composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/09985. For ease of reference, part of that list is now provided: macrophage inhibitory and/or T cell inhibitory activity and thus, anti-inflammatory activity; anti-immune activity, i.e. inhibitory effects against a cellular and/or humoral immune response, including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory-related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus .disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferate vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and/or side effects, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.
SULPHAMATE COMPOUND PREPARATION
The sulphamate compounds of the present invention may be prepared by reacting an appropriate alcohol with a suitable chloride. By way of example, the sulphamate compounds of the present invention may be prepared by reacting an appropriate alcohol with a suitable sulfamoyl chloride, of the formula R9R10NSO2CI.
Typical conditions for carrying out the reaction are as follows.
Sodium hydride and a sulfamoyl chloride are added to a stirred solution of the alcohol in anhydrous dimethyl formamide at 0°C. Subsequently, the reaction is allowed to warm to room temperature whereupon stirring is continued for a further 24 hours. The reaction mixture is poured onto a cold saturated solution of sodium bicarbonate and the resulting aqueous phase is extracted with dichloromethane. The combined organic extracts are dried over anhydrous MgSO4. Filtration followed by solvent evaporation in vacuo and co-evaporated with toluene affords a crude residue which is further purified by flash chromatography. '
Preferably, the alcohol is derivatised, as appropriate, prior to reaction with the sulfamoyl chloride. Where necessary, functional groups in the alcohol may be protected in known manner and the protecting group or groups removed at the end of the reaction.
Preferably, the sulphamate compounds are prepared according to the teachings of Page ef al (1990 Tetrahedron 46; 2059-2068).
The phosphonate compounds may be prepared by suitably combining the teachings of Page et al (1990 Tetrahedron 46; 2059-2068) and PCT/GB92/01586.
The sulphonate compounds may be prepared by suitably adapting the teachings of Page ef a/ (1990 Tetrahedron 46; 2059-2068) and PCT/GB92/01586.
The thiophosphonate compounds may be prepared by suitably adapting the teachings of Page ef al (1990 Tetrahedron 46; 2059-2068) and PCT/GB91/00270.
Preferred preparations are also presented in the following text
SUMMARY
In summation, the present invention provides compounds for use as steroid dehydrogenase inhibitors, and pharmaceutical compositions for the same. EXAMPLES
The present invention will now be described only by way of example.
Example
SECTION 1
16-(4-Dim©thylamino-fe©ri2yli ©ne)-3-hy ro3 y-13-methyl-@, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta[a]phenanthren-17-one STX477 (CAB02143, GMA01044)
Figure imgf000087_0001
To a stirred suspension of estrone (4.05g, 15mmol) and 4-dimethylaminobenzaldehyde (2.24g, 15mmol) in ethanol (120cm3) was added sodium hydroxide (3.0g, 75mmol) and the resulting yellow solution was stirred at room temperature for 72h. To this solution was added glacial acetic acid (20cm3) and the resulting yellow precipitate was collected by filtration and washed with water (100cm3), ethanol (50cm3), diethyl ether (50cm3) and hexane (100cm3) before being dried under reduced pressure. Yield 66%. 1H NMR (400MHz, DMSO-de) δ 0.86 (s, 3H), 1.34-1.61 (m, 6H), 1.82-1.86 (m, 1H), 1.98-2.02 (m, 1H), 2.15-2.19 (m, 1H), 2.32-2.34 (m, 1H), 2.73-2.86 (m, 3H), 2.97 (s, 6H), 6.45 (d, J=2.7Hz, 1H), 6.51 (appdd, J=8.4, 2.5Hz, 1H), 6.74 (d, J=8.6Hz, 2H), 7.04 (d, J=8.6Hz, 1H), 7.20 (s, 1H), 7.48 (d, J=8.6Hz, 2H), 9.02 (s, 1H); MS (FAB+) calcd. 401.2, obsd. 402.2 (M+H+)
16-(4-Dimethylamino-bensylidene)-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- dscsthydro-SH-cyclopen aJaJphenani'hrene-S.IJ- iol STX511 (CAB02158, GMA01006)
Figure imgf000088_0001
To a cooled (ice bath) solution of 16-(4-dimethylaminobenzylidene estrone (0.402g, 1mmol) in THF (20cm3) and ethanol (20cm3) was added NaBH4 (0.1 OOg, 2.6mmol) and the reaction was allowed to warm to room temperature and stirred overnight. The mixture was concentrated in vacuo until precipitate began to appear before water (50cm3) was added to precipitate the product as a white powder. This powder was collected by filtration and washed with water (50cm3), methanol (20cm3) and diethyl ether (50cm3) before being dried under reduced pressure. Yield 86%. 1H NMR (400MHz, DMSO-d6) δ 0.59 (s, 3H), 1.24-1.43 (m, 6H), 1.84-1.95 (m, 1H), 2.08-2.20 (m, 2H), 2.26- 2.34 (m, 1H), 2.55-2.65 (m, 1H), 2.70-2.80 (m, 2H), 2.89 (s, 6H), 3.95 (s, 1H), 4.99 (d, J=6.2Hz, 1H), 6.27 (d, J=2.0Hz, 1H), 6.45 (d, J=2.7Hz, 1H), 6.51 (appdd, J=8.4, 2.5Hz, 1H), 6.70 (d, J=9.0Hz, 2H), 7.05 (d, J=8.6Hz, 1H), 7.22 (d, J=9.0Hz, 2H), 9.01 (s, 1H); HPLC (98%, Rt=3.52, 10:90 H20:MeOH); MS (FAB+) calcd. 403.3, obsd. 403.2 (M+)
General procedure for hydrogenation reactions:
The starting 16-methylene estradiol was dissolved in a volume of THF and the same volume of ethanol added to the solution. The solution was then degassed by bubbling nitrogen through for 40min before Pd/C (5%wt., catalytic) was added and hydrogen gas (balioon) was passed over the reaction. The reaction was stirred under hydrogen at room temperature overnight before being filtered through celite.
16-(4-Dimethylamino-benzyl)-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro- 6H-cyclopenta[a]phenanthrene-3, 17-diol STX594 (GMA01008)
Figure imgf000088_0002
Purification by column chromatography using hexane followed by 20% ethyl acetate in hexane as eluent gave the product, Rf 0.33 (20% EtoAc in hexane), in 45%yield. H NMR (400MHz, DMSO-d6) δ 0.75 (S, 3H), 0.92-1.08 (M, 2H), 1.10-1.36 (M, 4H), 1.49- 1.55 (m, 1H), 1.66-1.71 (m, 1H), 1.87 (d, J=12.1Hz, 1H), 2.13 (d, J=13.3H∑, 1 H), 2.19- 2.29 (m, 2H), 2.58-2.73 (m, 2H), 2.83 (s, 6H), 2.88-2.93 (m, 1H), 3.63-3.66 (m, 1H), 4.60 (d, J=4.7Hz, 1H), 6.41 (d, J=2.3Hz, 1 H), 6.49 (dd, J=8.2, 2.7Hz), 6.64 (d, J=9.0Hz, 2H), 6.99 (d, J=8.6H∑, 2H), 7.03 (d, J=8.2Hz, 1H), 8.98 (s, 1 H, OH); HPLC (92%, Rt=3.28, 10:90 H20:MeOH); MS (FAB+) calcd 405.2, obsd 405.2 (M+), Ace. Mass (FAB+) calcd 405.2668, obsd 405.2668 (M+)
13-Methyl-16-pyridin-2-ylmethyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[D]phenanthrene-3-17-diol
STX 567 (GMA01018)
Figure imgf000089_0001
Purification by column chromatography using 100% hexane to 100% ethyl acetate as eluent gave the product, Rf 0.1 (25% EtOAc 75% hexane), in 57% yield. 1H NMR (400MHz, DMSO-d6) δ D0.76 (s, 3H), 1.04-1.07 (m, 2H), 1.15-1.36 (m, 6H), 1.55-1.60 (m, 1H), 1.64-1.71 (m, 1H), 1.87 (d, J=2.1Hz, 1H), 2.20-2.25 (m, 1H), 2.60-2.72 (m, 3H), 3.12 (d, J=8,6Hz, 1H), 3.68-3.71 (m, 1H), 4.97 (d, J=4.7Hz, 1 H), 6.41 (d, J=2.7Hz, 1H), 6.49 (dd, J=8.4, 2.5Hz, 1 H), 7.04 (d, J=8.2Hz, 1H), 7.15-7.19 (m, 1 H), 7.25 (d, J=7.8Hz, 1H), 7.67 (dt, J=7.6, 2.0Hz, 1H), 8.44-8.465 (m, 1H), 8.99 (s, 1H); HPLC (98%, Rt=2.62, 10:90 H20:MeOH); MS (FAB+) calcd 363.2, obsd 364.1 (M+H+), Ace. Mass (FAB+) calcd 364.2276, obsd 364.2297 (M+H+)
13-Met yl-16-pyridin-3-ylm®thyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-d®eahydrø-6W- cyclopenta[a]phenanthrene-3, 17-diol ST2 542 (GRM0 O22)
Figure imgf000090_0001
Purification by column chromatography using 100% hexane to 100% ethyl acetate as eluent gave the product, (Rf 0.1 (25% EtOac in hexane) in 88% yield. 1H NMR (270MH∑, DMSO-d6) δ D0.76 (s, 3H), 1.00-1.04 (m, 2H), 1.20-1.35 (m, 5H), 1.54-1,56 (m, 1 H), 1.73-1.90 (m, 2H), 2.13-2.32 (m, 3H), 2.64 (m, 2H), 3.01 (d, J=9.4H∑, 1H), 4.77 (d, J=4.5Hz, 1H), 6.41 (d, J=2.5Hz, 1H), 6.49 (dd, J=8.4, 2.5H∑, 1 H), 7.03 (d, J=8.4Hz, 1 H), 7.25-7.31 (m, 1H), 7.60-7.64 (m, 1 H), 8.34-8.39 (m, 1H), 8.41 (d, J=1.7Hz, 1H), 9.02 (s, 1H); HPLC (97%, Rt=2.50, 10:90 H20:MeOH); MS (FAB+) calcd 363.2, obsd 364.1 (M+H+), Ace. Mass (FAB+) calcd 364.2276, obsd 364.2287 (M+H+)
13-Methyl-16-pyridin-4-ylmethyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6tf- cyclopenta[a]phenanthrene-3, 17-diol STX 543 (GMA01020)
Figure imgf000090_0002
Purification by column chromatography using 100% hexane to 100% ethyl acetate as eluent. Gave the product, (Rf 0.1 (25% EtOAc 75% hexane) in 96% yield; 1H NMR (400MHz, DMSO-d6) δD0.76 (s, 3H), 0.94-1.10 (m, 2H), 1.12-1.36 (m, 5H), 1.54-1.60 (m, 1H), 1.65-1.71 (m, 1H), 1.85-1.89 (m, 1H), 2.20-2.42 (m, 3H), 2.64-2.68 (m, 2H), 2.98-3.02 (m, 1H), 3.66-3.70 (m, 1H), 4.75 (d, J=4.7Hz, 1H), 6.40 (d, J=2.3Hz, 1 H), 6.49 (dd, J=8.4, 2.5Hz, 1H), 7.03 (d, J=8.2Hz, 1H), 7.23 (d, J=6.2Hz, 2H), 8.41 (d, J=5.9Hz, 2H), 8.99 (s, 1H); HPLC (92%, Rt=2.44, 10:90 H20:MeOH); MS (FAB+) calcd 363.2, obsd 364.1 (M+H+), Ace. Mass (FAB+) calcd 364.2276, obsd 364,2294 (M+H+)
Acetic acid 8-acetyl-6a-methyl-4b, 5, β, βa, 8, 10, 10a, 10b, 11, 12-decahydr©-7, 8- diaza-pentaleno[2,1-a]phenanthene-2-yl ester (GMA01110)
Figure imgf000091_0001
To a solution of the starting pyrazole (0.598g, 2mmol) in dry pyridine (8cm3) was added acetic anhydride (2cm3) and the mixture was refluxed under nitrogen for 16h. The mixture was then cooled and poured onto ice and the resulting cream coloured precipitate was collected by filtration and purified by column chromatography using ethyl acetate/hexane (1/1), Rf 0.48. Yield: 40%. 1H NMR (270MHz, CDCI3) δ D1.07 (s, 3H), 1.40-1.60 (m, H), 1.62-2.10 (m, 5H), 2.28 (s, 3H, OAc), 2.28-2.48 (m, 4H), 2.64 (s, 3H, OAc), 2.64-2.72 (m, 1H), 2.87-2.96 (m, 2H), 6.82 (d, J=2.2Hz, 1H), 6.86 (appdd , J=8.4, 2.5Hz, 1 H), 7.30 (d=8.4Hz, 1H), 7.86 (s, 1 H); LCMS (ES+) 380.23 (M+2H+)
Acetic acid 8-acetyl-6a-methyl-12-oxo-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12- decahydro-7, 8-diaza-pentaleno-[2,1-a]phenanthren-2-yl ester (GMA01120)
Figure imgf000091_0002
To a solution of the bis-acetate protected pyrazole starting material (0.121g, 0.32mmol) in acetonitrile (2.5cm3) was added t-butylhydroperoxide (0.14cm3 of a 70%wt. solution in water, 1mmol) and chromium hexacarbonyl (0.021g, 0.09mmol). The solution was heated to reflux for 24h before being cooled to room temperature. Water (10cm3) was added and the products extracted with diethyl ether (3x10cm3). The ether extracts were combined, washed with water, aqueous saturated bicarbonate and brine before being dried (MgSO4) and concentrated in vacuo. The desired product was isolated by flash column chromatography using ethyl acetate/hexane (1/1) as eluent (Rf 0.4). Yield 23%. 1H NMR (400MHz, CDCI3) δ D1.09 (s, 3H), 1.8-2.0 (m, 2H), 2.2-2.5 (m, 4H), 2.33 (s, 3H), 2.5-2.9 (m, 5H), 2.65 (s, 3H), 7.34 (dd, J=2.5, 8.4Hz, 1H), 7.65 (d, J=8.6Hz, 1H), 7.79 (d, J=2.3Hz, 1H), 7.88 (s, 1H)
2-HydrQ--y-6a-methyl-5, ®, 6a, 8, 10, 10a, 10b, 11-octahyc-rø-4bH-7, 8-diasa- pentaleno[2,1-a]phenanthren-12-on© STCζ 737 (GM-A01138)
Figure imgf000092_0001
The bis-acetate protected starting material (0.0137g) was dissolved in ethanol (1cm3): To this was added a solution of KOH (0.004g) in ethanol/water (2cm3 of 1/1) and the mixture was shaken then left at room temperature for 30min. TLC at this stage showed no starting material remaining therefore the mixture was acidified with glacial acetic acid, concentrated in vacuo and water added. The solution was left standing overnight during which time a pale orange precipitate formed. This powder was collected by filtration. Yield 46%. 1H NMR (400MHz, DMSO-d6) δ 00.91 (s, 3H), 1.60-1.82 (m, 2H), 2.1-2.35 (m, 4H), 2.4-2.7 (m, 5H), 7.02 (dd, J=8.6, 2.7Hz, 1H), 7.29 (d, J=2.7Hz, 1H), 7.34 (d, J=8.6Hz, 1H), 9.65 (s, 1H), 12.04 (bs, 1H); LCMS: (ES+) 309.99 (M+2H+); Ace. Mass (FAB+) calcd 309.16030, obsd. 309.16132 (M+H+)
6a-Methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno[2,1- a]phenanthrene-2, 9-diol STX 820 (GMA02024)
Figure imgf000092_0002
To a suspension of the starting ester (0.657g, 2mmol) in toluene (10cm3) was added hydrazine monohydrate (0.11cm3, 2.2mmol) and the mixture was heated at 150°C in a sealed tube for 4h. During this time the starting material dissolved then another white precipitate formed. The reaction was then cooled to room temperature, acidified with glacial acetic acid and water was added to ensure that all product precipitated. The white powder was collected by filtration and washed with water, a small amount of ethanol, diethyl ether and hexane. LCMS showed two compounds with masses corresponding to 5 the desired product (312) and the hydrazone derivative of estrone (285). Flash chromatography using 10% methanol in DCM to 100% methanol failed to separate these. Recrystallisation was attempted by suspending the solid in ethanol then slowly adding aqueous NaOH until the solid dissolved, followed by acidification to pH3 with glacial acetic acid. No precipitate formed initially therefore a small amount of water was 0 added until a fine white powder precipitated. This was shown by LCMS and HPLC to be the desired product. Yield of pure product, 18%; total product recovered, 50%. 1H NMR
• (400MHz, DMSO-d6) δ D0.89 (s, 3H), 1.30-1.70 (m, 4H), 1.80-1.87 (m, 1H), 1.93-2.11
(m, 3H), 2.17-2.40 (m, 3H), 2.71-2.78 (m, 2H), 6.43 (d, J=2.2Hz, 1 H), 6.50 (dd, J=8.1 ,
2.6Hz, 1 H), 7.03 (d, J=8.4Hz, 1H), 9.01 (s, 1H), 10.3 (bs, 1H); HPLC (100%, Rt= 2.33, 5 20:80 H20:MeOH); LCMS (APCI) 311.29 (M+H+), Ace. Mass (FAB+) calcd. 311.1759, obsd. 311.1761 (M+H+)
General procedure for amide coupling using a Radleys GreenHouse Synthesiser.
0 To a solution of the acid starting material (0.107g, 0.25mmol) in dry DCM (2cm3) and triethylamine (0.02cm3, 0.18mmol) was added a solution of EDC (0.058g, 0.3mmol) triethylamine (0.017cm3, 0.16mmo() and DMAP (catalytic) in dry DCM (1cm3) under nitrogen and the reaction was stirred at room temperature for 30min. To this solution was then added amine (1.1eq) and stirring under nitrogen was continued for 12h, 5 followed by standing under nitrogen for 14h. The solution was then washed with saturated aqueous NaHCO3 before separation and concentration of the organic layer and purification by flash chromatography.
2-Benzyloxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- 0 pentaleno[2, 1-a]phenanthrene-9-carfoo3cyIic acid isopropylamide
(GUA02038- )
Figure imgf000094_0001
Yield 16%. Rf 0.72 (EtOAc); 1H NMR (270MHz, CDCI3) δ 0.97 (s, 3H), 1.18 (s, 3H), 1.20 (s, 3H), 1.37-1.92 (m, 5H), 2.08-2.46 (m, 5H), 2.71 (dd, J=13.9, 6.2Hz, 1H), 2.83-2.90 (m, 2H), 4.13-4.26 (m, 1H), 4.98 (S, 2H), 5.83 (bs, 1H), 6.68 (d, J=2.9Hz, 1H), 6.73 (dd, J=8.4, 2.9Hz, 1H), 7.15 (d, J=8.4Hz, 1H), 7.22-7.38 (m, 5H); LCMS (ES+) 470.37 (M+H+)
2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid isopropyl amide STX857 (G A02046)
Figure imgf000094_0002
A solution of the benzyl protected starting material (0.020g, 0.04mmol) in THF <5cm3) and ethanol (5cm3) was degassed by bubbling nitrogen through for 30min before Pd/C (5%wt., catalytic) was added and hydrogen gas (balloon) was passed over the reaction. Stirring at room temperature under a hydrogen blanket was continued overnight before the mixture was filtered through celite and the celite washed with ethyl acetate and methanol. The filtrate was concentrated in vacuo to give a white solid, Rf 0.60 (EtOAc). Yield 0.096g, 63%. 1H NMR (270MHz, CDCI3) δ 0.97 (s, 3H), 1.25 (s, 3H), 1.27 (s, 3H), 1.47-1.95 (m, 4H), 2.13-2.53 (m, 6H), 2.77 (dd, J=13.9, 6.2Hz, 1H), 2.87-2.94 (m, 2H), 4.19-4.30 (m, 1H), 5.88 (d, J=8.1Hz, 1H), 6.60 (d, J=2.93Hz, 1H), 6.65 (dd, J=8.3, 2.93Hz, 1H), 7.16 (d, J=8.4Hz, 1H); HPLC (95%, Rt=1.72, 4:96 H20:MeOH); LCMS (AP-) 378.35 (M-H+)
2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7,8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (pyridin-3-ylmethyl)-amide STX 860 (GMA02056/3)
Figure imgf000095_0001
To a suspension of acid starting material (0.170g, O.δmmol) in dry DCM (10cm3) under nitrogen was added DMAP (catalytic), EDC (0.116g, O.δmmol) and triethylamine (0.06cm3, Q.55mol) and the solution was stirred at room temperature for 20min. To this mixture was added S-(aminomethyl) pyridine (0.06cm3, O.δδmmol), followed by dry DMF (10cm3) to ensure complete dissolution of starting materials, and the reaction was stirred at room temperature under nitrogen for 24h. The solution was washed with saturated aqueous bicarbonate and the organic layer separated and concentrated in vacuo. To the resulting concentrated solution (in DMF) was added hexane followed by a small amount of DCM. The resulting cream-coloured precipitate was collected by filtration and purified by flash chromatography using DCM followed by 10% MeOH in DCM as eluent, giving a white crystalline solid. Rf 0.40 (10% MeOH in DCM), Yield 0.034g, 16%. 1H NMR (400MHz, DMSO-d6) δ D0.92 (s, 3H), 1.37-2.36 (m, 10H), 2.63-2.89 (m, 3H), 4.41 (appd, J=22.3Hz, 2H), 6.44 (d, J=2.3Hz, 1H), 6.51 (dd, J=8.2, 2.3Hz, 1H), 7.04 (d, J=8.2Hz, 1H), 7.33 (apps, 1H), 7.69 (apps, 1H), 7.69-8.63 (m, 3H), 9.02 (s, 1H), 12.65 & 12.92 (s, 0.5H, tautomers NH on pyrazole ring); HPLC (98%, Rt=2.04, 10:90 H20:MeOH); LCMS (ES+) 429.19 (M+H+)
Figure imgf000095_0002
Route to STX 820
Figure imgf000096_0001
Route to STX 737
Figure imgf000097_0001
NH2NH2.H20 EtOH
Figure imgf000097_0002
Routes to ST3C 857 and STIC 860 G A03034 [3-(tert-Buty|-dimethyl-silanyloxy)-2-ethyl-13-methyl-17-oxo-
6,7,8,9,11, 12,13,14,15,17-decahydro-cyclopenta[a]phenanthren-16-ylideneJ- hydro∑sy-acetic acid ©thyl ©st©r
Figure imgf000098_0001
To a stirred solution of TBDMS protected estrone (1.275 g, 3.1 mmol) in toluene (30 cm3) was added diethyl oxalate (0.84 cm3, 6.2 mmol) and potassium t-butoxide (0.45 g, 4 mmol) and the reaction was stirred at rt overnight. The reaction was acidified with glacial acetic acid and concentrated in vacuo before ethyl acetate (30 cm3) was added. The solution was washed with water (30 cm3) and brine (30 cm3) before being concentrated in vacuo to give a whife powder, This powder was washed with ethanol/water and collected by filtration. Yield quantitative.
1H NMR δ (270 MHz, CDCI3) 0.21 (s, 6H, (CH3)2Si), 0.98 (s, 3H, 18-CH3), 0.99 (s, 9H, (CH3)3C), 1.15 (t, J = 7.5 Hz, 3*H, ArCH2CH3), 1.38 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.54- 1.69 (m, 6H), 2.00-2.05 (m, 2H), 2.27-2.47 (m, 3H), 2.55 (q, J = 7.5 Hz, 2H, ArCH2CH3), 2.80-2.86 (m, 2H), 3.03-3.11 (m, 1H), 4.35 (q, J = 7.2 Hz, 2H, OC V2CH3), 6.48 (s, 1H, 4- H), 7.03 (s, 1H, 1-H); FAB-LRMS (MH+) 513.3 m/z; FAB-HRMS calcd for C^H^Si 512.2958 found (M+) 512.2965 m/z
G A03035 2-(tert-Butyl-dimethyl-silanyloxy)-3-ethyl-6a-methyl-
4b,5,6,6a,8, 10,10a, 10b, 11 , 12-decahydro-7,8-diaza-pentaleno[2,1 -a]phenanthrene-9- carboxylic acid ethyl ester
Figure imgf000098_0002
To a stirred suspension of sm (1.53 g, 3 mmol) in ethanol (25 cm3) was added hydrazine monohydrate (0.17 cm3, 3.4 mmol) at which time the sm dissolved. Stirring at room temperature was continued overnight during which time the solution became cloudy. To this was added p-toluene sulphonic acid (~0.1 g) and the mixture was heated for 5 min to aromatise the pyrazole ring. The solution was concentrated in vacuo until precipitate began to form before water was added and the white powder was collected by filtration, washed with water and air-dried. Yield ~quantitative. 1H NMR δ (270 MHz, CDCI3) 0.22 (s, 6H, (CH3)2Si), 0.98 (s, 3H, 18-CH3), 0.99 (s, 9H, (CH3)3C), 1.16 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.37 (t, J = 7.1 Hz, 3H, OCH2CH3), 1.40-2.18 (6H), 2.30-2.50 (4H), 2.56 (q, J = 7.5 Hz, 2H, ArCH2CH3), 2.81-2.96 (m, 3H), 4.35 (q, J = 7.1 Hz, 2H, OCH2CH3), 6.49 (s, 1H, 4-H), 7.06 (s, 1H, 1-H); HPLC > 96% (f?( = 6.69, 100% MeCN); FAB-LRMS (MH+) 509.2 m/z; FAB-HRMS calcd for C34H41N2O3 509.3168 found (MH+) 509.3178 m/z
STX 1066, GMA02187 3-Ethyl-2-hydroxy-6a-methyl-4b,5,6,6a,8, 10, 10a, 10b, 11 , 12-decahydro-7,8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid
Figure imgf000099_0001
To a stirred suspension of sm (1.27 g, 2.64 mmol) in MeOH (30 cm3) was added aqueous NaOH (0.211 g in 16 cm3) and the mixture was refluxed overnight (the sm dissolved on heating). The reaction was cooled, acidified with glacial acetic acid and concentrated in vacuo until precipitate began to form (~ half volume). Water (20 cm3) was added and the resulting precipitate was collected by filtration. This was dissolved in methanol, concentrated and dried in vacuo. Yield 57%.
(400 MHz, CD3OD) 1.02 (s, 3H, 18-CH3), 1.16 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.36-1.47 (m, 1H), 1.56-1.83 (3H), 1.93-1.99 (m, 1H), 2.02-2.15 (m, 1H), 2.22-2.28 (m, 2H), 2.36- 2.43 (m, 2H), 2.55 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.72-2.90 (m, 3H), 6.44 (s, 1H, 4-H), 6.95 (s, 1H, 1-H); HPLC > 92% (Rt = 1.49, 70% MeCN in H2O); ES-ve-MS (M-H+) 365.16 m/z; FAB-HRMS calcd for C22H27N2O3367.2022 found (MH+) 367.2010 m/z General procedure for amide coupling:
To a stirred suspension of acid sm (0.045 g, 0.123 mmol) in dry DCM (8 cm3) under N2 was added DMAP (catalytic), EDC (0.071 g, 0.37 mmol) and NEt3 (0.02 cm3) and the solution was stirred at rt for 30 min. To this was then added amine (0.02 cm3) and stirring was continued for 4 d. The solution was washed with saturated bicarbonate, the organic layer separated and the aqueous layer washed with DCM. The organic layers were combined and concentrated in vacuo and the product purified by flash chromatography (Flashmaster II) using an elution gradient of DCM to 10%' eOH in DCM.
STX 1013, GMA02188
3-Ethy l-2-hydroxy-6a-methy l-4b,5,6,6a,8, 10, 10a, 10b, 11 , 12-decahydro-7,8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (pyridin-3-ylmethyl)-amide
Figure imgf000100_0001
Yield 39%; 1H NMR δ (270 MHz, CD3OD) 1.03 (s, 3H, 18-CH3), 1.15 (t, J = 7.4 Hz, 3H, ArCH2CrV3), 1.39-1.52 (m, 1H), 1.59-1.87 (m, 3H), 1.94-2.00 (m, 1H), 2.14-2.50 (5H), 2.55 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.78-2.90 (3H), 4.58 (s, 2H), 6.45 (s, 1H, 4-H), 6.97 (s, 1H, 1-H), 7.38-7.43 (m, 1H), 7.85 (d, J = 7.7 Hz, 1H), 8.43 (d, J = 3.7 Hz, 1H), 8.56 (s, 1H); HPLC > 97% (Rt 2.11, 70% MeCN in H2O); ES+ve-MS (MH+) 457.38 m/z FAB- HRMS calcd for C28H33N4O2457.2603 found (MH+) 457.2589 m/z
STX 1144, GW1A03074-1
3-Ethyl-2-hydroxy-6a-methyl-4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-dia2a- pental@no[2,1 -a]ph@nanthren@-9-carbo..ylic acid (pyridin-2-ylm©thyl)-amid©
Figure imgf000101_0001
Yield 41%; 1H NMR δ (270 MHz, CD3OD) 1.01 (s, 3H, 18-CH3), 1.14 (t, J = 7.4 Hz, 3H, ArCH2CW3), 1.31-1.47 (m, 1H), 1.57-1.83 (m, 3H), 1.93-2.00 (m, 1H), 2.14-2.50 (5H), 2.54 (q, J = 7.4 Hz, 2H, ArCrV2CH3), 2.71-2.86 (3H), 4.64 (s, 2H), 6.44 (s, 1H, 4-H), 6.95 (s, 1H, 1-H), 7.27-7.31 (m, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.78 (dt, J = 7.7, 1.6 Hz, 1H), 8.48 (d, J = 4.7 Hz, 1H); HPLC > 92% (Rt 2.15, 80% MeCN in H2O); ES-ve-MS (M-H+) 455.36 m/z FAB-HRMS calcd for C28H33N4O2457.2603 found (MH+) 457.2607 m/z
STX 1084, G A03056-2
3-Ethy l-2-hydroxy-6a-methyl-4b,5,6,6a,8, 10, 10a, 10b, 11 , 12-decahydro-7,8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (2-pyridin-2-yl-ethyl)-amide
Figure imgf000101_0002
Yield 46%; 1H NMR δ (270 MHz, CD3OD) 1.00 (s, 3H, 18-CH3), 1.14 (t, J = 7.5 Hz, 3H, ArCH2CW3), 1.33-1.49 (m, 1H), 1.54-1.83 (3H), 1.93-2.02 (m, 1H), 2.04-2.43 (5H), 2.54 (q, J = 7.5 Hz, 2H, CH2CH3), 2.71-2.90 (3H), 3.07 (t, J = 7.1 Hz, 2H, -CrV2CH2-), 3.70 (dt, J = 6.9, 1.7 Hz, 2H, -CH2CH2), 6.45 (s, 1H, 4-H), 6.96 (s, 1H, 1-H), 7.26-7.31 (m, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.77 (dt, J = 7.7, 1.7 Hz, 1H), 8.47-8.50 (m, 1H); HPLC > 97% (R, 3.41, 70% MeCN in H2O); ES-ve-MS (M-H+) 469.55 m/z; FAB-HRMS calcd for C29H35N4O2471.2760 found (MH+) 471.2747 m/z
BVX 1085, GMA03056-1
3-Ethy l-2-hydroxy-6a-methyl-4b,5,6,6a,8, 10, 10a, 10b, 11 , 12-decahydro-7,8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (2-pyridin-3-yl-ethyl)-amide
Figure imgf000102_0001
Yield 51%; 1H NMR δ (270 MHz, CD3OD) 1.02 (s, 3H, 18-CH3), 1.15 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.30-1.49 (m, 1H), 1.59-1.86 (m, 3H), 1.93-2.01 (m, 1H), 2.10-2.45 (5H), 2.55 (q, J = 7.4 Hz, 2H, -CH2CH3), 2.72-2.85 (3H), 2.95 (t, J=7.1 Hz, 2H, -CH2CH2-), 3.61 (t, J = 7.3Hz, 2H, -CH2CH2-), 6.46 (s, 1H, 4-H), 6.97 (s, 1H, 1-H), 7.37-7.42 (m, 1H), 7.79 (d, J = 7.7 Hz, 1H), 8.40 (d, J = 4.2 Hz, 1H), 8.45 (s, 1H); HPLC > 98%; (Rt 3.09, 70% MeCN in H2O); ES-ve-MS (M-H+) 469.49 m/z; FAB-HRMS calcd for C29H35N4O2 471.2760 found (MH+) 471.2756 m/z
STX 1145, GMA03074-2
3-Ethyl-2-hydroxy-6a-methyl-4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (2-methoxy-ethyl)-amide
Figure imgf000102_0002
Yield 36%; 1H NMR δ (270 MHz, CD3OD) 1.02 (s, 3H, 18-CH3), 1.15 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.34-1.50 (m, 1H), 1.53-1.85 (m, 3H), 1.93-2.00 (m, 1H), 2.17-2.47 (5H), 2.54 (q, J = 7.4 Hz, 2H, -CW2CH3), 2.73-2.87 (3H), 3.3-3.4 (4H, -CH2CH2-), 3.53 (s, 3H, O-CH3), 6.45 (s, 1H, 4-H), 6.96 (s, 1H, 1-H), HPLC > 96%; (/?, 2.11, 80% MeCN in H2O); ES-ve-MS (M-H+) 422.35 m/z; FAB-HRMS calcd for C25H34N3O3 424.2600 found (MH+) 424.2606 m/z
STX 1146, GW1A03074-4
3-Ethyl-2-hydrø2.y-6a-m©thyl-4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-dia2a- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)- amide
Figure imgf000103_0001
Yield 42%; 1H NMR δ (270 MHz, CD3OD) 1.01 (s, 3H, 18-CH3), 1.14 (t J = 7.4 Hz, 3H, ArCH2CH3), 1.31-1.47 (m, 1H), 1.52-1.83 (m, 3H), 1.92-2.00 (m, 1H), 2.15-2.58 (7H), 2.52 (s, 3H, ArCH3), 2.69-2.86 (3H), 4.64 (s, 2H), 6.44 (s, 1 H, 4-H), 6.95 (s, 1 H, 1-H), 7.27-7.31 (m, 1H), 8.46 (d, J = 1.1 Hz, 1H), 8.49 (s, 1H); HPLC > 94% (Rt 2.08, 80% MeCN in H2O); ES-ve-MS (M-H+) 470.35 m/z FAB-HRMS calcd for C28H34N5O2 472.2713 found (MH+) 472.2718 m/z.
STX 1166, GMA03092
3-Ethyl-2-hydroxy-6a-methyl-4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (benzo[1,3]dioxol-5-ylmethyl)- amide
Figure imgf000103_0002
Yield 56%; 1H NMR δ (400 MHz, CD3OD) 1.08 (s, 3H, 18-CH3), 1.19 (t, J = 7.4 Hz, 3H, ArCH2Cf ), 1.48-2.52 (10H), 2.59 (q, J = 7.8 Hz, 2H), 2.80 - 2.95 (m, 3H), 4.48 (s, 2H), 5.95 (s, 2H), 6.49 (s, 1 H), 6.79-6.89 (m, 3H), 7.01 (s, 1 H); HPLC > 98% (R, 7.29, 90% MeCN in H2O); APCI (M-H+) 498.32 m/z; FAB-HRMS calcd for C28H34N5O2 found (MH+) m/z.
GMA03028 (3-Benzyloxy-2-ethyl-13-methyl-17-oxo-6,7,8,9,11,12,13,14,15,17- ecahydro- cycl©p©n£a[ajphenanthren-16-ylid©n©)-hydr©xy-acetie acid ethyl ester
Figure imgf000104_0001
To a stirred solution of benzyl protected 2-ethyl estrone (2.00 g, 5.15 mmol) in toluene (20 cm3) was added diethyl oxalate (1.40 cm3, 10.20 mmol) followed by potassium t- butoxide (0.693 g, 6.18 mmol) and the reaction was stirred at rt for 16 h. The solution was acidified with glacial acetic acid before being concentrated in vacuo. To the residue was added ethyl acetate (30 cm3) and the solution washed with water (30 cm3) and brine (30 cm3). The organic layer was concentrated in vacuo to give a white powder, which was washed with ethanol/water and collected by filtration. Yield 97%; 1H NMR δ (270 MHz, CDCI3) 0.99 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.38 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.41-1.69 (6H), 2.00-2.05 (m, 2H), 2.30-2.50 (m, 3H), 2.66 (q, J = 7.5 Hz, 2H, ArCH2CH3), 2.88-2.91 (m, 2H), 3.04-3.10 (m, 1 H), 4.35 (q, J = 7.2 Hz, 2H, OCH2CH3), 5.04 (s, 2H, ArCrV2O), 6.64 (s, 1H, 4-H), 7.09 (s, 1H, 1-H), 7.26-7.45 (m, 5H); HPLC > 97% (Rt = 2.86, 98% MeCN in H2O); ES-ve-MS (M-H+) 487 m/z; FAB-HRMS calcd for C31H36O5 488.2563 found (M+) 488.2569 m/z.
GMA03029
2-Benzyloxy-3-ethyl-6a-methyl-4b,5,6,6a,8, 10, 10a, 10b, 11 , 12-decahydro-7,8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid ethyl ester
Figure imgf000104_0002
To a stirred suspension of sm (2.44 g, 5 mmol) in ethanol (50 cm3) was added hydrazine monohydrate (0.3 cm3, 6 mmol) at which time the sm dissolved. Stirring at room temperature was continued overnight before the white precipitate was collected by filtration and washed with water. The filtrate was acidified with glacial acetic acid and more precipitate formed and was collected. The precipitates were combined, and suspended in ethanol before p-toluene sulphonic acid (-0.1 g) was added. The mixture was heated for 5 min to aromatise the pyrazole ring then stirred at rt for 1 h. The pale yellow solution was concentrated in vacuo until precipitate began to form before water was added and the white powder was collected by filtration, washed with water and air- dried. Yield 96%; 1H NMR δ (270 MHz, CDCI3) 1.03 (s, 3H, 18-CH3), 1.21 (t, J = 7.5 H∑, 3H, ArCH2CH3), 1.37 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.45-2.20 (6H), 2.32-2.50 (m, 4H), 2.67 (q, J = 7.5 Hz, ' 2H, ArCH2CH3), 2.82-2.96 (m, 3H), 4.35 (q, J = 7.2 Hz, 2H, OCW2CH3), 5.05 (s, 2H, ArCH2O), 6.65 (s, 1H, 4-H), 7.12 (s, 1H, 1-H), 7.28-7.46 (m, 5H); HPLC > 92% (Rt = 5.17, 94% MeCN in H2O); ES-ve-MS (M-H+) 483 m/z FAB-HRMS calcd for C31H36N2O3484.2726 found (M+) 484.2730 m/z.
STX 1057, GMA03032/2, GMA03062
3-Ethyl-2-hydroxy-6a-methyl-4b,5,6,6a,8, 10, 10a, 10b, 11 , 12-decahydro-7,8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid ethyl ester
Figure imgf000105_0001
A suspension of sm (3.40 g, 7 mmol) in MeOH (70 cm3) was degassed by bubbling nitrogen through for 30 min. To this was added Pd/C (5 % wt., catalytic) and degassing was continued for a further 10 min before hydrogen gas was passed over the reaction and stirring under hydrogen was continued for a further 3 d. The mixture was filtered through celite, the celite washed with ethyl acetate and methanol and the filtrate concentrated in vacuo. The product was purified by flash chromatography (Flashmaster II) using a gradient elution of hexane to 1:1 ethyl acetate:hexane. Rf (1/1 ethyl acetate/hexane) 0.24. Yield 89%;. Η NMR δ (270 MHz, CD3OD) 1.03 (s, 3H, 18-CH3), 1.15 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.36 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.39-2.20 (6H), 2.26-2.44 (m, 4H), 2.53 (q, J = 7.3 Hz, 2H, ArCH2CH3), 2.79-2.86 (m, 3H), 4.34 (q, J = 7.1 Hz, 2H, OCrV2CH3), 6.46 (s, 1H, 4-H), 6.97 (s, 1H, 1-H); FAB-HRMS calcd for for C24H30N2O3395.2335 found (M+) 395.2312 m/z. STX 1024, GMA03008, GMA03044/3, GMA03063 3-Ethyl-6a-methyl-2-sulfamoyloxy-4h,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8- dia2a-pentaleno[2,1-a]phenanthrene-9-carfoo2^ylic acid ethyl ester
Figure imgf000106_0001
A solution of sulfamoyl chloride (30 cm3 of a 0.68 M solution in toluene, 20.4 mmol) was concentrated in vacuo without heating then cooled on ice under nitrogen. To this was added DMA (15 cm3) and this mixture added to a cooled (ice/acetone bath) solution of 0 sm (2.42 g, 6.13 mmol) in DMA (10 cm3). The stirred solution was allowed to warm to rt overnight before being diluted with water (60 cm3) and the product extracted with ethyl acetate (100 cm3), giving an oily solution in DMA. This was redissolved in ethyl acetate
' and washed again with water before concentration to give a cream coloured crystalline solid. Recrystallisation from DCM/hexane gave 2g, 69% yield of product. 1H NMR δ (270 5 MHz, CD3OD) 1.04 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.36 (t, J = 7.1
Hz, 3H, OCH2CW3), 1.39-2.20 (6H), 2.29-2.50 (m, 4H), 2.71 (q, J = 7.5 Hz, 2H,
ArCH2CH3), 2.81-2.92 (m, 3H), 4.33 (q, J = 7.1 Hz, 2H, OCrV2CH3), 7.09 (s, 1H, 4-H),
• 7.22 (s, 1H, 1-H); HPLC > 97% (Rt = 2.40, 80% MeCN in H2O); FAB-LRMS (MH+) 474 m/z; FAB-HRMS calcd for for C24H32N3O5S 474.2063 found (MH+) 474.2067 m/z. 0
GMA03064
Figure imgf000106_0002
5 To a suspension of 2-chlorotrityl chloride resin (2.27g, 1.1 mmol/g theoretical loading) swollen in anhydrous DCM (12 cm3) and DIPEA (5.34 cm3, 29.5 mmol) under nitrogen was added sm (2.00 g, 4.22 mmol) as a solid in portions followed by a further volume of DCM (5 cm3). The reaction was shaken at rt for 96 h before the resin was collected by filtration and washed three times with DCM, methanol and again with DCM.
GRM03O68
Figure imgf000107_0001
To the loaded resin (3.74 g) swollen in THF (25 cm3) was added slowly aqueous sodium hydroxide (30 cm3' of 1 N) and the mixture was shaken at rt for 24 h. The resin was collected by filtration, washed with THF/water (1/1) and washed with MeOH and DCM before being dried in vacuo.
STX 1080, GMA03052-1
3-Ethyl-6a-methyl-2-sulfamoyloxy-4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8- diaza-pentaleno[2,1-a]phenanthrene-9-carboxylic acid
Figure imgf000107_0002
The loaded resin (0.03 g) was placed in a fritted syringe and washed with TFA/DCM (20 cm3 of 1/1, added portion-wise with 5 min between washings) followed by DCM. The washings were combined and concentrated in vacuo. The resin loading from the weight of the crude was calculated to be 0.8 mmol/g.
Purification by flash chromatography (Flashmaster II) using a gradient elution of DCM to 10% MeOH in DCM gave the product Rf 0.6 (10% MeOH in DCM). 1H NMR δ (270 MHz, CD3OD) 1.04 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.46-1.90 (5H), 1.99-2.07 (m, 1H), 2.12-2.23 (m, 1H), 2.31-2.51 (4H), 2.71 (q, J = 7.5 Hz, 2H, ArCW2CH3), 2.82-2.92 (3H), 7.09 (s, 1H, 4-H), 7.22 (s, 1H, 1-H); HPLC > 93% (Rt 1.45, 70% MeCN in H2O); ES+ve-MS (MH+) 468 m/z; FAB-HRMS calcd for for C22H28N3O5S 446.1750 found (MH+) 446.1713 m/z.
.General procedure for amide coupling on resin-bound starting material:
To the loaded resin (0.200 g) swollen in dry DCM (3 cm3) under N2 was added bromo- tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP, 0.260 g, 0.5 mmol) and N- hydroxybenzotriazole (HOBt, 0.070 g, 0.5 mmol) followed by DIPEA (0.18 cm3, 1.0 mmol). The mixture was shaken for 10 min before addition of amine (0.06 cm3) and shaking under N2 at rt was continued for 72 h. The resin was collected in a fritted syringe and washed with DMF, DCM and MeOH (x3) and again with DCM (x3) before cleavage of the product using TFA/DCM (1/1). Purification by flash chromatography (Flashmaster II) using a gradient elution of DCM to 10% MeOH in DCM yielded the sulfamates in yields of 1 - 53% and HPLC purities of 88-98%.
STX 1083, GMA03052-2/3
Sulfamic acid 3-ethyl-6a-methyl-9-[(pyridin-2-ylmethyl)-carbamoyl]-
4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza-pentaleno[2,1-a]phenanthren-2-yl ester
Figure imgf000108_0001
Yield 53 %; 1H NMR δ (270 MHz, CD3OD) 1.04 (s, 3H, 18-CH3), 1.19 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.39-1.55 (m, 1H), 1.59-1.88 (m, 3H), 1.98-2.04 (m, 1H), 2.14-2.58 (5H), 2.70 (q, J = 7.5 Hz, 2H, ArCH2CH3), 2.78-2.98 (3H), 4.64 (s, 2H), 7.08 (s, 1H, 4-H), 7.20 (s, 1H, 1-H), 7.28-7.33 (m, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.80 (dt, J = 7.8, 1.7 Hz, 1H), 8.49 (d, J = 4.5 Hz, 1H); HPLC > 97% (R, 2.11, 70% MeCN in H2O); ES-ve-MS (M-H+) 534 m/z; FAB-HRMS calcd for for C28H34N5O4S 536.2332 found (MH+) 536.2330 m/z.
STX 1082 GMA03052-3/3 Sulfamic acid 3-ethyl-6a-methyl-9-(2-pyridin-3-yl-ethylcarbamoyl)-
4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza-pentaleno[2,1-a]phenanthren-2-yl
©sier
Figure imgf000109_0001
Yield 7 %; 1H NMR δ (270 MHz, CD3OD) 1.02 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.46-1.89 (4H), 1.98-2.04 (m, 1H), 2.15-2.52 (5H), 2.65-2.98 (7H), 3.60 (t, J = 6.7 Hz, 2H, -CH2CH2), 7.04 (s, 1H, 4-H), 7.21 (s, 1H, 1-H), 7.36-7.41 (m, 1.H), 7.78 (dt, J = 6.2, .7 Hz, 1 H), 8.39 (dd, J. = 4.8, 1.7 Hz, 1 H), 8.44 (d, J = 1.7 Hz, 1 H); LC purity > 95%; ES-ve-MS (M-H+) 548 m/z; FAB-HRMS calcd for for C29H36N5O4S 550.2488 found (MH+) 550.2499 m/z.
STX 1081, G A03052-4/3 Sulfamic acid 3-ethyl-6a-methyl-9-(2-pyridin-2-yl-ethylcarbamoyl)-
4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza-pentaleno[2,1-a]phenanthren-2-yl ester
Figure imgf000109_0002
Yield 25 %; 1H NMR δ (270 MHz, CD3OD) 1.02 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2C V3), 1.46-1.88 (4H), 1.98-2.05 (m, 1 H), 2.12-2.52 (5H), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 2.77-2.92 (3H), 3.07 (t, J = 6.9 Hz, 2H, -CW2CH2)f 3.71 (dt, J = 7.2, 1.8 Hz, 2H, -CH2CH2), 7.09 (s, 1H, 4-H), 7.20 (s, 1H, 1-H), 7.26-7.29 (m, 1H), 7.36 (d, J = 7.9 Hz, 1 H), 7.77 (dt, J = 7.7, 1.9 Hz, 1H), 8.47-8.50 (m, 1H); HPLC > 98% (R, 1.96, 90% MeCN in H2O); ES-ve-MS (M-H+) 548 m/z; FAB-HRMS calcd for for C29H36N5O4S 550.2488 found (MH+) 550.2483 m/z. STX 1123 GMA03072-1/2 Sulfamic acid 3-ethyl-6a-methyl-9-[(pyridin-3-ylmethyl)-carbamoyl]-
4b,5,6,6aι,8,10,10a,10b,11,1- -decahydro-7,8-dia2a-pentaleno[2,1-a3phenanthren-2-yl
©st©r
Figure imgf000110_0001
Yield 8 %; 1H NMR δ (400 MHz, CD3OD) 1.06 (s, 3H, 18-CH3), 1.21 (t, J = 7.4 Hz, 3H, ArCH2CrV3), 1.47-1.57 (m, 1H), 1.65-1.90 (3H), 2.04-2.07 (m, 1H), 2.14-2.33 (2H), 2.39- 2.53 (3H), 2.72 (q, J = 7.4 Hz, 2H, ArCr 2CH3), 2.90-2.94 (3H), 4.58 (s, 2H), 7.09 (s, 1H, 4-H), 7.21 (s, 1H, 1-H), 7.40-7.43 (m, 1H), 7.85 (d, J = 7.8 Hz, 1H), 8.42 (d, J = 4.7 Hz, 1H), 8.54 (d, J = 2.0 Hz, 1H); HPLC > 99% (Rt 1.91, 80% MeCN in H2O); ES+ve-MS (MH+) 536.31 m/z; FAB-HRMS calcd for for C28H34N5O4S 536.2332 found (MH+) 536.2330 m/z.
STX 1124, GMA03072-2/2
Sulfamic acid 3-ethyl-6a-methyl-9-[(5-methyl-pyrazin-2-ylmethyl)-carbamoyl]-
4b,5,6,6a,8, 10, 10a, 10b, 11 , 12-decahydro-7,8-diaza-pentaleno[2, 1 -a]phenanthren-2-y I ester
Figure imgf000110_0002
Yield 8 %; 1H NMR δ (400 MHz, CD3OD) 1.06 (s, 3H, 18-CH3), 1.22 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.47-1.57 (m, 1H), 1.65-1.90 (3H), 2.03-2.07 (m, 1H), 2.14-2.55 (5H), 2.72 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.90-2.94 (3H), 4.66 (s, 2H), 7.09 (s, 1H, 4-H), 7.21 (s, 1H, 1-H), 8.48 (s, 1H), 8.49 (s, 1H); HPLC > 99% (R, 1.93, 80% MeCN in H2O); ES+ve- MS (MH+) 551.18 m/z; FAB-HRMS calcd for for C28H35N6O4S 551.2441 found (MH+) 551.2447 m/z STX 1125, G lA03072-4a
Sulfamic acid 3-ethyl-9-(2-metho2«y-e hylcarbamo l)-6a-methyl-
4bl5,6J@a,8,10,10a>10b,11,12-d©c®hydro-7,8-dia2a-p©ntal©no[2,1-a3ph©nanthr©n-2-yl ester
Figure imgf000111_0001
Yield 10 %; 1H NMR δ (400 MHz, CD3OD) 1.06 (s, 3H, 18-CH3), 1.22 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.47-1.57 (m, 1H), 1.65-1.90 (3H), 2.03-2.09 (m, 1H), 2.18-2.5δ (δH), 2.72 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.84-2.96 (3H), 3.3-3.55 (7H), 7.09 (s, 1H, 4-H), 7.22 (s, 1H, 1-H); HPLC > 88% ( ?, 3.28, 5 to 95% MeCN in H2O over 10 min); ES+ve-MS (MH+) 503.24 m/z; FAB-HRMS calcd for for C25H35N4O5S 503.2328 found (MH+) 503.2326 m/z.
STX 1126, GMA03072-5/2
Sulfamic acid 3-ethyl-9-(ethyl-methyl-carbamoyl)-6a-methyl-
4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza-pentaleno[2,1-a]phenanthren-2-yl ester
Figure imgf000111_0002
Yield 5 %; 1H NMR δ (400 MHz, CD3OD) 1.08 (s, 3H, 18-CH3), 1.20-1.25 (6H), 1.30-1.6 (1H), 1.65-1.90 (3H), 2.01-2.09 (m, 1H), 2.16-2.50 (5H), 2.72 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.84-2.93 (3H), 3.06-3.26 (3H), 3.46-3.64 (m, 2H), 7.09 (s, 1H, 4-H), 7.22 (s, 1H, 1-H); HPLC > 98% {R, 2.43, 5 to 95% MeCN in H2O over 10 min); ES+ve-MS (MH+) 487.24 m/z; FAB-HRMS calcd for for C25H35N4O4S 487.2379 found (MH+) 487.2396 m/z.
STX 1152, GMA03076-1 Sulfamic acid 3-ethyl-9-[(furan-2-ylmethyl)-carbamoyl]-6a-methyl-
4b,5,6,6a,8,10,10a,10b,11,12- decahydro-7,8-dia2a-pentaleno[2,1-a]phenanthren-2-yl
Figure imgf000112_0001
Yield 6 %; 1H NMR δ (270 MHz, CD3OD) 1.02 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.46-1.89 (4H), 1.95-2.06 (m, 1H), 2.12-2.52 (5H), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 2.81-2.91 (3H), 4.52 (s, 2H), 6.28-6.36 (m, 2H), 7.09 (s, 1H, 4-H), 7.21 (s, 1H, 1-H), 7.43 (s, 1H); HPLC > 91 % (R, 2.17, 70 % MeCN in H2O); ES-ve-MS (M-H+) 523.27 m/z; FAB-HRMS calcd for for C27H33N4O5S 525.2172 found (MH+) 525.2176 m/z
STX 1153, GMA03076-3
Sulfamic acid 9-[(benzo[1 ,3]dioxol-5-ylmethyl)-carbamoyl]-3-ethy l-6a-methyl- 4b,5,6,6a,8, 10, 10a, 10b, 11 , 12-decahydro-7,8-diaza-pentaleno[2, 1 -a]phenanthren-2-yl ester
Figure imgf000112_0002
Yield 1 %; 1H NMR δ (270 MHz, CD3OD) 1.06 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.46-2.54 (10H), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 2.81-3 (3H), 4.45 (s, 2H), 5.91 (s, 2H), 6.74-6.85 (2H), 7.09 (s, 1 H, 4-H), 7.21 (s, 1 H, 1-H); HPLC > 93 % (R, 5.39, 5 to 95 % MeCN in H2O over 10 min); ES-ve-MS (M-H+) 577.26 m/z; FAB-HRMS calcd for for C30H35N4O6S 579.2277 found (MH+) 579.2257 m/z
STX 1154, G A03076-4
Sulfamic acid 3-ethyl-9-[2-(1 H-indol-3-yl)-ethylcarbamoylJ-@a-methyl-
4b,5,6,6a,8, 10, 10a, 10b, 11 , 12-decahydro-7,8-diaza-pentaleno[2,1 -a]phenanthren-2-yl ester
Figure imgf000113_0001
Yield 3 %; 1H NMR δ (270 MHz, CD3OD) 1.00 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CW3), 1.46-2.52 (11H), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 2.90-2.92 (2H), 3.03- 3.08 (2H, -CH2CH2), 3.63-3.67 (2H, -CH2CW2), 6.95-7.12 (4H), 7.21 (s, 1H, 1-H), 7.34 (d, J = 8.0 Hz, 1 H), 7.56-7.59 (1H); HPLC > 98 % (R, 2.22, 70% MeCN in H2O); ES-ve-MS (M-H+) 586.33 m/z FAB-HRMS calcd for for C32H38N5O4S 588.2645 found (MH+) 588.2659 m/z
STX 1155, GMA03076-6
Sulfamic acid 3-ethyl-6a-methyl-9-phenethylcarbamoyl-
4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza-pentaleno[2,1-a]phenanthren-2-yl ester
Figure imgf000113_0002
Yield 9 %; 1H NMR δ (270 MHz, CD3OD) 1.03 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CW3), 1.46-1.90 (4H), 1.98-2.05 (m, 1 H), 2.12-2.52 (5H), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 2.77-2.99 (5H), 3.55-3.61 (2H, -C/-/2CH2), 7.10 (s, 1H, 4-H), 7.20-7.30 (6H); HPLC > 93 % {Rt 2.83, 70% MeCN in H2O); ES-ve-MS (M-H+) 547.27 m/z; FAB-HRMS calcd for for C30H37N4O4S 549.2536 found (MH+) 549.2532 m/z.
GRHA03036 2-Bensylo^y-7-(2-cyano-@thyl)-3-ethyl-4b,5,6,6a,7,10,10a,10b,11,12-d©cahydro-7,8- diazapentaleno[2,1-a]phenanthrene-9-carboxylic acid ethyl ester
Figure imgf000114_0001
To a stirred suspension of sm (0.17 g, 0.35 mmol) in ethanol (10 cm3) was added 2- cyanoethylhydrazine (0.05 cm3) and the dark orange solution was stirred at rt overnight. 5 The resulting dark brown solution was acidified with glacial acetic acid and water (10 cm3) was added. The solution was concentrated in vacuo to ~ 1 cm3 before more water was added until the product precipitated as a yellowish powder. This was collected by filtration and washed with water. 1H NMR at this stage showed that the pyrazole ring was still hydrated therefore the product was redissolved in ethanol, p-toluene sulphonic acid 0 monohydrate was added and the solution heated for a few min. The solution was then concentrated in vacuo until precipitate began to form, water (20 cm3) was added and the resulting orange powder was collected by filtration. The product was purified by flash chromatography using a gradient elution of hexane to 1 :1 ethyl acetate:hexane. Yield
■ 0.032g, 17% 5 1H NMR δ (270 MHz, CDCI3) 1.08 (s, 3H, 18-CH3), 1.21 (t, J = 7.5 Hz, 3H, ArCH2CrV3), 1.38 (t, J = 7.1 Hz, 3H, OCH2CH3), 1.35-2.08 (6H), 2.30-2.58 (4H), 2.67 (q, J = 7.6 Hz, 2H, ArCr72CH3), 2.80-2.91 (3H), 3.05-3.08 (m, 2H), 4.27-4.41 (4H), 5.04 (s, 2H, ArCH2O), 6.65 (s, 1 H, 4-H), 7.08 (s, 1H, 1-H), 7.27-7.45 (m, 5H); ES+ve-MS (MH+) 560 m/z. 0
GMA03043, STX 1079
7-(2-cyano-ethyl)-3-ethy l-4b,5,6,6a,7, 10, 10a, 10b, 11 , 12-decahydro-7,8- dia∑apentaleno[2,1-a]phenanthrene-@-carfooj{ylic acid ethyl ester
Figure imgf000115_0001
A stirred solution of sm (0.03 g, 0.06 mmol) in THF (2 cm3) and ethanol (3 cm3) was degassed by bubbling N2 through for 20 min before Pd/C (5 % wt., catalytic) was added. Degassing was continued for a further 10 min before H2 gas was passed over the reaction and the reaction was stirred under a hydrogen blanket overnight. The mixture was filtered through celite and the product purified by flash chromatography using a gradient elution of DCM to 10% MeOH in DCM. Yield 0.025 g, 93%; 1H NMR δ (270 MHz, CDCI3) 1.07 (s, 3H, 18-CH3), 1.21 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.37 (t, J = 7.0 Hz, 3H, OCH2CH3), 1.31- 1.52 (m, 1H), 1.57 - 2.08 (5H), 2.23-2.47 (4H), 2.60 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.78-2.87 (3H), 2.97-3.14 (m, 2H), 4.23-4.41 (4H), 6.51 (s, 1H, 4-H), 7.00 (s, 1H, 1-H); HPLC > 94 % (Rt 2.67, 80% MeCN in H2O); ES+ve-MS (MH+) 448 m/z
GMA02180
Figure imgf000115_0002
To a solution of benzyl protected estrone (1.08 g, 3.0 mmol) in dry THF (20 cm3) under N2, cooled to -78 °C (dry ice/acetone bath) was added slowly drop-wise LDA (6.0 cm3 of a 1.8 M soln, 3.3 mmol) and the reaction was stirred at - 78 °C for 30 min. To this was then added a solution of succinic anhydride (0.30 g, 3.0 mmol) in dry THF (15 cm3) and the reaction was allowed to warm slowly to rt overnight. The mixture was poured into 5 % HCl (120 cm3) and the products extracted with diethyl ether (2 x 60 cm3). The ether extracts were combined and concentrated in vacuo. Flash chromatography using a gradient elution of DCM to 10 % MeOH in DCM resulted in separation of starting material (2.7 mmol) from a mixture which was used without further purification for the following reaction.
GMA02181
Figure imgf000116_0001
To a solution of crude sm (0.29g, assume 0.3 mmol) in EtOH (5 cm3) was added hydrazine monohydrate (0.07 cm3, 1.4 mmol) and the reaction was stirred at rt for 3h then heated to reflux for 30 min to ensure complete reaction and aromatisation of the pyrazole ring. The reaction was cooled, acidified with glacial acetic acid and water was added until the product precipitated as a pale orange powder. Yield 0.098 g; 1H NMR δ (270 MHz, DMSO-d6) 0.89 (s, 3H, 18-CH3), 1.24 - 1.69 (5H), 2.09-2.57 (5H), 2.73-2.84 (m, 4H), 5.06 (s, 2H), 6.73-6.79 (2H), 7.19 (d, J = 8.9 Hz, 1H), 7.32-7.45 (5H); HPLC > 80% {R, = 1.89, 80% MeCN in H2O); FAB-LRMS (MH+) 457 mz; FAB-HRMS calcd for for C29H33N2O3457.2491 found (MH+) 457.2479 m/z.
G A02190
Figure imgf000116_0002
To a stirred solution of sm (0.090 g, assume 0.2 mmol) in dry DCM (5 cm3) was added DMAP (catalytic), EDC (0.046 g, 0.24 mmol) and NEt3 (0.02 cm3) and the solution was stirred for 15 min before addition of tetrahydrofurfuryl amine (0.023 cm3, 0.22 mmol).
The reaction was stirred for 40 h before being diluted with DCM (15 cm3) and the solution washed with sat. aq. bicarb. Flash chromatography using a gradient elution of DCM to 10%) MeOH in DCM yielded 0.035 g, 0.064 mmol of product. NMR δ (270 MHz, CD3OD) 0.96 (s, 3H, 18-CH3), 1.33 - 2.66 (16H), 2.82-2.91 (4H), 3.16-3.29 (3H), 3.56-3.93 (3H), 4.98 (s, 2H), 6.67 (d, J = 2.5 Hz, 1H), 6.73 (dd, J = 8.5, 2.5 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.27-7.41 (5H); FAB-LRMS (MH+) 540 m/z
GMA03014, $721 1035
Figure imgf000117_0001
A stirred solution of sm (0.034 g, 0.06 mmol) in THF (2 cm3) and ethanol (3 cm3) was degassed by bubbling N2 through for 20 min before Pd/C (5 % wt., catalytic) was added. Degassing was continued for a further 10 min before H2 gas was passed over the reaction and the reaction was stirred under a hydrogen blanket overnight. Tic showed the presence of sm therefore stirring under H2 was continued for a further 72h. The mixture was filtered through celite and the product purified by flash chromatography using a gradient elution of DCM to 10% MeOH in DCM. Yield 0.012 g, 44%; 1H NMR δ (270 MHz, CD3OD) 0.99 (s, 3H, 18-CH3), 1.28-2.12 (9H), 2.21-2.46 (4H), 2.53 (t, J = 7.4 Hz, 2H ) 2.62 (dd, J = 13.4, 6.2 Hz, 1H), 2.82-2.84 (m, 2H), 2.90 (t, J = 7.4 Hz, 2H), 3.22-3.3 (m, 3H), 3.67-3.96 (3H), 6.50 (d, J = 2.5 Hz, 1H), 6.55 (dd, J = 8.4, 2.7 Hz, 1H), 7.09 (s, 1H); HPLC > 97 % (Rt 2.03, 70% MeCN in H2O); ES+ve-MS (MH+) 450 m/z; FAB-HRMS calcd for for C27H36N3O3450.2757 found (MH+) 450.2746 m/z.
SECTION 2
3-Benzyloxy-13-methyl-6, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta [a] phenanthren-17-one (JAC01002).
Figure imgf000118_0001
A solution of 15 g (55 mmol) of estrone in DMF (110 mL) was cooled to 0°C and 2.44 g (61 mmol) of NaH (60% in oil) were added slowly. The mixture was stirred at 0°C under N2 for 1 h before addition of 13.2 mL (111 mmol) of benzyl bromine dropwise at 0°C. The reaction was then heated to 80°C for 4 h. After cooling, the mixture was poured into water. The suspension was extracted by diethyl ether (5 times). The mixed organic layer were washed with 2N HCl (3 times) and brine, dried over Na2SO4 and concentrated in vacuo. Recrystallisation of the solid residue in ethanol gave 12.33 g (62%) of JAC01002 as beige solid. 1H NMR (400 MHz, CDCI3) δ 0.92 (s, 3H, CH3); 1.49-1.63 (m, 6H); 1.99- 2.05 (m, 3H); 2.17 (dAB, J = 18.9, 8.9 Hz, 1 H); 2.23 (m, 1 H); 2.40 (m, 1H); 2.51 (dAB, J = 18.9, 8.9, 1 H); 2.85 (m, 2H, CH2 6); 5.05 (s, 2H, CH2Ph); 6.74 (d, J = 2.8 Hz, 1 H, H4); 6.80 (dd, J = 8.5, 2.8 Hz, 1 H, H2); 7.21 (d, J = 8.5 Hz, 1 H, H,); 7.30-7.45 (m, 5H); Acq. Mass (FAB+) calcd 360.2089, obsd 360.21675 (M+).
3-Benzyloxy-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a] phenanthrene-16-carboxylic acid methyl ester (JAC01011). [J.Org. Chem. 1991 , 65, 6199-6205].
Figure imgf000118_0002
3.48 g (87 mmol) of NaH (60% in oil) were covered with anhydrous THF (50 mL), treated with 6.11 mL (72 mmol) of dimethylcarbonate, and heated to reflux while being magnetically stirred under N2. A solution of benzylestrone (10.45 g, 29 mmol) in THF (50 mL) was added dropwise, and the reaction mixture was refluxed for 8 h, cooled to 0°C, and treated with 3M acetic acid (50 mL) in advance of being poured into brine. The product was extracted into CHCI3 (3 times), dried over Na2SO4 and evaporated. Recrystallisation of the solid residue in methanol gave 9.8 g (81 %) of JAC01011 as beige solid. 1H NMR (400 MHz, CDCI3) δ D0.97 (s, 1.05H, CH3); 0.99 (s, 1.95H, CH3); 1.34-1.74 (m, 6H); 1.90-2.50 (m, 6H); 2.92 (m, 2H, CH2 6); 3.22 (dd, J = 10.1 , 8.6 Hz, 0.65H, CHCO); 3.58 (dd, J = 9.4, 1.7 H∑, 0.35H, CHCO); 3.77 (s, 3H, OCH3); 5.03 (s, 2H, CH2Ph); 6.73 (d, J = 2.5 Hz, 1 H, H4); 6.80 (dd, J = 8.6, 2.5 Hz, 1 H, H2); 7.20 (d, 1H, J = 8.6 Hz, 1H, H,); 7.30-7.50 (m, 5H); MS (FAB+) calcd 418.21 , obsd 418.20 (M+).
3-Ben2yl©2sy-17©,p-hydro∑jy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro- 6H-cycl©pβnta[aJphenanthren -16-carboxylie acid methyl ester (JACΘ1013, STJ ' 569) and (JAC01013B, STX 570).
Figure imgf000119_0001
Solid NaBH4 (456 mg, 12 mmol) was added to a cold (0°C), magnetically stirred solution of JAC01011 (5g, 12 mmol) in THF/MeOH (90:10), and the reaction mixture was stirred for 4h at 0°C, treated with 2N HCl until acidic pH and extracted with ether (twice). The combine organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuo. The solid was purified by column chromatography using CHCI3/ EtOAc (90:10) as eluant to give 2.48 g of STX 669 (48%) and 0.91 g of STX 570 (18%) as white solids.
3-Benzyloxy-17β-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-16-carboxylic acid methyl ester (JAC01013, STX 569)
1H NMR (400 MHz, CDCI3): δ 0.85 (s, 3H, CH3); 1.19 (ddd, J = 12.9, 10.9, 6.2 Hz, 1 H); 1.29-1.43 (m, 2H); 1.46-1.56 (m, 2H); 1.76 (dt, J = 12.9, 8.6 Hz, 1H); 1.90 (m, 1H); 2.00- 2.13 (m, 2H); 2.23 (m, 1H); 2.33 (m, 1 H); 2.86 (m, 2H, CH2 6); 3.15 (q, J = 9.8 Hz, 1H, CHCOO); 3.34 (d, J = 8.2 Hz, 1 H, OH); 3.72 (s, 3H, OCH3); 3.89 (dd, J = 9.8, 8.2 Hz, 1H, CHO); 5.04 (s, 2H, CH2Ph); 6.72 (d, J = 2.7 Hz, 1H, H4); 6.79 (dd, J = 8.4, 2.7 Hz, 1H, H2); 7.21 (d, J = 8.6 Hz, 1 H, H,); 7.30-7.46 (m, 5H, Ph); Acq. Mass (FAB÷) calcd 420.2301 , obsd 420.2298; Anal. Calcd for C27H32O4: C, 77.11%; H, 7.67%; obsd C, 76.70%; H, 7.62%; purity: 99%. 3-Benzyloxy-17α-hydroxy-13-meth l-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-
6H-cyclopenta[a]phenanthrene-16-carbθ2sylic acid methyl ester (JAC0 013B, STIC 570).
1H NMR (400 MHz, CDCI3): δ 0.84 (s, 3H, CH3); 1.20-1.60 (m, 5H); 1.72 (app. q, J = 12.4 Hz, 1H), 1.85-1.88 (m, 1H); 1.97 (dt, J = 12.1, 3.3 Hz, 1H); 2.02-2.10 (m, 2H); 2.22 (m, 1H); 2.32 (m, 1H); 2.77 (ddd, J = 12.1, 8.4, 4.0 Hz, 1H, CHCOO); 2.86 (m, 2H, CH2 6); 3.75 (s, 3H, OCH3); 3.89 ( dd, J = 8.1, 4.4 Hz, 1H, CHO); 5.04 (s, 2H, CH2Ph); 6.73 (d, J = 2.9 Hz, 1H, H4); 6.79 (dd, J = 8.4, 2.6 Hz, 1H, H2); 7.21 (d, J = 8.1 Hz, 1H, Ht); 7.30- 7.46 (m, 5H, Ph); Acq. Mass (FAB+) calcd 420.2301, obsd 420.2301; Anal. Calcd for C27H32O4: C, 77.11%; H, 7.67%; obsd C, 76.60%; H, 7.71%; purity: 99%.
3-Benzyloxy-16-hydroxymethyl-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthren-17-ol (JAC01023C).
Figure imgf000120_0001
Solid NaBH4 (452 mg, 12 mmol) was added to a cold (0°C), magnetically stirred solution of JAC01011 (5g, 12 mmol) in THF/MeOH (90:10), and the reaction mixture was stirred for 18h at 0-20°C, treated with 2N HCl until acidic pH and extracted with ether (twice).
The combine organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuo. The solid was purified by column chromatography using CHCI3/ EtOAc (90:10) as eluant to give 1.95 g of STX 569 (39%), 0.55 g of STX 570 (18%) and 0.63g of JAC01023C (13%) as white solids. 1H NMR (270 MHz, DMSO-d6): δ 0.68 (s, 3H, CH3);
0.95-1.45 (m, 4H); 1.70-1.90 (m, 4H); 2.00-2.35 (m, 4H); 2.75 (m, 2H, CH2 6); 3.29 (m,
8H); 3.67 (m, 2H); 4.13 (dd, J = 5.9, 4.4 Hz, 1H, CHO); 4.60 (d, J = 4.4 Hz, 1H, OH);
5.03 (s, 2H, CH2Ph); 6.69 (d, J = 2.7 Hz, 1H, H4); 6.74 (dd, J = 8.4, 2.7 Hz, 1H, H2); 7.14
(d, J = 8.6 Hz, 1H, H ; 7.25-7.46 (m, 5H, Ph); Acq. Mass (FAB+) calcd 392.2351, obsd 392.2342. 3-Benzyloxy-16-(1,3-dioxo-1,3-dihydro-isoindol-2-ylmethyl)-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthrene-16- carbo-ς lic acid methyl ester (JAC01159).
Figure imgf000121_0001
57 mg NaH (60% in oil, 1.43 mmol) was suspended in 2 mL dry THF. The suspension was cooled to 0°C in an ice bath and 500 mg of JAC01011 (1.19 mmol) solubilized in 5 mL dry THF were added dropwise. The mixture was stirred for 30 min and the N- 0 (Bromomethyl)phtalimide (344 mg, 1.43 mmol) in 3 mL dry THF was added dropwise.
The reaction was stirred at 0°C for 1 h and allowed to react at room temperature for a
, , further 12h. 1 N HCl was added to the mixture and the acidic layer was extracted with diethylether (3 times). The combined organic layers were washed with sat. NaHCO3 (3 times), brine, dried over Na2SO4 and evaporated in vacuo to give JAC01159 as a white 5 solid. This compound was used without further purification. 1H NMR (270 MHz, CDCI3): δ 0.86 (s, 3H, CH3); 1.30-1.70 (m, δH); 1.85-1.95 (m, 2H); 2.10-2.40 (m, 4H); 2.79 (m, 2H, CH2 6); 3.75 (s, 3H, OCH3); 3.96 (d, J = 14.1 Hz, 1 H, NCH2); 4.17 (d, J = 14.1 Hz, 1 H, NCH2); 4.96 (s, 2H, CH2Ph); 6.66 (d, J = 2.6 Hz, 1 H, H4); 6.73 (dd, J = 8.4, 2.6 Hz, 1 H, H2); 7.12 (d, J = 8.4 Hz, 1H, Hi); 7.24-7.37 (m, 5H); 7.67 (dd, J = 5.1 , 2.7 Hz, 2H); 7.77 0 (dd, J = 5.1 , 2.7 Hz, 2H).
Representative procedure for debenzylation:
3-Hydroxy-13-methyl-17-OXO-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopentafa] phenanthrene-16-carboxylic acid methyl ester (JAC01012, STX 568). 5
Figure imgf000121_0002
2.5 g (5.97 mmol) of JAC01011 were solubilized in THF (30 mL). A catalytic amount of 10% Pd/C was added and the reaction mixture was stirred at room temperature under H2 for 8 h. Pd/C was filtered over celite and the filtrat was evaporated in vacuo. Recrysfallisation of the solid residue in methanol gave 1.82 g (93%) of STX 568 as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 0.87 (s, 1.95H, CH3); 0.91 (s, 1.05H, CH3); 1.20-2.40 (m, 11 H); 2.75 (m, 2H, CH2 6); 3.39 (dd, J = 9.8, 8.6 Hz, 0.65H, CHCOO); 3.65 (s, 1.05H, OCH3); 3.65 (s, 1.95H, OCH3); 3.80 (dd, J = 9.8, 8.6 Hz, 0.35H, CHCOO) 6.45 (d, J = 2.3 Hz, 1 H, H4); 6.05 (dd, J = 8.6, 2.7, 1H, H2); 7.04 (d, J = 8.2 Hz, 1H, hi,) 9.04 (s, 1 H, OH); mp: 215-217°C; Acq. Mass (FAB+) calcd 328.1675, obsd 328.1684 Anal. Calcd for C20H32O4: C, 73.15%; H, 7.37%; obsd C, 72.9%; H, 7.34%; purity: 97%.
This same procedure afforded the following phenols in the indicated yields: 3,17-Dihydroxy-l3-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid methyl ester (JAC01027, STX 572).
Figure imgf000122_0001
Recrystallisation of the solid residue in methanol/water. Yield = 92%, white solid. 1H NMR (400 MHz, DMSO-d6): δ 0.75 (s, 3H, CH3); 1.04-1.46 (m, 5H); 1.64-1.86 (m, 4H); 2.08 (td, J = 10.5, 3.3 Hz, 1H); 2.22 (m, 1H); 2.70 (m, 2H, CH2 6); 3.05 (q, J = 9.0 Hz, 1H, CHCO); 3.56 (s, 3H, OCH3); 3.83 (dd, J = 10.5, 5.5 Hz, 1 H, CHO); 5.02 (d, J = 5.0 Hz, 1 H, OH); 6.42 (d, J = 2.7 Hz, 1 H, H4); 6.48 (dd, J = 8.2, 2.7 Hz, 1 H, H2); 7.03 (d, J = 8.6 Hz, 1H, H ; 8.99 (s, 1 H, phenol); Acq. Mass (FAB+) calcd 330.1831 , obsd 330.1831 ; mp: 222-224°C; Anal. Calcd for C20H26O4: C, 72.70%; H, 7.93%; obsd C, 72.90%; H, 7.90%; purity: 95%.
16-Hydroxymeth l-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-3,17-diol (JACΘ1049, STX 612).
Figure imgf000123_0001
Yield = 78%, white solid. 1H NMR (400 MHz, DMSO-d6): δ 0,72 (s, 3H, CH3); 1.06 (m, 2H); 1.16-1.42 (m, 4H); 1.84 (m, 3H); 2.10 (td, J = 10.0, 3.3 Hz, 1 H); 2.24 (m, 2H); 2.72 (m, 2H, CH2 6); 3.34 (m, 1 H); 3.70 (m, 2H); 4.18 (dd, J = 6.2, 4.8 Hz, 1H, CHO); 4.64 (d, J = 4.8 Hz, OH); 6.45 (d, J = 2.6 H∑, 1H, H4); 6.52 (dd, J = 8.4, 2.6 H∑, 1 H, H2); 7.06 (d, J = 8.4 H∑, 1H, H ; 9.02 (s, 1H, phenol); mp: decomposition; MS (FAB+) calcd 302.19, obsd 302.1(M+); Anal. Calcd for C19H26O3: C, 75.46%; H, 8.67%; obsd C, 75.2%; H, 8.7%; purity: 100%.
16-(1,3-Dioxo-1,3-dihydro-isoindol-2-ylmethyl)-3-hydroxy-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthrene-16-carboxylic acid methyl ester (JAC01171-4, STX 780).
Figure imgf000123_0002
Purification by column chromatography using flash column ISOLUTE SI 1 g and an elution gradient Hexane / Ethyl Acetate. The white solid was precipitated in Ethyl acetate / Hexane. Yield = 6.9 %, white solid. Η NMR (270 MHz, MeOH-d3): δ 0.89 (s, 3H, CH3); 1.20-1.75 (m, 5H); 1.85-2.05 (m, 2H); 2.10-2.50 (m, 4H); 2.80 (m, 2H, CH2 6); 3.72 (s, 3H, OCH3); 4.08 (d, J = 5.5 Hz, 1 H, NCH2); 6.66 (d, J = 2.6 Hz, 1H, H4); 6.73 (dd, J = 8.4, 2.6 Hz, 1H, H2); 7.12 (d, J = 8.4 Hz, 1H, H,); 7.75-7.90 (m, 4H); MS (APCI+) calcd 487.20, obsd 489.36(M+2); purity: 88%.
3-Benzyloxy-17-hy roxy-13-mothyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a3phenanthrene-1@-carbθ2iylic acid (JAC01083).
Figure imgf000124_0001
2 g (4.75 mmol) of STX 569 were solubilized in 20 mL THF and 285 mg (7.13 mmol) NaOH solubilized in 5 mL water were added to the solution which was stirred at room temperature for 24 h. The solvents were evaporated and 6N HCl was added to the residue. The acidic layer was extracted by diethyl ether (5 times). The combined organic layers were extracted with 10% K2CO3 (5 times). This basic solution was made acidic adding 6N HCl and extracted by ethyl acetate (5 times). The combine organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuo. The white solid was suspended in hexane and filtered to give 720 mg (37%) of JAC01083. 1H NMR (270 MHz, DMSO-d6): δ 0.68 (s, 1.05H, CH3); 0.72 (s, 1.95H, CH3); 1.00-1.50 (m, 6H); 1.60-
1.90 (m, 3H); 2.11 (m, 1H); 2.26 (m, 1H); 2.74 (m, 2H, CH2 6); 2.92 (q, J = 8.9 Hz, 0.65H, CHCO); 3.68 (dd, J, = 7.2, 5.3 Hz, 0.35H, CHCO); 3.80 (d, J = 10.5 Hz, 0.65H, CHO);
4.91 (d, J = 5.3, 0.35H, CHO); 6.03 (s, 2H, CH2Ph); 6.68 (s, 1H, H4); 6.74 (dd, J = 8.6, 2.6 Hz, 1H, H2); 7.14 (d, J = 8.6 Hz, 1H, H,); 7.25-7.50 (m, 5H, Ph).
3-Hydroxy-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-16-carboxylic acid (JAC01043, STX 610).
Figure imgf000124_0002
2.14 g (6.51 mmol) of STX 568 were suspended in 46 mL of 5N NaOH and stirred at room temperature for 72h until complete solubilization. After cooling to 0°C, 6N HCl was added dropwise to the solution until pH 1. The acidic layer was extracted by diethyl ether (3 times). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuum at room temperature. The solid was suspended in hexane, filtered, washed with hexane and dried in vacuo to give 1.56 g (76%) of STX 610 as beige solid, which was used without further purification. 1H NMR (270 MHz, DMSO-d6): δ 0.84 (s, 1.95H, CH3); 0.88 (s, 1.05H, CH3); 1.20-1.55 (m, 4H); 1.70-2.00 (m, 3H); 2.10- 2.35 (m, 4H); 2.74 (m, 2H, CH2 6); 3.22 (t, J = 8.6 Hz, 0.65H, CHCOO); 3.60 (t, J = 8.6 Hz, 0.35H, CHCOO); 6.43 (d, J = 2.4 Hz, 1H, H4); 6.50 (dd, J = 8.4, 2.4 Hz, 1H, H2); 7.03
(d, J = 8.4 H∑, 1H, H,); 9-02 (s, 1H, phenol); Acq. Mass (FAB+) calcd 314.1518, obsd
314.1524; purity: 95%.
NB: decomposed with time to give estrone at room temperature.
3,17-Dihydrøxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a] pheήanthrene-16-carboxylic acid (JAC01041, STX 611).
Figure imgf000125_0001
1.1 g (3.20 mmol) of STX 572 were suspended in 10 mL MeOH and 0.58 g (14 mmol) NaOH solubilized in 3 mL water were added to the suspension. The solution was stirred at room temperature for 5 h. The solvents were evaporated and 6N HCl was added to the residue. The acidic layer was extracted by ethyl acetate (5 times) and the combined organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuo. Recrystallisation of the solid residue in acetonitrile gave 470 mg (46%) of STX 611 as a white solid. H NMR (400 MHz, DMSO-d6): δ 0.69 (s, 1.86H, CH3,); 0.73 (s, 1.14H, CH3); 1.20-1.40 (m, 5H); 1.45-1.85 (m, 4H); 2.10 (m, 1 H); 2.20 (m, 1H); 2.45 (m, 0.62H, CHCOO); 275 (m, 2H, CH2 6); 2.95 (q, J = 9.0 Hz, 0.38H, CHCOO); 3.69 (d, J = 8.00 Hz, 0.62H, CHO); 3.60 (d, J = 10.8 Hz, 0.38H, CHO); 4.92 (bs, 1 H, OH); 6.42 (d, J = 2.7 Hz, 1H, H4); 6.48 (dd, J = 8.2, 2.7 Hz, 1H, H2); 7.03 (d, J = 8.2 Hz, 1H, H,); 8.99 (s, 1H, phenol); mp: decomp.; Acq. Mass (FAB+) calcd 316.1675, obsd 316.1677; purity: 95%.
Solid phase chemistry
Figure imgf000125_0002
Loading = 1.1 mmol/g resin Loading = 0.82 mmol/g resin Loading of estrone on 2-chloro trityl resin.
500 mg of 2-chloro-trityl resin (loading of 1.1 mmol/g resin) were swollen in 7 mL dry DCM and 3 mL DIEA for 15 min. before addition of estrone. The mixture was shacked for 72h at room temperature under N2. The resin was filtered, washed with DCM, DMF, MeOH (3 cycles) and MeOH (5 times) and dried in vacuo. IR (cm"1) 1736 (C=O, ketone). Determination of estrone's loading. 51.4 mg of loaded resin were swollen in 1 ml DCM in a filter funnel, 10 mL DCM/MeOH/TFA (7:1:2) were added (5 fractions of 2 mL) and the filtrat was evaporated in vacuo to give 11.5 mg of a white solid corresponding to estrone when structure checked by 1H NMR. The loading was then determined: 0.82 mmol/g resin. NB: When addition DCM/MeOHTFA, the resin became purple.
Combinatorial library construction:
Method A:
Stepl: Loading ofJAC01041 on oxime resin.
Figure imgf000126_0001
Loading = 1.06 mmol/g resin Maximum loading = 1.06 mmol/g resin
500 mg of oxime resin (loading of 1.06 mmol/g resin) were swollen in 7 mL dry DMF for 15 min after which 251 mg of JAC01041 were added followed by the addition of 41 μL DIC and 358 mg HOBT. The mixture was shaken for 48h at room temperature under inert atmosphere and the resin was filtered, washed with DCM, DMF, MeOH (3 cycles) and MeOH (5 times) and dried in vacuo. IR (cm-1) 1750 (C=O, ester); 1600 (C=N, oxime).
Step2: Amidification. 200 mg of oxime resin loaded with JAC01041 (maximum loading of 1.06 mmol/g resin) were swollen in 4 mL dry DCM and 47μL (0.53 mmol) furfurylamine were added to the suspension. The mixture was shacked at 40°C under nitrogen atmosphere for 24h. After filtration, the resin was washed with DCM (5 times) and the filtrated was evaporated in vacuo.
M έhod ES: Step 1: Active ester formation.
3-Bβnzylθ2sy-17-hydro2-y-13-mo hyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydrø-@H- cyclopenta [a]phenanthrene-1β-carboxylic acid 4-nitro-phenyl ester (JAC01177).
Figure imgf000127_0001
[Helv. Chim. Act. 1963, 795-804]. 400 mg of JAC01083 were solubilized in 4 mL dry DMF and 0.14 mL triethylamine (0.98 mmol) and 299 mg bis-(4-nitrophenyl) carbonate (0.98 mmol) were successively added. The solution was stirred at room temperature for 2h under N2 atmosphere. The solution was cooled to 0°C and 2N HCl was added. The mixture was extracted with DCM (3 times). The combined organic layers were washed with sat. NaHCO3 (5 times) and brine, dried over Na2SO4 and evaporated in vacuo. The residue was purified by column chromatography ISOLUTE SI 20g using an elution gradient Hexane / Ethyl Acetate to give 270 mg of JAC01177 as a yellow solid (52%). 1H NMR (270 MHz CDCI3>): δ 0.87 (s, 3H, CH3); 1.27-1.50 (m, 4H); 1.80-2.05 (m, 4H); 2.10- 2.40 (m, 3H); 2.84 (m, 2H, CH2 6); 3.05 (ddd, J = 11.7, 7.7, 3.7 Hz, 1H, CHCO); 4.06 (dd, J = 8.1, 5.1 Hz, 1H, CHO); 5.01 (s, 2H, CH2Ph); 6.70 (d, J = 2.6 Hz, 1H, H4); 6.76 (dd, J = 8.8, 2.6 Hz, 1H, H2); 7.19 (d, J = 8.8 Hz, 1H, H ; 7.25-7.45 (m, 7H); 8.27 (d, J = 12.1 Hz, 2H); MS (FAB+) calcd 527.23, obsd 527.4 (M+); 389.4 (M - pNO2PhO ); 91.1 (PhCH2 +).
Step 2: General procedure for Amidification. The active ester JAC01177 (0.17 mmol) was solubilized in 2mL dry acetonitrile and the amine (2 eq) was added to the mixture. The solution was stirred overnight at room temperature and the solvents were evaporated in vacuo. The residue was purified by column chromatography using flash column ISOLUTE SI 5g and an elution gradient Hexane / Ethyl Acetate. <# Step 3: General procedure for debenzylation: The benzylated amide was solubilized in THF (3 mL). A catalytic amount of 5% Pd/C was added and the reaction mixture was stirred at room temperature under H2 for 24 h. Pd/C was filtered over paper and the filtrat was evaporated in vacuo. The final product was precipitated in a mixture Ethyl Acetate / Hexane.
Figure imgf000128_0002
-
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid (tetrahydro-furan-2-ylmethyl)- amide (JAC01163-3).
Figure imgf000128_0001
Method B, step2. Yield = 96%. 1H NMR (270 MHz, CDCI3): δ 0.74 (s, 3H, CH3); 1.10- 1.60 (m, 7H); 1.70-2.00 (m, 5H); 2.06-2.30 (m, 3H); 2.50 (m, 1H); 2.74 (m, 2H, CH2 6); 3.09 (m, 1H); 3.50 (m, 1H); 3.60-3.85 (m, 3H); 3.90 (m, 1H), 4.94 (s, 2H, CH2Ph); 6.42 (t, J = 5.5 Hz, 1H, NH); 6.63 (s, 1H, H4); 6.68 (dd, J = 8.4, 2.2 Hz, 1H, H2); 7.10 (d, J = 8.4 Hz, 1H, HO; 7.20-7.40 (m, 5H).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a3phenanthrene- 6-carbo∑sylic acid (pyridin-2-ylmethyl)-amide (JAC01179-1, STX 789).
Figure imgf000129_0001
Method B, step2. Yield = 94%. H NMR (270 MHz, CDCI3): δ 0.78 (s, 3H, CH3); 1.20- 1.60 (m, 8H); 1.70-1.90 (m, 2H); 2.05-2.30 (m, 3H); 2.63 (ddd, J = 11.0, 8.0, 3.7 H∑, 1H, CHCO); 2.75 (m, 2H, CH2 6); 3.73 (d, J = 8.0 H∑, 1H, CHO); 4.30-4.43 (dAB, J = 16.5, 4.8 Hz, 1H, NCH2); 4.60-4.75 (dAB, J = 16.5, 6.6 Hz, 1H, NCH2); 4,95 (s, 2H, CH2Ph); 6.64 (d, J = 2.9 Hz, 1H, H4); 6.70 (dd, J = 8.4, 2.9 Hz, 1 H, H2); 6.90 (t, J = 5.1 Hz, 1H, NH); 7.10 (d, J = 8.4 Hz, 1H, HO; 7.13-7.40 (m, 6H); 7.59 (td, J = 7.7, 1.8 Hz, 1H, H5.); 8.43 (d, J = 5.1 Hz, 1H, H3); MS (FλB+) calcd 496.27, obsd 497.3 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid (pyridin-3-ylmethyl)-amide (JAC01179-2, STX 790).
Figure imgf000129_0002
Method B, step2. Yield = 82%. 1H NMR (270 MHz, CDCI3): δ 0.81 (s, 3H, CH3); 1.10- 1.60 (m, 8H); 1.79 (m, 2H); 2.05-2.30 (m, 3H); 2.57 (ddd, J = 11.3, 8.4, 3.3 Hz, 1H, CHCO); 2.75 (m, 2H, CH2 6); 3.72 (d, J = 8.4 Hz, 1 H, CHO); 4.25-4.55 (dAB, J = 15.0, 6.2, 5.5 Hz, 2H, NCH2); 4.95 (s, 2H, CH2Ph); 6.63 (d, J = 2.6 Hz, 1H, H4); 6.70 (dd, J = 8.4, 2.6 Hz, 1H, H2); 7.10 (d, J = 8.4 Hz, 1 H, HO; 7.14-7.40 (m, 6H); 7.58 (d, J = 7.7 Hz, 1 H, H4.); 8.40 (dd, J = 4.8, 1.4 Hz, 1 H, H6.); 8.42 (d, J = 1.8 Hz, 1H, H2); MS (FAB+) calcd 496.3, obsd 497.3 (M+1 ).
3-B®n∑yloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydrø-@H- cyclopenta[a]phenanthrene-16-yl)-(4-methyl-piperazin-1-yl)-methanone (JAC01179-3, STX 791).
Figure imgf000130_0001
Method B, stepl. Yield = 86%. 1H NMR (270 MHz, CDCI3): δ 0.83 (s, 3H, CH3); 1.30-
5 1.60 (m, 7H); 1.70-1.85 (m, 2H); 2.15-2.45 (m, 10H); 2.75-2.95 (m, 3H); 3.40-3.80 (m,
4H); 4.20 (d, J = 7.0 Hz, 1H); 5.00 (s, 2H, CH2Ph); 6.68 (d, J = 2.6 Hz, 1H,' H4); 6.75
(dd, J = 8.8, 2.6 Hz, 1H, H2); 7.15 (d, J = 8.8 Hz, 1H, HO; 7.29-7.42 (m, 5H); MS (FAB+)
' calcd 488.3, obsd 489.3 (M+1).
0 3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid ethyl-methyl-amide (JAC01179-4, , STX 794).
Figure imgf000130_0002
5
Method B, step2. Yield = 35%. 1H NMR (400 MHz, CDCI3): δ 0.88 (s, 3H, CH3); 1.13 (t, J = 7.0 Hz, 1.5H, CH2CHO; 1.21 (t, J = 7.0 Hz, 1.5H, CH2CH3); 1.32-1.60 (m, 8H); 1.68- 1.88 (m, 4H); 1.98 (m, 1H); 2.24 (m, 1H); 2.33 (m, 1H, CHCO); 2.70 (m, 1H); 2.84 (m, 2H, CH2 6); 2.96 (s, 1.5H, NCH3); 3.03 (s, 1.5H, NCH3); 3.30-3.48 (m, 2H, CϋCHs); 4.24 0 (app.t, J = 7.4 Hz, 1H, CHO); 5.04 (s, 2H, CH2Ph); 6.72 (d, J = 2.4 Hz, 1H, H4); 6.79 (dd, J = 8.6, 2.4 Hz, 1H, H2); 7.22 (d, J = 8.6 Hz, 1H, HO; 7.36-7.48 (m, 5H); MS (FAB+) calcd 447.3, obsd 448.3 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- 5 cyclopenta [aJphenanthren@-16-carboj.ylic acid isopropylamid© (JAC01179-5, ST2 795).
Figure imgf000131_0001
Method B, step2. Yield = 92%. 1H NMR (270 MHz, CDCLJ: δ 0.80 (s, 3H, CH3); 1.14 (d, J = 7.0 Hz, 6H, 2 x CH3); 1.28-1.60 (m, 6H); 1.80-1.92 (m, 3H); 2.10-2.36 (m, 3H); 2.48 (ddd, J = 11.7, 8.4, 4.0 Hz, 1H, CHCO); 2.81 (m, 2H, CH2 6); 3.76 (dd, J = 8.0, 5.1 Hz, 1H, CHO); 4.09 (septuplet, J = 7.0 Hz, 1H, CH(CH3)2); 5.01 (s, 2H, CH2Ph); 5.72 (d, J = 8.1 Hz, 1H, NH); 6.70 (d, J = 2.7 Hz, 1H, H4); 6.75 (dd, J = 8.4, 2.7 Hz, 1H, H2); 7.17 (d, J = 8.4 Hz, 1H, HO; 7.29-7.43 (m, 5H); MS (FAB+) calcd 447.3, obsd 448.3 (M+1).
1
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-16-carboxylic acid (1 -methyl- 1H-pyrrόl-2-y I methyl) - amide ( JAC01179-6, STX 793).
Figure imgf000131_0002
Method B, step2. Yield = 86%. 1H NMR (270 MHz, CDCI3): δ 0.79 (s, 3H, CH3); 1.27- 1.65 (m, 6H); 1.80-1.92 (m, 3H); 2.10-2.36 (m, 3H); 2.54 (ddd, J = 11.7, 8.4, 4.0 Hz, 1H, CHCO); 2.82 (m, 2H, CH2 6); 4.44 (t, J = 5.1 Hz, 1H, CH2N); 5.01 ,(s, 2H, CH2Ph); 5.98 (t, J = 5.1 Hz, 1H, NH); 6.04 (m, 2H, H„ Hff); 6.59 (t, J = 2.2 Hz, H4); 6.70 (d, J = 2.6 Hz, 1H, H4); 6.76 (dd, J = 8.8, 2.6 Hz, 1H, H2); 7.17 (d, J = 8.8 Hz, 1H, HO; 7.29-7.43 (m, 5H); MS (FAB+) calcd 498.3, obsd 499.3 (M+1).
3-Benzyl©∑ty-17-hydrøκy-13-m hyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a3phenanthrene-1 -carboxylic acid (5-methyl-pyra2in-2-ylmethyl)- amide (JAC0 1 9-7, STO 792).
Figure imgf000132_0001
Method B, step2. Yield = 85%. 1H NMR (270 MHz, CDCLJ: δ 0.83 (s, 3H, CH3); 1.20- 1.60 (m, 9H); 1.87 (m, 1H); 2.10-2.35 (m, 1H); 2.50 (d, J = 5.1 Hz, 1H, OH); 2.54 (s, 3H, HarCH3); 2.65 (m, 1H, CHCO); 2.81 (m, 2H, CH2 6); 3.75 (dd, J = 8.4, 5.1Hz, CHO); 4.40- 4.80 (dAB, J = 16.0, 6.6, 4.8 Hz, NCH2); 5.01 (s, 2H, CH2Ph); 6.70 (m, 2H, H4 + NH); 6.74 (dd, J = 8.4, 2.6 Hz, 1H, H2); 7.17 (d, J = 8.4 Hz, 1H, HO; 7.28-7.43 (m, 5H); 8.36 (s, 1 H); 8.47 (s, 1 H); MS (FAB+) calcd 511.3, obsd 512.3 (M+1 ).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-16-carboxylic acid (pyridin-3-ylethyl)-amide
(JAC01185-1. STX 814).
Figure imgf000132_0002
Method B, step2. Yield = 58%. 1H NMR (270 MHz, CDCI3): δ 0.73 (s, 3H, CH3); 1.10- 1.60 (m, 7H); 1.81 (m, 2H); 2.05-2.30 (m, 3H); 2.45 (ddd, J = 11.7, 8.4, 3.7 Hz, 1H, CHCO); 2.77 (m, 2H, CH2 6); 3.30-3.65 (m, 3H); 4.94 (s, 2H, CH2Ph); 6.19 (t, J = 5.5 Hz, 1 H, NH); 6.64 (d, J = 2.6 Hz, 1 H, H4); 6.68 (dd, J = 8.4, 2.6 Hz, 1 H, H2); 7.10 (d, J = 8.4 Hz, 1H, HO; 7.15-7.38 (m, 6H); 7.49 (m, 1H); 8.36 (m, 2H); MS (FAB+) calcd 510.3, obsd 511.3 (M+1).
3-Ben2yloxy-17-hydr©xy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-16-carboxylic acid (l-phenyl-ethyl)-amide (JAC01185- 4A, STIC 815).
Figure imgf000133_0001
Method B, step2. Yield = 46%. 1H NMR (270 MHz, CDCI3): δ 0.80 (s, 3H, CH3); 1.20- 1.60 (m, 10H); 1.84 (m, 2H); 2.10-2.35 (m, 3H); 2.57 (ddd, J = 11.7, 8.4, 3.7 Hz, 1H, CHCO); 2.81 (m, 2H, CH2 6); 3.71 (dd, J = 8.8, 5.5 Hz, 0.71 H, CHO); 3.80 (m, 0.28H, CHO); 5.00 (s, 2H, CH2Ph); 5.12 (m, 1H, CHPh); 6.22 (m, 1H, NH); 6.69 (d, J = 2.9 Hz, 1H, H4); 6.76 (dd, J = 8.5, 2.9 Hz, 1H, H2); 7.12 (d, J = 8.5 Hz, 1H, HO; 7.15-7.45 (m, 10H); MS (FAB+) calcd 509.3, obsd 510.3 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-16-carboxylic acid (l-phenyl-ethyl)-amide (JAC01185- 4B, STX 816).
Figure imgf000133_0002
Method B, step2. Yield = 24%. 1H NMR (270 MHz, CDCI3): δ 0.79 (s, 3H, CH3); 1.20- 1.60 (m, 10H); 1:87 (m, 3H); 2.10-2.38 (m, 3H); 2.53 (ddd, J = 11.0, 8.1, 3.3 Hz, 1H, CHCO); 2.80 (m, 2H, CH2 6); 3.81 (dd, J = 8.1, 5.1 Hz, 1H, CHO); 5.00 (s, 2H, CH2Ph); 5.12 (m, 1H, CHPh); 6.20 (m, 1H, NH); 6.69 (d, J = 2.6 Hz, 1H, H4); 6.75 (dd, J = 8.6, 2.6 Hz, 1H, H2); 7.12 (d, J = 8.6 Hz, 1H, HO; 7.20-7.50 (m, 10H); MS (FAB+) calcd 509.3, obsd 510.3 (M+1). This second isomer has been isolated from the same reaction as STX 815.
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclop©n£a[aJph@nan£hr©n©-1@-carbo. ylic acid (2-pipera2in-1-yl-ethyi)-amϊde (JAC01185-2, STX 817).
Figure imgf000134_0001
Method B, step2. Yield = 73%. 1H NMR (270 MHz, CDCLJ: δ 0.81 (s, 3H, CH3); 1.20- 1.60 (m, 6H); 1.80-2.05 (m, 2H); 2.10-2.60 (m, 11H); 2.75-2.95 (m, 6H); 3.10-3.40 (m, 1H); 3.55-3.70 (m, 2H); 5.00 (s, 2H, CH2Ph); 6.01 (b s, 1H, NH); 6.69 (d, J = 2.6 Hz, 1H, H4); 6.75 (dd, J = 8.4, 2.6 Hz, 1 H, H2); 7.17 (d, J = 8.4 Hz, 1H, HO; 7.30-7.35'(m, 10H); MS (FAB+) calcd 517.3, obsd 518.4 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-16-carboxylic acid (pyridin-4-ylmethyl)-amide (JAC01191-1, STX 818).
Figure imgf000134_0002
Method B, step2. Yield = 92%. 1H NMR (270 MHz, CDCI3): δ 0.83 (s, 3H, CH3); 1.20- 1.65 (m, 7H); 1.80-1.95 (m, 2H); 2.15-2.40 (m, 3H); 2.64 (ddd, J = 12.4, 8.4, 3.6 Hz, 1H, CHCO); 2.82 (m, 2H, CH2 6); 3.79 (d, J = 8.4 Hz, 1H, CHO); 4.35-4.60 (dAB, J = 16.1, 8.4, 6.2 Hz, 2H, NCH2); 5.01 (s, 2H, CH2Ph); 6.44 (m , 1H, NH); 6.70 (d, J = 2.2 Hz, 1H, H4); 6.75 (dd, J = 8.4, 2.2 Hz, 1H, H2); 7.15-7.45 (m, 8H); 8.51 (d, J = 5.8 Hz, 2H, H2., H5.); MS (FAB+) calcd 496.3, obsd 497.3 (M+1 ).
3,17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopentafaj phenan hrene-16-carboxylic acid (furan-2-ylmethyl)-amide (JAC01081B, STX 679).
Figure imgf000135_0001
Method A, step2. The brown oil was purified by column chromatography using EtOAc/Hexane (50:50) as eluant gave 3 mg (3.6% over 2 steps) of STX 679 as a yellow solid. 1H NMR (400 MHz, CDCLJ: δ 0.82 (s, 2.49H, CH3); 0.84 (s, 0.51 H, CH3); 1.08-1.60 (m, 6H); 1.80 (m, 2H); 2.20 (m, 2H); 2.30 (m, 1H); 2.58 (m, 1H, CHCO); 2.80 (m, 2H, CH2 6); 3.74 (d, J = 8.4 Hz, 0.83H, CHO); 3.80 (d, J = 8.9 Hz, 0.17H, CHO); 4.41 (d, J = 5.7 Hz, 2H, CH2N); 6.15 (dd, J = 3.2, 1.7 Hz, 1H, furane H3); 6.25 (m, 1H, furane H4); 6.50 (d, J = 2.7 Hz, 1H, H4); 6.55 (dd, J = 8.4, 2.7 Hz, 1H, H2); 7.04 (d, J = 8.1 Hz, 1H, HO; 7.28 (dd, J = 2.0; 1.0 Hz, 1H, furane H5); MS (FAB+) calcd 395.21, obsd 396.18 (M+1); purity: 98%.
3,17-Dihydroxy-13rmethyl-7, 8, 9, 11, 42, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]henanthrene-16-carboxylic acid (tetrahydro-furan-2-ylmethyl)-amide (JAC01171-3, STX 779).
Figure imgf000135_0002
Method B, step3. Yield = 12 %, white solid. 1H NMR (MeOH-d3, 270 MHz): δ 0.81 (s, 3H, CH3); 1.26-1.45 (m, 6H); 1.50-1.70 (m, 3H); 1.80-2.00 (m, 6H); 2.15 (m, 1H); 2.30 (m, 1H); 2.60 (ddd, J = 11.4, 8.1, 3.7 Hz, 1H, CHCO); 2.78 (m, 2H, CH2 6); 3.70-3.95 (m, 3H); 3.98 (m, 1H); 6.45 (d, J = 2.6 Hz, 1H, H4); 6.52 (dd, J = 8.4, 2.6 Hz, 1H, H2); 7.06 (d, J = 8.4 Hz, 1H, HO; MS (APCI+) calcd 399.53, obsd 401.34 (M+2); Purity: 99%
3,17-Dihydroxy-13- ©thyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxyiic acid (pyridin-3-ylmethyl)-amide (JAC01181-2, STX 798).
Figure imgf000136_0001
Method B, stepS. Yield = 36%. 1H NMR (270 MHz, DMSO-d6): δ 0.69 (s, 3H, CH3); 1.10- 1.40 (m, 7H); 1.45-1.60 (m, 2H); 1.60-1.85 (m, 4H); 2.20 (m, 2H); 2.67 (m, 2H, CH2 e); 2.86 (m, 2H); 3.65 (m, 1H); 4.29 (d, J = 5.5 Hz, 1H); 4.80-5.05 (m, 1H); 6.41 (s, 1H, H4); 6.48 (dd, J = 8.4, 2.2 Hz, 1H, H2); 7.02 (d, J = 8.4 Hz, 1H, HO; 7.33 (m, 0.5H); 7.62 (m, 1H); 8.26 (m, 0.5H); 8.42 (dd, J = 4.8, 1.8 Hz, 1H); 8.47 (m, 1H); 9.00 (bs, 1H, phenol). MS (APCI+) calcd 406.5, obsd 408.3 (M+2); Purity: 100%
3,17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-yl)-(4-methyl-piperazin-1-yl)-methanone
(JAC01181-3, STX799).
Figure imgf000136_0002
Method B, step3. Yield = 58%. 1H NMR (270 MHz, CDCI3): δ 0.83 (s, 3H, CH3); 1.26- 1.55 (m, 6H); 1.65-1.85 (m, 3H); 1.89 (m, 1H); 2.25 (m, 1H); 2.30 (s, 3H, NCH3); 2.40 (m, 4H); 2.78 (m, 2H, CH2 6); 2.88 (m, 1H, CHCO); 3.40-3.80 (m, 4H); 4.17 (d, J = 7.7 Hz, CHO); 6.53 (d, J = 2.7 Hz, 1H, H4); 6.61 (dd, J = 8.4, 2.7 Hz, 1H, H2); 7.12 (d, J = 8.4 Hz, 1H, HO- MS (APCI+) calcd 398.5, obsd 400.3 (M+2); Purity: 100%.
3,17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid ethyl-methyl-amide (JAC01181-4, SIX 800).
Figure imgf000137_0001
Method B, step3. Yield = 75%. H NMR (400 MHz, DMSO-d6): δ 0.70 (s, 3H, CH3); 0.98 (t, J = 6.8 Hz, 1.5H, CHaCHg); 1.07 (t, J = 7.0 Hz, 1.5H, CHgCHO; 1.10-1.30 (m, 6H); 1.45-1.85 (m, 4H); 2.05 (m, 1H); 2.20 (m, 1H); 2.67 (m, 2H, CH2 6); 2.79 (s, 1.5H, NCH3); 2.88 (m, 1H, CHCO); 2.96 (s, 1.5H, NCH3); 3.30-3.48 (m, 2H, CJiCHO; 3.78 (dd, J = 12.8, 7.0 Hz, 1H); 4.76 (t, J = 5.3 Hz, NH); 6.41 (d, J = 2.6 Hz, 1H, H4); 6.47 (dd, J = 8.2, 2.6 Hz, 1H, H2); 7.02 (d, J = 8.2 Hz, 1H, HO; 8.99 (s, 1H, phenol). MS (APCI+) calcd 357.5, obsd 359.3 (M+2); Purity: 100%.
3-,17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid isopropylamide (JAC01181-5, STX 801).
Figure imgf000137_0002
Method B, step3. Yield = 50%. 1H NMR (270 MHz, CDCI3): δ 0.79 (s, 3H, CH3); 1.14 (d, J = 6.6 Hz, 6H, 2 x CH3); 1.20-1.60 (m, 6H); 1.84 (m, 2H); 2.09-2.20 (m, 2H); 2.22-2.34 (m, 1H); 2.48 (ddd, J = 12.1, 8.4, 4.0 Hz, 1H, CHCO); 2.77 (m, 2H, CH2 6); 3.76 (d, J = 8.4 Hz, 1H); 4.09 (m, 1H, CH(CH3)2); 5.73 (app. d, J = 7.3 Hz, 1H, NH); 6.54 (d, J = 2.7 Hz, 1H, H4); 6.61 (dd, J = 8.4, 2.7 Hz, 1H, H2); 7.11 (d, J = 8.4 Hz, 1H, HO- MS (APCI+) calcd 357.5, obsd 359.2 (M+2); Purity: 100%.
3,17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]ph@nanthren@-16-carbo2-ylic acid (1-methyl-1H-pyrrol-2-ylmethyl) - amide (JAC01181-6, ST3C 802).
Figure imgf000138_0001
Method B, step3. Yield = 45%. 1H NMR (270 MHz,' CDCI3): δ 0.78 (s, 3H, CH3); 1.20- 1.65 (m, 8H); 1.80-1.92 (m, 3H); 2.10-2.20 (m, 2H); 2.25 (m, 1H); 2.50 (ddd, J = 11.7, 8.4, 4.0 Hz, 1H, CHCO); 2.84 (m, 2H, CH2 6); 3.57 (s, 3H); 3.78 (d, J = 8.1 Hz, 1H, CHO); 4.44 (t, J = 5.8 Hz, 1H, CH2N); 5.99 (t, J = 5.1 Hz, 1H, NH); 6.05 (m, 2H); 6.54 (d, J = 2.9 Hz, 1H, H4); 6.57-6.64 ( , 2H); 7.10 (d, J = 8.4 Hz, 1H, HO- MS (APCI+) calcd 408.5, obsd 408.3 (M+), 410.3 (M+2); Purity: 100%.
3,17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)- a ide (JAC01181-7, STX 803).
Figure imgf000138_0002
Method B, step3. Yield = 26%. 1H NMR (270 MHz, CDCI3): δ 0.82 (s, 3H, CH3); 1.20- 1.65 (m, 8H); 1.75-1.95 (m, 2H); 2.05-2.40 (m, 3H); 2.54 (s, 3H, HarCH3); 2.64 (m, 1H, CHCO); 2.81 (m, 2H, CH2 6); 3.77 (d, J = 8.4 Hz, CHO); 4.40-4.80 (ddAB, J = 16.5, 7.0, 5.1 Hz, NCH2); 6.54 (app. s, 1H, H4); 6.60 (dd, J = 8.4, 2.6 Hz, 1H, H2); 6.72 (t, J = 5.1 Hz, 1H, NH); 7.10 (d, J = 8.4 Hz, 1H, HO; 8.36 (s, 1H); 8.48 (s, 1H). MS (APCI+) calcd 421.54, obsd 423.3 (M+2); Purity: 100%.
3,17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a3phenanthrene-16-carboJ ylic acid (l-phenyl-ethyl)-amide (JAC01 93- 3, STX 844).
Figure imgf000139_0001
[Starting material was STX 815]. Method B, step3. Yield = 7.3%. 1H NMR (270 MHz, DMSO-d6): δ 0.70 (s, 3H, CH3); 1.10-1.38 (m, 9H); 1.65-1.85 (m, 3H); 2.05 (m, 1H); 2.20 (m, 1H); 2.68 (m, 2H, CH2 6); 3.60 (m, 1 H, CHCO); 4.80-5.00 (m, 2H); 6.41 (app. s, 1H, H4); 6.48 (dd, J = 8.1 , 2.9 Hz, 1H, H2); 7.02 (d, J = 8.1 Hz, 1 H, HO; 7.20 (m, 1H); 7.25- 7.40 (m, 5H); 8.03 (d, J = 8.4 Hz, 1H, NH); 8.97 (s, 1H, OH); MS (APCI+) calcd 419.25, obsd 420.42, Purity: 98%.
3,17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-16-carboxylic acid (pyridin-4-ylmethyl)-amide (JAC001193-5, STX 845).
Figure imgf000139_0002
Method B, step3. Yield = 17 %. 1H NMR (270 MHz, DMSO-d6): δ 0.60 (s, 0.86H, CH3); 0.63 (s, 2.14H, CH3); 1.10-1.25 (m, 5H); 1.35-1.80 (m, 4H); 2.02 (m, 1H); 2.13 (m, 1H); 2.60 (m, 2H, CH2 6); 2.81 (m, 1H); 3.60 (dd, J = 8.1 , 4.4 Hz, 1H, CHO); 4.10-4.30 (dAB, J = 16.1 , 6.2 Hz, 2H, NCH2); 4.76 (d, J = 5.9 Hz, 0.29H, CHO); 4.92 (d, J = 4.4 Hz, 0.71 H, CHO); 6.33 (app. s, 1H, H4); 6.40 (app. d, J = 8.4 Hz, 1 H, H2); 6.94 (d, J = 8.4 Hz, 1H, HO; 7.16 (app. d, J = 5.5 Hz, 2H); 8.22 (t, J = 5.8 Hz, 1H, NH); 8.39 (dd, J = 4.7, 1.4 Hz, 2H); 8.92 (s, 1H, OH); MS (APCI+) calcd 406.2, obsd 407.3 (M+1); Purity: 96%
SECTION 3 Scheme 1: Synthesis of common intermediate acids (5 and 1) from Estrone
Figure imgf000140_0001
Experimental
General Methods. Unless otherwise noted, all reactions were conducted in oven-dried glassware using HPLC grade solvents. Purification of compounds was carried out using Argonaut pre-packed columns (Flash Column Chromatography) using flash vacuum system. Thin-layer chromatography (TLC) was performed on plates Kieselgel 60 F254 (Merck). 1H NMR was measured with a Jeol 270 MHz NMR spectrometer or 400MHz Bruker NMR spectrometer in ofeuterated-solvents as the internal reference. Low resolution mass spectral data were obtained either on a Micromass platform LCZ (ES +, APCI +) or FAB. High resolution mass was obtained using FAB technique Waters Alliance-HT-2790 system with a column Symmetry R C18 was used to determine the purity using HPLC spectra with 10% water in Methanol as eluent, Rτ values are quoted for these conditions.
2-Bensyl estrone 2 ( DS01-0S2):
To a solution of 3-hydroxy estrone (10.00 g, 0.036 mol) in THF (180 ml) under nitrogen, was added sodium hydride (1.056 g, 0.044 mol) portion wise and stirred at room temperature for 30 minutes. Benzyl bromide (7.52 g, 0.044 mol) was added at r.t. and the reaction mixture was refluxed overnight. The completion of the reaction was monitored by tic (ethyl acetate:hexane, 3 : 7). The crude mixture was cooled to r.t., quenched by addition of water (drop wise). The organic phase was extracted with DCM (2 x 100 ml), followed by ethyl acetate (2 x 100 ml) and the combined organic extracts were dried and concentrated in vacuo to obtain a colourless solid. The crude solid was dissolved in EtOH containing 5% EtOAc, left in a refrigerator to obtain the required product 2 (10.711 g, 82%) as a crystalline colourless solid. Analytical data see JAC01- 002
3-Benzyloxy-16-methylene estrone carboxylic acid ethyl ester 3 (HDS01-086 and HDS01-126): ' '
To a solution of Lithium Diisopropylamide (5.66 ml, 1.8M solution in heptanes, THF, and ethyl benzene) in THF (20 ml) was added a solution of estrone benzyl ether 2 (3.90 g, 10.8 mmol) in THF (30 ml) at 0 °C. The reaction mixture was stirred at 0 °C for approximately 15 minutes and cooled to - 45 °C. A solution of ethyl bromoacetate (5.344 g, 32 mmol) in THF (10.0 ml) was added drop wise over 10 minutes and stirred at - 45 °C for a period of 8 h and room temperature overnight. The reaction was monitored by tic. The crude reaction mixture was quenched with saturated NH4CI, extracted the product into DCM (2 x 100 ml), dried and concentrated in vacuo to obtain a yellow oily product. The crude product was purified by Flash Chromatography (SiO2, ethyl acetate hexane gradient elution) to obtain the required product 3 (4.91 g, 70%, a mixture of diastereoisomers) as a pale yellow solid: 1H NMR (CDCI3) δ 0.928 and 1.002 (2 x s, 3H each, Me), 1.295 (t, 3H, CH3), 1.41-1.61 (m, 6H), 1.77-1.82 (m, 1H), 1.93-2.03 (m, 3H), 2.26-2.30 (broad m, 1H), 2.38-2.41 (dd, J = 8.2, 5.4 Hz, 1H), 2.42-2.55 (m, 1H), 2.76- 2.81 (dAB, J = 16.0, 3.90 Hz, 1H), 2.88-2.92 (broad m, 2H), 2.98-3.53 (m, 1H), 4.17-4.19 (q, 2H, CH2), 5.04 (s, 1H, benzylic), 6.74-6.75 (app d, 1 H, aromatic), 6.79-6.82 (2 x d, J = 2.7 Hz each, 1H, aromatic) 7.332-7.456 (m, 6H); LCMS [(M+2)+ = 448], (M+H2O) = 465; HPLC (99%, Rt= 6.11)
3-Hydroxy-16-methylene estrone carboxylic acid ethyl ester 4 (STX 573, HDS01- 090):
A solution of 3-Benzyloxy-16-methylenβ carboxylic acid ethyl ester 3 (1.50 g, 3.36 mmol) was dissolved in EtOH (15 ml) and the mixture was degassed approximately 20 minutes and debenzylation was carried out under a hydrogen balloon at r.t. for 48 hours. The completion of the reaction was monitored using tic (EtOAc:hexane, 3:7). The crude reaction mixture was filtered through a pad of celite, the filtrate was concentrated to obtain a sticky white solid which was then purified by column chromatography (5 -20% ethyl acetate in hexane) to obtain the debenzylated product 4 (0.828 g, 70%, mixture of 2 diastereomers 3:1) as a white solid: 1H NMR (CDCI3) [chemical shifts of major to minor isomer ratio approximately 3:1 was determined using Me signals at 13-position], δ 0.916 and 0.988 (major isomer) [2 x s, 3H, 2 x Me], 1.27-1.29 (t, integrating to more than 3H, J = 7.0 Hz, CH3), 1.40-1.60 (m, 6H), 1.75-1.80 (m, 1H), 1.92-2.23 (m, 6H), 2.28-2.35 (dAB, J = 16.4 and 10.1 Hz, 1H), 2.36-2.39 (m, 1H), 2.47-2.54 (dAB, J = 18.7, 8.5 Hz, 1H, minor isomer), 2.74-2.79 (dd, J = 3.9, 4.2 Hz, 1H), 2.85-2.88 (m, 2H), 2.99-3.02 (m, 1H), 4.15-4.17 (q, 2H, CH2), 4.63 (1H, OH), 6.57-6.58 (appd, 1H, aromatic), '6.62-6.65 (dd, = 8.5, 2.7 Hz, 1H), 7.13-7.16 (dd, J = 8.5, 3.9 Hz, 1H); Mass (FAB) (M+1)+ = 357; Ace. Mass (FAB+) calcd 357.2065, obsd 357.2054; HPLC (99%, Rt = 2.01 ).
3-Hydroxy-17-hydroxy-16-methylene estrone carboxylic acid ethyl ester 6 (STX 574, HDS01-094):
A solution of 3-Hydroxy-16-methylene carboxylic acid ethyl ester 4 (1.20 g, 3.3 mmol) in MeOH (15 ml) was cooled to 0 °C and added NaBH4 (0.191 g, 5.0 mmol) over 15 minutes. The reaction mixture was warmed to r.t., stirred overnight. The reaction was monitored by tic (EtOAC:Hexane, 1:1). The crude mixture was quenched with water and extracted the aqueous layer with Ethyl Acetate and DCM. The combined organics were washed with saturated NaCI, dried and concentrated to obtain a white solid and then recrystalised with DCM and Hexane to obtain pure compound 6 (0.791 g, 71%) as a white solid: 1H NMR (CDCI3) [chemical shifts of major to minor isomer ratio approximately 3:1 was determined using Me signals at 13-position] δ 0.784 and 0.852 (major isomer) [2 x s, 3H each, 2 x Me], 1.280 (t, J = 7.4 Hz, integrating to more than 3H, CH3), 1.30-1.48 (m, 6H), 1.71-1.88 (m, 3H), 1.96-2.05 (1H, m), 2.24-2.56 (m, 4H) 2.54-2.56 (dd, J = 7.4, 2.3 Hz, approximately 1.5H), 2.79-2.83(m, 3H), 3.01-3.02 (d, J = 3.9 Hz, 1H), 3.37-3.40 (dd, J = 7.4, 3.5 Hz, 1H), 4.13-4.18 (q, J = 7.0 Hz, 2H), 4.65-4.70 (broad 2 x s, 1H), 6.55-6.56 (appd, 1H, aromatic), 6.61-6.64 (dd, J = 8.5, 2.7Hz, 1H, aromatic) 7.14-7.16 (d, J = 8.2 Hz, 1H, aromatic); (FAB) (M+1)+ = 359; Ace. Mass (FAB+) calcd 359.2222, obsd 357.2220; HPLC (99%, Rt= 1.
3-Hydro.jy-17-hydro-.y-16-m©thylene estrone carboxylic acid 7 (STX @03, HDS01- 096): The Ethyl Ester 6 (0.780 g, 2.1 mmol) was dissolved in a mixture of THF:MeOH:H2O (1:1:0.5, 10 ml) in a seal tube and added NaOH (043 g, 10.5 mmol). The mixture was heated to 80 °C overnight. The reaction was monitored by tic (EtOAc: Hexane, 1:1). The crude mixture was acidified to pH = 3 - 4, extracted with EtOAc and DCM. The combined organics were dried (MgSO4) and concentrated to obtain a white solid. The solid was recrystalised with MeOH and hexane to obtain pure acid 7 (0.700g, 101%) as a white solid: H NMR (CD3)2-SO) [chemical shifts of major to minor isomer ratio approximately 3:1 was determined using Me signals at 13-position] δ 0.649 and 0.688 (2 x s, 3H, 2 x CH3) 1.148-1.308 (m, 8H), 1.47-1.56 (q, J = 12.4, 1H), 1.90-2.19 (m, 6H), 2.65-2.68 (m, approximately 3H), 3.15-3.16 (broad m, 1H), 4.673-4.682 (broad d, 1H) 6.40-6.41 (appd, 1H, aromatic), 6.47-6.49 (dd, J = 8.2, 2.3 Hz, 1H, aromatic), 7.01-7.03 (d, J = 8.5 Hz, aromatic), 8.98 (s, 1H), 11.9 (broad s, 1H); (FAB) (M)+ = 330; Ace. Mass (FAB+) calcd 330.1831, obsd 330.1832; HPLC (> 95%, Rt= 2.21).
3-Hydroxy-17-oxo-16-methylene estrone carboxylic acid 5 (STX 604, HDS01-098):
The reaction was carried out using the procedure outlined for compound 7 with 3- Hydroxy-16-methylene carboxylic acid ethyl ester 4 (1.00g, 2.8 mmol), NaOH (0.56 g, 1.4 mmol) and THF:MeOH:H2O (1:1:0.5, 10 ml). The product 5 (0.940 g, 102%) was isolated as a white solid after recrystalisation: 1H NMR (CD3)2-SO) [chemical shifts of major to minor isomer ratio approximately 3:1 was determined using Me signals at 13- position] δ 0.786 [major isomer] and 0.896 (2 x s, 3H each, 2 x Me), 1.31-1.52 (broad m, 8H), 1.74-1.77 (m, 1H), 1.88-1.91 (m„1.5H), 2.16-2.22 (m, 3H), 2.27-2.33 (m, 3H), 2.48- 2.49 (2 x d, J = 195 Hz, 1H), 2.72-2.74 (m, 2.5H), 6.43 6.44 (appd, 1H, aromatic), 6.48- 6.51 (d, J = 8.5, 2.4 Hz, aromatic), 7.02-7.04 (d, J = 8.5 Hz, aromatic), 9.0 (broad s, 1 H); (FAB) (M+1)+ = 329; Ace. Mass (FAB+) calcd 329.1752, obsd 330.1751; HPLC (> 95%, Rt= 2.60).
3-Hydroxy-17-oxo-16-methylene estrone carboxylic acid 8 (STX 666, HDS01-109):
The reaction was carried out using the procedure outlined for compound 7 with 3- benzyloxy-16-methylene carboxylic acid ethyl ester 3 (1.714 g, 3.8 mmol), NaOH (1.537 g, 38.4 mmol) and THF:MeOH:H2O (1:1:0.5, 10 ml). The product 8 (1.114 g, 90%) was isolated as a white solid after recrystalisation: Diagnostic signals 1H NMR (CD3)2-SO) δ 0.81, 0.84 (2 x s, CH3), 1.36-1.91 (m, 6H), 2.19-2.25 (m, 2H), 2.33-2.37 (dd, J = 9.3 Hz, 2H), 2.59-2.63 ( 2 x d, J = .2 Hz each, 0.5 H), 2.81-2.83 (m, 2H), 5.06 (s, 2H, benzylic), 6.72 (appd, 1H, aromatic), 6.73-6.77 (dd, J = 8.6 and 5.8 Hz, 1H, aromatic), 7.17-7.19 (d, J = 8.5 Hz, 1H, aromatic), 7.31-7.44 (5H, aromatic); LCMS [(M)+ = 417]; (FAB) (M+1)+ = 418; Ace. Mass (FAB+) calcd 418.2099, obsd 418.2148; HPLC (99%, Rt =
1.89).
Figure imgf000144_0001
Scheme 3: Parallel synthesis of amides in solution phase Method-B
Figure imgf000144_0002
Synthesis of resin hound intermediate 9 (HDS01-104):
The Oxime resin (0.600 g, loading 1.06mmol/g) was swollen in a minimum amount of DMF (7 ml), added the estrone carboxylic acid 5 (SΩ( 604!) [0.521 g, 5.3 mmol) followed by Di-isopropyl carbodiimide [DIC] (0.667 g, 5.3 mmol) and Hydroxy benztriazole [HOBt] (0.715 g, 5.3 mmol). The reaction mixture was shaken using a flask shaker for approximately 72 hours under N2. The yellow reaction mixture obtained after 72 hours was filtered, and washed with DCM, DMF, MeOH (3 cycles with each solvent), finally MeOH (5 times) and dried in vacuo to obtain resin bound intermediate 8 as yellow beads: IR (cm"1) 1662 (C=O), 1739 (CO-0-N=), 3502 (OH).
Parallel synthesis of estrone-amides; M®thodA
The resin bound estrone intermediate 9 (0.100 g, 0.106 mmol) was suspended in anhydrous DCM (3.0 ml) under N2 in a glass tube in RadleysGreenHouse Synthesiser, added the corresponding amines (5 molar equivalents) and heated at 40 °C under N2for 72 h. The reaction mixtures were filtered and the filtrates were evaporated using Genevac to obtain a pale yellow solid. The crude compounds were purified using the Flash vacuum system and pre-packed columns (5 or 10 g) with DCM: MeOH (gradient elution, starting with 2% MeOH in DCM) to obtain pure compounds (average recovery 14 mg / 100mg of resin bound intermediate).
Parallel synthesis of 16-methylene estrone-amides; Method B:
The carboxylic acid 5 (0.050 g, 0.15 mmol) or 7 (0.60 g, 0.15 mmol) was suspended in DCM (1.0 ml) in a glass tube in Radleys GreenHouse Parallel Synthesiser, added a solution of EDC (0.043 g, 0.227 mmol), DMAP (catalytic amount, 5 mg, and Et3N (0.100 ml) in DCM (1.0 ml). The reaction mixture was stirred approximately for 30 minutes and the corresponding amines (5 molar equivalents) in DCM (1.0 ml) were added and stirred overnight. [The total volume of DCM used in each reaction was 3.0 ml\. The completion of the reactions were monitored by tic (EtOAc:Hexane, 2:7). The crude mixtures were evaporated in the Genevac and purified using the Flash vacuum system and pre-packed columns (5 or 10 g) with DCM:MeOH (gradient elution, starting with 2% MeOH in DCM) to obtain pure compounds.
Synthesis of 3-Hydroxy-17-oxo-16-methylene estrone carboxy-furfuryl amide (STX 672, HDS01-112-3).
Figure imgf000145_0001
Method B, Yield = 33%. 1H NMR (CDCI3) δ 0.908, 0.964, 1.021 (3 x s, 3 x Me), 1.39- 1.59 (broad m, 6H), 1.94-1.97 (m, 2H), 2.23-2.37 (m, 2H), 2.82-2.87 (m, 1.5H) 4.28-4.44 (d, J = 5.4 Hz, CH2), 6.23-6.24 (m, 2H, aromatic), 6.57-6.65 (m, 2H, aromatic), 7.10-7.12 (m, 1H, aromatic), 6.34-6.35 (appd, 1H, aromatic), LCMS [(M+1)+ = 407]; Ace. Mass (FAB+-M+1) calcd 408.3172, obsd 408.2174; HPLC (90%, Rt= 1.47).
Synthesis of 3-Hydr©∑sy-17-©rø-1@-methylene estrone carboxy-tetrahydro-2-fur l amide (STX 671, HDS01-112-6)
Figure imgf000146_0001
Method B, Yield = 40%. 1H NMR (CDCI3) δ 0.69, 0.73,0.83,0.87 and 0.87 (4 x s, approximately 3H each, Methyl signals), 1.2-1.55 (broad m, 7H), 1.74-1.94 (broad m, 4H), 2.08-2.29 (broad m, 2H), 2.29-2.78 (m, 3H), 3.01-3.07 (m, 1H), 3.50-3.92 (m, 2.5H),
6.15-6.57 (m, 3.5H, aromatic), 6.79-7.08 (m, 1.5H, aromatic); LCMS [(M+1)+ = 411]; Ace.
Mass (FAB+' M+1 ) calcd 412.2487, obsd 408.2491 ; HPLC (90%, Rt= 1.49).
Synthesis of 3-Hydroxy-17-oxo-16-methylene estrone carboxy-3-pyridylamide (STX 670, HDS01-112-2 and HDS01-168).
Figure imgf000146_0002
Wlethod B, Yield = 50%, 1H NMR δ (CD3)2-SO), 0.778-0.883 (4 x s, 3H), 1.26-1.55 (m, 6.5H), 1.61-1.84 (m, 6H), 2.04-2.41 (m, 6H), 2.29-2.89 (m, 6H), 4.13-4.17 (d, J = 15.6
Hz, 1H), 4.27-4.29 (d, J = 5.8 Hz, 2H), 4.35-4.37 (d, J = 7.0 Hz, 0.5H), 4.48-4.52 (d, J =
15.2, 1H), 5.76(s, 1H), 6.39 (s,1H), 6.04-6.50 (m, 3H, aromatic), 6.9 (2 x d, J = 8.2 Hz,
1 H), 7.0 (d, J = 8.5 Hz, 0.5H), 7.28-7.36 (m, 2H), 7.63-7.65 and 7.73-7.75 (2 x d, J = 8.2 Hz each), 8.35-8.54 (m, 2H), 8.9 9.0 (2 x s, 2H); LCMS [(M+1)+ = 419, -(Rt = 8:58)]; HPLC (100%, Rt= 2.08).
A single dias ereo er of ST2 670 shows the following analytical data. MP = 210-211 °C, 1H NMR δ (CD3OD), 0.86-0.88 (s, 3H, Me), 1.28 - 1.57 (broad m, 5H), 1.88 - 2.15 (broad m, 2H), 2.60-2.82 (broad m, 4H), 4.42 (s, 1H), 6.49 - 6.51 (appd, 1H, aromatic), 6.54 - 6.55 (d, J = 2.47 Hz, aromatic), 7.05 - 7.08 (d, J = 8.16 Hz, 1H, aromatic), 7.39 - 7.44 (dd, J = 7.91 and 5.19 Hz, 1H, aromatic), 7.80-7.83 (d, J = 7.91 Hz, 1H, aromatic), 8.43 - 8.45 (appd, 1H, aromatic), 8.51 (s, 1H, aromatic) LCMS [(M+1)+ = 419, Rt = 6.70]; HPLC (100%, Rt= 2.26).
Synthesis of 3-Hydroxy-17-hydroxy-16-methylene estrone carboxy-3-pyridylamide (S7X 669, HDS01-112-1).
Figure imgf000147_0001
Method B, Yield = 40%, 1H NMR δ (CDCI3), 0.778-0.883 (4 x s, 3H), 1.26-1.55 (m, 6.5H), 1.61-1.84 (m, 6H), 2.04-2.41(m, 6H), 2.29-2.89 (m, 6H), 4.13-4.17 (d, J = 15.6 Hz, 1H), 4.27-4.29 (d, J = 5.8 Hz, 2H), 4.35-4.37 (d, J = 7.0 Hz, 0.5H), 4.48-4.52 (d, J = 15.2, 1H), 5.76(8, 1H), 6.39 (s, 1H), 6.04-6.50 (m, 3H, aromatic), 6.9 (2 x d, J = 8.2 Hz, 1H), 7.0 (d, J = 8.5 Hz, 0.5H), 7.28-7.35 (m, 2H), 7.63-7.65 and 7.73-7.75 (2 x d, J = 8.2 Hz each), 8.35-8.54 (m, 2H), 8.9 9.0.(2 x s, 2H); LCMS [(M)+ = 418]; Ace. Mass (FAB+' M+1) calcd 419.2334, obsd 419.2349; HPLC (100%, Rt= 1.83).
Synthesis of 3-Hydroxy-17-hydroxy-16-methylene estrone carboxy-N, N- dimethylpropyl amide (STX 688, HDS01-116-1).
Figure imgf000147_0002
Method A. 1H NMR (CDCI3), δ 0.768 (Major isomer) and 0.90 (2 x s, 6H, 2 x Me), 1.2 - 1.61 (broad m, ,8H), 1.71-1.99 (broad m, 5H), 2.25 and 2.93 (2 x s, 6H each, 2 x Me), 2.30-2.36 (m, 2H), 2.39-2.56 (m, 2H), 2.56-2.78 (broad m, 2H), 2.89-2.90 (d, J = 3.1 Hz, 1H) 2.93-2.94 (d, J = 3.1 Hz, 1-H), 3.40-3.67 (m, 4H), 6.52 (appd, 1H, aromatic), 6.54- 6.56 (dd, J = 8.5, 2.7 H∑), 7.04-7.06 (d, J = 8.2 Hz, 1 H, aromatic); LCMS [(M)+ = 411 and (M + 1)+ = 412]; HPLC (98%, Rt= 1.51).
Synthesis of 3-Hydroxy-17-hydro∑sy-16-methylene estrone carbo_£y-ethyl- methoxya ide (STX 689, HDS01-116-3).
Figure imgf000148_0001
Method A. 1H NMR (CDCI3), δ 0.766, 0.852,- 0.898 and 1.182 (Me signals, approximately), 1.2,10- 1.475 (broad m, 8H), 1.57-2.30 (m, - 13H), 2.50-2.82 (m, 5H), 3.27-3.39 (4 x s, OMe), 4.85-4.93 (broad s, 1H), 5.93-6.08 (2 x s, 2H), 6.50-6.55 (m, 1H, aromatic), 6.55-6.57 (m, 1H, aromatic), 7.05-7.07 (m, 1H, aromatic); LCMS [(M + 1)+ = 386]; HPLC (99%, Rt= 1.47).
Synthesis of 3-Hydroxy-17-hydroxy-16-methylene estrone carboxy-ethyl- methoxyamide (HDS01-100-2).
Figure imgf000148_0002
Rfethod B. 1H NMR (CDCI3), δ 0.768 (Major isomer) and 0.90 (2 x s, 6H, 2 x Me), 1.2 - 1.61 (broad m, 8H), 1.71-1.99 (broad m, 5H), 2.25 and 2.93 (2 x s, 6H), 2.30-2.36 (m, 2H), 2.39-2.56 (m, 2H), 2.56-2.78 (broad m, 2H), 2.89-2.90 (d, J = 3.1 Hz, 2.93-2.94 (d, J = 3.1 Hz, 1H), 3.40-3.67 (m, 4H), 6.5 (appd, 1H, aromatic), 6.54-6.56 (dd, J = 8.5, 2.7 Hz), 7.04-7.06 (d, J = 8.2 Hz, 1H, aromatic); FAB [(M + 1)+ = 399; Ace. Mass (FAB+- M+1) calcd 399.2647, obsd 399.2627; HPLC (99%, Rf= 1.51). Synthesis of 3-Hydro2iy-17-ox©-16-methylen® estrone carbo∑ty-αc-me hyl ben∑yl amid© (STXβSO, HDS01 -116-4).
Figure imgf000149_0001
Method A. This compound exists as a mixture of 4 diastereomers. Diagnostic signals of 1H NMR (CDCI3), δ 0.69, 0.73, 0.88, 0.89 (4 x s, Methyl signals), 1.41-1.44 (m, benzylic methyl), 2.76-2.78 (m, 2H), 5.01-5.06 (m, 1H, Benzyic), 6.50 (appd, 1 H, aromatic), 6.55- 6.57 (broad d, 2H, aromatic), 7.03-7.06 (m, 1 H, aromatic), 7.17-7.29 (m, aromatic); LCMS [(M + 1 )+ = 432]; HPLC (99%, Rt= 1.51).
Synthesis of 3-Hydroxy-17-oxo-16-methylene estrone carboxy-2-ethyl pyridylamide (STX 691, HDS01-116-5).
Figure imgf000149_0002
Method A. Diagnostic signals of 1H NMR (CDCI3), δ 0.75, 0.80 and 0.92 and 1.0 (3 x Me signals), 1.20-2.84 (broad m, approximately 14 H), 3.0-3.03 (m, 2H), 3.36-3.62 (m, 0.5H), 3.64-3.69 (m, 2H), 6.57-6.59 (m, 1 H, aromatic), 6.63-6.64 and 6.65-6.66 (d, J = 2.7 Hz each, 1H, aromatic), 6.72-6.75 (m, 0.5H), 7.09-7.30 (m, 4.5H, aromatic), 7.64- 7.68 (m, 2H, aromatic), 8.40 - 8.42 (m, 0.5H, aromatic), 8.52-8.53 (m, 1H, aromatic); LCMS [(M + 1)+ = 433 and (M - OH)+ = 415]; FAB [(M + 1)+ = 433; HPLC (99%, Rt = 1.51 ).
Synthesis of 3-Hydroxy-17-hydroxy-16-methylen© estrone carbo∑-y-tetrahydro-2- furyl amide (STX 668, HDS01-110-6).
Figure imgf000150_0001
Method B, Yield = 33%. 1H NMR (CDCI3) δ 0.78 (2 x s, 3H, 2 x Me), 0.86-0.89 (m, 1H), 1.19-1.55 (broad m, 7H), 1.69-1.96 (m, 2H), 2.10 - 2.32 (broad m, 3H), 2.74-2.84 (m, 2H), 3.73-3.74 (m, 1H), 3.73-3.74 (d, J = 5.8 Hz, 1H), 4.46-4,48 (broad m, 1H), 6.57 (appd, 1H, aromatic), 6.62-6.65 (m, 1H, aromatic), 7.13-7.17 (m, 1H) LCMS [(M- C5H9NO)+ = 313]; HPLC (95%, Rt= 1.57).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-furfuryl amide (STX 667, HDS01-110-3).
Figure imgf000150_0002
Method B, Yield = 37%. 1H NMR (CDCI3) δ 0.076, 0.77, 0.79, (3 x s, approximately 3H each, Methyl signals), 1.25-1.58 (broad m, 14H), 1.86-1.95 (m, 1H), 2.02-2.42 (m, 3.5H), 2.47-2.49 (m, 0.5H), 2.78-2.87 (3H, m), 3.41-3.43 (dd, J = 2.3 and 1.9Hz, 1H), 4.44-4.45 (d, J = 5.4 Hz, 1H), 6.01 (broad s, 1H), 6.24-6.25 (d, J = 2.3 Hz, 1H, aromatic), 6.33-6.64 (2 sets of multiplets, 4H, aromatic), 7.14-7.16 (d, J = 8.5 Hz, 1.5H, aromatic), 7.28-7.35 (m, 1H, aromatic), 7.36-7.37 (m, 1H, aromatic); LCMS [(M-1)+ = 408 (Rt = 8.58)]; HPLC (100%, Rt= 2.08); Ace. Mass (FAB+ M+1) calcd 410.2331, obsd 410.2317; HPLC (90%, Rt= 1.82 and 1.90).
Figure imgf000151_0001
Synthesis of resin bound intermediate 12 (HDS01-120):
The Oxime resin (2.0 g, loading T.06mmol/g) was swollen in a minimum amount of DMF (7 ml), added the estrone carboxylic acid 8 (1.428 g, 3.18 mmol) followed by Di-isopropyl carbodiimide [DIC] (1.33 g, 10.6 mmol) and Hydroxy benztriazole [HOBt] (1.431 g, 10.6 mmol). The reaction mixture was shaken using a flask shaker for approximately 72 hours under N2. The yellow reaction mixture obtained after 72 hours was filtered, and washed with DCM, DMF, MeOH (3 cycles with each solvent), finally MeOH (5 times) and dried in vacuo to obtain resin bound intermediate 8 (2.391 g) as yellow beads: IR (cm"1) 1662 (OO), 1739 (CO-O-N=), 3502 (OH).
Parallel synthesis of estrone-amides; Method C
The resin bound estrone intermediate 12 (Scheme 4) (0.100 g, 0.106 mmol) was suspended in anhydrous DCM (3.0 ml) under N2 in a glass tube in Radleys GreenHouse Synthesiser, added the corresponding amines (5 molar equivalents) and heated at 40 °C under N2 for 72 h. The reaction mixtures were filtered and the filtrates were evaporated using Genevac to obtain a pale yellow solid. The crude compounds were purified using the Flash vacuum system and pre-packed columns (5 or 10 g) with DCM: MeOH (gradient elution, starting with 2% MeOH in DCM) to obtain pure benzylated intermediates (average recovery 15 mg/100 mg of resin bound intermediate, analysed by 1H nmr). These intermediates were then debenzylated under a balloon of H2 in the GreenHouse Synthesiser to obtain final compounds in average 90% purity. Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-methyl-N- ethyl amide (SΩ(741, HDS01-128-1).
Figure imgf000152_0001
C, Yield = 40%. 1H NMR (CDCI3) δ 0.81 and 0.9 0 (2 x s, 3H, 2 x CH3, 3:1), 1.01-1.24 (m, 5H), 1.66- 2.60 (m, 6H), 2.75-80 (m, 2.5H), 2.86-2.90 (2 x s, 4.5H, 2 x CH3), 3.26-3.28 (m, 1H), 4.79 (broad s, 1H), 6.50 (app d, 1H, aromatic), 6.51-6.66 (2 x d, J = 2.9 and 2.5 Hz, 1H, aromatic), 7.05-7.08 (d, J = 8.4 Hz, 1H, aromatic); LCMS [(M+2)+ = 371 , (Rt = 6.08)]; HPLC (85%, Rt= 2.29).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N,N-diethyl amide (STX 742, HDS01-128-2).
Figure imgf000152_0002
Method C, Yield = 40%. 1H NMR (CDCI3) δ 0.78 (major isomer) and 0.89 (2 x s, 2 x CH3), 1.03-1.13 (m, approximately,6H), 1.39-1.59 (m, 5H), 1.89-1.94 (m, 1.5H) 2.13-2.91 (m, 5H), 3.20-3.36 (m, 4H), 5.05-5.27 (broad s, 1H), 6.51-6.62 (appd, 1H, aromatic), 6.56-6.59 (dd, J = 2.9 and 2.5 Hz, 1 H, aromatic), 7.05-7.08 (d, J = 8.8 Hz, 1 H, aromatic); LCMS [(M+2)+ = 385, Rt = 6.55)]; HPLC (86%, Rt = 2.37).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-isopropyl amide (STX 743, HDS01-128-4).
Figure imgf000152_0003
Method C, Yield = 40%. 1H NMR (CDCI3) δ 0.83 [Major isomer] and 0.95 (2 x s, CH3 signals, 3:1), 1.12 (d, J = 1.4Hz, CH3), 1.14-1.15 (d, J = 2.20 Hz, CH3), 1.90-2.64 (m, 2H), 2.80-2.85 (m, 2H), 4.91 (broad s, 1H), 6.56 (app d, 1H, aromatic), 6.59-6.60 and 6.62-6.63 (dd, J = 2.9 and 2.5 Hz, 1H, aromatic), 7.10-7.13 (d, J = 8.0 Hz, 1H, aromatic); LCMS [(M+2)+ = 371 , (R, = 5.83)]; HPLC (93%, Rt = 2.27).
Synthesis of 3-hydroxy-17-hydroxy-16-methyl©n© estrone carbo-ty-N-@th.yl amide (STX 744, HDS01-128-5).
Figure imgf000153_0001
, Method C, Yield = 40%. 1H NMR (CDCI3) δ 0.78 [Major isomer] and 0.81 (2 x s, CH3 signals, 3:1), 1.05 -1.10 (t, J = 6.9 Hz, CH3), 1.35-.45 (m, approximately 4H), 1.89-2.64 (m, 11H), 2.79-2.80 (m, 2H), 3.18-3.24 (m, 2H, CH2), 4.83 (broad s, 1H), 6.51 (appd, 1H, aromatic), 6.54-6.55 and 6.57-6.59 (dd, J - 2.5 and 2.9 Hz, 1H, aromatic), 7.05-7.08 (d,
J = 8.4Hz, 1H, aromatic); LCMS [(M+2)+ = 357, [(M+Na)+ = 379]; HPLC (87%, Rt= 2.17).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-methyl amide (STX 745, HDS01 -128-6).
Figure imgf000153_0002
Method C, Yield = 40%. 1H NMR (CDCI3) δ 0.77 [major isomer], 0.85, 0.90 (3 x s, CH3 signals), 1.43-1.52 (m, 5H), 1.89-1.93 (m, 2H), 2.15-2.31 (m, 3H), 2.49-2.68 (m, 2H), 2.72 and 2.74 (2 x s, 3H, Me), 2.75-2.80 (m, 2H), 4.94-4.95 (broad s, 1H), 5.91 (broad s, 1H), 6.52 (app d, 1H, aromatic), 6.55-6.56 and 6.58-6.59 (dd, J = 2.9 Hz each, 1H, aromatic), 7.04-7.08 (d, J = 8.4 Hz, 1H, aromatic), LCMS [(M-30)+ = 311]; HPLC (97%, R,= 2.12). Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-methyl amide
(SM 74$, HDS01-128-9).
Figure imgf000154_0001
Method C, Yield = 40%. 1H NMR (CDCI3) δ 0.88 (Major isomer) and 0.93 (s, CH3), 1.4- 1.66 (m, 6H), 1.97-2.00 (m, 2H), 2.39-3.15 (m, 6H), 4.94 (broad s, 1H), 6.51-6.53 (d, J = 3.6 Hz, 1H, aromatic) 6.70 (app d, 1H, aromatic), 6.71-6.74 and 6.77-6.78 (2 x d, J = 2.5 Hz each, 1H, aromatic), 7.04-7.08 (d, J = 8.4 Hz, 1H, aromatic), LCMS [(M)+ = 412]; HPLC (77%, f?,= 2.16).
Synthesis of 3-hydroxy-17-hydrόxy-16-methylene estrone carboxy-N-methyl-N- ethyl amide (STX 771, HDS01 -138-1)
Figure imgf000154_0002
Method C, Yield = 40%. 1H NMR (CDCI3) δ 0.78 (Major isomer) 0.85 (s, 2 x CH3, 2:1), 1.08-1.19 (m, δH), 1.79 - 2.66 (m, 9.5H), 2.77 - 2.80 (m, 4H), 2.90 and 2.96 (2 x s, 4H approximately, CH3), 3.27-3.41 (m, 4H) , 3.81-3.94 (m, 1.5H), 4.46 (s, 1.5H), 6.54 (appd, 1H, aromatic), 6.58-6.62 (dd, J = 8.4 and 2.6Hz, 7.12-7.15 (d, J = 8.4 Hz, aromatic); LCMS [(M - 29)+ = 341]; HPLC (92%, Rt= 2.32).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-N-diethyl amide (STX 772, HDS01 -138-2).
Figure imgf000155_0001
Method C, Yield = 40%. 1H NMR (CDCI3) δ 0.74 and 0.80 (2 x s, 2 x CH3, 2:1), 1.02 - 1.15 (m, 6H), 1.70-2.65 (m, approximately 16H), 3.22-3.32 (m, 4H), 4.05 (appd, 1H), 4.69 and 4.99 (2 x s, 1H), 6.48 (appd, 1H, aromatic), 6.49-6.56 (dd, J = 8.4 and 5.8 Hz, 1H, aromatic), 7.07-7.10 (d, J = 8.0 Hz, 1H, aromatic); LCMS [(M + 2)+ = 387]; HPLC (99%, Rt- 2/
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-N-diethyl amide (STX 773, HDS01-138-4).
Figure imgf000155_0002
Method C, Yield = 40%. 1H NMR (CDCI3) δ 0.77 and 0.83 (2 x s, 2 x CH3 2:1), 1.16-1.15 (2 x d, J = 2.5 Hz each, isopropyl), 1.82-2.80 (m, approximately 14 H), 3.82-3.85 (2 x appd, 2H), 4.02-4.14 (m, 4H), 4.27-4.28 (appd, 1H), 4.59 (s, 2H), 5.3-5.4 (broad s, 2H), 6.53 (appd, 1H, aromatic), 6.54-6.62 (dd, J = 11.0 and 2.9Hz, 1H, aromatic), 7.12-7.15 (d, J = 8.4 Hz, 1H, aromatic); LCMS [(M + 2)+ = 373]; HPLC (87%, R,= 2.31).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-N-diethyl amide (STX 774, HDS01-138-5).
Figure imgf000155_0003
Method C, Yield = 40%. 1H NMR (CDCI3) δ 0.73 and 0.78 (2 x s, 2 x CH3, 2:1), 1.05-1.10 (t, J = 14.0, 6.9 Hz, CH3), 1.78-2.77 (approximately 16 H), 3.17-3.27 (m, 4H), 3.77-3.81 (d, J = 9.5 Hz 1H), 5.55 (broad s, 1H), 6.48 (appd, 1H, aromatic), 6.49-6.56 (dd, J = 8.4 and 5.5 Hz, 1 H, aromatic), 7.10-7.07 (d, J = 8.8 Hz, 1H, aromatic); LCMS [(M + 2)+ = 359]; HPLC (90%, Rt- 2.10).
Synthesis ©f 3-hydro--y-17-hydro-ty-1β-rn©£hylen© estrone carboxy-M-M-diethyl amide (STX 775, HDS01 -138-6)
Figure imgf000156_0001
Method C, Yield = 40%. H NMR (CDCI3) δ 0.73 (s, 3H, CH3), 1.78-2.77 (approximately 16 H), 2.75-2.77 (2 x s, 2 x CH3), 3.77 - 3.82 (d, J = 9.5 Hz, 1H), 4.04-4.06 (broad s, 1 ), 5.56 (broad s, 1H), 6.48 (appd, 1H, aromatic), 6.49-6.56 (dd, J = 8.0 and 5.5 Hz, 1H, aromatic), 7.07-7.10 (d, J = 8.4 Hz, 1H, aromatic); LCMS [(M - CH3)+ = 327]; HPLC (90%, ft, = 2.10).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-propylamide (STX 776, HDS01 -138-8).
Figure imgf000156_0002
Method C, Yield = 40%. 1H NMR (CDCI3) δ 0.91 (s, 3H, CH3), 0.84-0.96 (m, 3H), 1.23- 1.26 (t, 2H), 1.48- 1.51 (m, 8H), 2.13-2.33 (m, 4H), 2.85-2.88 (m, 2H), 3.16-3.23 (m, 2H), 4.98 (s, CH2), 5.27-5.43 (broad s, 1H), 5.97 (broad s, 1H), 6.71 (appd, 1H, aromatic), 6.77-6.78 (dd, J = 8.0 and 5.5 Hz, 1 H, aromatic), 7.18-7.21 (d, J = 8.4 Hz, 1H, aromatic) 7.29 - 7.41 (m, 5H, aromatic); HPLC (90%, Rt= 2.10). Scheme 1: Synthetic route to s lfamate and phenol libraries using a solid support
Figure imgf000157_0001
Reagents are: a) Benzyl bromide, K2C03, DMF, r.t. b) LDA, THF, Ethyl bromoacetate, -78 °C - r.t. c) H2) PdC, MeOHTHF (1:1) d) Sulfamoyl chloride, DMA, 0 °C to r.t. e) Triryl-Cl resin, DIPEA, DCM, 72h. f) NaOH, THF:H20 (1:1), r.t. g) EDCI, HOBt, PyBop, DIPEA, DCM, R-NH2, r.t., O N. h) TFA:DCM (50:50). i) Piperazine, THF. j) 50% TFA in DCM 20 min. k) Piperazine, THF, 70 °C, 72 h.
Synthesis of 2-ethyl benzyl estrone (HDS02-054)
To a stirred solution of 2-ethyl estrone 1 (10.00 g, 33.5 mmol) in anhydrous DMF (100 ml) under inert atmosphere was added potassium carbonate (14.0 g, 100 mmol) followed by Benzyl bromide (4.78 ml, 40 mmol). The reaction was left to stir at r.t. over 2 days. The solid obtained was filtered using a Buchner funnel, dissolved in DCM, filtered to remove any inorganics and concentrated to obtain a white solid. The white solid obtained was re-crystallised with DCM:MeOH (90:10) to obtain the pure product the benzyl ether estrone derivative 2 (12.00 g, 92%) also as a white solid: 1H NMR (270 MHz, CDCI3) δ 0.90 (s, 3H, 18-CH3), 1.17-1.23 (t, J = 7.4 Hz, CH2-CH3), 1.51-2.29 (m, 14H), 2.45-2.55 (m, 4H, 16-CH2), 2.62-2.70 (q, J = 7.6 Hz, 2H, CHZ-CH2), 2.84-2.90 (m, 2H, 6-CH2), 5.04 (s, 2H, CH2-phenyl) 6.64 (s, 4-H), 7.10 (s, 1-CH), 7.28-7.45 (m, 5H); FAB-HRMS calcd for C27H32O2 388.2357 found (M+) 388.2389.
Synthesis of benzyl ethyl ester (HDS02-098, HDS02-108) 2
To a stirred solution of 2-Ethylbenzyl estrone (5.00 g, 12.8 mmol) in anhydrous THF under inert conditions was added a solution of LDA (4.07 ml, 1.8 M solution in heptane, THF, and Ethylbenzene) at -10 °C over a period of 20 min. The reaction mixture was cooled to -60 °C, stirred 15 min, added ethyl bromoacetate drop wise over 10 min. and allowed to warm to rt overnight. Diluted the mixture with DCM, added saturated NH4CI and extracted with DCM. The organic phases were combined, dried, and concentrated to obtain a yellow solid which was then purified using Flash Master-11 (50 g column, DCM:MeOH, gradient elution) to obtain the pure product (6.101g, 99% yield) as a pale yellow solid: 1H NMR (270 MHz, CDCl3) δ 0.869 and 0.978(2 x s, 3H, 18-CH3), 1.17- 1.23 (t, J = 7.6 Hz, 3H, CH2-CH3), 1.24-1.29 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.32-2.59 (broad m, 12H), 2.62-2.70 (q, J = 7.4 Hz, 2H, 2-CH3-CH2), 2.78-3.03 (broad m, 2H, 6- CH2), 4.11-4.19 (q, J = 7.17 Hz, 2H, CH3-CrV2-O), 5.0 (s, 2H, CH2-phenyl), 6.63 (s, 4- CH), 7.09 (s, 1-CH), 7.30-7.45 (m, 5H); FAB-MS (M+) 474 m/z.
Synthesis of 3-Hydroxy estrone derivative (HDS02-100, HDS02-110) 3
A solution of 2 (2.80g, 5.9 mmol) in THF:MeOH (1 :1, 40 ml) was degassed for 20 min. bubbling nitrogen gas. Added 5% Pd on C (10% Wt, 300 mg) and debenzylation was carried out using a balloon of H2 for 24 h. The reaction was monitored using TLC (EtOAC: Hexane, 90:10). The crude mixture was filtered using a pad of celite, concentrated to obtain the debenzylated product 3 (2.210 g, 97%) as a colourless solid: 1H NMR (270 MHz, CDC/3) δ 0.85 and 0.96 (2 x s, 3H, 18-CH3), 1.17-1.28 (2 x t overlapped, 6H, 2 x CH2-CW3), 1.49-2.49 (m, 12H), 2.54-2.62 (q, J = 7.4 Hz, 2H, 2-CH3- CH2), 2.72-3.02 (m, 4H), 4,11-4.16 (q, J = 7.1 Hz, 2H, CH3-CrV2-O), 4.54 (s, 1H, CH2- Phenyl), 6.50 (s, 1H, 4-CH), 7.03 (s, 1 H, 1-CH); HPLC > 97% (Rt = 6.53, 70% MeCN/water); ES-ve-MS (M-H+) 383 m/z; (FAB-HRMS calcd for C24H32O4 384.2255 found (M+) 384.2288 m/z. Synthesis of sulfamate estrone derivative (HDS02-102) 4
A solution of sulfamoyl chloride (57 ml, 0.68M solution in toluene) was evaporated bellow 30 °C using a rotary evaporator. The brown oil obtained was cooled to 0 °C under inert atmosphere, dissolved in anhydrous DMA (30 ml) and treated dropwise with 3 in anhydrous DMA (30 ml) using a cannula. The reaction mixture was left to stir at 0 °C, warmed to r.t. overnight. TLC indicated the completion of the reaction (DCM:MeOH, 98:2). The crude mixture was diluted with EtOAC, washed with water (5 X) and brine. The organic phase was dried and concentrated to obtain a pale yellow solid. The crude product was purified using Flash Master-11 (50 g column, DCM:MeOH, gradient elution) to obtain pure product 4 (5.901 g, 98%) as a colourless foamy solid: 1H NMR (270 MHz, DMSO-dβ) δ 0.81 and 0.92 (2 x s, 3H each, 18-CH3), 1.09-1.20 (2 x t, J = 4.9 and 7.4 Hz, 6H, 2 x CH2-CH3), 1.33-2.47 (m, 12H), 2.58-2.66 (q, J = 7.6 Hz, 2H, 2-C V2-CH3), 2.89- 2.97 (m, 3H), 4.04-4.09 (q, J = 6.9 Hz, 2H, 2-CH3-CH2) 7.02 (s, 1H, 4-CH), 7.21 (s, 1H, 1-CH), 7.95 (s, 2H, SO2NH2); HPLC > 90% (Rt = 6.53, 70% MeCN/water); ES-ve-MS (M- H+) 462 m/z.
Synthesis of resin bound estrone sulfamate derivative (HDS02-114) 5
The trityl chloride resin (2.60 g, Novabiochem, loading 1.1 mmol/g) was swollen in anhydrous DCM (25 ml) under inert atmosphere. Added estrone sulfamate (2.00 g, 4.31 mmol) followed by DIPEA (7.50 ml, 43.1 mmol). The mixture was shaken for approximately 75 h. The acidic minicleavage (50% TFA/DCM, 20 min.) of a sample from the reaction confirmed the completion of the reaction. The crude mixture was filtered, washed with DMF, DCM, MeOH (5 cycles each) and finally with MeOH (3 cycles). After drying under vacuum this resin bound intermediate 4 (3.78 g, loading 0.86 mmol/g, pale yellow) was stored in a refrigerator until taken to the next stage. The 1H NMR and other analysis of the cleaved compound was same as that is reported for 4.
Synthesis of resin bound library precursor estrone sulfamate (HDS02- 12) 6
The resin 5 (6.00 g, loading 0.86 mmol/g) was swollen in THF (60 ml), and aqueous solution of NaOH (4M, 7 ml) was added slowely. The resin was shaken at r.t. for approximately 30 h, then filtered and washed with THF/H2O (1 :1 , 3 x 50 ml), THF, DCM and MeOH (5 cycles each). Dried under vacuum to a constant weight to obtain resin bound acid 6 (5.25 g, 0.86 mmol/g loading) as pale yellow beads. Acidic minicleavage (50% TFA/DCM, 20 min.) of a sample of this resin confirmed the complete hydrolysis of the ester to the acid. This compound exist as a mixture of diastereomers 1H NMR of this compound is same as that reported for compound ΘA.
Synthesis of sulfamate acid 10 A (EC BΘ1-137)
Resin 6 (200 mg, 0.154 mmol) was rinsed with a 50:50 solution of TFA CH2CI2 (10 mL, over a period of 20 min.) and the filtrate was evaporated under reduced pressure. The residue was recrystaUized from hot methanol to yield 0A (50 mg , 74%) as a white solid: Mixture of Diastereomers 1H NMR (270MHz, DMSO-dβ) δ 0.81 (3H, s, 18-CH3), 1.13-1.15 (t, J = 7.5 Hz, 2-CH2CH3), 1.45-2.50 (m, 13H), 2.57-2.64- (2H, q, J = 7.5 Hz, 2- Cr 2CH3), 2.66-2.83 (m, 2H), 7.22 (s, 1H), 7.03 (s, 1H), 7.96 (s, 2H, NH2); HPLC > 94% (Rf = 1.89, 10% water/acetonitrile); ES-ve-MS (M-H+) 434 m/z.
Synthesis of Acid (KRB01-139)
Nucleophilic Cleavage to form 10B: To resin 6 (300 mg, 0.231 mmol) was added piperazine (199 mg, 2.31 mmol) and anhydrous THF (5 mL) in a round bottom flask. The mixture was heated under N2 to 65 °C for 48 h (without stirring), after which time the mixture was filtered and the resin was rinsed with MeOH and CH2CI2 (5x). The combined washings were evaporated under reduced pressure and purified by flash chromatography on silica gel (eluted with 5%→15% methanol in CH2CI2, piperazine sticks to baseline) to afford of 10B (60 mg, 73%) as a white solid: 1H NMR signals (270MHz, CDCI3) δ 0.98 and 0.87 (s, 18-CH3, two diastereomers in approx. 1:3 ratio), 1.21 (3H, t, J = 7.5 Hz, 2-CH2CH3), 1.20-1.38 (t, J = 7.5 Hz, 3H, 2-CH2Cr/3), 1.45-2.50 (m, 13H), 2.54-2.64 (q, J = 7.5 Hz, 2H, 2-CH2CH3), 2.79-2.82 (m, 2H), 2.96-2.98 (1H, dd, J = 16.3, 4.2 Hz, 16-rV), 6.61 (s, 1H), 7.03 (s, 1H); HPLC >99% (10% water/acetonitrile); ES-ve-MS (M-H+) 356 m/z.
General procedure for synthesis of suifamat© amide library
A stock solution of PyBOP (Benzotriazole-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate) was prepared in 14 ml of DCM and 2.0 ml used for each reaction. A stock solution of HOBt ((N-hydroxy benzotriazole, 0.185 g/reaction) and DIPEA (0.297 ml/reaction) prepared in 9.0 ml of DCM and 1.0 ml used for each reaction. The resin bound acid 6 (0.400 g, loading 0.86 mmol/g) was suspended in DCM (4.0 ml) under inert atmosphere in a 25 ml round bottom flask, added the activating agent PyBOP (0.715 g, 1.37 mmol,), followed by HOBt and DIPEA mixture (HOBt 0.185 g, 1.37 mmol, DIPEA 0.297 ml), and the amines (4 molar equivalents). The mixture was shaken for approximately 72 h using a flask shaker. The crude resins were then filtered, washed with DMF (5 x), DCM (5 x), MeOH (5 x) using Argonaut Flash Vacuum Manifold and fritted syringes. The washed resins were dried under vacuum, one half of each resin was then transferred to Radleys Green House Synthesiser tubes to generate the phenol library. The remaining half was treated with 50% TFA in DCM over 20 min., TFA was evaporated using the Genevac and the products were purified using Flash Master-11 (DCM/MeOH, gradient elution, 5 or 10 g pre-packed columns) to generate the sulfamate library. The compounds generated exist as a mixture of diastereomers.
General procedure for synthesis of phenol library.
The resin bound sulfamate amides (0.200 g, 0.86 mmol/g) were suspended in anhydrous THF (4 ml) under inert atmosphere (Radleys Green House Parallel Synthesiser was used to carry out these reactions), treated with piperazine (0.75 g, 8.7 mmol) and refluxed at 70 °C for approximately 70 h. The resins were filtered, the filtrates were collected in to tubes, concentrated using the Genevac. These compounds were pre-absorbed onto silica gel, purified using the Flash Master-11 (Ethyl acetate/Hexane gradient elution) to give pure products as colourless solids. These compounds exist as a mixture of diastereomers.
Figure imgf000161_0001
Figure imgf000161_0002
Figure imgf000162_0001
Figure imgf000163_0001
Compound 1 (STIC 1039) HDS02-0S8 (TFA Salt): 1H NMR (270 MHz, DMSO-d6) δ 0.45 and 0.56 (2 x s, 3H each, 18-CH3), 0.75-0.79 (t, J = 7.3 H∑, 3H, CH2-CrV3), 1.33-2.47 (m, 12H), 2.26-2.29 (q, J = 7.3 Hz, 2H, CH2-CH3), 2.42-2.59 (m, 3H), 3.98-4.00 (m, 2H, 2- 5 CH3-CW2), 6.65 (s, 1H, 4-CH), 6.84 (s, 1H, 1-CH), 7.20 (broad s, 1H) 7.52 (broad m, 1H), 7.59 (s, 2H, NH2, disappeared on D2O shake), 8.21-8.17 (m, 3H, aromatic); HPLC > 95 (Rt = 12.13, 5% MeCN/water to 95% MeCN/water); ES+ve-MS (M+H+) 526 m/z; (FAB-HRMS calcd for C28H35N3O5S 526.2375 found (M+H+) 526.2379.
0 Compound 12 HDS02-128-8 (TFA Salt): 1H NMR (270 MHz, DMSO-d6) 0.81 (s, 3H, 18- CH3), 1.09-1.15 ( t, J = 7.1 Hz, 3H, CH2-CW3), 1.33-2.47 (m, 12H), 2.61-2.64 (q, J = 7.3 Hz, 2H, CH2-CH3), 2.82-2.90 (m, 3H), 4.36-4.38 (m, 2H, 2-CH3-CW2), 7.02 (s, 1H, 4-CH), 7.21 (s, 1H, 1-CH), 7.28-7.30 (m, 2H, aromatic) 7.52 (broad m, 1H), 7.96 (s, 2H, NH2), 8.48-8.51 (m, 2H, aromatic); HPLC > 91% (Rt = 2.12, 70% MeCN/water); ES+ve-MS ,5 (M+H+) 526 m/z; (FAB-HRMS calcd for C28H35N3O5S 526.2375 found (M+H+) 526.2374
Compound 13 HDS02124-2 (TFA Salt): 1H NMR (400 MHz, DMSO-d6) 0.805 (s, 3H, 18- CH3), 1.10-1.13 (t, J = 7.4 Hz, 3H, CH2-CH3), 1.37-2.45 (m, 12H), 2.58-2.62 (q, J = 7.8 Hz, 2H, CW2-CH3), 2.80-2.87 (m, 3H), 4.34-4.36 (m, 2H, CH3-CH2), 6.99 (s, 1H, 4-CH), 0 7.18 (s, 1H, 1-CH), 1H), 7.92 (s, 2H, NH2), 8.43 (2 x s, 2H, aromatic) 8.51-8.84 (t, J = 5.85 Hz, 1H, NH); ES+ve-MS (M+H+) 541, (M+H-18+) 523 m/z; HPLC > 99% (Rt = 2.0, 80% MeCN/water); FAB-HRMS calcd for C28H36N4O5S 541.2484 found (M+H+) 541.2478
Compound 15 HDS02128-2 (TFA Salt): 1H NMR (270 MHz, DMSO-dβ) 0.80 and 0.83 (s, 5 3H, 18-CH3), 1.09-1.14 (t, J = 7.4 Hz, 3H, CH2-CH3), 1.32-1.37 (d, J = 6.8 Hz, 3H, CH- CH3), 1.39-2.45 (m, 12H), 2.60-2.63 (q, J = 7.6 Hz, 2H, Cry2-CH3), 2.80-2.81 (m, 3H), 4.95-4,99 (m, 1H, CH3-CH), 7.01 (s, 1H, CH), 7.20-7.22 (m, 2H, aromatic), 7.30-7.32 (m, 4H, aromatic), 7.95 (broad m, 2H, aromatic), 8.35-8.39 (broad t, 1H, NH); ES+ve-MS (M+H+) 539 m/z; HPLC > 91% (Rt = 2.0, 70% MeCN /water); FAB-HRMS calcd for CsoHgs sOgS 539.2579 found (M+H+) 539.2579
Compound 16 HDS0212S-3 (TFA Salt): 1H NMR (270 MHz, DMSO~dβ) 0.80 and 0.83 (2 x s, 3H, 18-CH3), 1.09-1.15 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.32-1.35 (d, J = 6.8 H∑, 3H, CH-CH3), 1.39-2.45 (m, 12H), 2.57-2.66 (q, J = 7.9 H∑, 2H, CH2-CH3), 2.80-2.84 (m, 3H), 4.91-4.94 (m, 1 H, CH3-CH), 7.02 (s, 1 H, CH), 7.21-7.23 (m, 2H, aromatic), 7.30-7.32 (m, 4H, aromatic), 7.94 (broad m, 2H, aromatic), 8.31-8.35 (broad t, J = 3.57 H∑, 1H, NH); ES+ve-MS (M+H+) 539 m/z; HPLC > 98% (R, = 2.69, 70% MeCN /water); FAB- HRMS calcd for C30H38N2O5S 535.2679 found (M+H+) 539.2574
Compound 17 HDS02120-2 (TFA Salt): 1H NMR (400 MHz, DMSO-d6) 0.81 and 0.89 (2 x s, 3H, I8-CH3), 1.09-1.13 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.23-2.45 (m, 16H), 2.58-2.63 (q, J = 7.4 Hz, 2H, CH2-CH3), 2.79-2.81 (m, 3H), 3.10-3.12 (m, 2H), 3.62-3.80 (m, 3H), 6.99 (s, 1 H, CH), 7.18 (s, 1 H, aromatic), 7.92 (broad m, 2H, aromatic); ES+ve-MS (M+H+) 619 m/z; HPLC > 90% (R, = 2.42, 70% MeCN/ water).
Compound 18 HDS02128-1 (TFA Salt): 1H NMR (270 MHz, DMSO-d6) 0.80 and 0.89 (2 x s, 3H, I8-CH3), 0.98-1.03 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.09-1.16 (t, J = 7.4 Hz, 3H, CH2-CH3), 1.23-2.46 (m, 12H), 2.57-2.66 (q, J = 7.4 Hz, 2H, CH2-CH3), 2.79-2.82 (m, 3H), 3.02-3.08 (q, J = 7.4 Hz, CH2-CH3), 7.01 (s, 1 H, CH), 7.21 (s, 1H, aromatic), 7.88 (broad t, 1 H, NH), 7.95 (s, 2H, NH2); ES+ve-MS (M+H+) 463 m/z; HPLC > 89% (R, = 3.61 , 5% MeCN/water to 95% MeCN/water); FAB-HRMS calcd for C24H34N2O5S 463.2266 found (M+H+) 463.2282
Compound 19 HDS02128-4 (TFA Salt): 1H NMR (270 MHz, DMSO-dβ) 0.82 and 0.91(2 x s, 3H, 18-CH3), 0.96-1.02 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.09-1.15 (t, J = 7.4 Hz, 3H, CH2- CH3), 1.23-2.45 (m, 12H), 2.58-2.66 (q, J = 7.4 Hz, 2H, CH2-CH3), 2.79-2.82 (m, 4H), 2.92 (s, N-CH3), 7.01 (s, 1H, CH), 7.21 (s, 1H, aromatic), 7.95 (s, 2H, NH2); ES+ve-MS (M+H+) 477 m/z; HPLC > 95% (Rt = 2.44, 70% MeCN/water); FAB-HRMS calcd for C2gH36N2OgS 477.2423 found (M+H+) 477.2414
Compound 20 HDS02128-5 (TFA Salt): Diagnostic signals 1H NMR (270 MHz, DMSO- d6) 0.81 (s, 3H, I8-CH3), 0.83-0.90 (m, 6H) 1.09-1.15 (t, J = 7.1 Hz, 3H, CH2-CrV3), 1.23- 2.45 (m, 12H), 2.59-2.66 (q, J = 7.4 Hz, 2H, CW2-CH3), 2.79-2.82 (m, 3H), 3.00-3.06 (m,
2H) 7.02 (s, 1 H, CH), 7.21 (s, 1H, aromatic), 7.82-7.87 (m, 1H, NH), 7.96 (s, 2H, NH2); ES+ve-MS (M+H+) 491 m/z; HPLC > 92% (R, = 2.45, 70% MeCN/water); FAB-HRMS calcd for C26H38N2O5S 491.2579 found (M+H+) 491.2581
Compound 21J-SDS02128-S (TFA Salt): 1H NMR (270 MHz, DMSO-dβ) 0.82 and 0.91(2 x s, 3H, 18-CH3), 0.96-1.03 (t, J = 6.8 Hz, 3H, CH2-CH3), 1.05-1.15 (m, 6H, 2 x CH2-CH3), 1.23-2.45 (m, 12H), 2.58-2.63 (q, J = 7.6 Hz, 2H, CH2-CH3), 2.79-2.82 (m, 3H), 3.25- 3.10 (q, J = 7.1 Hz, CH2-CH3) 7.01 (s, 1H, CH), 7.21 (s, 1H, aromatic), 7.94 (s, 2H, NH2); ES+ve-MS (M+H+) 491 m/z; HPLC > 97% (Rt = 2.65, 70% MeCN/water); FAB-HRMS calcd for C26H38N2O5S 491.2575 found (M+H+) 491.2568
Compound 22 (STX 1040) HDS02-080-1: 1H NMR (270 MHz, CDCI3) 0.79 (s, 3H, 18- CH3), 1.17-1.22 ( t, J = 7.6 Hz, 3H, CH2-CH3), 1.32-2.40 (m, 13H), 2.54-2.63 (q, J = 7.6 Hz, 2H, CH2-CH3), 2.67-2.82 (m, 3H), 4.43-4.46 (m, 2H, NH-CH2), 6.12 (broad s, 1H, OH), 6.41 (broad t, 1H, NH), 6.51 (s, 1H, aromatic), 6.39-6.41 (appt, J = 5.9 Hz, 1H, NH), 6.70-6.72 (appt t, J = 5.9 Hz, 1H, NH), 7.01 (s, 1H, CH), 7.28-7.30 (m, 1H, aromatic) 7.64-7.67 (appd, J = 7.6 Hz, 1H, aromatic), 8.50-8.53 (m, 2H, aromatic); HPLC > 99% (R, = 5.20, 5% MeCN/water to 95% MeCN/water); ES-ve-MS (M+H+) 445 m/z.
Compound 23 HDS02-086-1: 1H NMR (270 MHz, CDCI3) 0.46 (s, 3H, 18-CH3), 0.81-0.88 (m, 2H), 1.14-1.24 (m, 4H), 1.32-2.85 (m, 15H), 4.39-4.90 (m, 2H, NH-CH2), 6.48 (s, 1H, aromatic), 6.90-6.95 (broad t, 1H, NH), 6.97 (s, 1H, aromatic), 7.01-7.06 (s, 1H, aromatic), 7.27-7.39 (m, 1H, aromatic), 7.67-7.74 (m, 1H, aromatic), 8.52-8.54 (m, 1H, aromatic); HPLC > 99% (Rt = 2.25 and 2.76, 75% MeCN/water); ES-ve-MS (M+H+) 445 m/z; FAB-HRMS calcd for C28H34N2O3447.2647 found (M+H+) 447.2655
Compound 24 HDS02-086-4: 1H NMR (270 MHz, CDCI3) 0.81 and 0.84 (2 x s, 3H, 18- CH3), 1.17-1.22 ( m, 3H, 2 x CH2-CH3), 1.32-2.40 (m, 13H), 2.54-2.63 (q, J = 7.6 Hz, 2H, CH2-CH3), 2.67-2.82 (m, 3H), 4.55-4.57 and 4.80 4.82 (m, 2H, NH-CH2), 6.19 (broad s, 1H, OH), 6.49 (s, 1H, CH), 7.02 (s, 1H, CH), 8.24, 8.37, 8.47, 8.60,8.61 (4 x s, 2H, CH); HPLC > 95% (Rt = 2.16 and 2.38, 80% MeCN/water); ES-ve-MS (M+H+) 445 m/z; FAB- HRMS calcd for C28H3sN3O3 462.4756 found (M+H+) 462.2744.
Compound 25 HDS02-134-2: 1H NMR (270 MHz, CDCI3) 0.74 and 0.94 (2 x s, 3H, 18-
CH3), 1.16-1.22 (t, J = 7.6 Hz, 3H, CH2-CW3), 1.46-1.49 (d, J = 6.8 Hz, 3H, CH-CH3), 2.65-2.72 (dd, J = 5.4 and 5.8 Hz, 1H), 1.32-2.40 (m, 12H), 2.53-2.61 (q, J = 7.6 Hz, 2H,
Ctf2-CH3), 2.67-2.82 (m, 3H), 4.9 and 5.0 (2 x s, 1H, OH), 5.08-5.13 (m, CW-CH3), 6.06- 6.03 (d, J = 8.2 Hz, 1H, NH), 6.30-6.33 (d, J = 7.9 Hz, 1H, NH), 6.50 (s, 1H, CH), 7.02 (s, 1H, CH), 7.32-7.40 (m, 5H, aromatic); HPLC > 99% (Rt = 3.36, 80% MeCN/water); ES+ve-MS (M+H+) 460 m/∑; FAB-HRMS calcd for C30H37NO3 460.2851 found (M+H+) 460.2841
Compound 26 HDS02-134-3: 1H NMR (270 MHz, CDCI3) 0.84 and 0.94 (2 x s, 3H, 18- CH3), 1.17-1.22 (t, J = 7.4 Hz, 3H, CH2-CW3), 1.46-1.49 (d, J = 6.8 Hz, 3H, CH-CH3), 1.32-2.40 (m, 12H), 2.53-2.61 (q, J = 7.6 Hz, 2H, CW2-CH3), 2.65-2.72 (dd, J = 5.4 and 5.8 Hz, 1H) 2.79-2.82 (m, 3H), 4.71 and 4.75 (2 x s, 1 H, OH), 5.06-5.11 (hi, CH-CH3), 5.59-6.03 (d, J = 8.2 Hz, 1H, NH), 6.30-6.33 (d, J = 7.9 Hz, 1H, NH), 6.50 (s; 1H, CH), 7.02 (s, 1H, CH), 7.31-7.40 (m, 5H, aromatic); HPLC > 99% (Rt = 3.36, 70% MeCN/water); ES+ve-MS (M+H+) 460 m/z; FAB-HRMS calcd for C30H37NO3 460.2851 found (M+H+) 460.2842
Compound 27 HDS02-134-1: 1H NMR (270 MHz, CDCI3) 0.81 and 0.95 (2 x s, 3H, 18- CH3), 1.10-1.16 ( t, J = 7.4 Hz, 3H, CH2-CH3), 1.16-1.12 ( t, J = 7.4 Hz, 3H, CH2-CH3), 1.42-2.43 (m, 13H), 2.55-2.62 (q, J = 7.9 Hz, 2H, CH2-CH3), 2.65-2.82 (m, 3H), 3.22- 3.32 (m, 2H, N-CH2-CH3), 4.94 and 5.03 (2 x s, 1 H, OH), 5.72 and 5.93 (2 x broad t, 1H, NH), 6.51 (s, 1H, CH), 7.01 (s, 1H, CH); HPLC > 95% (Rt = 5.20, 70% MeCN/water); ES+ve-MS (M+H+) 384 m/z; FAB-HRMS calcd for C24H33NO3 385.2538 found (M+H+) 384.2532
Compound 28 HDS02-134-4: 1H NMR (270 MHz, CDCI3) 0.82 and 0.94 (2 x s, 3H, 18- CH3), 1.06-1.12 ( t, J = 7.1 Hz, 3H, CH2-CH3), 1.16-1.22 ( t, J = 7.6 Hz, 3H, CH2-CH3), 1.92-2.43 (m, 13H), 2.53-2.61 (q, J = 7.6 Hz, 2H, CrV2-CH3), 2.78-2.82 (m, 3H), 2.91- 2.95 (2 x s, 3H, N-CH3), 3.31-3.38 (m, 1H), 4.78-4.82 (broad s, 1H, OH), 6.51 (s, 1H, CH), 7.02 (s, 1H, CH); HPLC > 99% (Rt = 5.20, 70% MeCN/water); ES+ve-MS (M+H+) 398 m/z; FAB-HRMS calcd for C25H35NO3 398.2695 found (M+H+) 398.2695
Compound 29 HDS02-134-5: Diagnostic signals 1H NMR (270 MHz, CDC/3) 0.82 (s, 3H, 18-CH3), 0.91-0.94 (m, 3H), 1.17-1.22 ( t, J = 7.6 Hz, 3H, CH2-CrV3), 1.29-2.43 (m, 15H), 2.53-2.62 (q, J = 7.9 Hz, 2H, CH2-CH3), 2.78-2.82 (m, 3H), 3.20-3.25 (m, 1 H), 4.82 and 4.89 (2 x broad s, 1H, OH), 5.72 and 5.96 (2 x broad t, 1H, NH), 6.51 (s, 1H, CH), 7.02 (s, 1 H, CH); HPLC > 99% (Rt = 2.96, 70% MeCN/water); ES+ve-MS (M+H+) 412 m/z; FAB-HRMS calcd for C26H37NO3 412.2851 found (M+H+) 412.2857 Compound 30 HDS02-134-6: 1H NMR (270 MHz, CDCI3) 0.82 and 0.94 (2 x s, 3H, 18- CH3), 1.08-1.22 ( 2 x t, J = 7.1 and 7.4 Hz, 3H each, 2 x CH2-CW3), CH2-CH3), 1.92-2.43 (m, 13H), 2.53-2.62 (q, J = 7.6 Hz, 2H, CH2-CH3), 2.78-2.81 (m, 3H), 3.25-3.42 (m, 4H, N-CW2-CH3), 4.77 and 4.81 (broad s, 1H, OH), 6.51 (s, 1H, CH), 7.02 (s, 1H, CH); HPLC
> 96% (R( = 13.41, 5% MeCN/water to 95% MeCN/water); ES+ve-MS (M+H+) 412 m/z; FAB-HRMS calcd for C26H37NO3 412.2851 found (M+H+) 412.2854
Compound 31 HDS02-134-7 Mixture of diastereomers 1H NMR (270 MHz, CDCI3) 0.79 and 0.93 (2 x s, 3 18-CH3), 1.12-1.16 (2 x d, J = 6.3 Hz each, 6H, 2 x CH3), 1.19-1.21 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.92-2.43 (m, 13H), 2.53-2.62 (m, 2H, CH2-CH3), 2.78-2.85 (m, 3H), 4.04-4.08 (m, 4H, NH-CH-(CH3)2), 5.28 and 5.42 (2 x s, 1H, 2 x OH), 5.57-5.59 and 5.83-5.86 (2 6, J - 7.6 Hz each, 1H, NH), 6.52 (s, 1H, CH), 7.01 (s, 1H, CH); HPLC > 84% (Rt =■ 3.90, 5% MeCN/water to 96% MeCN/water); ES+ve-MS (M+H+) 398 m/z; FAB-HRMS calcd for C25H35NO3398.2695 found (M+H+) 398.2692
Synthesis of 2-Methoxy estrone amide.
Figure imgf000167_0001
S nthesis of benzyl ether (HDS02-170) 32
The 2-Methoxy estrone (0.521 g, 0.00173 mol) was dissolved in anhydrous DMF under nitrogen atmosphere, added K2CO3 (0.718 g, 0.00225 mol) and stirred at r.t. for approximately 40 h. The crude mixture was diluted with EtOAc, washed with water and brine. The organics were dried (MgSO4) and concentrated. The crude product was pre- absorbed onto silica gel, purified using Flash Master-2 system to give the benzylated compound 32 (0.651 g, 96%) as a chalky white solid: TLC (Rt, 0.4, EtOAc:Hexane, 3:7); 1H NMR (270 MHz, CDCI3) δ 0.90 (s, 3H, 18-CH3 diastereomeric methyl signals), 1.24- 1.27 (t, J = 3.4 Hz, 3H, CH2-CH3), 1.24-1.29 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.39-2.44 5 (broad m, 12H), 2.44-2.54 (dd, J = 18.8 and 8.6 Hz, 1H), 2.77-2.83 (m, 2H), 3.84-3.87 (s, 3H, OMe), 5.0 (s, 2H, PhCH2), 6.6 (s, 1H, aromatic), 6.77 (s, 1H, aromatic), 3.87-3.84 (s, 3H, OMe), 4.10-4.16 (q, J = 7.0 Hz, 2H, CH3-CH2-O), 5.09 (s, 2H, CH2-phenyl), 6.61 (s, 1H, 4-CH), 6.82 (s, 1H, aromatic 1-CH), 7.41-7.43 (m, 5H).
0 Synthesis of th© ethyl ester (HDS02-174) 33 ' '
To a stirred solution of 2-Methoxy benzyl estrone 32 (150 mg, 0.38 mmol) in anhydrous THF under inert conditions was added a solution of LDA (0.234 ml, 1.8 M solution in heptane, THF, and Ethylbenzene) at -10 °C over a period of 20 min. The reaction 5 mixture was cooled to -60 °C, stirred 15 min, added ethyl bromoacetaite (0.052 ml, 0.46 mmol) drop wise over 10 min. and allowed to warm to rt overnight. Diluted the mixture with DCM, added saturated NH4CI and extracted with DCM. The organic phases were combined, dried, and concentrated to obtain a yellow solid which was then purified using Flash Master-2 (10 g column, DCM:MeOH, gradient elution) to obtain the pure product 0 (0.120g, 65% yield) as a colourless glassy solid: 1H NMR (270 MHz, CDCI3) δ 0.99 and
0.97 (2 x s, 3H, 18-CH3diastereomeric methyl signals), 1.24-1,27 (t, J = 3.4 Hz, 3H, CH2-
' CH3), 1.24-1.29 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.47-3.19 (broad m, 15H), 3.87-3.84 (s,
3H, OMe), 4.10-4.16 (q, J = 7.0 Hz, 2H, CH3-CH2-O), 5.09 (s, 2H, CH2-phenyl), 6.61 (s,
1H, 4-CH), 6.82 (s, 1H, aromatic 1-CH), 7.41-7.43 (m, 5H). 5
Synthesis of the acid (HDS02-182) 34
The estrone ester 33 (0.120 g, 0.252 mmol) was dissolved in a mixture of MeOH:THF (1:1, 6 ml), added NaOH (4M, 3.0 ml) and stirred at room temperature for 30 h. The 0 completion of the reaction was determined by t.l.c (EtOAc: Hexane 3:7, product-base line spot). The crude mixture was acidified to pH = 2 to 3, extracted with DCM, washed with water and brine. The organic phase was dried (MgSO4) and concentrated to obtain the product as a pale yellow solid. This product 33 (0.092 g, 0.205 mmol) was dissolved in a mixture of MeOH:THF (1:1, 7 ml), degassed the mixture using N2, added 5% Pd on 5 C (10 mg) then hydrogenated under a of balloon of Hydrogen for approximately for 15 h. The crude mixture was filtered using a pad of celite, purified (SiO2, EtOAchexane gradient elution using Flash Master-2) to obtain the product 34 (0.081 g, 100 % ) as a pale yellow solid: The diagnostic signals of the 1H NMR (400 MHz, CDCI3) δ 0.93 (s, 3H, 18-CH3), 1.47-2.65 (broad m, 13H), 2.83-2.85 (m, 2H, CH2), 2.96-3.01 (dd, J = 16.3 and 4.6 Hz, CH-diastereotopic) 3.89 (s, 3H, OMe), 5.33 ( broad s, 1H, OH), 6.69 (s, 1H, aromatic 4-CH), 6.81 (s, 1H, aromatic 1-CH); HPLC > 99% (Rt = 1.59, 70% MeCN in water); ES+ve-MS (M+H+) 357 m/z.
Synthesis of th© amide (HDS02-190) 35 (STX 1191).
The starting material acid 34 ( 0.038 g, 0.106 mmol) was suspended in DCM ( 5.0 ml), added PyBop (0.062 g, 0.12 mmol), HOBt ( 0.016 g, 0.12 mmol) stirred for 20 minutes and added DIPEA (0.076 ml) followed by 3-amino methyl pyridine (0.026 g, 0.24 mmol). The reaction was left to stir 20 h, quenched with sat. Na2CO3, washed with brine. The organics were separated, dried concentrated to obtain a pale yellow oil. This crude oil was purified using the Flash Master-2 system to obtain a white solid. The product obtained at this point was re-crystalised with hexane/EtOAc to give the required product amide 35 also as a white solid: The diagnostic signals of the mixture of diastereomers of 35 1H NMR (400 MHz, CDCI3) δ 0.84 and 0.97 (2 x s, 3H, 18-CH3), 1.47-2.81 (broad m, 13H), 3.11-3.17 (m, 2H, CH2), 3.84 (s, 3H, OMe), 4.44-4.46 (m, 2H, NH-CH2-), 5.46 (broad s, 1H, OH), 6.61 (s, 1H, aromatic 4-CH), 6.75 (s, 1H, aromatic CH), 7.63 (m, 1H, CH aromatic), 8.53 (m, 2H, 2H); HPLC > 90% (R, = 1.59, 90% MeCN in water); ES+ve- MS (M+H+) 448 m/z.
SECTION 4
3-O-Acetyl-estrone (DSF 02124)
Figure imgf000169_0001
Acetic acid (60 mL, 1.05 mmol) was added dropwise over 45 minutes to a stirred solution of estrone (3 g, 11.1 mmol) in anhydrous pyridine (200 mL) at 0°C under an atmosphere of N2. The resulting yellow mixture was heated to reflux for 1 hour. After cooling down, this was poured into H2O and ice (300 mL) and acidified with HCl 5M. The organics were extracted with EtOAc (300 mL), washed with H2O (200 mL), aq. NaHCO3 (200 mL), then brine (200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crystalline orange crude (3.88 g) was recrystaUized from IPA to give light yellow needles (3.25 g, 94%): TLC (chloroform/EtOAc, 4:1) R 0.82 cf. Rf 0.68 (E1); δH (CDCI3, 400 MHz) 0.91 (3H, s, C-18-H3), 1.45-2.54 (13H, m), 2.29 (3H, s, OAc), 2,89- 2.92 (2H, m, C-6-H2), 6.81 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6.85 (1H, dd, JC-I-H,C-2-H = 8.3 Hz and JC-4.H,C-2-H = 2.6 Hz, C-2-H) and 7.28 (1H, d, JC-2-H,C-I-H = 8.3 Hz, C-1-H).
6-Oxo-3-0-ac©tyl-©strone (DSF 02128) '
Figure imgf000170_0001
A solution of CrO3 (673 mg, 6.73 mmol) in acetic acid 10% (4 mL) was added dropwise over 2 hours to a stirred solution of 3-O-acetyl-estrone (500 mg, 1.60 mmol) in acetic acid at 10-15°C in an ice/water bath. The resulting dark brown solution was stirred overnight at room temperature. The solvent was then removed under reduced pressure and H2O (100 mL) added. The organics were extracted with EtOAc (100 mL), washed with H2O (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a green foam. This was recrystaUized from IPA to give grey crystals (103 mg, 20%): TLC (chloroform/EtOAc, 4:1) Rf 0.53 cf. Rf 0.86; δH (CDCI3, 400 MHz) 0.92 (3H, s, C-18-H3), 1.20-2.63 (12H, m), 2.32 (3H, s, OAc), 2.88 (1H, dd, JAB= 16.8 Hz, J -8-H,C-7-HB = 3.1 Hz, C-7-HB), 7.28 (1 H, dd, JC-I-H,C-2-H = 8-6 Hz and Jc- -H,c-2-H = 2.6 Hz, C-2-H), 7.46 (1H, d, JC-2-H,C-I-H = 8.6 Hz, C-1-H) and 7.77 (1 H, d, JC-2.H,C-4-H = 2.6 Hz, C-4-H).
6-Oxo-estrone (DSF 02130, STX 416)
Figure imgf000170_0002
A solution of KOH (247 mg, 4.40 mmol) in MeOH (1.5 mL) was added dropwise to a stirred solution of 6-oxo-3-O-acetyl-estrone (80 mg, 0.24 mmol) in MeOH (1.5 mL). The resulting brown solution was stirred for 2 hours at room temperature. The solvent was then concentrated under reduced pressure and H2O (20 mL) added, followed by HCl 5M. The organics were extracted with EtOAc (50 mL), washed with H2O (20 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product (74 mg) was purified by flash chromatography with chloroform/EtOAc (7:3) as eluent and gave the product as a light pink powder (41 mg, 58%): mp 241-243°C; TLC (chloroform/EtOAc, 4:1) Rf 0.39 cf. R, 0.64; IR (KBr) 3345 (br, OH), 2940-2880 (aliph CH), 1720 (C=O), 1680 (C=O), 1610-1495 (arom C=C) cm"1; δH (CDCI3, 400 MHz) 0.93 (3H, s, C-18-Hg), 1.24-2.59 (12H, m), 2.87 (1H, dd, JC-8-H,C-7-HB = 3.3 Hz, JAB= 17.0 Hz, C- 7-HB), 5.74 (1H, s, exchanged with D2O, OH), 7.10 (1H, dd, 'JC-I-H,C-2-H = 8.5 Hz and Jc-4- H,C-2-H = 2.8 Hz, C-2-H), 7.33 (1H, d, JC-2-H,c-ι-H = 8.5 Hz, C-1-H) and 7.60 (1H, d, Jc-2-H.c- H = 2.8 Hz, C-4-H); MS m/z (FAB+) 369.2 [50], 285.1 [95, (M+H)+], 113.1 [68], 84.0 [94]; Ace MS m/z (FAB+) 285.14917, C18H21O3 requires 285.14907. HPLC (methanol/water, 90:10, λmax = 223.9 nm) Rt = 2.34 min, 100%.
6,17-Bis-oxime-estrone (DSF 02169, STX 454)
Figure imgf000171_0001
NaOAc (293 mg, 3.58 mmol) followed by hydroxylamine hydrochloride (274 mg, 3.94 mmol) were added to a solution of 6-oxo-estrone (100 mg, 0.35 mmol) in a mixture of MeOH/H2O (5:1 , 18 mL). The resulting solution was stirred at room temperature overnight. The solvent was then removed under reduced pressure and H2O added (50 mL). The organics were extracted with EtOAc (50 mL + 20 mL), washed with H2O (2x20 mL), then brine (2x20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a light brown crude (120 mg). This was purified by flash chromatography using a gradient of chloroform/EtOAc (7:3 to 1 :1) then chloroform/EtOAc/acetone (2:2:1 to 1 :1 :2) as eluent and gave the product as a creamy powder (46 mg, 41%): mp 341-344°C; TLC (chloroform/EtOAc, 7:3) R, 0.11 cf. RA 0.44; IR (KBr) 3410, 3265-3050 (br, NOH, OH), 2930-2850 (aliph CH), 1705 (C=N), 1580- 1495 (arom C=C) cm"1; δ H (DMSO-d6, 400 MHz) 0.83 (3H, s, C-18-H3), 1.14-2.42 (12H, m), 3.06 (1H, m, C-7-HB), 6.73 (1H, dd, JC-1.H,C-2-H = 8.3 Hz and C-4-H,C-2-H = 2.5 Hz, C-2- H), 7.14 (1H, d, JC-2-H,C-I-H = 8.3 Hz, C-1-H), 7.29 (1 H, d, C-2-H,C-4-H = 2.8 Hz, C-4-H) and 9.28, 10.15, 11.08 (each 1H, s, 2χNOH, OH); MS /z (FAB+) 315.1 {82, (M÷H)+], 73 [100]; Ace MS m/z (FAB+) 315.17149, C18H23 2O3 requires 315.17087.
6,17-Bis-0-methyl-o∑ me-©strone (DSF 02183, SOC 515)
Figure imgf000172_0001
NaOAc (146 mg, 1.79 mmol) followed by O-methyl-hydroxylamine hydrochloride (164 mg, 1.97 mmol) were added to a solution of 6-oxo-estrone (50 mg, 0.18 mmol) in a mixture of MeOH/H2O (5:1 , 9 mL). The, resulting solution was stirred at room temperature overnight. The solvent was then removed under reduced pressure and H2O added (30 mL). The organics were extracted with EtOAc (20 mL + 10 mL), washed with H2O (2x10 mL) then brine (2x10 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The white crystalline crude was recrystaUized from EtOAc/hexane to give the product as white crystals (55 mg, 92%): mp 206-208°C; TLC (chloroform/EtOAc, 8:2) Rf 0.70 cf. Rf 0.28; IR (KBr) 3134 (br, OH), 2995 (arom CH), 2935-2890 (aliph CH), 1570-1490 (arom C=C) cm"1; δH (CDCI3, 400 MHz) 0.92 (3H, s, C- 18-H3), 1.24-2.58 (12H, m), 3.12 (1 H, dd, JC-8-H,C-7-HB = 4.5 Hz, AB= 18.1 Hz, C-7-HB), 3.84 (3H, s, OMe), 3.99 (3H, s, OMe), 4.85 (1H, br s, exchanged with D2O, OH), 6.84 (1H, dd, Jc-ι-H,c-2-H = 8.6 Hz and JC-4-H,c-2-H = 2.7 Hz, C-2-H), 7.20 (1H, d, JC-2-H,c-ι-H = 8.6 Hz, C-1-H) and 7.29 (1H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H); MS m/z (FAB+) 343.2 [100, (M+H)+]; Ace MS m/z (FAB+) 343.20329, C20H27N2O3 requires 343.20217. Found: C, 70.20; H, 7.57; N, 8.14. C20H26N2O3 requires: C, 70.15; H, 7.65; N, 8.18.
16-Oximino-esfrone (DSF 02036/ST 327)
Figure imgf000173_0001
Estrone (200 mg, 740 mmol) was added to a stirred solution of potassium ei -butoxide, freshly prepared by dissolving potassium metal (80 mg, 2.05 mmol) in terf-butanol (2 mL), under an atmosphere of N2. The reaction mixture was stirred for 1 hour at room temperature before addition of isoamyl nitrite (180 μL, 1.34 mmol) in a dropwise manner. The deep red mixture obtained was stirred overnight, then poured into H2O (20 mL). The resulting solution was extracted with ether (2x20 mL) and the aqueous layer was acidified with glacial acetic acid (10 mL) to give a light yellow precipitate. After standing for 2h, the solid was isolated by filtration to give a yellow powder (140 mg, 63%). For analysis, a sample was recrystaUized from acetone to give white crystals: mp 223-225°C [lit.1 (aq. MeOH) 214-215°C]; TLC (chloroform/acetone, 9:1) R, 0.27 cf. R, 0.69 (E1 ); IR (KBr) 3385 (NOH), 2920-2860 (aliph CH), 1735 (C=O), 1605-1500 (arom C=C) cm"1; δ H (DMSO-d6, 400 MHz) 0.89 (3H, s, C-18-H3), 1.30-2.85 (11H, m), 2.70-2.81 (2H, m, C-6- H2), 6.46 (1H, d, Jc-2-H,c-4-H = 2.3 Hz, C-4-H), 6.52 (1H, dd, JC-1-H,C-2-H = 8.3 Hz and JC-4-H,C-2- H = 2.3 Hz, C-2-H), 7.05 (1 H, d, JC-2-H,C-I-H = 8.3 Hz, C-1-H), 9.05 (1H, s, exchanged with D2O, OH) and 12.39 (1 H, s, exchanged with D2O, NOH); δc (DMSO-d6, 100.4 MHz) 14.09 (q, C-18), 25.09 (t), 25.46 (t), 26.18 (t), 29.02 (t), 30.92 (t), 37.20 (d), 43.20 (d), 44.59 (d), 48.50 (s, C-13), 112.70 (d), 114.83 (d), 125.82 (d), 129.59 (s), 136.88 (s), 154.84 (s, C-3 or C-16), 155.23 (s, C-3 or C-16) and 204.64 (s, C=O); MS m/z (FAB+) 453.2 [30, (M+H+NBA)*], 300.1 [100, (M+H)+]; MS m/z (FAB-) 451.3 [38, (M-H+NBA)"], 298.2 [100, (M-H)']; Ace MS m/z (FAB+) 300.15963, C18H22NO3 requires 300.15997. Found: C, 71.30; H, 7.08; N, 4.35. C18H21NO3.(AcOH)1/6 requires: C, 71.17; H, 7.06; N, 4.53.
1 Huffman, M. N.; Darby, H. H. J. Am. Chem. Soc. 1944, 66, 150
16, 17-Bis-oximino-estrone (DSF 02110, STX 338)
Figure imgf000174_0001
NaOAc (556 mg, 6.79 mmol) followed by hydroxylamine hydrochloride (520 mg, 7.48 mmol) were added to a solution of 16-oximino-estrone (200 mg, 0.67 mmol) in a mixture of MeOH/H2O (5:1 , 36 mL). The resulting pale yellow solution was stirred at room temperature overnight. The solvent was then removed under reduced pressure and brine added (100 mL). The organics were extracted with EtOAc (100 mL), washed with brine (2x50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product (236 mg). This was recrystaUized from acetone to give creamy crystals (109 mg, '52%): mp 245-247°C; TLC (chloroform/acetone, 8:2) Rf 0.13 cf. R, 0.60; IR (KBr) 3420-3200 (br, NOH, OH), 3020 (arom CH), 2935-2870 (aliph CH), 1705 (ON), 1620 (C=N or arom C=C), 1585-1500 (arom C=C) cm"1; δH (DMSO-d6, 400 MHz) 0.97 (3H, s, C-18-H3), 1.43-2.83 (13H, m), 6.44 (1 H, m, C-4-H), 6.51 (1H, dd, ΛC-I-H,C-2-H = 8.2 Hz and JC-4-H,C-2-H = 2.3 Hz, C-2-H), 7.04 (,1H, d, JC-2-H,C-I-H = 8.2 Hz, C-1-H), 9.02 (1H, s, exchanged with D2O, OH), 10.81 (1H, s, exchanged with D2O, NOH) and 11.19 (1H, s, exchanged with D2O, NOH); MS m/z (FAB+) 315.2 [100, (M+H)+], 133.1 [21]; MS m/z (FAB-) 466.2 [66, (M-H-NBAV], 313.2 [100, (M-HV], 276.1 [80]; Ace MS m/z (FAB+) 315.17108, C18H23N2O3 requires 315.17087. HPLC (methanol/water, 70:30, λmax = 252.1 nm) Rt = 2.71 min, 99.2%. Found: C, 67.30; H, 7.41; N, 7.59. C18H23N2O3.(CH3)2O requires: C, 67.72; H, 7.58; N, 7.52.
17-Oxime-estrone (DSF 03007).
Figure imgf000174_0002
NaOAc (1.5 g, 18.80 mmol) followed by hydroxylamine hydrochloride (1.4 g, 20.72 mmol) were added to a suspension of E1 (500 mg, 1.85 mmol) in a mixture of MeOH/H2O (5:1 , 90 mL). The resulting suspension was stirred at room temperature overnight. The solvent was then removed under reduced pressure and H2O added (100 mL). The organics were extracted with EtOAc (100 mL + 50 mL), washed with H2O (50 mL), then brine (50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a white crystalline crude product (627 mg). This was recrystaUized from MeOH to give white crystals (426 mg, 81%): TLC (chloroform/EtOAc, 4:1) Rf 0.13 cf. Rf 0.55 (E1); IR (KBr) 3415 (NOH), 3270 (OH), 2930 (aliph CH), 1620 (C=N or arom OC), 1585-1460 (arom C=C) cm-1; δ H (DMSO-d6, 400 MHz) 0.85 (3H, s, C-18-H3), 1.32-2.41 (13H, m), 2.65-2.80 (2H, m, C-6-H2), 6.44 (1H, d, P-2-H,C-4-H = 2.6 Hz, C-4-H), 6.50 (1H, dd, JC-I-H,C-2-H = 8.5 Hz and JC-4-H,c-2-H = 2.6 Hz, C-2-H), 7.05 (1H, d, JC-2-H,C-I-H = 8.5 Hz, C- 1-H), 9.01 (1H, s, exchanged with D2O, OH) and 10.10 (1H, s, exchanged with D2O, NOH); MS m/z (FAB+) 286.1 [100, (M+H)+], 268.1 [23, (M+H-H2O)+]; Ace MS m/z (FAB+) 286.18093, C18H24NO2 requires 286.18070.
3-O-Benzyl-Marrianolic acid (DSF 01042)
Figure imgf000175_0001
A solution of iodine (7.6 g, 29.9 mmol) in 95 mL of MeOH and a solution of KOH (13.7 g) in 27 mL of H2O and 61 mL of MeOH were added dropwise and alternatively to a stirred solution of 3-O-benzyl-estrone (3.8 g, 10.5 mmol) in MeOH (1 L) so that the colour of the mix remains orange/brown. The addition was carried out over 45 minutes and the resulting light yellow solution was stirred overnight at room temperature under an atmosphere of N2. The resulting eleariight yellow solution was then concentrated under reduced pressure and poured into H2O (800 mL). After acidification with 5M HCl, the organics were extracted with ether (600 mL), washed with aq. Na2S2O3 (4x100 mL), H2O (4x100 mL), dried (MgSO4), filtered and concentrated under reduced pressure. The resulting yellow foam (4.54 g) was then dissolved in a solution of KOH (7.6 g) in MeOH/H2O (1:2, 228 mL) and heated to reflux for 4 hours. The resulting orange solution was poured into H2O (800 mL) and after acidification with 5M HCl the organics were extracted with EtOAc (300 mL). After washing with brine (4x200 mL), the organic layer was dried (MgSO4), filtered and evaporated under reduced pressure to give a yellow residue (4.32 g). This was recrystaUized from CHCI3/Hexane 5:3 to give a creamy powder (3.25 g, 75%): mp 212-215°C [lit.2 (aq. MeOH) 226-227°C]; TLC (chloroform/methanol, 5:1) R, 0.37 cf. R, 0.88; IR (KBr) 3050-2650 (CO2H), 1700 (C=O), 1600-1500 (arom C=C) cm"1; δH (DMSO-d6, 400 MHz) 1.02 (3H, s, C-18-H3), 1.20-2.78 (11H, m), 2.72-2.76 (2H, m, C-6-H2), 5.05 (2H, s, OCH2Ar), 6.68 (1 H, d, Joanc-m = 2.5 Hz, C-4-H), 6.75 (1H, dd, JC-I-H,C-2-H = 8.7 Hz and
Figure imgf000176_0001
= 2.5 Hz, C-2-H), 7.18 (1H, d, C-2-H,C-I-H = 8.7 Hz, C-1-H), 7.30-7.42 (5H, m, C6H5) and 12.14 (2H, s, exchanged with D20, C02H); Dc (DMSO-d6, 100.4 MH∑) 15.37 (q, C-18), 25.84 (t), 26.53 (t), 29.73 (t), 35.77 (t), 36.10 (t), 40.73 (d), 41.84 (d), 42.55 (d), 46.21 (s, C-13), 68.93 (t, OCH2Ar), 112.35 (d), 114.02 (d)', 126.32 (d), 127.29 (2xd), 127.49 (d), 128.19 (2χd), 131.64 (s), 137.18 (2xs), 155.96 (s, C-3), 173.93 (s, CO2H) and 178.60 (s, CO2H); MS m/z (FAB+) 408.2 [41 , M+], 91.1 [100, (CH2Ar)+]; Ace MS m/z (FAB+) 408.19404, C25H28O5 requires 408.19367.
2 Heer, J.; Miescher, K. Helv. Chim. Acta 1945, 28, 156.
Marrianolic acid (DSF 02136, STX 417)
Figure imgf000176_0002
A suspension of Pd-C (10%, 90 mg) in THF (2 mL) was added to a stirred solution of 3- benzyl-O-marrianolic acid (200 mg, 0.61 mmol) in a mixture of MeOH/THF (2:1 , 30 mL). The resulting suspension was hydrogenated at room temperature for 2 hours using a hydrogen-filled balloon. After removal of the supported catalyst by filtration and evaporation of the filtrate under reduced pressure, the resulting yellow foam (142 mg) was recrystaUized from EtOAc/hexane: mp 211-213°C [lit.2 (EtOH), 223-224°C]; TLC (chloroform/MeOH, 5:1 ) Rf 0.40 cf. Rf 0.53; IR (KBr) 3600-3350 (br, CO2H), 3080-3005 (arom CH), 2940-2860 (aliph CH), 1715 (C=O), 1695 (C=O), 1610-1450 (arom C=C) cπr 1; δ H (DMSO-d6, 400 MHz) 1.01 (3H, s, C-18-H3), 1.18-2.34 (11H, m), 2.65-2.71 (2H, m, C-6-H2), 6.41 (1 H, d, Jc-2-H,c-4-H = 2.3 Hz, C-4-H), 6.51 (1H, dd, JC-1.H,C-2-H = 8.6 Hz and Jc.4- H,C-2-H = 2.3 Hz, C-2-H), 7.06 (1H, d, JC-2-H,C-I-H = 8.6 Hz, C-1-H), 9.03 (1 H, s, exchanged with D2O, OH) and 12.12 (2H, br s, exchanged with D2O, 2χCO2H); MS m/z (FAB+) 318.1 [100, (M+H)+], 301.1 [80, (M-OH)+]; Ace MS m/z (FAB+) 318.14635, C18H22O5 requires 318.14672.
2 Heer, J.; Miescher, K. Helv. Chim. Acta 1945, 28, 156. 3-te/ϊ-Butyl-dimethylsilyl-estrone (DSF 03034)
Figure imgf000177_0001
Imidazole (6.3 g, 92.4 mmol) and tert-butyl-dime.thylsilyl chloride (7.8 g, 51.8 mmol) were added to a stirred solution of E1 (10 g, 37.0 mmol) in anhydrous DMF (300 mL) at room temperature, under an atmosphere of N2. The resulting solution was stirred overnight to give a white suspension. This was poured into H2O (500 mL) and the organics were extracted with EtOAc (500 mL + 300 mL), washed with H2O (2x200 mL), then brine (2x200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude was recrystaUized from EtOH to give white needles (~14g, 100%): TLC (chloroform/EtOAc, 9:1) R, 0.91 cf. R, 0.61 (E1); δH (CDCI3, 400 MHz) 0.19 (6H, s, Si(CH3)2), 0.91 (3H, s, C-18-H3), 0.98 (9H, s, C(CH3)3), 1.37-2.55 (13H, m), 2.81-2.89 (2H, m, C-6-H2), 6.57 (1 H, d, JC.2-H,C-4-H = 2.6 Hz, C-4-H), 6.62 (1H, dd, JC.I-H,C-2-H = 8.3 Hz and Jc-4.H,c-2-H = 2.6 Hz, C-2-H) and 7.12 (1H, d, JC-2-H,C-I-H = 8.3 Hz, C-1-H).
3-0-tert-Butyl-dimethylsilyl-16-formyl-estrone (DSF 03066)
Figure imgf000177_0002
NaOMe (3.8 g, 70.2 mmol) was added portionwise to a stirred solution of 3-fert-butyl- dimethylsilyl-estrone (9 g, 23.4 mmol) in dry toluene (250 mL) at room temperature, under an atmosphere of N2. Ethyl formate (13.2 mL, 164 mmol) was then added and the resulting bright yellow solution stirred overnight. The final thick yellow mixture was poured into H2O (300 mL), acidified with HCl 5M. The organics were extracted with EtOAc (2x500 mL), washed with water (3x200 mL), then brine (2x200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a creamy crude (9.49 g, 98%). For analysis a sample was recrystaUized from EtOH to give white crystals: TLC (chloroform/EtOAc, 9:1 ) Rf 0.61 cf. Rf 0.84; δH (DMSO-d6, 400 MHz) 0.17 (6H, s, Si(CH3)2), 0.83 (3H, s, C-18-H3), 0.96 (9H, s, C(CH3)3), 1.32-2.63 (12H, m), 2.77-2.85 (2H, m, C-6-H2), 6.55 (1H, d, JC-2-H|M-H = 2.6 Hz, C-4-H), 6.61 (1H, dd, JC-1.H,C-2-H = 8.3 Hz and JC-4-H,C-2-H = 2.6 Hz, C-2-H) , 7.14 (1H, d, JC-2-H,C-I-H = 8.3 Hz, C-1-H), 7.40 (1H, s,
=CH) and 10.69 (1 H, s, exchanged with D2O, =COH); MS m/z (FAB÷) 413.2 [68, (M+H)+], 355.1 [33, (M-C(CH3)3)+], 72.9 [100]; Ace MS m/z (FAB+) 413.24921 , C25H37O3Si requires 413.25120.
3-Hydro2jy-16-formyl-estr©ne (DSF 03093, STX 486)
Figure imgf000178_0001
A 1.0 M solution of tetrabutyl ammonium fluoride in dry THF (21.3 mL, 21.3 mmol) was added to a stirred solution of 3-O-fert-butyl-dimethylsilyl-16-hydroxymethylene-estrone (4.4 g, 10.7 mmol) in dry THF (100 mL) at room temperature, under an atmosphere of N2. The mixture was stirred overnight to give a brown solution. After removal of the solvent under reduced pressure, H2O was added (300 mL) and the organics were extracted with EtOAc (2x200 mL), washed with H2O (200 mL), then brine (200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The light yellow crude was precipitated from boiling EtOAc, yielding a pale yellow powder (2.21 g, 69%): TLC (chloroform/EtOAc, 8:2) Rf 0.32 cf. Rf 0.68; δH (DMSO-d6, 400 MHz) 0.80 (3H, s, C-18- H3), 1.28-2.61 (11 H, m), 2.69-2.78 (2H, m, C-6-H2), 6.44 (1 H, d, JC-2-H,C-4-H = 2.6 Hz, C-4- H), 6.51 (1H, dd, JC-1-H,C-2-H = 8.5 Hz and JC.4-H,C-2-H = 2.6 Hz, C-2-H), 7.04 (1H, d, JC-2-H,C-I-H = 8.5 Hz, C-1-H), 7.38 (1H, s, =CH), 9.02 (1H, s, exchanged with D2O, C-3-OH) and 10.69 (1H, br s, exchanged with D2O, =COH); MS m/z (FAB+) 299.2 [36, (M+H)+], 242.3 [100]; Ace MS m/z (FAB+) 299.16505, C19H23O3 requires 299.16472.
3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (CAB 02156/DSF 03060, STX 509)
Figure imgf000179_0001
Hydrazine hydrate (48 μL, 1.00 mmol) was added to a solution of 16-hydroxymethylene- estrone (200 mg, 0.67 mmol) in absolute EtOH (10 mL) at room temperature, under an atmosphere of N2. The resulting intense yellow solution was heated to reflux for 30 minutes. The solvent was then removed under reduced pressure, H2O (50 mL) added and the mixture acidified with HCl 5M. The organics were extracted with EtOAc (100 mL + 50 mL), washed with H2O (2x50 mL), then brine (2x50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a pale yellow crude (212 mg). This was recrystaUized from absolute EtOH to give pale yellow flaky crystals (125 mg, 64%): mp 326-328°C [lit.3 320-328°C]; TLC (chloroform/EtOAc, 7:3) Rf 0.11 cf. R, 0.28; δ H (DMSO-d6, 400 MHz) 0.90 (3H, s, C-18-H3), 1.32-2.58 (11 H, m), 2.69-2.84 (2H, m, C-6- H2), 6.45 (1 H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6.52 (1 H, dd, JC-I-H,C-2-H = 8.4 Hz and JM-H,C-2- H = 2.6 Hz, C-2-H), 7.06 (1H, d, JC-2.H,C-I-H = .8.4 Hz, C-1-H), 7.26 (1H, s, C-5'-H), 9.02 (1 H, s, exchanged with D2O, C-3-OH) and 12.00 (1H, d, J = 3.5 Hz, exchanged with D2O, NH); MS m/z (FAB+) 295.0 [100, (M+H)+]; Ace MS m/z (FAB+) 295.18078, Cι9H23N2O requires 295.18104.
3 Sweet, F; Boyd, J.; Medina, O.; Konderski, L.; Murdock, G. Biochem. Biophys. Res. Comm. 1991, 180, 1057-1063
16-Cyano-estradiol (DSF 03019/STX 561)
Figure imgf000179_0002
Sodium borohydride (50 mg, 1.32 mmol) was added portionwise to a stirred solution of 16-nitrile-estrone (150 mg, 0.51 mmol) in a mixture of THF/EtOH (3:2, 5 mL) at room temperature. The resulting pale yellow solution was stirred for 30 minutes. The solvent was then removed under reduced pressure, and H2O added (20 mL). The organics were extracted with EtOAc (20 mL + TO mL), washed with H2O (20 mL), then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a light yellow crude (159 mg). This was recrystaUized from IPA/H2O to give white crystals (16 mg) and a further crop of the product (40 mg) was obtained from the residue of the mother liquor upon recrysfallizaiion from absolute EtOH (overall yield 37%): mp 252-254°C; IR (KBr) 3430-3235 (OH), 2925-2850 (aliph CH), 2255 (CN), 1610-1500 (arom C=C) cm'1; δH (DMSO-d6, 400 MHz) 0.79 (3H, s, C-18-H3), 1.10-2.27 (11H, m), 2.66-2.76 (2H, m, C-6- H2), -3.33 (~1H, m, C-16-H, under solvent peaks), 3.68 (1H, dd, C-I6-H,C-I7-H = 9.4 Hz, Jc_ 17-OH.C-17-H = 4.7 Hz, C-17-H), 5.50 (1H, d, Jc.ι7-H,c-i7.oH = 4.7 Hz, C-17-OH), 6.43 (1H, d, Jc- 2-H,C-4-H = 2.5 Hz, C-4-H), 6.50 (1H, dd, C-I-H,C-2-H = 8.5 Hz and JC-4.H,C-2-H = 2.5 Hz, C-2-H), 7.03 (1H, d, JC-2-H,C-I-H = 8.5 Hz, C-1-H) and 9.01 (1H, s, exchanged with D2O,' C-3-OH); MS m/z (FAB+) 297.1 [100, M+], 242.1 [50]; Ace MS m/z (FAB+) 297.17239, C19H23NO2 requires 297.17288.
16-lsobutylidene-estrone (DSF 03029, STX 571)
Figure imgf000180_0001
A solution of estrone (420 mg, 1.55 mmol) in dry THF (5 mL) was added dropwise to a stirred solution of LDA (2.47 mL of a 1.8 M solution in heptane/THF/ethyl benzene, 4.44 mmol) in dry THF (2 mL) at -78°C, under an atmosphere of N2. After 2 hours of stirring at -78°C, isobutyraldehyde (185 μL, 2.03 mmol, freshly distilled from Na2SO4) was added. The resulting mixture was stirred overnight during which it was allowed to warm to room temperature. The solvent was then removed under reduced pressure and H2O added (50 mL). The organics were extracted with EtOAc (50 mL + 20 mL), washed with H2O (20 mL) then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a crude white foam (602 mg). This was purified by flash chromatography with chloroform/EtOAc (9:1 ) as eluent and gave the product as a white foam (401 mg, 79%). This was recrystaUized from EtOAc/hexane to give white crystals (325 mg, 64%): mp 188-190°C; TLC (chloroform/EtOAc, 8:2) Rf 0.65 cf. Rf 0.74 (E1); IR (KBr) 3370 (OH), 2930-2890 (aliph CH), 1710 (C=O), 1645-1445 (arom C=C and exocyclic C=C) cm"1; δ H (CDCI3, 400 MHz) 0.92 (3H, s, C-18-H3), 1.06 (3H, d, JC-Z-H.C^-H = 6.6 Hz, C-3'-H3), 1.04 (3H, d, C^.C^H = 6.6 Hz, C-4'-H3), 1.40-2.70 (12H, m), 2.84- 2.90 (2H, m, C-6-H2), 4.67 (1H, s, exchanged with D2O, OH), 6.46 (1 H, ddd, JC-2'-H,c-r-H = 9.8 Hz, Jc-i5-H,c-r-H = 1-9 Hz, C-f-H), 6.59 (1H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H), 6.64 (1H, dd, JC-I-H,C-2-H = 8.3 Hz and JC-4-H,G-2-H = 2.7 Hz, C-2-H) and 7.16 (1H, d, JC-2-H,c-ι-H = 8.3 Hz, C-1-H);DDC (CDCIs, 100.4 MHz) 14,99 (q, C-18), 22.34 (2xq), 26.42 (t), 26.44 (t), 27.18 (t), 29.69 (d), 29.94 (i), 31.98 (t), 38.32 (d), 44.38 (d), 48.34 (d), 48.79 (s, C-13), 113.12 (d), 115.54 (d), 126.66 (d), 132.20 (s), 134.73 (s), 138.12 (s), 144.25 (d, C-1'), 153.78 (s, C-3), and 210.04 (s, C=O); MS m/z (FAB+) 325.1 [100, (M+H)+]; Ace MS m/z (FAB+) 325.21663, C22H29O2 requires 325.21675. Found: C, 81.30; H, 8.71 ; N, 0. C22H28O2 requires: C, 81.44; H, 8.70; N, 0.
16-lsobutyiidene-estradiøl (DSF 03046, STX 614)
Figure imgf000181_0001
A solution of sodium borohydride (57 mg, 1.51 mmol) in H2O (3 mL) was added dropwise to a stirred solutio of 16-isobutylidene-estrone (100 mg, 0.31 mmol) in a mixture of MeOH/THF (3:1, 9 mL) at 0°C. The resulting solution was stirred at 0°C for 20 minutes. 5 Drops of AcOH glacial were then added, followed by aq. NaCI (10% solution, 20 mL) and the white precipitate that formed was filtered and dried (102 mg). This was recrystaUized from acetone/hexane to give white crystals (60 mg, 60%): TLC (chloroform/EtOAc, 8:2) Rf 0.72 cf. R, 0.82; IR (KBr) 3435-3326 (OH), 2955-2865 (aliph CH), 1695-1500 (arom C=C and exocyclic C=C) cm-1; δH (CDCI3, 400 MHz) 0.67 (3H, s, C-18-H3), 0.96 (3H, d, JC- -H,c-3'-H = 6.6 Hz, C-3'-H3), 0.99 (3H, d, C-2.H,C-4-H = 6.6 Hz, C-4'- H3), 1.22-2.02 (14H, m), 2.81-2.87 (2H, m, C-6-H2), 3.91-3.98 (1H, m, C-17-H), 4.66 (1H, s, exchanged with D2O, OH), 5.32 (1H, ddd, JC-2-H,C-I'-H = 9-4 Hz, ' JC-i5-H,c-r-H = 2.3 Hz, C- 1'-H), 6.56 (1H, d, JC.2-H,C-4-H = 2.7 Hz, C-4-H), 6.63 (1H, dd, JCM-H^-H = 8.6 Hz and JC-4.H,C- 2.H = 2.7 Hz, C-2-H) and 7.16 (1H, d, JC.2-H,C-I-H = 8.6 Hz, C-1-H); MS m/z (FAB+) 326.2 [95, Ml, 309.2 [100, (M-OH)+], 283.1 [50, (M-C3H7)+J; Ace MS m/z (FAB+) 326.22520, C22H3oO2 requires 326.22458.
16-(2',2'-Dimethyl)-propylidene-estrone (DSF 03069A/STX 748)
Figure imgf000182_0001
A solution of estrone (500 mg, 1.85 mmol) in dry THF (5 mL) was added dropwise to a stirred solution of LDA (2.47 mL of a 1.8 M solution in heptane/THF/ethyl benzene, 4.44 mmol) in dry THF (2 mL) at -78°C, under an atmosphere of N2. After 2 hours of stirring at -78°C, 2,2-dimethyl-propionaldehyde (261 μL, 2.40 mmol, freshly distilled from Na2SO4) was added. The resulting mixture was stirred for 50 hours during which it was allowed to warm to room temperature. The solvent was then removed under reduced pressure and H2O added (50 mL). The organics were extracted with EtOAc (50 mL + 20 mL), washed with H2O (20 mL), then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The light yellow crude was purified by flash chromatography with a gradient of chloroform/EtOAc (9:1 to 8:2) as eluent and gave the product as a white crystalline solid (264 mg, 42%). This was recrystaUized from EtOAc to give white crystals (113 mg, 21 %): TLC (chloroform/EtOAc; 8:2) R, 0.71 cf. Rf 0.61 (E1); δH (CDCI3, 400 MHz) 0.90 (3H, s, C-18-H3), 1.17 (9H, s, C(CH3)3), 1.41-2.45 (11H, m), 2.83-2.91 (2H, m, C-6-H2), 4.72 (1H, s, exchanged with D2O, OH), 6.59 (1 H, d, JG-2-H.C-4-H = 2.7 Hz, C-4-H), 6.63-6.65 (2H, m, C-2-H and C-1'-H) and 7.16 (1 H, d, C-2-H,C-I-H = 8.0 Hz, C-1-H); MS m/z (FAB+) 677.5 [54, (2M+H)+]( 339.2 [100, (M+H)+], 492.2 [24, (M+H+NBA)+]; Ace MS m/z (FAB+) 339.23256, C23H31O2 requires 339.23241. Found: C, 81.50; H, 8.96; N, 0. C23H30O2 requires: C, 81.61 ; H, 8.93; N, 0.
16-(1'-Hydroxy-propyl)-estrone (DSF 03044B, STX 665)
Figure imgf000182_0002
A solution of estrone (500 mg, 1.85 mmol) in dry THF (30 mL) was added dropwise to a stirred solution of LDA (2.47 mL of a 1.8 M solution in heptane/THF/ethyl benzene, 4.44 mmol) in dry THF (20 mL) at -78°C, under an atmosphere of N2. After 2 hours of stirring at -78°C, propionaldehyde (160*μL, 2.40 mmol, dried on CaCI2 and freshly distilled from Na2SO4) was added. The resulting mixture was stirred overnight during which it was allowed to warm to room temperature. The solvent was then removed under reduced pressure and H2O added (100 mL). The organics were extracted with EtOAc (2x100 mL), washed with H2O (100 mL), then brine (100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a creamy crude oil that crystallizes (971 mg). This was purified by flash chromatography with chloroform/EtOAc (8:2) as eluent and gave the product as a white solid (363 mg, 60%). This was recrystaUized from EtOAc to give white crystals (49 mg) and a further crop of the product (170 mg) was obtained from the residue of the mother liquor upon recrystalli∑ation from EtOAc/hexane (overall yield 36%): TLC (chloroform/EtOAc, 8:2) Rf 0.38 cf. R, 0.61 (E1); δH (CDCI3, 400 MH∑) 0.94 (3H, s, C-18-H3), 1.01 (3H, t, JO*-H.W-H = 7.5 Hz, C-3'-H3), 1.32-2.44 (14H, m), 2.83-2.91 (2H, m, C-6-H2), 3.65-3.72 (1H, m, C-1'-H), 3.99 (1 H, s, OH), 4.69 (1H, s, OH), 6.59 (1H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H), 6.65 (1H, dd, JC-I-H,C-2-H = 8.4 Hz and JC-4-H,C- 2-H = 2.7 Hz, C-2-H) and 7.15 (1 H, d, JC-2-H,C-I-H = 8.4 Hz, C-1-H); MS m/z (FAB+) 329.2 [100, (M+H)+], 311.2 [30, (M-OH)+]; Ace MS m/z (FAB+) 329.21188, C21H29O3 requires 329.21167.
3-0-Acetyl-16-methylene-estrone (DSF 03023)
Figure imgf000183_0001
Paraformaldehyde (480 mg, 16.0 mmol for one unit) and dimethylamine hydrochloride (1.6 g, 19.6 mmol) were added to' a stirred solution of 3-O-acetyl-estrone in anhydrous isoamyl alcohol (8 mL). The resulting mixture was heated to reflux for 24 hours. The resulting light yellow solution was poured into H2O (30 mL) and acidified with HCl 5M. The organics were extracted with EtOAc (2x50 mL), washed with NaHCO3 sat. (20 mL), H2O (20 mL), then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a light yellow oil. This was distilled in a kugelrohr until no more isoamyl alcohol was distilled. The oily residue crystallized overnight and was purified by flash chromatography with chloroform/EtOAc (95:5) as eluent to give the product as a white solid (527 mg, 51%). For analysis some crystals were obtained by adding hexane to the distilled oily crude and leaving standing for 1 h: δH (CDCI3, 400 MHz) 0.93 (3H, s, C-18-H3), 1.41-2.72 (11H, m), 2.29 (3H, s, OAc), 2.90-2.95 (2H, m, C-6-H2), 5.41-5.43 (1 H, m, C-1'-Htrans), 6.09-6.12 (1 H, m, C-1'-Hcis), 6.81 (1H, d, JC-2-H,C-4-H = 2.5 Hz, C-4- H), 6.86 (1 H, dd, JC-ι-H,c-2-H = 8.3 Hz and JC-4-H,c-2-H = 2.5 Hz, C-2-H) and 7.29 (1 H, d, Jc-2- H.c-1-H = 8.3 Hz, C-1-H); MS m/z (FAB+) 325.2 [100, (M+H)+J, 282.2 [78, (M+H-CH3CO)+]; Ace MS m/z (FAB+) 325.18224, C21H25O3 requires 325.18037.
16-E hoxymethyl-©stron© (DSF 0303@B, STIC 664)
Figure imgf000184_0001
A solution of KOH (41 mg, 0.74 mmol) in H2O (2 mL) was added dropwise to a stirred solution of 16-methylene-estrone (20 mg, 0.62) in absolute EtOH (20 mL) at 0°C. The resulting light yellow solution was stirred for 30 minutes at 0°C. The solvent was then removed under reduced pressure, H2O added (40 mL) followed by a few drops of HCl 5M. The white precipitate that formed was filtered and dried (131 mg). This was purified by flash chromatography with chloroform/EtOAc (95:5) as eluent and gave the product as a light yellow solid (26 mg, 13%). This was recrystaUized from EtOH/H2O to give light yellow crystals (13 mg, 6%): mp 208-210°C; TLC (chloroform/EtOAc, 8:2) R, 0.40 cf. R, 0.86; δH (CDCI3, 400 MHz) 0.89 (3H, s, C-18-H3), 1.16 (3H, t, ^- .H.C-S-H = 7.0 Hz, C-3'- H3), 1.36-2.42 (12H, m), 2.81-2.85 (2H, m, C-6-H2), 3.42-3.50 (2H, m, C-2'-H2), 3.59 (2H, m, C-1'-H2), 4.66 (1H, s, OH), 6.58 (1 H, d, C-2-H,C-4-H = 2.8 Hz, C-4-H), 6.64 (1H, dd, JC-ι- H,C-2-H = 8.1 Hz and JC-4-H,C-2-H = 2.8 Hz, C-2-H) and 7.15 (1H, d, JC-2-H,C-I-H = 8.1 Hz, C-1-H); MS m/z (FAB+) 329.1 [100, (M+H)+]; Ace MS m/z (FAB+) 329.21242, C21H29O3 requires 329.21167. Found: C, 76.60; H, 8.62; N, 0. C21H28O3 requires: C, 76.79; H, 8.59; N, 0.
17-O-Allyl-oxime-estrone (DSF 03048)
Figure imgf000184_0002
NaOAc (3 g, 10.17 mmol) followed by O-allyl-hydroxylamine hydrochloride (4.5 g, 41.4 mmol) were added to a solution of estrone (1 g, 3.70 mmol) in a mixture of MeOH/H2O (5:1 , 180 mL). The resulting solution was stirred at room temperature overnight. The solvent was then removed under reduced pressure and water added (200 mL). The organics were extracted with EtOAc (200 mL + 100 mL), washed with water (2x100 mL) then brine (2x100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a white crude (1.37 g). This was recrystaUized from MeOH/H2O and gave the product as white crystals (1.13 g, 94%): TLC (chloroform/EtOAc, 8:2) Rf 0.66 cf. R, 0.56; δ H (CDCIg, 400 MH∑) 0.94 (3H, s, C-18-H3), 1.35-2.60 (13H, m), 2.80-2.87 (2H, m, C-6-H2), 4.55 (2H, ddd, JAB = 5.5 H∑, JC-2'.H,c-ι-H = 2.7 H∑ and JC-3'-H,C-I'-H = 1-4 Hz, C-1'-H2), 4.74 (1 H, s, echanged with D2O, OH), 5.19 (1H, ddd, cis = 10.3 Hz, JO.V- .O9* = 1.4 Hz, C-3'-Ha), 5.28 (1H, d, Jlrans = 17.2 Hz, Jc-r-H,c-3'-H = 1 -6 Hz, C-3'-Hb), 5.59-6.01 (1H, m, C-2'-H), 6.56 (1H, d, C-2.H,C-4-H = 2.7 H∑, C-4-H), 6.63 (1H, dd, C-I-H,C-2-H = 8.2 Hz and JC-4-H,C-2-H = 2.7 Hz, C-2-H) and 7.15 (1H, d, JC-2-H,C-I-H = 8.2 Hz, C-1-HJ; MS m/z (FAB.+) 326.1 [100, (M+H)+], 268.1 [30, OCH2CH=CH2 +]; Ace MS m/z (FAB+) 326.21157, C21H28NO2 requires 326.21200. Found: C, 76.40; H, 8.24; N, 4.36. C21H27NO2 requires: C, 76.23; H, 8.50; N, 4.17.
3-Hydroxy-estra-1, 3, 5 (10)-triene-(17,16)-[1]pyrindine (DSF 03073, STX 663)
Figure imgf000185_0001
17-O-allyl-oxime (2.45 g, 7.53 mmol) was stirred and heated to 230°C (using a sand bath) for 46 hours. The resulting dark brown solid was cooled and EtOH was added until most of the solid was dissolved. The remaining undissolved material was crushed to a fine powder, silica was added and the solvent removed under reduced pressure. The light brown powder was then transferred onto a flash chromatography column (wet packed) and the column eluted with chloroform/EtOAc (9:1). A dark orange solid was recovered (200 mg, 9%) which was purified by performing another flash chromatography with chloroform/EtOAc (96:6) as eluent and gave a light orange powder (146 mg, 6%): TLC (chloroform/EtOAc, 8:2) Rf 0.26 cf. Rf 0.70; δH (CDCI3, 400 MHz) 1.01 (3H, s, C-18- H3), 1.43-2.68 (11H, m), 2.80-2.96 (2H, m, C-6-H2), 6.08 (1H, s, OH), 6.61 (1H, d, JC.2-H,C- 4.H = 2.6 Hz, C-4-H), 6.67 (1H, dd, JC-I-H,G-2-H = 8.4 Hz and C.4.H,C-2-H = 2.5 Hz, C-2-H), 7.04 (1H, dd, Jc-4'-H,c-5--H = 7.4 Hz and JC.6-H,C-3'-H = 4.8 Hz, C-5'-H), 7.18 (1H, d, C.2-H,C-I-H = 8.4 Hz, C-1-H), 7.54 (1 H, d, C.S.H,C-4'-H = 7-4 Hz, C-4'-H) and 8.33 (1H, d, JC-5-H,C-6'-H = 4.8 Hz, C-2'-H); MS m/z (FAB+) 306.1 [85, (M+H)+], 207.0 [95], 114.9 [100]; Ace MS m/z (FAB+) 306.18645, C21H24NO requires 306.18579. 3-0-Acetyl-16-acetoxymethylene-estrone (DSF 03054)
Figure imgf000186_0001
Acetic anhydride (8.34 mL, 88.4 mmol) was added dropwise over 10 minutes to a stirred solution of 16-formyl-estrone (230 mg, 0.77 mmol) in dry pyridine (15 mL) at 0°C, under an atmosphere of N2. The resulting yellow mixture was then heated to reflux for 1 hour. The final brown solution was cooled, then poured into H2O (50 mL) and ice and acidified wifh HCl 5M. The organics were extracted with EtOAc (2x50 mL), washed with H2O (30 mL), Na2CO3 10% (30 mL), then brine (30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give an orange foam (250 mg). This was purified by flash chromatography with chloroform/EtOAc (95:5) as eluent and gave the product as a pale yellow oil that crystalizes (140 mg, 47%): TLC (chloroform/EtOAc, 9:1) Rf 0.70 cf. R, 0.30; δH (CDCI3, 400 MHz) 0.93 (3H, s, C-18,H3), 1.42-2.84 (11H, m), 2.24 (3H, s, OAc), 2.29 (3H, s, OAc), 2.89-2.96 (2H, m, C-6-H2), 6.81 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6.86 (1H, dd, JC-I-H,C-2-H = 8.3 Hz and ΛC-4-H,C-2-H = 2.6 Hz, C-2-H), 7.29 (1H, d, JC-2-H,C-I-H = 8.3 Hz, C-1-H) and 8.15 (1 H, dd, JC-ι5-Ha,c-r-H = 2.9 Hz, JG-ι5.Hb,c-i'-H = 1 - Hz, =CH); MS m/z (FAB+) 383.0 [100, (M+H)+], 341.0 [86, (M+H-CH2C=O)+]; Ace MS m/z (FAB+) 383.18523, C23H27O5 requires 383.18585.
6-Oxo-3-0-acetyl-16-acetoxymethylene-estrone (DSF 03062)
Figure imgf000186_0002
A solution of CrO3 (132 mg, 1.32 mmol) in acetic acid 10% (750 μL) was added dropwise over 30 minutes to a stirred solution of 3-O-acetyl-16-acetoxymethylene-estrone (120 mg, 0.31 mmol) in acetic acid at 10-15°C in an ice/water bath. The resulting dark brown solution was stirred for 40 hours at room temperature. The solvent was then removed under reduced pressure and H2O (50 mL) and ice added. The organics were extracted with EtOAc (2x50 mL), washed with H2O (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a brown crude. This was purified by flash chromatography with chloroform/EtOAc (8:2) as eluent an gave the product as a light yellow solid (13 mg, 10%): TLC (chloroform/EtOAc, 4:1) P 0.40 cf. Rf 0.63; δH (CDCIg, 400 MHz) 0.95 (3H, s, C-18-H3), 1.24-2.92 (11H, m), 2.26 (3H, s, OAc), 2.32 (3H, s, OAc), 7.29 (1H, dd, JC-I-H,C-2-H = 8.4 Hz and JC-4-H,C-2-H = 2.6 Hz, C-2-H), 7.46 (1 H, d, JC-2-H,C-I-H = 8.4 Hz, C-1-H), 7.77 (1H, d, C-2-H,C-4-H = 2.6 Hz, C-4-H) and 8.17 (1H, dd, Jc-i5-H ,oi'-H = 2.9 Hz, JC-i5-Hb,c-i'-H = 1 -7 Hz, =CH); MS m/z (FAB+) 663.5 [48], 397.2 [38, (M+H)+], 355.2 [30], 73.0 [100]; Ace MS m/z (FAB+) 397.16700, C23H25O6 requires 397.16511.
β-Oxo-16-ethoxymethylene-estrone (DSF 03083A, STX 749)
Figure imgf000187_0001
A solution of KOH (27 mg, 0.48 mmol) in H2O (0.8 mL) was added dropwise to a stirred solution of 6-oxo-3-O-acetyl-16-acetoxymethylene-estrone (80 mg, 0.20 mmol) in absolute EtOH at 0°C. The resulting bright yellow mixture was stirred at 0°C for 30 minutes. The mixture was then acidified with 3 drops of 5M HCl and the solvent removed under reduced pressure. H2O was added (50 mL) and the organics were extracted with EtOAc (2x50 mL), washed with H2O (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a pale yellow crude (52 mg). This was purified by flash chromatography with DCM/EtOAc (75:25) as eluent and gave the product as a white solid (12 mg, 18%): mp 314-316°C (dec.); TLC (chloroform/EtOAc, 8:2) R, 0.11 cf. Rf 0.82; δH (CDCI3, 270 MHz) 0.94 (3H, s, C-18-H3), 1.10-2.72 (10H, m), 1.35 (3H, t, JC-2'-H,C-3'-H = 7.0 Hz, C-3'-H3), 2.85 (1H, dd, JC-8-H,C-7-HB = 3.1 Hz, JAB= 16.7Hz, C-7-HB), 4.05-4,13 (2H, m, C-2'-H2), 7.09 (1H, dd, JC.I-H,C-2-H = 8.6 Hz and C-4-H,C-2-H = 2.7 Hz, C-2-H), 7.31 (1H, s, C-1'-H), 7.34 (1H, m, C-1-H) and 7.57 (1H, d, C-2-H,C-4-H = 8.4 Hz, C-4-H); MS m/z (FAB+) 341.2 [100, (M+H)+]; Ace MS m/z (FAB+) 341.17466, C21H25O4 requires 341.17528.
6-Oxo-16-formyl-estrone (DSF 03105, STX 784)
Figure imgf000188_0001
A solution of K2CO3 (63 mg, 0.46 mmol) in H2O (1 mL) was added dropwise to a stirred solution of 6-oxo-3-O-acetyl-16-acetoxymethylene-estrone (30 mg, 0.08 mmol) in MeOH (10 mL). The resulting pale yellow solution was stirred for 45 minutes. The final pale brown mixture was then acidified with 3 drops of HCl 5M and the solvent removed under reduced pressure. H2O was added (30 mL) and the organics were extracted with EtOAc (2x50 mL), washed with H2O (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The beige crude was recystallized from EtOAc/hexane to give a white powder (13 mg, 54%): mp 224-227°C; δH (DMSO-d6, 400 MHz) 0.80 (3H, s, C-18-H3), 1.41-2.65 (11H, m), 7.01 (1H, dd, JC-1-H,C-2-H - 8.4 Hz and Jc. 4-H,C-2-H = 2.7 Hz, C-2-H), 7.28 (1H, d, JC-2-H,c- H = 2.7 Hz, C-4-H), 7.31 (1H, d, JC-2-H,c-ι-H = 8.4 Hz, C-1-H), 7.41 (1H, s, C-1'-H), 9.64 (1H, s, exchanged with D2O, C-3-OH) and 10.77 (1H, br s, exchanged with D2O, C-1'-OH); MS m/z (FAB+) 313.2 [6, (M+H)+], 149.1 [100]; MS m/z (FAB-) 311.2 [100, (M-H)"]; Ace MS m/z (FAB+) 313.14503, C19H21O4 requires 313.14398.
3-0-Benzyl-16-formyl-estrone (DSF 03091)
Figure imgf000188_0002
Potassium tert-butoxide (9.4 g, 84.1 mmol) was added portionwise to a stirred solution of 3-O-benzyl-estrone (10 g, 27.7 mmol) in dry toluene (250 mL) at room temperature, under an atmosphere of N2. After stirring for 20 minutes, ethyl formate (14.8 mL, 194 mmol) was added and the resulting suspension stirred for 2.5 hours. The final thick creamy suspension was poured into H2O (300 mL) and ice, and the organics extracted with EtOAc (2x200 mL), washed with H2O (3x100 mL), then brine (2x100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude was precipitated from boiling EtOAc to give a creamy solid (9.25 g, 86%): TLC (hexane/EtOAc, 1 :1) Rf 0.57 cf. Rf 0.78; δH (DMSO-d6, 400 MHz) 0.81 (3H, s, C-18-H3), 1.31-2.62 (11H, m), 2.79-2.85 (2H, m, C-6-H2), 5.05 (2H, s, OCH2Ar), 6.71 (1H, d, JC.2-H,C-4-H = 2.7 Hz, C-4-H), 6.75 (1H, dd, JC-I-H,C-2-H = 8.4 Hz and JC-4-H,C.2-H = 2.7 Hz, C-2-H), 7.16 (1H, d, JC-2-H,C-I-H = 8.4 Hz, C-1-H), 7.28-7.33 (1H, m, =CH), 7.35-7.43 (5H, m, C6H5) and 10.6-10.9 (1H, br s, exchanged with D2O, =COH); MS m/z (FAB+) 389.3 [31 , (M+H)+], 91.1 [100, (CH2Ar)+]; Ace MS m/z (FAB+) 389.20994, C26H29O3 requires 389.21167.
3-0-Benzyl-estra-1, 3, 5 (10)-trieno-(1 , 16-c)-pyrazole (DSF 03095)
Figure imgf000189_0001
Hydrazine hydrate (751 μL, 15.4 mmol) was added to a suspension of 3-O-benzyl-16- formyl-estrone (4 g, 10.3 mmol) in absolute EtOH (200 mL) at room temperature, under an atmosphere of N2. The resulting yellow solution was heated to reflux for 45 minutes. After acidification with HCl 5M, the solvent was removed under reduced pressure until precipitation of the product. H2O was then added (20 mL) and the creamy precipitate filtered and dried (3.73 g, 94%): TLC (hexane/EtOAc, 1 :1) Rf 0.20 cf. Rf 0.66; δH (DMSO- d6, 400 MHz) 0.91 (3H, s, C-18-H3), 1.36-2.58 (11H, m), 2.78-2.87 (2H, m, C-6-H2), 5.05 (2H, s, OCH2Ar), 6.72 (1H, d, JC.2-H,C-4-H = 2.7 Hz, C-4-H), 6.76 (1H, dd, JC-I-H,C-2-H = 8.6 Hz and JC-4-H,c.2-H = 2.7 Hz, C-2-H), 7.18 (1H, d, JC-2-H,C-I-H = 8.6 Hz, C-1-H), 7.27 (1H, s, C-5'- H), 7.29-7.44 (5H, m, C6H5) and 12.00 (1H, s, exchanged with D2O, NH); MS m/z (FAB+) 385.3 [75, (M+H)+], 91.1 [100, (CH2Ar)+]; Ace MS m/z (FAB+) 385.22796, C26H29N2O requires 385.22799.
General procedure for alkylation of D-rinq fused pyrazoles.
NaH (60% dispersion, 1.5 eq.) was added to a stirred solution of 3-O-benzyl-estra-1 , 3, 5 (10)-trieno-(17, 16-c)-pyrazole in DMF at 0°C, under an atmosphere of N2. After stirring for 20 minutes at 0°C, the alkylating agent was added (2 eq.) and the mixture was stirred at room temperature until completion of the reaction (followed by TLC). The mixture was then poured into H2O (50 mL) and the organics extracted with EtOAc (2x50 mL), washed with H2O (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography using DCM/EtOAc as eluent.
3-©-B©nε I-estø-1, 3, 5 (10)-triøno-( , 6-c)--W-m© hyl-pyra2θl© Following the general procedure, 3-O-benzyl-estra-1 , 3, 5 (10)-trieno-(1 , 16-e)- pyra∑ole (150 mg, 0.39 mmol) was treated with NaH (19 mg, 0.47 mmol) and the subsequent reaction with methyl iodide (57 μL, 0.78 mmol) was complete within 50 minutes. Purification of the crude by flash chromatography with a gradient of DCM/EtOAC (98:2 to 95:5) as eluent gave: 1 '-Methyl- 3-0-Benzyl-estra-1, 3, 5 (10)- trieno-(17, 16-c)-pyrøzol®, DSF 03098A, as a light yellow oil that crystallized on standing (52 mg, 33%): TLC (hexane/EtOAc, 1 :1) Rf 0.52 cf. Rf 0.38; δH (CDCI3, 400 MHz) 1.01 (3H, s, C-18-H3), 1.42-2.67 (11H, m), 2.81-2.99 (2H, m, C-6-H2), 3.84 (3H, s, N-Me), 5.03 (2H, s, OCH2Ar), 6.73 (1H, d, JC-2-H,C-4-H = 2.5 Hz, C-4-H), 6.79 (1H, dd, Jc-1. H,c-2-H = 8.5 Hz and JC-4-H,C-2-H = 2.5 Hz, C-2-H), 6.97 (1H, s, C-5'-H), 7.22 (1H, d, JC-2-H,C-I-H = 8.5 Hz, C-1-H) and 7.29-7.45 (5H, , C6H5); MS m/z (FAB+) 399.3 [74, (M+H)+], 91.1 [100, (CH2Ar)+], 73.0 [24]; Ace MS m/z (FAB+) 399.24467, C27H31N2O requires ,399.24364.
2'-Methyl- 3-O-Benzyl-estra-l, 3, 5 (10)-trieno-(17, 16-c)-pyrazole, DSF 03098B, was obtained as a creamy solid (55 mg, 35%): TLC (hexane/EtOAc, 1 :1 ) Rf 0.46 cf. Rf 0.38; δH (CDCI3, 400 MHz) 1.01 (3H, s, C-18-H3), 1.44-2.64 (11H, m), 2.85-2.98 (2H, m, C-6- H2), 3.82 (3H, s, N-Me), 5.04 (2H, s, OCH2Ar), 6.74 (1 H, d, Jc-2-H,c-4-H = 2.8 Hz, C-4-H), 6.80 (1H, dd, Jc.ι-H,c-2-H = 8.6 Hz and JC-4-H,C-2-H = 2.8 Hz, C-2-H), 7.15 (1H, s, C-3'-H), 7.20 (1H, d, JC-2-H,c-i-H = 8.6 Hz, C-1-H) and 7.30-7.45 (5H, m, C6H5); MS m/z (FAB+) 399.3 [55, (M+H)+], 91.1 [100, (CH2Ar)+]; Ace MS m/z (FAB+) 399.24341 , C27H31N2O requires 399.24364.
1'-lsobutyl- 3-0-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole and 2'-lsobutyl- 3-0-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 6-c)-pyrazole
Following the general procedure, 3-O-benzyl-estra-1 , 3, 5 (10)-trieno-(17, 16-c)- pyrazole (250 mg, 0.65 mmol) was treated with NaH (31 mg, 0.78 mmol) and the subsequent reaction with 1-bromo-2-methyl-propane (124 μL, 1.30 mmol) was -complete within 2 hours. Purification of the crude by flash chromatography with DCM/EtOAC (95:5) as eluent gave: 1'-lsobutyl- 3-©-Benzyl-estra-1, 3, 5 (10)-tri©no-(17, 16-c)- pyrazole, DSF 03111A, as a pale yellow oil (121 mg, 42%): TLC (DCM/EtOAc, 9:1) Rf 0.93 cf. Rf 0.26; δH (CDCI3, 400 MHz) 0.87-0.92 (6H, 2χd, JC-2"-H,C-3»-H = 6.6 Hz and JC-2"-H,c- 4».H = 6.6 Hz, C-3"-H3, C-4"-H3), 1.01 (3H, s, C-18-H3), 1.42-2.98 (14H, m), 3.81 (1H, dd, JBA = 13.8 Hz and JC-2 »-H,C-I »-H = 7.5 Hz, N-CHΛHB), 3.86 (1H, dd, JAB = 13.8 Hz and Jc-2»-H,c- -H = 7.5 Hz, N-CHAWβ), 5.04 (2H, s, OCH2Ar), 6.71-6.75 (1H, m, C-4-H), 6.79 (1H, dd, ΛC-I-H,C-2-H = -6 Hz and C-4-H,C-2-H = 2.7 Hz, C-2-H), 6.94-6.99 (1H, m, C-5'-H), 7.22 (2H, d, Jc- c-ι- = 8.6 Hz, C-1-H) and 7.31-7.46 (5H, m, C6H5).
2'-lsofoutyl- 3-O-Benzyl-estra-l, 3, 5 (10)-trieno-(1 , 16-c)-pyra∑ole, DSF 03 1 B, was obtained as a white crystalline solid (56 mg, 19%): mp 126-128°C; TLC (DCM/EtOAc, 9:1) Rf 0.64 cf. Rf 0.26; δ H (CDCI3, 400 MHz) 0.92 (3H, d, JC-2»-H,C-3"-H = 6-6 Hz, C-3"-H3), 0.95 (3H, d, JC-2"-H,C-4»-H = 6.6 Hz, C-4"-H3), 1.02 (3H, s, C-18-H3), 1.46-2.60 (12H, m), 2.89-2.94 (2H, m, C-6-H2), 3.74 (1H, dd, JBA = 13.3 Hz and JC.2».H,C-I»-H = 7.8 Hz, N-Crτ" ΛHB), 3.85 (1H, dd, JAB = 13.3 Hz and Jc.2»-H,c-i"-H = 7.4 Hz, N~CHAHβ), 5.04 (2H, s, OCH2Ar), 6.74 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6.79 (1 H, dd, JC-I-H,C-2-H = 8.6 Hz and Jc- H.C-2-H = 2.6 Hz, C-2-H), 7.17-7.21 (2H, m, C-1-H and C-3'-H) and 7.31-7.45 (5H, m, C6H5).
1'-Methylacetate-3-0-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole and 2'- Methylacetate-3-O-Benzyl-estra-l, 3, 5 (10)-trieno-(17, 16-c)-pyrazole Following the general procedure, 3-O-benzyl-estra-1 , 3, 5 (10)-trieno-(17, 16-c)- pyrazole (300 mg, 0.78 mmol) was treated with NaH (47 mg, 1.17 mmol) and the subsequent reaction with methyl-chloro-acetate (136 μL, 1.66 mmol) was complete within 2.6 hours. Purification of the crude by flash chromatography with a slow gradient of DCM to DCM/EtOAC (8:2) as eluent gave: 1'-Methylacetate-3-0-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole, DSF 03117-2A as a white crystalline solid (173 mg, 48%): TLC (DCM/EtOAc, 9:1) R, 0.53 cf. Rf 0.09; δw (CDCI3, 400 MHz) 1.03 (3H, s, C-18- H3), 1.44-2.69 (11H, m), 2.85-2.99 (2H, m, C-6-H2), 3.76 (3H, s, OMe), 4.86 (2H, s, N- CH2), 5.04 (2H, s, OCH2Ar), 6.74 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6.79 (1H, dd, JMMf 2-H = 8.6 Hz and JC-4-H,C-2-H = 2.6 Hz, C-2-H), 7.07 (1H, s, C-5'-H), 7.22 (1H, d, JC-2-H,C-I-H = 8.6 Hz, C-1-H) and 7.29-7.44 (5H, m, C6H5); MS m/z (FAB+) 457.3 [55, (M+H)+], 91.1 [100, (CH2Ar)+], 73.0 [72]; Ace MS m/z (FAB+) 457.24954, C29H33N2O3 requires 457.24912.
2'-Wiethylacetat©-3-0-Bensyl-©stra-1, 3, 5 (10)-trieno-(1 , 16-c)-pyrasole, DSF 03117-2B, was also obtained as a pale yellow oil (70 mg, 20%): TLC (DCM/EtOAc, 9:1) Rf 0.28 cf. Rf 0.09;ϋ δ H (CDCI3, 400 MHz) 1.02 (3H, s, C-18-H3), 1.42-2.64 (11H, m), 2.86-2.98 (2H, m, C-6-H2), 3.78 (3H, s, OMe), 4.69-4.88 (2H, m, N-CH2), 5.03 (2H, s, OCH2Ar), 6.74 (1 H, d, C-2-H,C-4-H = 2.6 Hz, C-4-H), 6.78 (1 H, dd, JC.1-H,C-2-H = 8.6 Hz and Jc. 4-H,C-2-H = 2.6 Hz, C-2-H), 7.18 (1H, d, JC-2.H,C-1-H = 8.6 Hz, C-1-H), 7.25 (1H, s, C-3'-H), and 7.31-7.44 (5H, m, C6H5); MS m/z (FAB+) 457.3 [64, (M+H)+], 91.1 [100, (CH2Ar)+], 73.0 [27]; Ace MS m/z (FAB+) 457.25071 , C29H33 2O3 requires 457.24912.
1,-(2-iHieth©∑sy©thyl)-3-0-B©n2yl-estr®-1, 3, 5 (10)-tri©n©-(1 , 1@-e)-pyraε©l©, DSF 03117-3A and 2,-(2-Mettιox βthyl)-3-0-Benz l-estra-1l 3, 5 (10)-4ri®no-(17, 16-c)- pyrasole, DSF 03117-3B.
Following the general procedure, 3-O-benzyl-estra-1 , 3, 5 (10)-trieno-(1 , 16-c)- pyrazole (300 mg, 0.78 mmol) was treated with NaH (47 mg, 1.17 mmol) and the subsequent reaction with 1-chloro-2-methoxy-ethane (142 μL, 1.56 mmol) was complete within 4 hours. Purification of the crude by flash chromatography with DCM/EtOAC (9:1) as eluent gave: 1'-(2-Methoxyethyl)-3-0-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)- pyrazole, DSF 03117-3A as acreamy solid (92 mg, 27%): TLC (DCM/EtOAc, 9:1 ) Rf 0.38 cf. Rf 0.12; δH (CDCI3, 400 MHz) 1.01 (3H, s, C-18-H3), 1.46-2.66 (11H, m), 2.84- 2.99 (2H, m, C-6-H2), 3.33 (~3H, s, OMe), 3.72 (2H, dt, J= 5.3 Hz, J = 1.3 Hz, C-1"-H2 or C-2"-H2), 4.23 (2H, t, J = 5.2 Hz, C-1 "-H2 or C-2"-H2), 5.04 (2H, s, OCH2Ar), 6.74 (1 H, d, JC-2-H,c-4-H = 2.8 Hz, C-4-H), 6.80 (1H, dd, JC-1-H,C-2-H = 8.3 Hz and JC-4-H,C-2-H = 2.8 Hz, C-
2-H), 7.09 (1 H, s, C-5'-H), 7.23 (1H, d, JC-2-H,C-I-H = 8.3 Hz, C-1-H) and 7.31-7,45 (5H, m, C6HS); MS m/z (FAB+) 443.3 [100, (M+H)+], 91.1 [80, (CH2Ar)+]; Ace MS m/z (FAB+)
443.27119, C29H35N2O2 requires 443.26985.
2'-(2-Methoxyethyl)-3-0-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole DSF 03117-3B, was obtained as a creamy solid (80 mg, 23%): TLC (DCM/EtOAc, 9:1) Rf 0.29 cf. Rf 0.12; δ H (CDCI3, 400 MHz) 1.04 (3H, s, C-18-H3), 1.42-2.66 (11 H, m), 2.84- 2.99 (2H, m, C-6-H2), 3.32 (~3H, s, OMe), 3.78 (2H, m, C-1"-H2 or C-2"-H2), 4.19 (2H, dt, J= 5.7 Hz, J = 2.9 Hz, C-1"-H2 or C-2"-H2), 5.04 (2H, s, OCH2Ar), 6.74 (1 H, d, Jc-2-H,c- 4.H = 2.7 Hz, C-4-H), 6.79 (1 H, dd, JC-I-H,C-2-H = 8.6 Hz and JC-4-H,C-2-H = 2.7 Hz, C-2-H), 7.19-7.21 (2H, m, C-1-H and C-3'-H) and 7.30-7.44 (5H, m, C6H5); MS m/z (FAB+) 443.3 [100, (M+H)+], 91.1 [95, (CH2Ar)+], 73.1 [44]; Ace MS m/z (FAB+) 443.26964, C29H35N2O2 requires 443.26985.
General procedure for hydrogenation of benzylated derivatives. A suspension of Pd-C (10%) in THF (2 mL) was added to a stirred solution of the benzylated precursor in MeOHTHF (2:1 , 30 mL) and the resulting suspension hydrogenated at room temperature using a hydrogen-filled balloon. After removal of the supported catalyst by filtration and evaporation of the filtrate in vacuo, the product obtained was purified by recrystallization to give the parent compound.
1'-M©thyl-3-Hydro y-©stra-1, 3, 5 (10)-trl®no-(1 , 16-c)-pyras©le (DSF 03100, STO 785)
Figure imgf000193_0001
Following the hydrogenation conditions, a suspension of 3-O-benzyl-estra-1, 3, 5 (10)- trieno-(17, 16-c)~N-methyl-pyrazole (60 mg, 0.12 mmol) and Pd-C (20 mg) was hydrogenated overnight to give the product as a pale yellow solid (31 mg). This was recrystaUized from EtOH/H2O to give creamy crystals (26 mg, 68%): TLC (chloroform/EtOAc, 9:1) Rf 0.16 cf. Rf 0.50; δH (DMSO-d6, 400 MHz) 0.89 (3H, s, C-18- H3), 1.32-2.57 (11H, m), 2.69-2.83 (2H, m, C76-H2), 3.73 (3H, s, N-Me), 6.46 (1H, d, Jc-2- H.C-4-H = 2.4 Hz, C-4-H), 6.62 (1H, dd, JC-I-H,C-2-H = 8.6 Hz and JC-4.H,C-2-H = 2.4 Hz, C-2-H), 7.06 (1 H, d, JC-2-H,C-I-H = 8.5 Hz, C-1-H), 7.24 (1H, s, C-δ'-H) and 9.02 (1H, s, OH); MS m/z (FAB+) 309.2 [100, (M+H)+], 219.2 [52]; Ace MS m/z (FAB+) 309.19747, C20H25N2O requires 309.19669.
2'-Methyl-3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (DSF 03102,.STX 786)
Figure imgf000193_0002
Following the hydrogenation conditions, a suspension of 3-O-benzyl-estra-1 , 3, 5 (10)- trieno-(17, 16-c)~N-methyl-pyrazole (50 mg, 0.12 mmol) and Pd-C (20 mg) was hydrogenated overnight to give the product as a white solid (40 mg). This was recrystaUized from EtOH to give white crystals (20 mg, 53%): TLC (chloroform/EtOAc, 9:1) Rf 0.13 cf. Rf 0.32; δ H (DMSO-d6, 400 MHz) 0.95 (3H, s, C-18-H3), 1.32-2.52 (11H, m), 2.69-2.83 (2H, m, C-6-H2), 3.73 (3H, s, N-Me), 6.45 (1 H, d, Jc-2-H,c-4-H = 2.6 Hz, C-4- H), 6.52 (1H, dd, C-I-H,C-2-H = 8.4 Hz and JC-4-H,C-2-H = 2.6 Hz, C-2-H), 7.04 (1H, s, C-3'-H), 7,06 (1H, d, Jc-2-H,c-ι-H = 8.4 Hz, C-1-H) and 9.03 (1H, s, OH); MS m/z (FAB+) 663.5 [24], 391.3 [28], 309.2 [100, (M+H)+]; Ace MS m/z (FAB+) 309.19737, C20H25N2O requires
309.19669.
2'-lsobutyl-3-Hydr©^y-estra-1, 3, 5 (10)-trfeno-(1 , 16-c)-pyrasol© (DSF 031 3, STIC 813)
Figure imgf000194_0001
Following the hydrogenation conditions, a suspension of 3-O-benzyl-estra-1 , 3, 5 (10)- trieno-(17, 16-c)-N-isobutyl-pyrazole (45 mg, 0.10 mmol) and Pd-C (20 mg) was , hydrogenated overnight to give the product as a light yellow solid (103 mg). This was recrystaUized from EtOAc/hexane to give creamy crystals (33 mg, 92%): TLC (chloroform/EtOAc, 9:1 ) Rf 0.30 cf. R, 0.57; δ H (CDCI3, 400 MHz) 0.92 (3H, d, C-2"-H,C-3»-H = 6.6 Hz, C-3"-H3), 0.95 (3H, d,
Figure imgf000194_0002
6-6 Hz, C-4"-H3), 1.03 (3H, s, C-18-H3), 1.41-2.63 (12H, m), 2.84-2.91 (2H, m, C-6-H2), 3.77 (1H, dd, JBA = 13.4 Hz and JC-2"-H,c-r- H = 8.2 Hz,
Figure imgf000194_0003
3.89 (1H, dd, AB = 13.4 Hz and C-2»-H,C-I''-H - 7.6 Hz, N-CHArVβ), -5.50 (~1H, br s, exchanged with D2O, OH), 6.60 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6.66 (1H, dd, Jc-1-H,c-2-H = 8.4 Hz and JC.4-H,C-2-H = 2.6 Hz, C-2-H), 7.14 (1H, d, JC-2-H,c-ι-H = 8.4 Hz, C-1-H) and 7.22 (1H, s, C-5'-H); MS m/z (FAB+) 351.3 [100, (M+H)+]; Ace MS m/z (FAB+) 351.24437, C23H31N2O requires 351.24364.
1'-lsobutyl-3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (DSF 03115, STX 812).
Figure imgf000194_0004
Following the hydrogenation conditions, a suspension of 3-O-benzyl-estra-1 , 3, 5 (10)- trieno-(17, 16-c)-N-isobutyl-pyrazole (110 mg, 0.25 mmol) and Pd-C (40 mg) was hydrogenated overnight to give the product as a white solid (78 mg). This was recrystaUized from MeOH to give colourless crystals (42 mg, 48%): mp 122-123°C; TLC (chloroform/EtOAc, 9:1) Rf 0.29 cf. Rf 0.72; δH (CDCI3, 400 MHz) 0.87 (3H, d, JC-2"-H,c-3»-H = 6.4 Hz, C-3"-H3), 0.89 (3H, d, C-2»-H,C-4»-H = 6.4 Hz, C-4"-H3), 1.01 (3H, s, C-18-H3), 1.42-2.65 (12H, m), 2.81-2.95 (2H, m, C-6-H2), 3.81 (1H, dd, JBA = 13.7 Hz and Jc^-H.c-r- H = 7.4 Hz, N-CWΛHB), 3.86 (1 H, dd, JAB = 13.7 Hz and JC-2"-H,c-ι»-H = 7.4 Hz, N-CHAH8), 5.61 (1H, s, exchanged with D2O, OH), 6.59 (1 H, d, C-2-H,C-4-H = 2.6 Hz, C-4-H), 6.65 (1 H, dd, JC-I-H,C-2-H = 8.4 Hz and JC-4-H,C-2-H = 2.6 Hz, C-2-H), 6.97 (1 H, s, C-3'-H) and 7.16 (1H, d, ^C-2-H,C-I-H = 8.4 Hz, C-1-H); MS m/z (FAB+) 351.3 [100, (M+H)+]; Aec MS m/z (FAB+) 351.24485, C23H31N2O requires 351.24364. Found: C, 75.20; H, 8.79; N, 7.42. C23H30N2O.MeOH requires: C, 75.35; H, 8.96; N, 7.32.
1'-Methyiacetate-3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (DSF 03129, STX 806).
Figure imgf000195_0001
Following the hydrogenation conditions, a suspension of 3-O-benzyl-estra-1 , 3, 5.(10)- trieno-(17, 16-c)~N-methylacetate-pyrazole (90 mg, 0.20 mmol) and Pd-C (40 mg) was hydrogenated for 6 hours to give the product as a white solid (66 mg). This was recrystaUized from MeOH to give colourless crystals (49 mg, 68%): TLC (DCM/EtOAc, 9:1) Rf 0.15 cf. Rf 0.55; δ H (CDCI3, 400 MHz) 1.03 (3H, s, C-18-H3), 1.41-2.69 (11H, m), 2.81-2.96 (2H, m, C-6-H2), 3.75 (3H, s, OMe), 4.83 (1H, d, JBA = 17.6 Hz, N-CHAHB), 4.88 (1H, d, JAB = 17.6 Hz, N-CHAHβ), 5.04 (1 H, s, OH), 6.58 (1 H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H) and 6.64 (1H, dd, JC-ι-H,c-2-H = 8.4 Hz and C.4.H,C-2-H = 2.7 Hz, C-2-H), 7.07 (1H, s, C-5'-H), 7.16 (1H, d, JC-2-H,C-I-H = 8.4 Hz, C-1-H); MS m/z (FAB+) 367.3 [100, (M+H)+], 176.1 [23], 73.1 [292]; Aec MS m/z (FAB+) 367.20383, C22H27N2O3 requires 367.20217.
2,- e hylac@ta ©-3-Hydro∑sy-estra-1, 3, 5 (10)-tri©n©-(17, 16-c)-pyrasol© (DSF 03131,
Figure imgf000196_0001
Following the hydrogenation conditions, a suspension of 3-O-ben∑yl-estra-1 , 3, 5 (10)- trieno-(17, 16-c)-N-methylacetate-pyrazole (60 mg, 0.13 mmol) and Pd-C (25 mg) was hydrogenated overnight to give the product as a light brown foam (34 mg). This was purified by flash chromatography with DCM/EtOAc (8 :2) as eluent and gave the prodcut as a light yellow foam (18 mg, 37%): TLC (DCM/EtOAc, 9:1) Rf 0.18 cf. Rf 0.37;D δ H (CDCI3, 400 MHz) 1.02 (3H, s, C-18-H3), 1.42-2.64 (12H, m), 2.84-2.91 (2H, m, C-6-H2), 3.77 (3H, s, OMe), 4.81 (1 H, d, JBA = 17.4 Hz, N-CHΛHB), 4.87 (1 H, d, JAB = 17.4 Hz, N- CHAHe), 6.59 (1H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H), 6.64 (1 H, dd, JC-I-H,C-2-H = 8.6 Hz and c-4- H,c-2-H = 2.7 Hz, C-2-H), 7.12 (1 H, d, C-2-H,C-I-H = 8.6 Hz, C-1-H) and -7.25 (-1 H, s, C-3'-H, under solvent peak).
1'-(2-Methoxyethyl)-3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (DSF 03133, STX 808)
Figure imgf000196_0002
Following the hydrogenation conditions, a suspension of 3-O-benzyl-estra-1 , 3, 5 (10)- trieno-(17, 16-c)-N-(2-methoxyethyl)-pyrazole (80 mg, 0.18 mmol) and Pd-C (35 mg) was hydrogenated for 6 hours to give the product as a white crystalline solid (58 mg). This was recrystaUized from MeOH to give white crystals (32 mg, 50%): TLC (DCM/EtOAc, 9:1) Rf 0.11 cf. Rf 0.28; δ H (CDCI3, 400 MHz) 1.02 (3H, s, C-18-H3), 1.43- 2.66 (11H, m), 2.81-2.93 (2H, m, C-6-H2), 3.33 (3H, s, OMe), 3.71 (2H, td, J = 5.5 Hz, J - 1 Hz, C-1"-H2 or C-2"-H2), 4.23 (2H, app t, J - 5 Hz, C-1"-H2 or C-2"-H2), 5.57 (1 H, br s, OH), 6.59 (1 H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H), 6.64 (1 H, dd, JC-1.H,C-2-H = 8.4 Hz and Jc-4- H,C-2-H = 2.7 Hz, C-2-H), 7.09 (1H, s, C-5'-H) and 7.16 (1H, d, JC-2-HIC-I-H = 8.4 Hz, C-1-H); MS m/z (FAB+) 353.3 [100, (M+H)+]. 2'-(2-Methoxyethyl)-3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (DSF
03135, STX 809).
Figure imgf000197_0001
Following the hydrogenation conditions, a suspension of 3-O-benzyl-estra-1 , 3, 5 (10)- trieno-(17, 16-c)-N-(2-methoxyethyl)-pyrazole (70 mg, 0.16 mmol) and Pd-C (30 mg) was hydrogenated for overnight to give the product as a creamy foam (50 mg). This was purified by flash chromatography using DCM/EtOAc (8:2) as eluent and gave the product as a creamy oil (25 mg, 45%): TLC (DCM/EtOAc, 9:1) R, 0.15 cf. Rf 0.27; δH (CDCI3, 400 , MHz) 1.05 (3H, s, C-18-H3), 1.42-2.61 (12H, m), 2.82-2.95 (2H, m, C-6-H2), 3.31 (3H, s, OMe), 3.74-3.82 (2H, m, C-1"-H2 or C-2"-H2), 4.15-4.24 (2H, m, C-1"-H2 or C-2"-H2), 6.60 (1H, d, JC-2-HIC-4-H = 2.6 Hz, C-4-H), 6.66 (1H, dd, JC-I.H,C-2-H = 8.6 Hz and JC-4-H,c-2-H = 2.6 Hz, C-2-H), 7.14 (1H, d, JC-2-H,C-I-H = 8.6 Hz, C-1-H) and 7.23 (1H, s, C-3'-H); MS m/z (FAB+) 353.3 [100, (M+H)+]; Ace MS m/z (FAB+) 353.22369, C22H29N2O2 requires 353.22290.
3-Benzyloxy-16-nitrile,16-methyl-estrone (DSF03151/DSF03163)
Figure imgf000197_0002
NaH (60% dispersion in mineral oil, 1.2 eq., 0.248 mmol, 10 mg) was added to a stirred solution of 3-ben∑yloxy-16-nitrile-estrone (0.207 mmol, 80 mg) in anhydrous DMF (5 mL) at 0°C under an atmosphere of N2. After 30 minutes of stirring, methyl iodide (0.310 mmol, 19 μL) was added to the white suspension and the mixture was stirred for 3 h, in which time it was allowed to warm to room temperature. The resulting light yellow solution was then poured into water (50 mL) and the organics were extracted with EtOAc (3x20 mL), washed with brine (2x20 mL), dried (Na2SO4), filtered and evaporated in vacuo. The crude product was purified by flash chromatography with CH2CI2/hexane (3:2) as eluent to give the product as a crystalline light yellow solid (60 mg, 72%): δH (DMSO-d6, 270 MHz) 1.02 (3H, s, C-18-H3), 1.25-2.54 (11H, m), 1.46 (~3H, s, C-16-H3), 2.79-2.83 (2H, m, C-6-H2), 5.06 (2H, s, OCH2Ar), 6.73-6.80 (2H, m, C-4-H and C-2-H), 7.18 (1H, d, JC-2.H, C-I-H = 8.4 Hz, C-1-H), 7.29-7.46 (5H, m, C6H5); Found: C, 80.80; H, 7.45; N, 3.46. C27H2gN02 requires: C, 81.17; H, 7.32; N, 3.51.
16-Mitrile,16-m©thyl-©stron© (DSF0317O, STX924)
Figure imgf000198_0001
Pd/C (10%, 40 mg) was added to a stirred solution of 3-benzyloxy-16-nitrile,16-methyl- estrone (90 mg, 0.225 mmol) in a mixture of MeOH/THF (1:1, 40 mL). The resulting suspension was hydrogenated at room temperature overnight using a hydrogen-filled balloon. The final mixture was then filtered, the filter cake washed several times with boiling MeOH and THF, and concentrated in vacuo to give a crude white foam. This was purified by flash chromatography with CH2CI2/EtOAc (9:1) as eluent to give the product as a white foam (46 mg, 66%). A sample was then triturated in hexane to give a creamy powder: δH (CDCI3, 270 MHz) 1.12 (3H, s, C-18-H3), 1.32-2.48 (11H, m), 1.51 (-3H, s, C- 16-H3), 2.82-2.92 (2H, m, C-6-H2), 4.55 (1H, s, C-3-OH), 6.73-6.58 (1H, app d, C-4-H), 6.63 (1 H, dd, C-I-H, C-2-H = 8.5 Hz, Jc.4-H, C-2-H = 2.5 Hz, C-2-H), 7.13 (1 H, d, Jc.2-H, C-I-H = 8.5 Hz, C-1-H); MS m/z (FAB+) 309.1 [60, M+], 97.1 [50], 83.0 [65]; Ace MS m/z (FAB+) 309.12761, C2oH23NO2 requires 309.12788. HPLC (methanol/water, 80:20, λmax = 280.5 nm) Rt = 2.53 min, 98%.
1'-Propionitrile-3-0-ferf-Butyl-dimethylsilyf-estra-1, 3, 5 (10)-trieno-(17, 6-c)- pyrazole (DSF03145A)
Figure imgf000198_0002
KOC(CH3)3 (0.807 mmol, 91 mg) was added to a stirred solution of 3-O-ter -l dimethylsilyl-estra-1 , 3, 5 (10)-trieno-(17, 16-c)-pyrazole (0.734 mmol, 300 mg) in anhydrous THF (10 mL) at 0°C under an_afmosphere of N2. After 20 minutes of stirring, acrylonitrile (0.881 mmol, 58 μL) was added and the resulting bright orange solution was stirred for 5 h, in which time at room temperature. The resulting dark orange cloudy mixture was then concentrated in vacuo and water (50 mL) was added. The organics were extracted with EtOAc (2 50 mL), washed with water (2 30 mL), then brine (2*30 mL), dried (Na2SO4), filtered and evaporated in vacuo. The crude product was purified by flash chromatography with CH2CI2/EtOAc (9:1) as eluent to give two products, one of which was DSF03145A, as a light yellow solid (29 mg, 8%): δH (CDCI3, 400 MHz) 0.20 (6H, s, Si(CH3)2), 0.98 (9H, s, C(CH3)3), 1.02 (3H, s, C-18-H3), 1.42-2.67 (11 H, m), 2.82- 2.91 (4H, m, C-6-H2 and CH2CN), 4.28-4.40 (2H, m, NCH2), 6.57 (1H, d, JC-2-H, C-4-H = 2.6 Hz), 6.63 (1 H, dd, JG-I-H, C-2-H = 8.5 Hz, JC-4-H, C-2-H = 2.6 Hz, C-2-H), 7.10 (1H, s, C-5'-H), 7.14 (1H, d, C-2-H, C-I-H = 8.5 Hz, C-1-H).
3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-N-propionitrile-pyrazole (DSF03153, STX921).
Figure imgf000199_0001
TBAF (0.110 mmol, 0.11 mL) was added to a stirred solution of 3-TBDMS-N-propionitrile
(25 mg, 0.050 mmol) in anhydrous THF (5 mL) at room temperature under an atmosphere of N2. The mixture was stirred for 3 h at room temperature. The solvent was then removed in vacuo and water added (60 mL).The organics were extracted with EtOAc (50 mL + 20 L), washed with water (20 mL), then brine (20 mL), dried (Na2SO4), filtered and evaporated in vacuo. The crude yellow oil was purified by flash chromatography with CH2CI2/EtOAc (8:2) as eluent to give a light yellow oil (12 mg, 63%). This was then precipitated from EtOAc/Hexane to give a light yellow powder (8 mg): δH (CDCI3, 400 MHz) 0.94 (3H, s, C-18-H3), 1.34-2.60 (H, m), 2.76-2.86 (4H, m, C- 6-H2 and CH2CN), 4.19-4.31 (2H, m, NCH2), 4.92 (1 H, br s, exchanged with D2O, C-3- OH), 6.50 (1H, d, C-2-H, C-4-H = 2.7 Hz), 6.55 (1H, dd, Jc.^, C-2-H = 8.4 Hz, JG-4 H. C-2-H = 2.7 Hz, C-2-H), 7.02 (1H, s, C-5'-H), 7.08 (1 H, d, JC-2-H, C-I-H = 8.4 Hz, C-1-H); MS m/z (APCI) 348.3 [32, (M+H)+], 141.0 [20]. HPLC (methanol/water, 90:10, λma.t = 229.8 nm) Rt = 2.27 min, 96%.
16-(2',2'-Dimethyl-pr©pyl«εten©)-estradi©l (ST2 948)
Figure imgf000200_0001
A solution of NaBH4 (44 mg, 1.16 mmol) in H2O (2.5 mL) was added to a stirred solution of STX748 in a mixture of MeOHTHF (2:1 , 15 mL) at 0°C. The colourless solution was i0 stirred for 1 hour at 0°C, after which AcOH glacial (4 drops) was added, followed by 10% aq. NaCI solution (15 mL). The resulting white precipitate was filtered, dried (79 mg) and recrystallised from EtOAc/Hexane (1 :8) to give 68 as white crystals (63 mg, 79%): mp 219-222°C; IR (KBr) 3550, 3410 (OH), 2960-2875 (aliph CH), 1610, 1506 (aliph C=C and arom C=C) cm;1; δH (CDCI3, 400 MHz) 0:65 (3H, s, C-18-.H3), 1.10 (9H, s, C(CH3)3),
15 1.18-2.59 (12H, m), 2.82-2.88 (2H, m, C-6-H2), 3.85-3.90 (1H, app d, J = 9.4 Hz, C-17- H), 4.60 (1H, s, exchanged with D2O, OH), 5.46 (1H, dd, J = 4.9 Hz, J = 2.5 Hz, C-1'-H), 6.56 (1H, d, J = 2.8 Hz, C-4-H), 6.61 (1H, dd, J = 8.5 Hz, J = 2.8 Hz, C-2-H) and 7.14 (1H, d, J = 8.5 Hz, C-1-H);δc (CDCI3, 100.4 MHz) 11.4 (q, C-18), 26.8 (t), 27.8 (t), 29.1 (t), 30.0 (t), 30.8 (3xq, C(CH3)3), 33.5 (s, C(CH3)3), 36.8 (t), 38.7 (d), 42.9 (s, C-13), 44.4
20 (d), 47.5 (d), 85.0 (d, C-17), 113.0 (d), 115.5 (d), 126.7 (d), 132.7 (s), 133.7 (d, C-1'), 138.3 (s), 140.2 (s) and 153.6 (s, C-3); MS m/z (FAB+) 340.2 [49, M+], 323.2 [100, (M- OH)+], 283.2 [92, (M-C(CH3)3)+] ; Aec MS m/z (FAB+) 340.2390, C23H32O2 requires 340.2402. HPLC (MeOH/H2O, 96:4, λmax = 280.5 nm) Rt = 2.54 min, 99.5%. Found: C, 81.00; H, 9.54. C^Oa requires: C, 81.13; H, 9.47%.
25
Synthesis of N-propionitrile f17-16-c -pyrazole derivatives
3-0- ef -Butyl-dim©thylsilyl-estrøne (1)
Figure imgf000200_0002
Imidazole (6.3 g, 92.4 mmol) and tert-butyl-dimethylsilyl chloride (7.8 g, 51.8 mmol) were added to a stirred solution of E1 (10.0 g, 37.0 mmol) in anhydrous DMF (300 mL) at room temperature, under an atmosphere of N2. After stirring overnight, the resulting white suspension was poured into H2O (500 mL). The organics were extracted with EtOAc (500 mL + 300 mL), washed with H2O (2x200 mL), then brine (2x200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was recrystallised from EtOH to give 1 as white needles (14.0 g, 98%): mp 171-173°C [lit.{Fevig, 1987} (EtOH) 170-172°C]; IR (KBr) 2960-2855 (aliph CH), 1730 (C=O), 1605, 1495 (arom C=C) cm"1; δH (CDCI3, 400 MHz) 0.19 (6H, s, Si(CH3)2), 0.91 (3H, s, C-18- H3), 0.98 (9H, s, C(CH3)3), 1.37-2.55 (13H, m), 2.81-2.89 (2H, m, C-6-H2), 6.57 (1H, d, J = 2.6, Hz, C-4-H), 6.62 (1H, dd, J = 8.3 Hz, J = 2.6 Hz, C-2-H) and 7.12 (1H, d, J = 8.3 Hz, C-1-H); MS m/z (FAB+) 384.2 [100, M+], 327.2 [44, (M-C(CH3)3)+], 73.0 [44]; Aec MS m/z (FAB+) 384.2477, C24H36O2Si requires 384.2485.
3-0-fert-Butyl-dimethylsilyl-16-hydroxymethylene-estrone (2)
Figure imgf000201_0001
NaOMe (4.8 g, 70.2 mmol) was added portionwise to a stirred solution of 1 (9 g, 23.4 mmol) in anhydrous toluene (250 mL) at room temperature, under an atmosphere of N2. After stirring for 20 minutes, ethyl formate (13.2 mL, 164 mmol) was added and the resulting bright yellow solution was stirred overnight. The final thick yellow suspension was poured into H2O (300 mL) and acidified with 5M HCl. The organics were extracted with EtOAc (2x500 mL), washed with H2O (3x200 mL), then brine (2x200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give an off-white crude product (9.49 g, 98%). For analysis a sample was recrystallised from EtOH to give 2 as white crystals: mp 168-171 °C; IR (KBr) 2930-2855 (aliph CH), 1710 (C=O), 1695-1600, 1495 (C=C and arom C=C) cm'1; δH (DMSO-d6, 400 MHz) 0.17 (6H, s, Si(CH3)2), 0.83 (3H, s, C-18-H3), 0.96 (9H, s, C(CH3)3), 1.32-2.63 (11H, m), 2.77-2.85 (2H, m, C-6-H2), 6.55 (1 H, d, J = 2.6 Hz, C-4-H), 6.61 (1H, dd, J = 8.3 Hz, J = 2.6 Hz, C-2-H), 7.14 (1H, d, J = 8.3 Hz, C-1-H), 7.40 (1H, s, =CHOH) and 10.69 (1H, s, exchanged with D2O, =CHOW); δc (DMSO-d6,100.4 MHz) -4.3 (2χq, Si(CH3)2), 14.5 (q, C-18), 18.0 (s, C(CH3)3), 24.1 (t), 25.6 (t and 3xq, C(CH3)3), 26.2 (t), 29.0 (t), 31.5 (t), 37.3 (d), 43.6 (d), 47.7 (s, C-13), 48.4 (d), 113.0 (s, C-16), 116.9 (d), 119.4 (d), 126.9 (d), 132.5 (s), 137.3 (s), 149.9 (d, C-1 '), 152.5 (s, C-3) and 208.2 (s, C=O); MS m/z (FAB+) 413.2 [68, (M+H)+], 355.1 [33, (M-C(CH3)3)+], 72.9 [100]; Aec MS m/z (FAB+) 413.2492, C25H37O3Si requires 413.2512. Found: C, 72.77; H, 8.79. C25H36O3Si requires: C, 72.40; H, 8.90%.
3-O-f©f1-Butyl-dimethylsilyl-©stra-1,3,5(10)-tri©n®-[17,16-c3-pyrasol© (3)
Figure imgf000202_0001
Hydrazine hydrate (440 μL, 9.1 mmol) was added to a suspension of 2 (2.5 g, 6.1 mmol) in EtOH (125 mL) at room temperature, under an atmosphere of N2. The resulting mixture was heated to reflux for 45 minutes. After cooling down, the solvent was removed under reduced pressure, H2O (200 mL) added and the mixture acidified with δM HCl. The organics were extracted with EtOAc (2*10 mL), washed with H2O (2x50 mL), then brine (2x50 mL), dried (Na2SO4), filtered and concentrated under reduced
, pressure to give a yellow foam (2.9 g). This was recrystallised from EtOH/H2O to give 3 as yellow crystals (2.0 g, 81%): mp 122-124°C; IR (KBr) 3190 (arom CH), 2930-2855 (aliph CH), 1605-1496 (C=N and arom C=C) cm"1; δH (DMSO-d6, 400 MHz) 0.18 (6H, s, Si(CH3)2), 0.95 (3H, s, C-18-H3), 0.96 (9H, s, C(CH3)3), 1.36-2.61 (11H, m), 2.79-2.87 (2H, m, C-6-H2), 6.55 (1H, d, J = 2.3 Hz, C-4-H), 6.62 (1H, dd, J = 8.5 Hz, J = 2.3 Hz, C- 2-H), 7.16 (1H, d, J = 8.5 Hz, C-1 -H), 7.28 (1H, s, C-5'-H) and 12.02 (1H, s, exchanged with D2O, NH); δc (CDCI3, 100.4 MHz) -3.8 (2xq, Si(CH3)2), 18.6 (s, C(CH3)3), 18.8 (q, C- 18), 24.3 (t), 26.2 (3xq, C(CH3)3), 26.6 (t), 27.9 (t), 30.0 (t), 34.5 (t), 38.0 (d), 41.1 (s, C- 13), 44.9 (d), 61.9 (d), 117.4 (d), 120.2 (d), 121.8 (s), 123.2 (s), 126.2 (d), 133.2 (s), 137.8 (s), 153.5 (s) and 168.6 (s); MS m/z (FAB+) 409.2 [100, (M+H)+], 73.0 [33]; Ace MS m/z (FAB+) 409.2662, C25H37N2OSi requires 409.2675. Found: C, 71.40; H, 8.93; N, 6.53. C25H36N2θSi.(H2θ)2/3 requires: C, 71.38; H, 8.95; N, 6.66%.
3-O-ferf-Butyl-dimethylsilyl-estra-1.3.5(10)-triene-ri7.16-cT-(15-propionitrile)- pyrazole (4) and 3-O-teft-Butyl-dimethylsilyl-estra-1.3.5(10)-triene-f17.16-c1-(2'- propionitrile)-pyragole (5)
'BuOK (91 mg, 807 μmol) was added to a stirred solution of 3 (300 mg, 734 μmol) in anhydrous THF (10 mL) at 0°C, under an atmosphere of N2. After 20 minutes of stirring, acrylonitrile (58 μL, 881 μmol) was added and the resulting bright orange solution was stirred for δ hours at room temperature. The resulting dark orange mixture was then concentrated under reduced pressure and H2O (50 mL) was added. The organics were extracted with EtOAc (2*50 mL), washed with H2O (2*30 mL), then brine (2*30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM/EtOAc, 9:1) to give two products:
Figure imgf000203_0001
The less polar fraction gave 4 as a light yellow oil (29 mg, 8%): IR (KBr) 2930-2860 (aliph CH), 2250 (CN), 1640-1495 (C=N and arom C=C), 1250 (C-O or Si(CH3)2) cm"1; δH (CDCI3, 400 MHz) '0.20 (6H, s, Si(CH3)2), 0.98 (9H, s, C(CH3)3), 1.02 (3H, s, C-18-H3), 1.42-2.67 (11H, m), 2.82-2.91 (4H, m, C-6-H2 and CH2CN), 4.28-4.40 (2H, m, N-CH2), 6.57 (1H, d, J = 2.6 Hz, C-4-H), 6.63 (1H, dd, J = 8.5 Hz, J = 2.6 Hz, C-2-H), 7.10 (1 H, s, C-5'-H) and 7.14 (1H, d, J = 8.5 Hz, C-1-H); MS m/z (FAB+) 462.2 [100, (M+H)+], 73.0 [55]; Aec MS m/z (FAB+) 462.2914, C28H40N3OSi requires 462,2941.
Figure imgf000203_0002
The more polar fraction gave 5 as a light brown oil that crystallises on standing (42 mg, 12%): mp 80-82°C; IR (KBr) 2930-2855 (aliph CH), 2255 (CN), 1705-1445 (C=N and arom C=C), 1285, 1250 (C-O or Si(CH3)2) cm"1; δH (CDCI3, 400 MHz) 0.20 (6H, s, Si(CH3)2), 0.98 (9H, s, C(CH3)3), 1.07 (3H, s, C-18-H3), 1.41-2.91 (13H, m), 2.99 (2H, t, J = 6.6 Hz, CH2CN), 4.21-4.38 (2H, m, N-CH2), 6.67 (1H, d, J = 2.4 Hz, C-4-H), 6.63 (1 H, dd, J = 8.7 Hz, J = 2.4 Hz, C-2-H), 7.11 (1 H, d, J = 8.7 Hz, C-1-H) and 7.25 (1 H, s, C-5'- H); MS m/z (FAB+) 462.1 [100, (M+H)+], 73.0 [79]; Aec MS m/z (FAB+) 462.2922, C28H40N3OSi requires 462.2941.
3-Hydroxy-estra-1 ,3,5(10)-triene-[1 , -c]-(1 '-propionitrile)-pyra2ole (ST1C921 )
Figure imgf000204_0001
A 1.0 M solution of TBAF in anhydrous THF (110 μL, 110 μmol) was added to a stirred solution of 4 (25 mg, 50 μmol) in anhydrous THF (5 mL) at room temperature, under an 5 atmosphere of N2. The mixture was stirred for 3 hours at room temperature, after which the solvent was removed under reduced pressure and H2O added (50 mL). The organics were extracted with EtOAc (70 mL), washed with H2O (20 mL), then brine (20 nL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude yellow oil was purified by flash chromatography (DCM/EtOAc, 8:2) to give STX921 as a light yellow oil 0 (12 mg, 63%). This was then triturated with EtOAc/hexane to give a light yellow powder
(8 mg): mp 92-94°C; lR (KBr) 3230 (OH), 3095 (arom CH), 2925-2860 (aliph CH), 2265
(CN), 1665-1500 (C=N and arom C=C), 1245 (C-O) cm"1; δH (CDCI3> 400 MHz) 0.94 (3H,
, , s, C-I8-H3), 1.34-2.60 (11H, m), 2.76-2.86 (4H, m, C-6-H2 and CH2CN), 4.19-4.31 (2H, m, N-CH2), 4.92 (1H, br s, exchanged with D2O, OH), 6,50 (1H, d, J = 2.7 Hz, C-4-H), 5 6.55 (1H, dd, J = 8.4 Hz, J = 2.7 Hz, C-2-H), 7.02 (1H, s, C-5'-H) and 7.08 (1H, d, J = 8.4 Hz, C-1-H); MS m/z (FAB+) 348.1 [90, (M+H)+], 147.1 [64], 73.0 [100]; Aec MS m/z (FAB+) 348.2072, C22H26N3O requires 348.2076. HPLC (MeOH/H2O, 90:10, λmax = 229.8 nm) Rt = 2.27 min, 96%.
0 3-Hydroxy-estra-1 ,3,5(10)-triene-[17, 16-c]-(2'-propionitrile)-pyrazole N
Figure imgf000204_0002
Method A: A 1.0 M solution of TBAF in anhydrous THF (150 μL, 150 μmol) was added to a stirred solution of 5 (35 mg, 76 μmol) in anhydrous THF (5 mL) at room temperature, under an atmosphere of N2. The mixture was stirred for 3 hours at room 5 temperature, after which the solvent was removed under reduced pressure and H2O added (50 mL). The organics were extracted with EtOAc (70 mL), washed with H2O (20 mL), then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude brown oil was purified by flash chromatography (DCM/EtOAc, 8:2) to give 3-Hydroxy-estra-1 ,3,5(10)-triene-[17,16-c]-(2'-propionitrile)-pyrazole as a light brown oil (10 mg, 38%).
ethod S3: A 1.0 M solution of TBAF in anhydrous THF (916 μL, 916 μmol) was added to a stirred solution of 6 (220 mg, 458 μmol) in anhydrous THF (20 mL) at room temperature, under an atmosphere of N2. The mixture was stirred overnight at room temperature after which the solvent was removed under reduced pressure and H2O added (50 mL). The organics were extracted with EtOAc (2*50 mL), washed with H2O (2*30 mL), then brine (2*30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (EtOAe/CHCI3, 9:1) to give 3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(2'-propionitrile)- pyrazole as a pale pink foam (136 mg, 86%). This was then triturated with EtOAc/Hexarie to give white crystals (99 mg, 62%): mp 198-200°C; IR (KBr) 3160 (br, OH), 2925-2860 (aliph CH), 2250 (CN), 1610-1500 (arom C=C) cm"1; δH (CDCIg, 400 MHz) 1.06 (3H, s, C-18-H3), 1.41-2.63 (11H, m), 2.82-2.91 (2H, m, C-6-H2), 2.99 (2H, app td, J = 6.8 Hz, J = 3.5 Hz, CH2CN), 4.22-4.34 (2H, m, N-CH2), 5.42 (1H, br s, exchanged with D2O, OH), 6.57 (1H, d, J = 2:4 Hz, C-4-H), 6.64 (1H, dd, J = 8.3 Hz, J = 2.4 Hz, C-2-H), 7.12 (1H, d, J = 8.3 Hz, C-1-H) and 7.25 (1H, s, C-δ'-H); δc (CDCI3, 100.4 MHz) 18.6 (q, C-18), 19.7 (t, CH2CN), 24.5 (t), 26.5 (t), 27.6 (t), 29.8 (t), 34.8 (t), 37.7 (d), 42.4 (s, C-13), 44.4 (d), 45.9 (t, N-CH2), 62.2 (d), 113.1 (d), 115.7 (d), 117.4 (s), 124.3 (s), 126.4 (d), 132.0 (s), 135.2 (d), 138.2 (s), 154.1 (s) and 157.98 (s); MS m/z (FAB+) 348.1 [100, (M+H)+], 147.0 [50], 85.1 [75], 73.0 [94]; Ace MS m/z (FAB+) 348.2060, C22H26N3O requires 348.2076. Found: C, 75.90; H, 7.17; N, 12.10. C22H25N3O requires: C, 76.05; H, 7.25; N, 12.09%.
3-0-fert-Butyl-dimethylsilyl-estra-1, 3,5(10)-triene-[17,16-c]-(2'-propionitrile-3',4'- dihydro)-pyrazol-3'-ol (6)
Figure imgf000205_0001
Cyanoethylhydrazine (177 μL, 2.18 mmol) was added to a stirred solution of 2 (600 mg, 1.45 mmol) in EtOH (50 mL) under an atmosphere of N2. After 4 hours of stirring at room temperature, the resulting orange mixture was concentrated under reduced pressure and H2O (100 mL) added, followed by 5M HCl. The organics were extracted with EtOAc (2*100 mL), washed with H2O (50 mL), then brine (50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (CHCI3/EtOAc, 7:3) to give 6 as a pale yellow solid (510 mg, 73%). This 5 was recrystallised from EtOH to give white needles (240 mg, 34%): IR (KBr) 3380 (OH), 2950-2860 (aliph CH), 2260 (CN), 1610, 1495 (C=N and arom C=C) cnrϊ1; δH (DMSO-d6, 400 MHz) 0.15 (6H, s, Si(CH3)2), 0.88 (3H, s, C-18-H3), 0.93 (9H, s, C(CH3)3), 1.19-3.16 (11H, m), 2.70-3.16 (7H, m), 5.97 (1H, s, exchanged with D2O, OH), 6.48 (1H, d, J = 2.7 Hz, C-4-H), 6.56 (1H, dd, J = 8.2 Hz, J = 2.7 Hz, C-2-H), 6.74 (1H, d, J = 1.9 Hz, C-5'-H) 0 and 7.10 (1H, d, J = 8.2 Hz, C-1-H);δc (CDCI3, 100.4 MHz) -4.4 (2xq, Si(CH3)2'), 15.6 (q, C-18), 17.1 (t, CH2CN), 18.0 (s, C(CH3)3), 25.6 (3xq, C(CH3)3), 25.8 (t), 27.0 (t), 29.1 (t), 29.4 (t), 31.4 (t), 38.6 (d), 43.2 (d), 43.9 (t, N-CH2), 47.1 (s, C-13), 49.5 (d), 56.7 (d), 104.2 (s), 116.8 (d), 119.4 (d), 119.9 (s, CN), 126.0 (d), 132.5 (s), 137.3 (s), 144.6 (s) and 152.4 (s); MS m/z (FAB+) 480.1 [100, (M+H)+], 462.1 [63, (M+H-H2O)+], 72.9 [77]; 5 MS m/z (FAB-) 632.3 [32, (M+NBA)"]; Aec MS m/z (FAB+) 480.3038 C28H42N3O2Si
, , requires 480.3046.
Synthesis of 16-hydroxymethylene derivatives and corresponding 5'-alkylated [17.16-c]-py razoles 0
3-Benzyloxy-16-( 1 '-hydroxy-ethylidene)-estrone (99
Figure imgf000206_0001
'BuOK (438 mg, 4.14 mmol) was added to a stirred solution of 3-benzyloxy-estrone (500 mg, 1.38 mmol) in anhydrous toluene (6 mL) and anhydrous DMSO (1.5 mL) at 0°C, 5 under an atmosphere of N2. Ethyl acetate (1.27 mL, 17.94 mmol) was then added and the mixture was heated to reflux for 1 hour. After cooling down, the resulting brown solution was poured into H2O (200 mL) and acidified with 5M HCl. The organics were extracted with EtOAc (2*150 mL), washed with H2O (2*100 mL), then brine (2*100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product 0 was purified by flash chromatography (DCM) to give 99 as a light yellow crystalline solid (447 mg, 80%). This was recrystallised from EtOH to give yellow crystals (351 mg, 63%): mp 126-128°C; IR (KBr) 2930-2860 (aliph CH), 1660 (C=O), 1615-1465 (C=C and arom C=C) cm"1; δH (CDCI3, 400 MHz) 0.83, 0.95 (3H, 2*s, C-18-H3), 1.38-2.49 (11H, m), 1.97, 2.37 (3H, 2*s, C-2'-H3), 2.87-2.92 (2H, m, C-6-H2), 3.43 (app dd, J = 9,4 Hz, J = 8.2 Hz, C-16-H), 5.02 (2H, s, OCH2Ar), 6.71 (1H, d, J = 2.8 Hz, C-4-H), 6.77 (1H, dd, J = 8.3 Hz, J = 2.8 Hz, C-2-H), 7.17 (1H, d, J =-8.3 Hz, C-1-H), 7.2S-.42 (5H, m, C6Hg) and 12.91 (br s, exchanged with D2O, C-1'-OH); MS m/z (FAB+) 403.1 [68, (M+H)+], 91.0 [100, (CH2Ar)+]; Aec MS m/z (FAB+) 403.2246, C27H31O3 requires 403.2273. Found: C, 80.20; H, 7.56. C27H30O3 requires: C, 80.56; H, 7.51%.
3-0-#e/,f-Butyl-dimethylsilyl-16-(1'-hydroxy-2',2,,2'-trifluoro-ethylidene)-estrone (100)
Figure imgf000207_0001
'BuOK (875 mg, 7.80 mmol) was added to a stirred solution of 1 (1.0 g, 2.60 mmol) in anhydrous toluene (20 mL) at 0°C, under an atmosphere of N2. After 20 minutes of stirring, ethyl trifluoroacetate (2.16 mL, 18.2 mmol) was added and the mixture heated to reflux for 2 hours. After cooling down, the resulting dark orange suspension was poured into H2O (50 mL) and acidified with 5M HCl. The organics were extracted with EtOAc (2*50 mL), washed with H2O (2*30 mL), then brine (2*30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude orange oil that crystallises on standing was used without further purification (1.4 g, quant.). An analytical sample was recrystallised from EtOH to give 100 as light yellow crystals: mp 145-146°C; IR (KBr) 2930-2860 (aliph CH), 1690 (C=O), 1640-1495 (C=C and arom C=C) cm"1; δH (CDCI3, 400 MHz) 0.19 (6H, s, Si(CH3)2), 0.98 (9H, s, C(CH3)3), 0.99 (3H, s, C-18-H3), 1.42-2.90 (13H, m), 6.57 (1H, d, J - 2.6 Hz, C-4-H), 6.63 (1H, dd, J = 8.4 Hz, J = 2.6 Hz, C-2-H) and 7.11 (1H, d, J = 8.4 Hz, C-1-H), C-V-OH not seen; δc (CDCI3, 100.4 MHz) -4.4 (2χq, Si(CH3)2), 14.8 (q, C-18), 18.1 (s, C(CH3)3), 25.2 (t), 25.6 (t), 25.7 (3xq, C(CH3)3), 26.7 (t), 29.3 (t), 31.0 (t), 37.5 (d), 43.9 (d), 48.5 (s, C-13), 49.5 (d), 110.9 (s, C-16), 117.4 (d), - 119 (app d, J = 272 Hz, CF3), 120.0 (d), 126.0 (d), 132.0 (s), 137.4 (s), 153.6 (s, C-3), - 154 (app d, J = 38 Hz, C-1') and 216.0 (s, C=O); MS m/z (FAB+) 480.1 [47, M+], 423.0 [38, (M-F3)+], 73.0 [100]; Aec MS m/z (FAB+) 480.2296, C26H35F3O3Si requires 480.2308. Found: C, 64.90; H, 7.13. C26H35F3O3Si requires: C, 64.97; H, 7.34%.
3-0-tert-Butyl-dimethylsilyl-16-(1'-hydroxy-1"-pyridin-3"-ylmethylene)-estrone (101)
Figure imgf000208_0001
'BuOK (875 mg, 7.80 mmol) was added to a sfirred solution of 1 (1.0 g, 2.60 mmol) in anhydrous toluene (30 mL) at 0°C, under an atmosphere of N2. After 20 minutes of stirring, ethyl nicotinate (2.48 mL, 18.2 mmol) was added and the mixture was heated to reflux for 1.5 hours. After cooling down, the resulting dark red suspension was poured into H2O (200 mL) and acidified with 5M HCl. The organics were extracted with EtOAc (2*100 mL), washed with H2O (100 mL), then brine (2*100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude yellow solid was recrystallised from EtOH to give' 101 as white flaky crystals (985 mg, 77%): mp 169-170°C; IR (KBr) 2930-2855 (aliph CH), 1660 (C=O), 1605, 1495 (C=C and arom C=C) cm-1; δH (CDCI3, 400 MHz) 0.21 (6H, s, Si(CH3)2), 0.99 (9H, s, C(CH3)3), 1.11 (3H, s, C-18-H3), 1.38-2.77 (11 H, m), 2.81-2.95 (2H, m, C-6-H2), 6.58 (1H, d, J = 2.6 Hz, C-4-H), 6.63 (1H, dd, J = 8.5 Hz, J = 2.6 Hz, C-2-H), 7.12 (1H, d, J = 8.5 Hz, C-1-H), 7.41 (1 H, ddd, J = 8.1 Hz, J = 4.9 Hz, J = 0.8 Hz, C-5"-H), 8.09 (1H, app dt, J = 8.1 Hz, J = 2.0 Hzi C-4"-H), 8.69 (1H, dd, J = 4.9 Hz, J = 1.9 Hz, C-6"-H), 8.97 (1H, app dd, J = 2.1 Hz, J = 0.8 Hz, C-2"- H) and 13.63 (1H, s, exchanged with D2O, C-1'-OH); MS m/z (FAB+) 490.1 [100, (M+H)+]; Ace MS m/z (FAB+) 490.2776, C30H40NO3Si requires 490.2777. Found: C, 64.90; H, 7.13. C26H35F3O3Si requires: C, 64.97; H, 7.34%.
16-(1 '-hydroxy-ethy lidene)-estrone (102)
Figure imgf000208_0002
Following method 2, a suspension of 99 (200 mg, 497 μmol) and Pd-C (10%o, 80 mg) in MeOH/THF 2:1 (30 mL) was hydrogenated for 2 hours to give 102 as a white solid (98 mg, 63%). This was recrystallised from acetone/hexane to give white crystals (72 mg, 46%): mp 211-214°C; IR (KBr) 3450 (OH), 2915-2860 (aliph CH), 1705 (C=O), 1655- 1510 (C=O, C=C and arom C=C) cm-1; δH (CDCI3, 400 MHz) 0.83, 0.96 (3H, 2*s, C-18- H3), 1.39-2.40 (11H, m), 1.98, 2.37 (3H, 2*s, C-2'-H3), 2.83-2.89 (2H, m, C-6-H2), 3.43 (dd, J = 9.4 Hz, J = 8.2 Hz, C-16-H), 4.70 (br s, exchanged with D2O, OH), 6.55-6.57 (1 H, m, C-4-H), 6.62 (1 H, dd, J = 8.0 Hz, J = 2.7 Hz, C-2-H) and 7.11 (1 H, d, J = 8.0 Hz, C-1-H), 1*OH not seen; MS m/z (FAB+) 313.1 [100, (M+H)+], 73.0 [19]; Aec MS m/z (FAB+) 313.1799, C20H25O3 requires 313.1804. Found: C, 76.60; H, 7.82. C20H24O3 requires: C, 76.89; H, 7.74%.
16-(1'-hydroxy-2, l2,,2>-trifluoro-ethylidene)-®stron® (103, ST2 946)
Figure imgf000209_0001
A 1.Q M solution of TBAF in anhydrous THF (4.90 mL, 4.90 mmol) was added to a stirred solution of 100 (1.18 g, 2.45 mmol, crude product) in anhydrous THF (50 mL) under an atmosphere of N2. The resulting light brown solution was stirred at room temperature for 4 hours, after which the solvent was removed under reduced pressure and H2O added (100 mL). The organics were extracted with EtOAc (2*100 mL), washed with H2O (2*80 mL), then brine (2*80 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude brown oil was purified by flash chromatography (DCM) to give 103 as a light brown oil (535 mg, 60%). This was crystallised from cold DCM to give white crystals (249 mg, 28%): mp 91-93°C; IR (KBr) 3430 (OH), 2930-2860 (aliph CH), 1685 (C=O), 1610, 1500 (C=C and arom C=C) cm"1; δH (CDCI3, 400 MHz) 1.02 (3H, s, C-18- H3), 1.43-2.91 (13H, m), 6.58 (1H, d, J = 2.7 Hz, C-4-H), 6.64 (1H, dd, J = 8.4 Hz, J = 2.7 Hz, C-2-H) and 7.14 (1H, d, J = 8.4 Hz, C-1-H), C-3-OH and C-1 '-OH not seen; MS m/z (FAB+) 366.1 [100, M+]; Aec MS m/z (FAB+) 366.1476, C20H21F3O3 requires 366.1443. HPLC (MeOH/H2O, 96:4, λmax = 280.5 nm) Rt = 1.66 min, 100%. Found: C, 57.60; H, 5.37. C2oH21F3O3.(CH2CI2)3M requires: C, 57.95; H, 5.27%.
16-(1 '-hydroxy-1 "-pyridin-3"-ylmethylene)-estrone (104)
Figure imgf000209_0002
A 1.0 M solution of TBAF in anhydrous THF (1.43 mL, 1.43 mmol) was added to a stirred solution of 101 (350 mg, 716 μmol) in anhydrous THF (10 mL) under an atmosphere of N2. The resulting light brown solution was stirred at room temperature for 3 hours, after which the solvent was removed under reduced pressure and H2O added (100 mL), followed by a few drops of AcOH glacial. The organics were extracted with EtOAc (100 mL), Et2O (100 mL) and CHCI3 (100 mL), washed with H2O (100 mL), then brine (100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was triturated with boiling EtOH to give 104 as a pale yellow solid (125 mg, 50%): mp 275-278°C; IR (KBr) 2950-2855 (aliph CH), 1645 (OO), 1610-1500 (C=C and arom C=C) cm"1; δH (C'DCI3, 400 MH∑) 1.09 (3H, s, C-18-H3), 1.39-2.76 (11 H, m), 2.85- 2.91 (2H, m, C-6-H2), 5.48 (1H, s, exchanged with D2O, C-3-OH), 6.59 (1H, d, J = 2.7 Hz, C-4-H), 6.65 (1H, dd, J = 8.4 H∑, J = 2.7 Hz, C-2-H), 7.15 (1H, d, J = 8.4 Hz, C-1-H), 7.41 (1H, ddd, J = 8.1 Hz, J = 4,9 Hz, J = 0.8 Hz, C-5"-H), 8.09 (1H, dt, J = 8.1 Hz, J = 1.8 Hz, C-4"-H), 8.67 (1H, dd, J = 4.9 Hz, J = 1.8 Hz, C-6"-H), 8.94 (1H, app d, J = 1.8 Hz, C-2"-H) and 13.58 (1 H, s, exchanged with D2O, C-1'-OH); MS m/z (FAB+) 376.0 [100, (M+H)+], 242.1 [54]; Aec MS m/z (FAB+) 376.1903, C24H26NO3 requires 376.1913. Found: C, 74.70; H, 6.80; N, 3.51. C24H25NO3.(H2O)1/2 requires: C, 74.98; H, 6.82; N, 3.64%.
3-O-tert-Butyl-dimethylsilyl-estra-1,3,5(10)-triene-[17,16-c]-[5'-(1"-pyridin-3"-yl)- 3',4'-dihydro)]-pyrazol-3'-ol (105)
Figure imgf000210_0001
Hydrazine hydrate (75 μL, 1.53 mmol) was added to a refiuxing solution of 101 (500 mg, 1.02 mmol) in EtOH (20 mL), under an atmosphere of N2. The resulting pale yellow solution was heated to reflux for 1 hour, after which the solvent was partially removed under reduced pressure and H2O (100 mL) added. The resulting white precipitate was filtered and dried (460 mg, 89%) and an analytical sample was recrystallised from EtOH/H2O to give 101 as white crystals: mp 144-145°C; IR (KBr) 3480, 3330, 3190 (NH and OH), 2940-2860 (aliph CH), 1610-1495 (C=N and arom C=C) cm-1; δH (DMSO-d6, 400 MHz) 0.14 (6H, s, Si(CH3)2), 0.92 (9H, s, C(CH3)3), 0.94 (3H, s, C-18-H3), 1.12-2.31 (11H, m), 2.63-2.71 (2H, m, C-6-H2), 3.43 (1H, app d, J = 9.0 Hz, C-16-H), 5.97 (1H, s, exchanged with D2O, C-17-OH), 6.46 (1H, d, J = 2.5 Hz, C-4-H), 6.55 (1H, dd, J = 8.4 Hz, J = 2.5 Hz, C-2-H), 7.10 (1H, d, J = 8.4 Hz, C-1-H), 7.26 (1 H, s, exchanged with D2O, NH), 7.35 (1H, app ddd, J = 8.1 Hz, J = 4.6 Hz, J = 0.8 Hz, C-5"-H), 7.91 (1H, app dt, J = 8.1 Hz, J = 1.7 Hz, C-4"-H), 8.43 (1 H, dd, J = 4,6 Hz, J = 1.7 Hz, C-6"-H) and 8.74 (1H, app d, J = 1.7 Hz, C-2"-H); MS m/z (FAB+) 504.1 [100, (M+H)+], 486.1 [27, (M+H-H2O)+]; Aec MS m/z (FAB+) 504.3045, C30H42N3O2Si requires 504.3046.
3-Hvdroιw-astra-l .3.5(1 ORrtenp-li 7-.16-c]-f S'-mothvO-pyrazol® (i 06)
Figure imgf000211_0001
Hydrazine hydrate (56 μL, 1.15 mmol) was added to a refiuxing solution of 102 (240 mg, 768 μmol) in EtOH (15 mL), under an atmosphere of N2. The resulting pale yellow 0 solution was heated to reflux for 45 minutes, after which the solvent was removed under reduced pressure, H2O (50 mL) added and the mixture acidified with 5M HCl. The organics were extracted with EtOAc (2*50 mL), washed with H2O (30 mL), then brine
(2*30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude
, product was purified by flash chromatography (DCM/EtOAc, 8:2) to give 106 as a pale 5 brown solid (84 mg, 35%). This was triturated with acetone to give a creamy solid (32 mg, 13%): mp 234-236°C; IR (KBr) 3295 (OH), 2930-2860 (aliph CH), 1620-1500 (C=N and arom C=C) cm"1; δH (DMSO-d6, 400 MHz) 1.01 (3H, s, C-18-H3), 1.34-2.64 (11 H, m),
2.71-2.85 (2H, m, C-6-H2), 2.28 (3H, s, C-1"-H3), 6.45 (1 H, d, J = 2.6 Hz, C-4-H), 6.52
, (1 H, dd, J = 8.6 Hz, J = 2.6 Hz, C-2-H), 7.04 (1H, d, J = 8.6 Hz, C-1-H) and 9.04 (1 H, s, 0 exchanged with D2O, NH or OH), NH or OH not seen; MS m/z (FAB+) 309.2 [100, (M+H)+], 95.1 [33], 69.0 [39]; Aec MS m/z (FAB+) 309.1978, C20H25N2O requires 309.1967. HPLC (MeOH/H2O, 96:4, λmax = 280.5 nm) Rt = 1.98 min, 99%.
3-Hvdroxy-estra-1.3.5(10)-triene-π 7.16-c]-(5'-trifluoro-methyl)-pyrazole (107. 5 STX949)
Figure imgf000211_0002
Hydrazine hydrate (32 μL, 655 μmol) was added to a refiuxing solution of 103 (160 mg,
437 μmol) in EtOH (15 mL), under an atmosphere of N2. The resulting pale yellow 0 solution was heated to reflux for 3 hours, after which p-toluene sulfonie acid (- 10 mg) was added. The mixture was then heated to reflux overnight and after cooling down, the solvent was removed under reduced pressure, H2O (50 mL) added, followed by 5M HCl. The organics were extracted with EtOAc (2*50 mL), washed with H2O (30 mL), then brine (2*30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was. purified by flash chromatography (DCM/EtOAc, 8:2) to give 107 as a pale yellow solid (95 mg, 60%). This was precipitated from DCM/Hexane (1:5) to give an off-white solid (51 mg,' 32%): mp 152-155°C; IR (KBr) 3220 (br, OH), 2930-2860 (aliph CH), 1610-1500 (C=N and arom C=C) cm'1; δH (CDCI3, 400 MHz) 1.01 (3H, s, C-18-H3), 1.40-2.74 (11H, m), 2.78-2.89 (2H, m, C-6-H2), 4.63 (1H, s, exchanged with D2O, C-3- OH), 6.53 (1H, d, J = 2.7 Hz, C-4-H), 6.59 (1H, dd, J = 8.2 Hz, J = 2.7 Hz, C-2-H) and 7.08 (1H, d, J = 8.2 Hz, C-1-H), NH not seen; MS m/z (FAB+) 363.1 [100, (M+H)+]; Aec MS m/z (FAB+) 363.1689, C20H22 2F3θ requires 363.1684. HPLC (MeOH/H2O, 85:15, λmax = 280.5 nm) Rt = 2.43 min, 98%.
3-Hydroxy-estra-1 ,3,5(10)-triene-[17,16-c]-[5'-(1 "-pyridin-3"-yl)]-pyrazole (108)
Figure imgf000212_0001
A 1.0 M solution of TBAF in anhydrous THF (436 μL, 436 μmol) was added to a stirred solution of 105 (110 mg, 218 μmol) in anhydrous THF (10 mL) at room temperature, under an atmosphere of N2. The resulting light brown solution was stirred overnight at room temperature, after which the solvent was removed under reduced pressure and H2O added (50 mL). The organics were extracted with EtOAc (2*50 mL), washed with H2O (2*30 mL), then brine (2*30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude foam was triturated with boiling EtOAc/CHCI3 (1:1) to give 108 as a white powder (61 mg, 75%): mp 298-300°C (dec); IR (KBr) 2980-2840 (aliph CH), 1610-1495 (C=N and arom C=C) cm'1; δH (DMSO-d6, 400 MHz) 0.98 (3H, s, C-18- H3), 1.37-2.87 (13H, m), 6.45 (1H, d, J = 2.6 Hz, C-4-H), 6.51 (1 H, dd, J = 8.5 Hz, J = 2.6 Hz, C-2-H), 7.05 (1H, d, J = 8.5 Hz, C-1-H), 7.37-7.48 (1H, m, C-4"-H or C-5"-H), 7.97-8.06 (1H, m, C-4"-H or C-5"-H), 8.42-8.49 (1H, m, C-2"-H or C-6"-H) and 8.84-8.89 (1H, m, C-2"-H or C-6"-H), 9.01 (1H, s, exchanged with D2O, OH), 12.65 and 12.72 (total 1H, 2*s, NH); MS m/z (FAB+) 372.0 [100, (M+H)+]; Aec MS /z (FAB+) 372.2080, C24H26N3O requires 372.2076. 3-O-fert-butyl-dimethylsilyl-estra-l ,3,5(10)-triene-[17, 16-c]-(5'-trif luoromethyl-3',4'- dihydro)-pyrazol-3'-ol (109)
Figure imgf000213_0001
Hydrazine hydrate (22 μL, 468 μmol) was added to a refiuxing solution of 100 (150 mg,
5 312 μmol) in absolute EtOH (8 mL), under an atmosphere of N2. The resulting pale yellow solution was heated to reflux for 1 hour, after which the solvent was removed in vacuo and H2O (50 mL) added, followed by HCl 5M (4 drops). The organics were extracted with EtOAc (3*30 mL), washed with H2O (2*30 mL), then brine (2*30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product 0 was purified by flash chromatography (CH2CI2 to CH2CI2/EtOAc, 98:2, gradient, flashmaster) to give a light brown crystalline solid (65 mg, 42%). This was recrystallised from EtOH/H2O to give creamy crystals (26 mg, 18%): mp 235-238°C; IR (KBr) 3336
' (NH), 3170 (br, OH), 2930-2860 (aliph CH), 1664, 1605, 1495 (C=N and arom C=C) cm" 1; δH (DMSO-d6, 400 MHz) 0.18 (6H, s, SiMe2), 0.96 (9H, s, C(CH3)3), 1.04 (3H, s, C-18- 5 H3), 1.32-2.39 (11H, m), 2.76-2.81 (2H, m, C-6-H2), 3.53-3.57 (1 H, m, C-16-H), 6.53 (1H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H), 6.60 (1H, dd, C-1.H,C-2-H = 8.4 Hz, JC-4-H,C-2-H = 2.7 Hz, C-2-H),
6.63 (~ 2H, s, exchanged with D2O, OH and NH) and 7.14 (1H, d, JC-2-H,C-I-H = 8.4 Hz, C-
' 1-H); MS m/z (FAB+) 495.0 [69, (M+H)+], 453.0 [54], 437.0 [51 , (M-F3)+], 73.0 [100]; Aec
MS m/z (FAB+) 495.26311 , C26H38N2F3O2Si requires 495.26547. 0
3-Hydroxy-estra-1,3,5(10)-triene-(17,16-c)-3'-trifluoromethyl-5'-hydroxy-pyrazole (110, STX947)
Figure imgf000213_0002
A 1.0 M solution of tetrabutyl ammonium fluoride in dry THF (404 μL, 404 μmol) was 5 added to a stirred solution of 109 (100 mg, 202 μmol) in THF (20 mL) at room temperature, under an atmosphere of N2. The resulting light brown solution was stirred for 4 hours at room temperature, after which the solvent was removed in vacuo and H2O added (50 mL). The organics were extracted with EtOAc (2*50 mL), washed with H2O (2*30 mL), then brine (2*30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The light brown crude oil was purified by flash chromatography (CH2CI2/EtOAc, 9:1 , flashmaster) to give a pale yellow oil (51 mg, 66%). This was then precipitated from Et2O/Hexane (1:5) to give a light yellow powder (7 mg): IR (KBr) 3470, 3415 (OH), 2930-2870 (aliph CH), 1640, 1615, 1500 (C=N and arom C=C) cm'1; δH (CDCI3, 400 MHz) 1.06 (3H, s, C-18-H3), 1.16-2.26 (11H, m), 2.70-2.84 (2H, m, C-6-H2), 3.64-3.71 (1H, app d, C-16-H), 4,39 (1H, br s, OH or NH), 5.88 (1H, s, OH or NH), 6.53 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6.61-6.65 (1 H, app dd, C-2-H) and 7.03 (1H, d, Jc-2-H,c- 1-H = 8.8 Hz, C-1-H); MS m/z (FAB+) 381.1 [100, (M+H)+], 85.1 [87]; MS m/z (FAB-) 533.3 [100, (M+NBA)-], 379.2 [98, (M-H)']; Aec MS m/z (FAB+) 381.17967, C20'H24N2O2F3 requires 381.17899. HPLC (methanol/water, 96:4, λmax = 280.5 nm) Rt = 1.66 min, 100%.
New Pathway to STX664
3-Benzyloxy-16-ethoxymethylene-estrone (127)
Figure imgf000214_0001
K2CO3 (595 mg, 4.31 mmol) was added portionwise to a stirred suspension of 3- benzyloxy-16-hydroxymethylene-estrone (250 mg, 643 mmol) in acetone (20 mL) at room temperature, under an atmosphere of N2. After 10 minutes of stirring, ethyl iodide (296 μL, 3.70 mmol) was added and the resulting mixture was stirred for 36 hours at room temperature. H2O was then added (100 mL) and the organics were extracted with DCM (3x50 mL), washed with H2O (50 mL), then brine (2*50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (EtOAc/hexane, 1:3) to give 127 as a white solid (215 mg, 80%). This was recrystallised from EtOAc to give white crystals (139 mg, 52%): mp 158-160°C; IR (KBr) 2935-2850 (aliph CH), 1 10 (C=O), 1640-1600 (aliph and arom C=C), 1235 (C- O) cm'1; δH (CDCI3, 400 MHz) 0.92 (3H, s, C-18-H3), 1.34 (3H, t, J = 7.2 Hz, C-3'-H3), 1.38-2.71 (11H, m), 2.84-2.93 (2H, m, C-6-H2), 4.02-4.10 (2H, m, C-2'-H2), 5.02 (2H, s, OCH2Ar), 6.71 (1 H, d, J = 2.6 Hz, C-4-H), 6.77 (1 H, dd, J = 8.6 Hz, J = 2.6 Hz, C-2-H), 7.18 (1H, d, J = 8.6 Hz, C-1-H) and 7.26-7.43 (6H, m, C-1'-H and C6H5); MS m/z (FAB+) 417.0 [100, (M+H)+]; Ace MS m/z (FAB+) 417.2425, C28H33O3 requires 417.2430. 16β-Ethojsymethyl-estrone (STX664)
A suspension of 127 (640 mgr1.54 mmol) and Pd-C (10%, 200 mg) in MeOH/THF (1:1, 60 mL) was hydrogenated (H2-filled balloon) at room temperature for 48 hours. After removal of the supported catalyst by filtration through celite and concentration of the filtrate under reduced pressure, the product obtained was purified by flash chromatography (EtOAc/hexane, 3:7) to give ST2 664 as a white crystalline solid (232 mg, 46%). An analytical sample was recrystallised from EtOH: mp 208-210°C; IR (KBr) 3370 (OH), 2970-2855 (aliph CH), 1730 (C=O), 1610, 1505 (arom C=C), 1225 (C-O) cm' 1;δH (CDCI3, 400 MHz) 0.89 (3H, s, C-18-H3), 1.16 (3H, t, JC.2.-H,C-3'.H - 7.0 Hz, C-3'-H3), 1.36-2.42 (12H, m), 2.81-2.85 (2H, m, C-6-H2), 3.42-3.50 (2H, m, C-2'-H2), 3.61 (1H, dd, AB = 9.4 Hz, J = 3'.9 Hz, C-1'-HAHβ), 3.65 (1H, dd, JBA = 9.4 Hz, J = 5.5 Hz, C-V-HAHB), 4.59 (1H, s, exchanged with D2O, OH), 6.56 (1H, d, J = 2.8 Hz, C-4-H), 6.62 (1H, dd, J = 8.5 Hz, J = 2.8 Hz, C-2-H) and 7.13 (1H, d, J = 8.5 Hz, C-1-H);δc (CDCI3, 100.4 MHz) 12.9 (q, C-3'), 15.0 (q, C-18), 25.8 (t), 25.9 (t), 26.6 (t), 29.4 (t), 32.0 (t), 37.7 (d), 44.0 (d), 48.2 (s, C-13), 48.9 (d), 50.2 (d), 66.4 (t, C-1' or C-2'), 69.3 (t, C-1' or C-2'), 112.6 (d), 116.0 (d), 126.2 (d), 131.9 (s), 137.8 (s) and 153.0 (s, C-3), C=O not seen; MS m/z (FAB+) 329.1 [100, (M+H)+]; Ace MS m/z (FAB+) 329.2124, C21H29O3 requires 329.2117. HPLC (MeOH/H2O, 90:10, λmax = 279.3 nm) Rt = 2.37 min, 98%. Found: C, 76.60; H, 8.62. C21H28O3 requires: C, 76.79; H, 8.59%.
Sulfamates
3-Sulfamoyloxy-16β-ethoxymethyl-estrone (20)
Figure imgf000215_0001
Anhydrous DMA (1.5 mL) was added to a freshly concentrated solution of sulfamoyl chloride (2.2 eq.) cooled to 0°C under an atmosphere of N2. ST2 664 (100 mg, 304 μmol) was then added and the resulting mixture was stirred at room temperature for 3 hours. The mixture was then poured into cold brine (15 mL), and the organics were extracted with EtOAc (2x20 mL), washed with brine (5x20 mL), dried (MgSO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (CHCI3/EtOAc, 8:2) to give 20 as a colourless oil (124 mg, 100%): IR (KBr) 3380, 3285 (br, NH2), 2970-2865 (aliph CH), 1730 (C=O), 1605-1495 (arom C=C), 1380, 1190 (SO2) cm'1;ππH (CDCI3, 400 MHz) 0.89 (3H, s, C-18-H3), 1.16 (3H, t, J = 7.0 H∑, C-3'-H3), 1.40r2.43 (12H, m), 2.89-2.94 (2H, m, C-6-H2), 3.43-3.50 (2H, m, C-2'-H2), 3.61 (1H, dd, JAB = 9.4 Hz, J = 3.7 Hz, C-1'-HAHH), 3.66 (1H, dd, JBA = 9.4 Hz, J = 5.5 Hz, C-V-HAHB), 4.87 (2H, s, exchanged with D2O, SO2NH2), 7.04 (1 H, d, J = 2.4 Hz* C-4-H), 7.07 (1H, dd, J = 8.4 Hz, J = 2.4 H∑, C-2-H) and 7.29 (1H, d, J = 8.4 Hz, C-1-H); Dc (CDCI3, 100.4 MH∑) 12.9 (q, C-3'), 15.1 (q, C-18), 25.8 (2*t), 26.4 (t), 29.4 (t), 32.0 (t), 37.4 (d), 44.3 (d), 48.2 (s, C-13), 49.0 (d), 50.3 (d), 66.6 (t, C-1' or C-2'), 69.3 (t, C-1' or C-2'), 119.1 (d), 122.1 (d), 126.8 (d), 138.8 (s), 139.1 (s), 148.0 (s, C-3) and 220.8 (s, C=O); MS m/z (FAB+) 408.3 [39, (M+H)+], 146.1 [63], 131.1 [61], 117.1 [57], 85.1 [100], 68.0 [50]; Ace MS m/z (FAB+) 408.1841 , C21H30NO5S requires 408,1845. HPLC (MeOH/H2O, 90:10, λmax = 267.5 nm) Rt = 2.28 min, 99%. Found: C, 59.65; H, 6.84; N, 3.36. C21H29NO5S.(CHCI3)1/6 requires: C, 59.48; H, 6.88; N, 3.28%.
3-Sulfamoyloxy-16-oximino-estrone (21)
Figure imgf000216_0001
Following an analogous procedure to that described for the preparation of 20, reaction of 16-oximino-estrone (200 mg, 668 μmol) with sulfamoyl chloride in DMA (1.5 mL) was complete within 3 hours. The crude product was purified by flash chromatography (CHCI3/acetone, 8:2) to give a white foam (93 mg, 37%). This was triturated with hexane to give 21 as a white powder (36 mg, 14%): mp 88-91 °C; IR (KBr) 3370-3250 (NH2, NOH), 2930-2860 (aliph CH), 1740 (CO), 1635-1496 (C=N and arom C=C), 1380, 1185 (SO2) cm'1; δH (DMSO-d6, 400 MHz) 0.90 (3H, s, C-18-H3), 1.32-2.48 (11H, m), 2.78-2.94 (2H, m, C-6-H2), 7.00 (1H, m, C-4-H), 7.03 (1H, dd, J = 8.6 Hz, J = 2.3 Hz, C-2-H), 7.36 (1H, d, J = 8.6 Hz, C-1-H), 7.92 (2H, s, exchanged with D2O, SO2NH2) and 12.41 (1H, s, exchanged with D2O, NOH); MS m/z (FAB+) 532.2 [32, (M+H+NBA)+], 379.1 [100, (M+H)+]; MS m/z (FAB-) 531.1 [40, (M+NBA)'], 377.1 [100, (M-H)']; Ace MS m/z (FAB+) 379.1320, C18H23N2O5S requires 379.1328. Found: C, 56.70; H, 6.02; N, 6.83. CιaH22N2O5S requires: C, 57.13; H, 5.86; N, 7.40%. 3,1'-Bis-sulfamoyloxy-estra-1,3,5(10)-triene-[17,16-c]-pyrazole (21) and 3- Sulfamoyloxy-estra-1 ,3,5(10)-triene-[17,16-cJ-pyrazole (22)
Following an analogous procedure to that described for the preparation of 20, reaction of 3-hydroxy-estra-1 ,3,5(10)-triene-[17,16-c]-pyra∑ole (170 mg, 577 μmol) with sulfamoyl chloride in DMA (2 mL) was complete within 4 hours. The crude product was purified by flash chromatography (CHCI3/EtOAc, 1:1) to give two products:
Figure imgf000217_0001
The less polar fraction gave 21 as a pale yellow solid (85 mg, 32%): IR (KBr) 3440, 3410 (NH2), 2930-2860 (aliph CH), 1635-1496 (C=N and arom C=C), 1385, 1185 (SO2) cm' 1;δH (DMSO-d6, 400 MHz) 0.99 (3H, s, C-18-H3), 1.40-2.68 (11H, m), 2.85-2.90 (2H, m, C-6-H2), 6.97 (1H, d, J = 2.3 Hz, C-4-H), 7.02 (1H, dd, J = 8.6 Hz, J = 2.3 Hz, C-2-H), 7.35 (1H, d, J = 8.6 Hz, C-1-H), 7.70 (1H, s,' C-5'-H), 7.88 (2H, s, exchanged with D2O, SO2NH2) and 8.30 (2H, s, exchanged with D2O, SO2NH2); MS m/z (FAB+) 452.8 [100, M+], 373.9 [80, (M+H-SO2NH2)+], 171.0 [26], 116.0 [29]; Aec MS m/z (FAB+) 452.1164, C19H24N4O5S2 requires 452.1188. HPLC (MeOH/H2O, 80:20, λmax = 220.4 nm) Rt = 1.95 min, 97%..
Figure imgf000217_0002
The more polar fraction gave 22 as a white solid (123 mg, 57%): IR (KBr) 3370, 3305 (NH2), 2975-2860 (aliph CH), 1590, 1495 (C=N and arom C=C), 1370, 1195 (SO2) cm' 1;δH (DMSO-d6, 400 MHz) 1.17 (3H, s, C-18-H3), 1.38-2.59 (11 H, m), 2.85-2.90 (2H, m, C-6-H2), 6.97 (1H, d, J = 2.7 Hz, C-4-H), 7.02 (1H, dd, J = 8.6 Hz, J = 2.7 Hz, C-2-H), 7.25 (1H, s, C-5'-H), 7.35 (1H, d, J = 8.6 Hz, C-1-H), 7.89 (2H, s, exchanged with D2O, SO2NH2) and 11.99 (1H, br s, exchanged with D2O, NH); MS m/z (FAB+) 747.3 [38, (2M+H)1, 527.2 [80, (M+H+NBA)+], 509.2 [30], 443.2 [28], 374.2 [100, (M+H)+]; Aec MS m/z (FAB+) 374.1544, C19H24N3O3S requires 374.1538. HPLC (MeOH/H2O, 80:20, λmax = 218.0 nm) Rt = 2.04 min, 100%. SECTION 5
3-( srt-Butyl-dime hyl-silanylθHy)-13-m© hyl-17-θ2∑©-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclop©nta[a]phenanthr©n@-16-carbonitril©-(3-(^o -Buftyl- dimethyl)silyl-16-cyano estrone) (CAB01044):
Figure imgf000218_0001
CAB01044
O,N-bistrifluoroacetyl-hydroxylamine (1.35 g, 6.0 mmol) was added to a solution of 3-
(tert-butyl-dimethyl)silyl-16-hydroxymethylene estrone (820 mg, 2.0 mmol, CAB01042) and pyridine (1.0 mL) in toluene (50 mL). The mixture was heated to reflux for 1 hour (120°C oilbath temperature) and then allowed to cool to room temperature (TLC-control). Ethyl acetate (50 mL) was added and the mixture was transferred into separation funnel, washed with water (50 mL) and brine (30 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica (eluent: ethyl acetate/hexane 1:2, Rf: 0.38) to give a light yellow oil, which was dissolved in a small amount of acetone (2 mL) and precipitated by addition of hexane. The light yellow solid was filtered off and dried under high vacuum. Yield: 585 mg (71%), mixture of 16α-cyano and 16β-cyano diastereomers). 1H-NMR(CDCI3, 400 MHz) δ: 0.19 (s, 6H), 0.98 (s, 9H, -C(CH3)3), 0.99 (s, ca. 1H, CH3-group of minor isomer), 1.02 (s, ca. 2H, CH3- group of major isomer), 1.40-1.71 (m, 5H), 1.94-2.10 (m, 3H), 2.23-2.34 (m,1H), 2.38- 2.48 (m,1H), 2.54-2.64 (m, 1H), 2.82-2.90 (m, 2H), 3.12 (dd, J = 9.8, 8.6 Hz, ca. 0.65 H, H-16 of major isomer), 3.58 (dd, J = 10.2, 1.6 Hz, ca. 0.35 H, H-16 of minor isomer), 6.58 (d, J = 2.3 Hz, 1H), 6.63 (dd, J = 8.6, 2.3 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H). LRMS (FAB+): 73.1 (100), 352.2 (65), 409.3 (85, [M]+). LRMS (FAB-): 408.3 (100, [M-H]'). HRMS (FAB+) 409.243294 C25H35 O2Si requires 409.243708
3-(tert-Butyl-dimethyl-silanylo y)-2-methoxy-13-methyl-17-oj«o-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-d©cahydro-6H-cyclop©nta[a]phenanthr©ne-16-car onitril© (Z~(tert- Butyl-dimethyl)silyl-2-methoxy-16-cyano estrone) (CAB01066):
Figure imgf000219_0001
Sodium ethoxide (340 mg, 5.0 mmol) was added to a solution of 2-methoxy-3-(tert-butyi- dimethyl)silyl estrone (829 mg, 2.0 mmol) and ethyl formate (2.0 mL) in toluene (20 mL). The mixture was stirred for 2 hours at room temperature (TLC-control) and acetic acid (1 mL) and ethyl acetate (60 mL) were added. The mixture was transferred into a separation funnel and washed with water (2 x 30 mL) and brine (30 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude 2- methoxy-3-(tert-butyl-dimethylsilyl)-16-hydroxymethylene estrone (785 mg, 1.773 mmol), that was obtained was dissolved in toluene (50 mL) and pyridine (1 mL) and O,N- bistrifluoroacetyl-hydroxylamine (675 mg, 3.0 mmol) were added. The mixture was heated to reflux for 1 hour and then allowed to cool to room tempereature (TLC-control). The reaction mixture was diluted with ethyl acetate (50 mL), transferred into a separation funnel, washed with water (2 x 50 mL) and brine (30 mL), dried over Na2SO4 and concentrated under reduced pressure. . The residue was purified by column chromatography on silica (eluent: ethyl acetate/hexane 1:2, Rf: 0.38) to give a pale yellow solid, which was dissolved in a small amount of acetone (2 mL) and precipitated by addition of hexane. The light yellow solid was filtered off and dried under high vacuum. Yield: 620 mg (71%, ca. 2 to 1 mixture of 16α-cyano and 16β-cyano diastereomers). 1H-NMR(CDCI3, 400 MHz) δ: 0.15 (s, 6H), 0.98 (s, 9H, -C(CH3)3), 0.99 (s, ca. 1H, CH3-group of minor isomer), 1.08 (s, ca. 2H, CH3-group of major isomer), 1.36-1.70 (m, 5H), 1.90-2.08 (m, 3H), 2.21-2.30 (m,1H), 2.34-2.42 (m,1H), 2.54-2.62 (m, 1H), 2.76-2.81 (m, 2H), 3.13 (dd, J = 9.8, 8.6 Hz, ca. 0.65 H, H-16 of major isomer), 3.60 (dd, J = 10.2, 1.6 Hz, ca. 0.35 H, H-16 of minor isomer), 3.77 (s, 3H, -OCH3), 6.57 (s,1H), 6.73 (s, 1H). LRMS (FAB+): 73.1 (100), 382.3 (65), 440.3 (20, [M+H]+). LRMS (FAB-): 438.4 (100, [M-H]'). HRMS (FAB+) 440.260025 C26H38NO3Si requires 440.262098.
STX207 3-Hydroxy-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-d©cahydro- 6H-cyclopenta[a]phenanthrene-16-carbonitrile (16-cyano-estrone, mixture of 16α and 16β diastereomers) (CAB01058):
Figure imgf000220_0001
CAB01058
Tetrabutyl ammonium fluoride (1.0 mL, 1 M in THF, 1.0 mmol) was added to a solution of 3-(tert-Butyl-dimethyl)silyl-16-cyano estrone (350 mg, 0.856 mmol, CAB01044) in THF (20 mL) at 0°C (icebath). The mixture was stirred for 30 minutes at this temperature, acetic acid (0.5 mL) and ethyl acetate (80 mL) were added and the mixture was transferred into a separation funnel. The organic phase was washed with water (2 x 25 mL) and brine (25 mL), dried over Na2SO4 and concentrated under reduced pressure. The solid residue was dissolved in ethyl acetate and precipitated by addition of hexane. The pale yellow solid was filtered off and dried under high vacuum. Yield: 223 mg (88%, ca. 2 to 1 mixture of 16α-cyano and 16β-cyano diastereomers). 1H-NMR(CDCI3, 400 MHz) δ: 0.97 (s, ca.1 H, CH3-group of minor isomer), 1.07 (s, ca. 2H, CH3-group of major isomer), 1.36-1.70 (m, 5H), 1.90-2.16 (m, 3H), 2.22-2.34 (m,1H), 2.38-2.46 (m,1H), 2.56-2.62 (m, 1H), 2.85-2.92 (m, 2H), 3.12 ,(dd, J = 9.8, 8.6 Hz, ca. 0.65 H, H-16 of major isomer), 3.60 (dd, J = 10.2, 1.6 Hz, ca. 0.35 H, H-16 of minor isomer),4.76 (bs, 1H, -OH), 6.59 (d, J = 2.3 Hz, 1H), 6.65 (dd, J = 8.6, 2.3 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1 H). LRMS (FAB+): 295.2 (100, [M+H]+).
STX208 3-Hydroxy-2-methoxy-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthrene-16-carbonitrile (2-methoxy-16-cyano- estrone, mixture of 16α and 16β diastereomers) (CAB01068).
Figure imgf000220_0002
CAB01068
Tetrabutyl ammonium fluoride (1.0 mL, 1M in THF, 1.0 mmol) was added to a solution of 3-(tert~Butyl-dimethyl)silyl-2-methoxy-16-cyano estrone (440 mg, 1.0 mmol, CAB01066) in THF (20 mL) at 0°C (icebath). The mixture was stirred for 30 minutes at this temperature, acetic acid (0.5 mL) and ethyl acetate (80 mL) were added and the mixture was transferred into a separation funnel. The organic phase was washed with water (2 x 25 mL) and brine (25 mL), dried over Na2SO4 and concentrated under reduced pressure. The solid residue was dissolved in ethyl acetate (ca. 2 mL) and precipitated by addition of hexane. The pale yellow solid was filtered off and dried under high vacuum. Yield: 233 mg (72%, ca. 2 to 1 mixture of 16α-cyano and 16β-cyano diastereomers). 1H- NMR(CDCI3, 400 MHz) δ: 0.98 (s, ca. 1 H, CH3-group of minor isomer), 1.07 (s, ca. 2H, CH3-group of major isomer), 1.38-1.68 (m, 5H), 1.92-2.12 (m, 3H), 2.24-2.32 (m,1H), 2.38-2.44 (m,1H), 2.54-2.62 (m, 1 H), 2.74-2.86 (m, 2H), 3.13 (dd, J = 9.8, 8.6 H∑, ca. 0.65 H, H-16 of major isomer), 3.60 (dd, J = 10.2, 1.6 Hz, ca. 0.35 H, H-16 of minor isomer), 3.86 (s, 3H, -OCH3), 5.48 (s, 1H, -OH), 6.57 (s,1H), 6.73 (s, 1H). LRMS (FAB+): 325.3 (100, [Mf). LRMS (FAB-): 324,3 (100, [M-H]'). HRMS (FAB+): 325.168983 C2oH23NO3S requires 325.167794
STX209 Sulfamic acid 16-cyano-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthren-3-yl ester (3-sulphamoyl-16-cyano- estrone, mixture of 16α and 16β diastereomers) (CAB01062):
Figure imgf000221_0001
Sulphamoyl chloride solufion (1.0 mL, 0.7M, 0.7 mmol) in toluene was concentrated under reduced pressure (30°C waterbath temperature) to ca. 0.5 mL volume. The residue was cooled to 0°C (icebath) and DMA(1.5 mL) was added to give a colourless solution. 16-cyano-estrone (89 mg, 0.30 mmol, CAB01058) was added at CC, the solution was allowed to warm to room temperature and was stirred overnight at this temperature. Ethyl acetate (50 mL) and water (26 mL) were added, the organic layer was separated and washed with water (2 x 25 mL) and brine (25 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in acetone (ca. 2 mL) and precipitated by addition of hexane. The white solid was filtered off and dried under high vacuum. Yield: 94 mg (84%, ca. 2 to 1 mixture of 16α-cyano and 16β-cyano diastereomers). H-NMR(CDCI3, 400 MHz) δ: 0.97 (s, ca.1 H, CH3-group of minor isomer), 1.07 (s, ca. 2H, CH3-group of major isomer), 1.42-1.70 (m, 5H), 1.94-2.18 (m, 3H), 2.26-2.38 (m,1H), 2.40-2.48 (m,1H), 2.56-2.64 (m, 1H), 2.88-3.00 (m, 2H), 3.14 (dd, J = 9.8, 8.6 Hz, ca. 0.65 H, H-16 of major isomer), 3.60 (dd, J = 10.2, 1.6 Hz, ca. 0.35 H, H-16 of minor isomer),4.92(bs, 2H, -NH2), 7.07 (d, J = 2.3 Hz, 1 H), 7.11 (dd, J = 8.6, 2.3 Hz, 1H), 7.30 (d, J = 8.6 Hz, 1H). LRMS (FAB+): 375.1 (100, [M]+). STX210 Sulfamic acid 16-cyano-2-methoxy-13-methyl-17-oxo-7, 8, 9, 11* 12, 13, 14, 15, 16, 17-decahydro-6H-cycloρenta[a]phenanthrert-3-yl ester (2-methoxy-3- sulpharnøyl-16-cyano-estron®, mixture of 1@os and 1ββ diastereomers) (CAB0 070):
Figure imgf000222_0001
Sulphamoyl chloride solution (1.0 mL, 0.7M, 0.7 mmol) in toluene was concentrated under reduced pressure (30°C waterbath temperature) to ca. 0.5 mL volume. The residue was cooled to 0°C (icebath) and DMA(1.5 mL) was added to give a colourless solution. 2-methoxy-16-cyano-estrone (89 mg, 0.30 mmol, CAB01068) was added at 0°C, the solution was allowed to warm to room temperature and was stirred overnight at this temperature. Ethyl acetate (50 mL) and water (25 mL) were added, the organic layer was separated and washed with water (2 x 25 mL) and brine (25 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in acetone (ca. 2 mL) and precipitated by addition of hexane, The white solid was filtered off and dried under high vacuum. Yield: 102 mg (82%, ca. 2 to 1 mixture of 16α-cyano and 16β-cyano diastereomers). 1H-NMR(CDCI3, 400 MHz) δ: 0.98 (s, ca.1H, CH3-group of minor isomer), 1.07 (s, ca. 2H, CH3-group of major isomer), 1.38-1.70 (m, 5H), 1.90-2.16 (m, 3H), 2.22-2.48 (m, 2H), 2,56-2.64 (m, 1H), 2.79-2.90 (m, 2H), 3.15 (dd, J = 9.8, 8.6 Hz, ca. 0.65 H, H-16 of major isomer), 3.62 (dd, J = 10.2, 1.6 Hz, ca. 0.35 H, H-16 of minor isomer), 3.88 (s, 3H, -OCH3), 5.07 (s, 2H, -NH2), 6.91 (s, 1H), 7.06 (s, 1H). LRMS (FAB+): 405.1 (100, [Mf).
(3-Benzyloxy-13-methyl-17-oxo-6, 7, 8, 9, 11, 12, 13, 14, 15, 17-decahydro- cyclopenta[a]phenanthren-16-ylidene)-hydroxy-acetic acid ethyl ester (CAB03048):
Figure imgf000222_0002
Potassium tert.-butoxide (2.24 g, 20.0.mmol) was added to a solution of 3-benzyl- estrone (5.77 g, 16.0 mmol) ind diethyl oxalate (5 mL) in toluene (100 mL). The resulting clear yellow solution was stirred overnight at room temperature. Acetic acid (5 mL) was added (colour changes from dark yellow to light yellow), the mixture was transferred into a separation funnel and ethyl acetate (50 mL) and water (100 mL) were added. The organic layer was separated and washed with water (100 mL) and brine (50 mL), dried over Na2SO4 and concentrated under reduced pressure. The light yellow solid residue was suspended in ethanol (50 mL) and heated to reflux for 5 minutes. The colourless crystalline solid was filtered off after cooling to room temperature and dried under high vacuum. Yield: 7.28 g (99%). 1H-NMR(CDCI3, 400 MHz) δ: 1.01 (s, 3H), 1.41 (t, J = 7.0 Hz, 3H), 1.44-1.72 (m, 5H), 1.98-2.08 (m, 2H), 2.26-2.35 (m, 1H), 2.40-2.50 (m, 2H), 2.89-2.98 (m, 2H), 3.10 (dd, J = 16.0, 5.9 H∑, 1H), 4.37 (q, J = 7.0 Hz, 2H), 5.05 (s, 2H), 6.75 (d, J = 2.7 Hz, 1H), 6.80 (dύ, J = 8.6, 2.7 Hz, 1H), 7.20 (d, J = 8.6 Hz, 1H), 7.30- 7.46 (m, 5H), 12.48 (S, 1H). LRMS (FAB+): 461.2 (100, [M+H]+).
2-Benzyloxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid ethyl ester (CAB03049).
Figure imgf000223_0001
Hydrazine hydrate (0.75, ca. 15 mmol) was added to a suspension of (3-Benzyloxy-13- methyl-17-oxo-6, 7, 8, 9, 11, 12, 13, 14, 15, 17-decahydro-cyclopenta[a]phenanthren-16- ylidene)-hydroxy-acetic acid ethyl ester (6.91 g, 15.0 mmol, CAB03048) in ethanol (100 mL) and dichloromethane (20 mL). The suspension turned into a clear, pale yellow solution after a few minutes, which was refluxed for 15 minutes and stirred overnight at room temperature. The solvents were removed under reduced pressure to give a white solid, which was dissolved in ethanol (50 mL) and p-toluene sulphonic hydrate (200 mg) was added. The mixture was heated to reflux for 5 minutes and transferred into a separation funnel after cooling to room temperature. Ethyl acetate (120 mL) and water (60 mL) were added, the organic layer was separated, washed with concentrated NaHCO3-solution (30 mL) and brine (30 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was crystallised from ethyl acetate/hexane. Yield: 5.43 g (79%) fine colourless crystals. 1H-NMR(CDCI3, 400 MHz) δ: 1.05 (s, 3H, -CH3), 1.39 (t, J = 7.0 Hz, 3H, OCH2CH3), 1.44-1.72 (m, 4H), 1.98-2.06 (m, 1H), 2.10-2.20 (m, 1 H), 2.30- 2.42 (m, 4H), 2.82-3.00 (m, 3H), 4.37 (q, J = 7.0 Hz, 2H, OCϋCHg), 5.05 (s, 2H, OCfcLPh), 6.73 (d, J = 2.3 Hz, 1 H), 6.81 (dd, J = 8.6, 2.3 Hz, 1 H), 7.23 (d, J = 8.6 Hz, 1H), 7.30-7.46 (m, 5H) NH-proton not visible, probably very broad signal at 11 ppm. LRMS (FAB+): 457.4 (100, [M+H]+).
2-B©nsyioxy-6a-m©thyI-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydr©-7, S-diasa- pental©no[2,1-a3phen'anthrene-9-carbθϊjylic acid ©thyl ©ster (CAES03051).
Figure imgf000224_0001
Sodium hydroxide solution (7.5 mL, 2N NaOH, 15 mmol) was added to a suspension of 2-Benzyloxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid ethyl ester (2.283 g, 5.0 mmol, CAB03049) in ethanol (40 mL). The mixture was heated to reflux for 30 minutes. Acetic acid (5 mL) was added after cooling to room temperature and the mixture was stirred for 2 hours, while the product precipitated. The product was filtered off, washed with water (50 mL) and ethanol (20 mL) and dried under high vacuum for 2 days. Yield: 2.130 g (99%) white powder. 1H-NMR(DMSO-d6, 400 MHz) δ: 0.95 (s, 3H, -CH3), 1.36-1.82 (m, 5H), 1.88-1.98 (m, 1H), 2.00-2.10 (m, 1H), 2.12-2.20 (m, 1H), 2.26-2.46 (m, 4H), 2.70 (dd, J = 14.6, 6.2 Hz, 1H), 2.80-2.94 (m, 2H), 5.06 (s, 2H, OCHzPh), 6.73 (d, J = 2.3 Hz, 1H), 6.78 (dd, J = 8.6, 2.3 Hz, 1 H), 7.20 (d, J = 8.6 Hz, 1 H), 7.30-7.45 (m, 5H), 12.90 (bs, 2H, NH + OH). LRMS (FAB+): 429.2 (100, [M+H]+).
STX782 2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8- diaza-pentaleno[2,1-a]phenanthrene-9-carboxylic acid ethyl ester (CAB03052):
Figure imgf000224_0002
CAB03052
Palladium on charcoal (100 mg, 5%Pd) was added to a solution of 2-Benzyloxy-6a- methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno[2, 1- a]phenanthrene-9-carboxylic acid ethyl ester (913 mg, 2.0 mmol, CAB03049) in THF (30 mL) and ethanol (30 mL). The resulting mixture was stirred under a hydrogen- atmosphere (balloon) for 72 hours, filtered through a 3 cm layer of celite and concentrated under reduced pressure. The residue was crystallised from acetone/cyclohexane. Yield: 602 mg (82%) pale yellow crystals. 1H-NMR(DMSO-d6, 400 MHz) δ: 0.92 (s, 3H, -CH3), 1.12-2.84 (m, 16H), 4.14-4.40 (m, 2H, OCϋCHg), 6.45 (d, J = 2.3 H∑, 1H), 6.50 (dd, J = 8.6, 2.3 Hz, 1H), 7.05 (d, J = 8.6 Hz, 1H), 9.01 (s, 1H, - OH), 13.07 (s, 1H, NH). LRMS (FAB+): 367.2 (100, [M+H]+). HRMS (FAB+): 367.203796 C22H27N2O3 requires 367.202168
ST 783 2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8- diaza-pentaleno[2,1-a]phenanthrene-9-carboxylic acid (CAB03053): ):
Figure imgf000225_0001
CAB03053
' Palladium on charcoal (50 mg, 5%Pd) was added to a solution 2-Benzyloxy-6a-methyl- 4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno[2, 1- a]phenanthrene-9-carboxylic acid ethyl ester (214 mg, 0.5 mmol, CAB03051) in THF (15 mL) and ethanol (15 mL). The resulting mixture was stirred under a hydrogen- atmosphere (balloon) for 24 hours, filtered through a 3 cm layer of celite and concentrated under reduced pressure. The residue was dried under high vacuum. Yield: 165 mg (98%) light grey solid. 1H-NMR(DMSO-d6, 400 MHz) δ: 0.92 (s, 3H, -CH3), 1.30-2.86 (m, 13H), 6.44 (d, J = 2.3 Hz, 1 H), 6.52 (dd, J = 8.6, 2.3 Hz, 1 H), 7.05 (d, J = 8.6 Hz, 1H), 9.02 (s, 1H, -OH), 12.90 (bs, 2H, -COOH and NH). LRMS (FAB+): 176.0 (100), 329.1 (45, [M+H]+). HRMS (FAB+): 339.171135 C2oH23N2O3 requires 339.170868
(2-Benzyloxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthren-9-yl)-methanol (CAB03058):
Figure imgf000225_0002
2-Benzyloxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-l ', 8-diaza- pentaleno[2, 1-a]phenanthrene-9-carboxylic acid ethyl ester (457 mg, 1.0 mmol, CAB03049) was added to a suspension of LiAIH4 (100 mg) in THF (20 mL). The mixture was stirred for 30 minutes at room temperature, then water (1 mL) was added (to destroy the excess of LiAIH4). Stirring was continued for 1 hour (light yellowgreen suspension), acetic acid (0.5 mL) was added, the inorganic solids were filtered of and washed carefully with THF (ca. 50 mL).The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was crystallised from ethyl acetate/diethyl ether. Yield: 215 mg (52%) colorless crystals. 1H-NMR(CDCI3, 400 MH∑) δ: 1.01 (s, 3H, -CH3), 1.40-2.46 (m, 10 H), 2.60 (dd, J = 13.7, 6.3 H∑, 1H), 2.82- 3.00 (m, 2H), 4.69 (s, 2H, -C bOH), 5.04 (s, 2H, -CH2Ph), 6.28 (bs, 2H, -OH and NH), 6.73 (d, J = 2.3 H∑, 1 H), 6.80 (dd, J = 8.6, 2.3 H∑, 1H), 7.21 (d, J = 8.6 H∑, H), 7.30- 7.45 (m, 5H). LRMS (FAB+): 91.1 (100), 415.3 (60, [M+H]+). HRMS (FAB+): 415.238434 C27H31N2O2 requires 415.238554
STX804 9-Hydroxymethyl-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro- 7, 8-diaza-pentaleno[2,1-a]phenanthren-2-ol (CAB03065): ):
Figure imgf000226_0001
Palladium on charcoal (50 mg, 5%Pd) was added to a solution (2-Benzyloxy-6a-methyl- 4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7 , 8-diaza-pentaleno[2, 1-a]phenanthren- 9-yl)-methanol (120 mg, 0.289 mmol, CAB03058) in THF (20 mL) and ethanol (20 mL). The resulting mixture was stirred under a hydrogen-atmosphere (balloon) for 24 hours, filtered through a 3 cm layer of celite and concentrated under reduced pressure. The residue was dried under high vacuum. Yield: 93 mg (99%) white solid. 1H-NMR(DMSO- d6, 270 MHz) δ: 0.82 (s, 3H, -CH3), 1.20-2.80 (m, 13H), 4.31 (s, 2H, -CϋOH), 4.95 (bs, 1 H, -OH), 6.37 (s, 1H), 6.44 (d, J = 8.5 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 8.92 (s, 1H, - OH), 11.78 (bs, 1H, NH). LRMS (FAB+): 325.2 (100, [M]+). HRMS (FAB+): 325.192528 C20H25N2O2 requires 325.191603.
ST2 319 3-Hydroκy-13-methyl-16-pyridin-2-ylmethylen©-6,7, 8,9,11,12,13,14,15,16- decahydro-cyclopenta[a]phenanthren-17-one (CAB01150):
Figure imgf000227_0001
CAB01150 Sodium hydroxide (1.0 g, 25 mmol) was added to a suspension of estrone (1.35 g, 5.0 mmol) and pyridine-2-carbaldehyde (595 mg, 5.0 mmol) in ethanol (40 mL) at room temperature. The resulting dark orange solution was stirred at room temperature for 4 hours. Then glacial acefic (ca. 10 mL) acid was added with stirring. The colour changed to light yellow and a light yellow solid precipitated. The solid was filtered off and washed with water (50 mL), ethanol (20 mL), diethyl ether (50 mL) and hexane (50 mL) and dried under high vacuum. Yield: 1.638 g (91 %) yellow powder.
1H-NMR(DMSO-d6', 400 MHz) δ: 0.89 (s, 3H, -CH3), 1.30-1.65 (m, 5H), 1.84-1.90 (m, 1 H), 1.93-2.00 (m, 1H), 2.14-2.23 (m, 1H), 2.30-2.38 (m, 1 H), 2.52-2.60 (m, 1 H), 2.70- 2.86 (m, 1H), 3.24-3.32 (m, 2H), 6.46 (d, J = 2.3 Hz, 1H), 6.62 (dd, J = 8.6, 2.3 Hz, 1H), 7.05 (d, J = 8.6 Hz, 1H), 7.30 (s, 1H), 7.36 (dd, J = 6.6, 4.7 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.86 (dt, J = 7.4, 1.6 Hz, 1 H), 8.70 (d, J = 3.9 Hz, 1H), 9.05 (s, 1H, -OH). LRMS (FAB+): 360.2 (100, [M+H]+). LRMS (FAB-): 358.2 (100, [M-H]'). HRMS (FAB+): 360.196640 C24H26NO2 requires 360.196354.
C24H25NO2 (359.46) calculated: C 80.19% H 7.01% N 3.90% found: C 79.8% H 7.00% N 3.92%
STX321 3-Hydroxy-13-methyl-16-pyridin-4-ylmethylene-6,7, 8,9,11,12,13,14,15,16- decahydro-cyclopenta[a]phenanthren-17-one (CAB01154):
Figure imgf000227_0002
CAB01154
Sodium hydroxide (1.0 g, 25 mmol) was added to a suspension of estrone (1.35 g, 5.0 mmol) and pyridine-4-carbaldehyde (595 mg, 5.0 mmol) in ethanol (40 mL) at room temperature. The resulting dark orange solution was stirred at room temperature for 4 hours. Then glacial acetic (ca. 10 mL) acid was added with stirring. The colour changed to light yellow and a light yellow solid precipitated. The solid was filtered off and washed with water (50 mL), ethanol (20 mL), diethyl ether (50 mL) and hexane (50 mL) and dried under high vacuum. Yield: 1.588 g (88%) light yellow solid. H-NMR(DMSO-d6, 400 MHz) δ: 0.92 (s, 3H, -CH3), 1.30-1.68 (m, 5H), 1.82-1.92 (m, 1H), 1.96-2.04 (m, 1H), 2.14-2.24 (m, 1H), 2.31-2.38 (m, 1H), 2.61-2.69 (m, 1H), 2.71-2.84 (m, 2H), 2.87-2.96 (m, 1H), 6.47 (d, J = 2.3 Hz, 1 H), 6.53 (dd, J = 8.6, 2.3 Hz, 1 H), 7.06 (d, J = 8.6 H∑, 1 H), 7.28 (s, 1H), 7.60 (d, J = 6.2 H∑, 2H), 8.65 (d, J = 6.2 Hz, 2H), 9.05 (s, 1H, -OH). LRMS (FAB+): 360.2 (100, [M+H]+). LRMS (FAB-): 358.2 (100, [M-H]'). HRMS (FAB+): 360.196986 C24H26NO2 requires 360.196354.
C24H25NO2 (359.46) calculated: C 80.19% H 7.01% N 3.90%. found: C 80.1% H 6.90% N 3.96%
STX320 3-Hydroκy-13-'mβt yl-16-pyridin-3-ylmethylene-6, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta[a]phenanthren-17-one (CAB01156):
Figure imgf000228_0001
CAB01156
Sodium hydroxide (1.0 g, 25 mmol) was added to a suspension of estrone (1.35 g, 5.0 mmol) and pyridine-3-carbaldehyde (696 mg, 5.0 mmol) in ethanol (40 mL) at room temperature. The resulting dark orange solution was stirred at room temperature for 4 hours. Then glacial acetic (ca. 10 mL) acid was added with stirring. The colour changed to light yellow and a light yellow solid precipitated. The solid was filtered off and washed with water (50 mL), ethanol (20 mL), diethyl ether (50 mL) and hexane (50 mL) and dried under high vacuum. Yield: 1.6313 g (90%) yellow powder. 1H-NMR(DMSO-d6, 400 MHz) δ: 0.90 (s, 3H, -CH3), 1.26-1.66 (m, 5H), 1.82-1.90 (m, 1H), 1.95-2.02 (m, 1H), 2.14-2.22 (m, 1 H), 2.30-2.38 (m, 1H), 2.57-2.66 (m, 1H), 2.70-2.81 (m, 2H), 2.83-2.92 (m, 1H), 6.45 (d, J = 2.3 Hz, 1 H), 6.51 (dd, J = 8.2, 2.3 Hz, 1 H), 7.04 (d, J = 8.2 Hz, 1H), 7.34 (s, 1H), 7.47 (dd, J = 8.2, 5.1 Hz, 1 H), 8.06 (d, J = 8.2 Hz, 1H), 8.56 (dd, J = 4.7, 1.2 Hz, 1H), 8.82 (d, J = 1.6 Hz, 1H), 9.03 (s, 1H, -OH). LRMS (FAB+): 360.2 (100, [M+H]+). LRMS (FAB-): 276.1 (100), 358.2 (90, [M-H]'). HRMS (FAB+): 360.196434 C24H26NO2 requires 360.196354
ST1C324 13-ftflβthyl-16-pyιidin-4-ylmeihylene-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol (CAB01158):
Figure imgf000229_0001
GAB01158 16-(pyridin-4-yl)methylene estrone (360 mg, 1.0 mmol, CAB01154) was dissolved in ethanol (20 mL) and THF (20 mL). The solution was cooled to 0°C (icebath) and sodium borohydride (100 mg) was added. The reaction mixture was allowed to warm to room temperature and was stirred overnight at this temperature. The clear and colourless solution was concentrated under reduced pressure to ca. 20 mL volume and water (50 mL) was added. The product was filtered off and washed with water (50 mL), ethanol (20 mL) and diethylether (20 mL) and dried under high vacuum. Yield: 312 mg (86%) white powder. 1H-NMR(DMSO-d6, 400 MHz) δ: 0.58 (s, 3H, -CH3), 1.30-1.40 (m, 5H), 1.84- 1.98 (m, 2H), 2.12-2.34 (m, 3H), 2.64-2.78 (m, 3H), 3.97-4.03 (m, 1H, H-17), 5.30 (d, J = 5.9 Hz, 1H, -OH), 6.38 (s, 1H) 6.44 (s, 1H), 6.50 (dd, J = 8.2, 2.4 Hz, 1 H), 7.03 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 5.6 Hz, 2H), 8.47 (d, J = 5.5 Hz, 2H), 9.00 (s, 1H, -OH). LRMS (FAB+): 362.2 (100, [M+HD.
STX322 13-Methyl-16-pyridin-2-ylmethylene-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol (CAB01160):
Figure imgf000229_0002
CAB01160
16-(pyridin-2-yl)methylene estrone (360 mg, 1.0 mmol, CAB01150) was dissolved in ethanol (20 mL) and THF (20 mL). The solution was cooled to 0°C (icebath) and sodium borohydride (100 mg) was added. The reaction mixture was allowed to warm to room temperature and was stirred overnight at this temperature. The clear and colourless solution was concentrated under reduced pressure to ca. 20 mL volume and water (50 mL) was added. The product was filtered off and washed with water (50 mL), methanol (20 mL) and diethylether (50 mL) and dried under high vacuum. Yield: 328 mg (91%) white powder. 1H-NMR(DMSO-d6, 400 MHz) δ: 0.58 (s, 3H, -CH3), 1.23-1.45 (m, 5H), 1.81-1.92 (m, 2H), 2.10-2.18 (m, 1H), 2.23-2.37 (m, 2H), 2.66-2.89 (m, 3H), 3.97-4,03 (m, 1H, H-17), 5.21 (d, J = 5.9 Hz, 1 H, -OH), 6.41 (d, J = 1.3 Hz, 1 H), 6.48-6.51 (m, 2H), 7.04 (d, J = 8.6 Hz, 1H), 7.11 (dd, J = 7.0, 4.7 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.69 (dt, J. = 7.8, 2.0 Hz, 1H), 8.53 (d, J = 3.5 Hz, 1H), 9.00 (s, 1H, -OH). LRMS (FAB+): 362.2 (100, [M+Hf). LRMS (FAB-): 360.2 (1000, [M-H]").
ST1C323 13-ftfl®t yl-16-pyr1din-3-ylm®t yl®n®-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- d©cahydro-6H-c clopenta[a]phenanthr©ne-3, 17-diol (CAB01162):
Figure imgf000230_0001
CAB01162 16-(pyridin-3-yl)methylene estrone (360 mg, 1.0 mmol, CAB01156) was dissolved in ethanol (20 mL) and THF (20 mL). The solufion was cooled to 0°C (icebath) and sodium borohydride (100 mg) was added. The reaction mixture was allowed to warm to room temperature and was stirred overnight at this temperature. The clear and colourless solution was concentrated under reduced pressure to ca. 20 mL volume and water (50 mL) was added. The product was filtered off and washed with water (50 mL), methanol (20 mL) and diethylether (50 mL) and dried under high vacuum. Yield: 333 mg (92%) white powder. 1H-NMR(DMSO-d6, 400 MHz) δ: 0.58 (s, 3H, -CH3), 1,15-1.43 (m, 5H), 1.83-1.96 (m, 2H), 2.12-2.31 (m, 3H), 2.62-2.80 (m, 3H), 3.99 (bs, 1H, H-17), 5.25 (bs, 1H, -OH), 6.41 (d, J = 2.0 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 6.49 (dd, J = 8.6, 2.3 Hz, 1 H), 7.04 (d, J = 8.6 Hz, 1H), 7.34 (dd, J = 8.2, 5.1 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 8.36 (dd, J = 4.7, 1.2 Hz, 1 H), 8.56 (d, J = 2.0 Hz, 1H), 9.00 (bs, 1H, -OH). LRMS (FAB+): 362.2 (100, [M+H]+). LRMS (FAB-): 360.2 (1000, [M-H]'). HRMS (FAB+): 362.211723 C24H28NO2 requires 362.212004
STX452 Sulfamic acid 13-methyl-17-oxo-16-pyridin-4-ylmethylene-7, 8, 9, 11, 12, 13, 14, 5, 16, 17-decahydro-6H-cyclopenta[a]phenanthren-3-yl ester (CAB02078):
Figure imgf000230_0002
CAB02078 A solutufion of sulphamoyl chloride (5 mL, 0.7M, 3.5 mmol) in toluene was concentrated under reduced pressure (waterbath temperaturen 30°C). The residue was cooled to 0°C and DMA (5 mL) was added. The resulting colourless solution was stirred for 5 minutes and 16-(pyridin-4-yl)methylene estrone (360 mg, 1.0 mmol, CAB01154) was added. The mixture was stirred overnight at room temperature and the product was precipitated by addition of diethyl ether (20 mL) and ethyl acetate (20 mL). The yellow solid was filtered off and purified by heating it with acetone (30 mL) to reflux (the product did not dissolve completely). After cooling to room temperature the solid was filtered off and dried under high vacuum. Yield: 341 mg (78%) yellow powder. 1H-NMR(DMSO-d6, 400 MHz) δ: 0.94 (s, 3H, -CH3), 1.37-1.70 (m, 5H), 2.01-2.06 (m, 1H), 2.29-2.34 (m,1H), 2.41-2.48 (m, 2H), 2.73-3.02 (m, 4H), 6.98 (d, J = 1.8 Hz, 1H), 7.02 (dd, J = 8.4, 1.8 Hz, 1H), 7.34 (d, J = 8.4 H∑, 1H), 7.45 (s, 1H), 7.92 (s, 2H, -NH2), 8.18 (d. J = 6.2 H∑, 2H), 8.89 (d, J = 6.2 Hz, 2H). LRMS'(FAB÷): 439.1 (100, [M+H]+).
STX453 Sulfamic acid 13-methyl-17-oxo-16-pyridin-3-ylmethylene-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthren-3-yl ester (CAB02085):
Figure imgf000231_0001
CAB02085
A solutufion of sulphamoyl chloride (5 mL, 0.7M, 3.5 mmol) in toluene was concentrated under reduced pressure (waterbath temperaturen 30°C). The residue was cooled to 0°C and DMA (8 mL) was added. The resulting colourless solution was stirred for 5 minutes and 16-(pyridin-3-yl)methylene estrone (360 mg, 1.0 mmol, CAB01156) was added. The mixture was stirred overnight at room temperature and the product was precipitated by addition of diethyl ether (20 mL) and ethyl acetate (20 mL). The yellow solid was filtered off and purified by heating it with acetone (30 mL) to reflux (the product did not dissolve completely). After cooling to room temperature the solid was filtered off and dried under high vacuum. Yield: 333 mg (76%) yellow powder. 1H-NMR(DMSO-d6, 400 MHz) δ:
0.93 (s, 3H, -CH3), 1.34-1.74 (m, 5H), 1.86-1.96 (m,1H), 2.00-2.09 (m, 1 H), 2.26-2.36
(m, 1 H), 2.38-2.46 (m, 1H), 2.66-2.76 (m, 1H), 2.82-3.00 (m, 3H), 6.99 (d, J = 2.3 Hz, 1H), 7.02 (dd, J = 8.6, 2.3 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 7.45 (s, 1H), 7.94 (s, 2H, -
NH2), 8.01 (dd, J = 8.2, 5.5 Hz, 1H), 8.68 (d, J = 8.2 Hz, 1H), 8.83 <d, J = 5.5 Hz, 1H),
9.12 (s, 1 H). LRMS (FAB+): 439.1 (100, [M+H]+).
STX474 16-Furan-2-ylmethylene-3-hydroxy-13-methyl-6, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta[a]phenanthr©n-17-on© (CAB02072):
Figure imgf000232_0001
GAB02072 Potassium hydroxide (2.0 g) was added to a suspension of estrone (2.704 g, 10 mmol) and furfuraldehyde (2 mL, ca. 20 mmol) in ethanol (40 mL). The resulting dark brown solution was stirred for 4 hours at room temperature, then acetic acid (δ mL) and water (5ml) were added.' After a few minutes the product started to precipitate. The precipitation was completed by addition of more water (ca. 10 mL). The yellow solid was filtered off, washed with water, dissolved in chloroform and dried over Na2SO4. The solvent was removed under reduced pressure and the residue was crystallised from ethyl acetate/hexane. Yield: 3.24 g (93%) light yellow crystals. 1H-NMR(CDCI3, 400 MHz) δ: 0.97 (s, 3H, -CH3), 1.42-1.76 (m, 5H), 2.00-2.20 (m, 2H), 2.22-2.36 (m, 1H), 2.36-2.61 (m, 2H), 2.82-3.00 (m, 2H), 3.14 (ddd, J = 15.2, 6.6, 1.6 Hz, 1H), 5.08 (s, 1 H, - OH), 6.52 (dd , J = 3.1 , 1.6 Hz, 1 H), 6.62 (d, J = 2.7 Hz, 1H), 6.64-6.70 (m, 2H), 7.16 (d, J = 8,2 Hz, 1 H), 7.22-7.27 (m, 1H), 7.67 (d, J, = 1.6 Hz, 1 H). LRMS (FAB+): 349.2 (100, [M+H]+). ;
STX475 3-Hydroxy-13-methyl-16-thiophen-2-ylmethylene-6, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta[a]phenanthren-17-one (CAB02073):
Figure imgf000232_0002
CAB02073
Potassium hydroxide (2.0 g) was added to a suspension of estrone (1.352 g, 5.0 mmol) and thiophene-2-carboxaldehyde (561 mg, 5.0 mmol) in ethanol (40 mL). The resulting dark brown solution was stirred for 4 hours at room temperature, then acetic acid (5 mL) and water (5ml) were added, the product started to precipitate after a few minutes and was filtered off, washed with water (20 mL), ethanol (10 mL) and diethyl ether (10 mL) and dried under high vacuum. Yield: 1.614 g (89%) yellow solid. 1H-NMR(CDC13, 400 MHz) δ: 0.98 (s, 3H, -CH3), 1.44-1.78 (m, 5H), 2.02-2.14 (m, 2H), 2.26-2.46 (m, 3H), 2.84-3.02 (m, 3H), 4.98 (s, 1H, -OH), 6.62 (d, J = 2.7 Hz, 1H), 6.67 (dd, J = 8.6, 2.7 Hz, 1H), 7.14 (dd, J = 5.1, 3.5 Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 7.35 (d, J = 3.5 Hz, 1H), 7.53 (d, J = 5.1 Hz, 1H), 7.67 (s, 1H). LRMS (FAB+): 365.2 (100, [M+H]+). STX507 13-Methyl-16-thiophen-2-ylmethylene-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- d©cahydro-6H-cyclopenta[a]phenanthr©ne-3, 17-diol (CAB02076):
Figure imgf000233_0001
GAB0207® Sodium borohydride (100 mg) was added to a solution of 16-(thiophenen-2- yl)methylene estrone (365 mg, 1.0 mmol, CAB02073) in THF (20 mL) and ethanol (40 mL) at 0°C. The solution was stirred overnight at room temperature and concentrated under reduced pressure. The residue was dissolved in ethyl acetate (40 mL), the organic layer was washed with water (40 mL) and brine (20 mL), dried over NA2SO4 and concentrated under reduced pressure. The white crude product was crystallised from ethyl acetate/hexane. Yield: 208 mg (57%) colourless crystals. 1H-NMR(CDCI3, 400 MHz) δ: 0.71 (s, 3H, -CH3), 1.38-1.58 (m, 6H), 1.98-2.06 (m, 2H), 2.12-2.22 (m, 1 H), 2.26-2.40 (m, 2H), 2.68 (dd, J = 16.9, 6.6 Hz, 1 H), 2.82-2.92 (m, 2H), 4.17 (d, J = 9.4 Hz, 1 H, H-17), 4.66 (s, 1 H, -OH), 6.59 (d, J = 2.3 Hz, 1 H), 6.64 (dd, J = 8.6, 2.3 Hz, 1 H), 6.81 (d, J = 2.3 Hz, 1 H), 7.00-7.06 (m, 2H), 7.18 (d, J = 8.6 Hz, 1 H), 7.27 (d, J = 4.7 Hz, 1 H). LRMS (FAB+): 366.2 (100, [M]+).
STX476 3-Hydroxy-13-methyl-16-(4-nitro-benzylidene)-6, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta[a]phenanthren-17-one (CAB02142):
Figure imgf000233_0002
CAB02142
Potassium hydroxide (2.0 g) was added to a suspension of estrone (1.352 g, 5.0 mmol) and 4-nitro-benzaldehyde (756 mg, 5.0 mmol) in ethanol (80 mL). The resulting dark brown solution was stirred for 4 hours at room temperature, then acetic acid (5 mL) and water (5ml) were added (colour changes to orange). The product precipitated and was filtered off, washed with water (50 mL), ethanol (50 mL) and diethyl ether (50 mL) and dried under high vacuum. Yield: 1.682 g (83%) yellow solid. 1H-NMR(DMSO-d6, 400 MHz) δ: 0.93 (s, 3H, -CH3), 1.30-1.68 (m, 5H), 1.86-1.92 (m, 1H), 1.96-2.04 (m, 1 H), 2.16-2.24 (m, 1H), 2.32-2.39 (m, 1H), 2.62-2.82 (m, 2H), 2.88-2.96 (m, 1H), 6.46 (d, J = 2.3 Hz, 1H), 6.52 (dd, J = 8.6, 2.3 Hz, 1 H), 7.06 (d, J = 8.6 Hz, 1H), 7.42 (s, 1H), 7.92 (d, J = 8.6 Hz, 2H), 8.27 (d, J = 8.6 Hz, 2H), 9.04 (s, 1H, -OH). LRMS (FAB+): 404.1 (100, +Hf).
STX478 4-(3-Hydroxy-13-methyl-17-oxo-6, 7, 8, 9, 11, 12, 13, 14, 15, 17-d©cahydro- cyclop©nta[a]phenanthr©n-16-ylidenem©th l)-ben2onitrile (CAB02144):
Figure imgf000234_0001
CAB02144
Potassium hydroxide (2.0 g) was added to a suspension of estrone (1.352 g, 5.0 mmol) and 4-cyano-benzaldehyde (655 mg, 5.0 mmol) in ethanol (40 mL). The resulting orange solution was stirred for 4 hours at room temperature, then acetic acid (5 mL) and water (5ml) were added (colour changes to yellow). The product precipitated and was filtered off, washed with water (50 mL), ethanol (50 mL) and diethyl ether (50 mL) and dried under high vacuum. Yield: 1.795 g (94%) pale yellow solid. H-NMR(DMSO-d6, 400 MHz) δ: 0.91 (s, 3H, -CH3), 1.30-1.66 (m, 5H), 1.82-1.90 (m, 1H), 1.94-2.02 (m, 1H), 2.14-2.22 (m, 1 H), 2.30-2.38 (m, 1H), 2.58-2.92 (m, 3H), 6.45 (d, J = 2.3 Hz, 1 H), 6.51 (dd, J = 8.6, 2.3 Hz, 1H), 7.04 (d, J = 8.6 Hz, 1H), 7.36 (s, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.90 (d, J = 8.6 Hz, 2H), 9.03 (s, 1H, -OH). LRMS (FAB+): 384.1 (100, [M+H]+). LRMS (FAB-): 383.2 (100, [M]').
STX508 13-Methyl-16-(4-nitro-benzylidene)-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthrene-3, 17-diol (CAB02155):
Figure imgf000234_0002
CAB02155 Sodium borohydride (100 mg) was added to a solufion of 16-(4"-nitrophenyl)methylene estrone (404 mg, 1.0 mmol, CAB02142) in THF (20 mL) and ethanol (20 mL) at 0°C. The solution was stirred overnight at room temperature and concentrated under reduced pressure to ca 10 mL volume. Water (50 mL) was added to the mixture, the precipitate was filtered off, washed with water (50 mL), ethanol (50 mL) and diethyl ether (50 mL) and dried under high vacuum. Yield: 401 mg (99%) pale yellow solid. 1H-NMR(DMSO- dβ, 400 MHz) δ: 0.60 (s, 3H, -CH3), 1.22-1.46 (m, 5H), 1.84-1.96 (m, 2H), 2.16-2.32 (m, 3H), 2.62-2.80 (m, 3H), 4,02 (bs, 1H, H-17), 5.36 (d, J = 5.9 Hz, 1H, -OH), 6.44 (d, J = 2.3 Hz, 1H), 6.52 (dd, J = 8.6, 2.3 Hz, 1H), 6.56 (d, J = 2.3 Hz, 1H), 7.05 (d, J = 8.6 H∑, 1H), 7.65 (d, J = 9.0 Hz, 2H), 8.18 (d, J = 9.0 Hz, 1H), 9.02 (s, 1H, -OH). LRMS (FAB+): 405.2 (100, [M]+).
STX510 4-(3,17-Dihydro2cy-13-methyl-6, 7, 8, 9, 11, 12, 13, 14, 15, 17-decahydro- cyciopenta[a]phenanthren-16-ylidenemethyi)-benzonitrile (CAB02157):
Figure imgf000235_0001
CAB02157
Sodium borohydride (100 mg) was added to a solution of 16-(4x-cyano- pheny!)methylene estrone (384 mg, 1.0 mmol, CAB02144) in THF (20 mL) and ethanol (20 mL) at 0°C. The solution was stirred overnight at room temperature and concentrated under reduced pressure to ca 10 mL volume. Water (50 mL) was added to the mixture, the precipitate was filtered off, washed with water (50 mL), ethanol (30 mL) and diethyl ether (50 mL) and dried under high vacuum. Yield: 366 mg (95%) white solid. 1H-NMR(DMSO-d6, 400 MHz) δ: 0.60 (s, 3H, -CH3), 1.30-1.42 (m, 5H), 1.84-1.96 (m, 2H), 2.14-2.34 (m, 3H), 2.65-2.79 (m, 3H), 3.98-4.02 (m, 1H, H-17), 5.31 (d, J = 5.9 Hz, 1H, -OH), 6.44 (d, J = 2.3 Hz, 1 H), 6.49-6.52 (m, 2H), 7.05 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 9.0 Hz, 1H), 9.01 (s, 1H, -OH). LRMS (FAB+): 385.2 (100, [M]+).
[3-(tert-Butyl-dimethyl-silanyloxy)-13-methyl-17-oxo-6, 7, 8, 9, 11, 12, 13, 14, 15, 17- decahydro-cyclop@nta[a]phenanthren-16-ylidene]-hydroxy-ac@tic acid ©thyl ©ster (CAB01072).
Figure imgf000236_0001
CAB0 072
Sodium ethoxide (340 mg, 5.0 mmol) was added to a solution of 3-O-TBDMS-estrone (700 mg, 1.82 mmol) and diethyl oxalate (1.0 ml) in toluene (20 ml). The resulting yellow solution was stirred for 2 hours at room temperature (TLC-control). The solution was transferred into a separation funnel and ethyl acetate (50 ml) and 6N hydrochloric acid (20 ml) were added. Intensive shaking resulted in a nearly colourless organic layer, which was separated and dried over sodium sulphate and concentrated under reduced pressure. The residue was crystallised from ethanol. Yield: 737 mg (83%) colourless crystals. 1H-NMR (CDCI3, 400 MHz) δ: 0.19 (s, 6H), 0.98 (s, 9H, -C(CH3)3), 1.00 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H), 1.43-1.71 (m, 5H), 1.98-2.06 (m, 2H), 2.24-2.34 (m, 1H), 2.38- 2.46 (m, 2H), 2.82-2.90 (m, 2H), 3.08 (dd, J = 16.0, 6.0 Hz, 1H), 4.36 (q, J = 7.2 Hz, 2H), 6.57 (d, J = 2.3 Hz„ 1H), 6.63 (dd, J = 8.6, 2.3 Hz, 1H), 7.11 (d, J = 8.6 Hz, 1H), 12.47 (bs, 1H). LRMS (FAB+): 73.1 (61), 485.3 (100, [M+Hf)- LRMS (FAB-): 483.2 (100, [M- H]'). HRMS (FAB+) 484.263771 C28H40O5Si requires 484.264503
Hydroxy-(3-hydroxy-13-methyl-17-oxo-6, 7, 8, 9, 11, 12, 13, 14, 15, 17-decahydro- cyclopenta[a]phenanthren-16-ylidene)-acetic acid ethyl ester (CAB01096, STX330).
Figure imgf000236_0002
CAB01096
Tetrabutylammonium fluoride (1.5 ml, 1.5 mmol, 1M solution in THF)was added to a 6o\ t\on o 3-(tert-Butyl-dimethyl-silanyloxy)-13~methyl-17-oxo-β, 7, 8, 9, 11, 12, 13, 14, 15, 17-decahydro-cyclopenta[a]phenanthren-16-ylidene]-hydroxy-acetic acid ethyl ester (485 mg, 1.0 mmol, CAB01072) in THF (25 ml) at 0°C (icebath). The resulting yellow solution was stirred for 1 h at this temperature, and then glacial acetic acid (1 ml) and ethyl acetate (75 ml) were added. The mixture was transferred into a separation funnel, washed with water (3 x 25 ml) and brine (25 ml), dried over sodium sulphate and concentrated under reduced pressure. The residue was crystallised from ethyl acetate/hexane. Yield: 303 mg (82%) colourless crystals. 1H-NMR (CDCI3, 400 MHz) δ: 1.00 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H), 1.42-1.70 (m, 2H), 2.22-2.32 (m, 1H), 2.36-2.48 (m, 2H), 2.83-2.91 (m, 2H), 3.09 (dd, J = 16.0, 6.0 H∑, 4.36 (q, J = 7.2 Hz, 2H), 4.61 (bs, 1H, -OH), 6.59 (d, J = 2.3 Hz, 1H), 6.64 (dd, J = 8.2, 2.3 Hz, 1H), 7.14 (d, J = 8.2 Hz, 1H), 12.47 (bs, 1H, -OH). 13C-NMR (CDCI3, 100 MHz) δ : 14.2, 15.0, 25.9, 26.7, 27.5, 29.4, 31.1, 37.7, 43.9, 48.8, 49.4, 62.2, 112.8, 115.2, 116.5, 126.2, 131.6, 134.0, 137.7, 152.4, 153.4, 162.6, 216.7. LRMS (FAB+): 370.2 (100, [M+Hf ).
1 -Benzy lidene-3-hydroxy-13-mβthyl-6,7, 8,9, 11 , 12, 13, 14, 15, 1 -decahydro- cyclopenta[a]phenanthren-17-one (CAB01122, STX312).
Figure imgf000237_0001
CAB01122
Potassium hydroxide (1.0 g) was added to a solution of estrone (1.350 g, 5.0 mmol) and benzaldehyde (1.0 g, 9.4 mmol) in methanol (50 ml) at room temperature. The resulting orange solution was stirred at this temperature for 4h. Then glacial acetic acid (ca. 5 ml) was added with stirring. The colour changed to light yellow and a white solid precipitated. The solid was filtered off and washed with water (50 ml), methanol (20 ml), diethyl ether (50 ml) and hexane and dried under high vacuum. Yield 1.578 g (88%). H-NMR (CDCI3, 400 MHz) δ: 1.00 (s, 3H), 1.40-1.77 (m, 5H), 2.04-2.12 (m, 2H), 2.26-2.34 (m, 1H), 2.38- 2.45 (m, 1H), 2.50-2.60 (m, 1H) 2.86-3.03 (m, 3H), 4.76 (s, 1H, -OH), 6.60 (d, J = 2.3 Hz, 1H), 6.66 (dd, J = 8.2, 2.3 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 7.34-7.45 (m, 3H), 7.48 (s, 1H), 7.54-7.69 (m, 2H). LRMS (FAB+): 359.2 (100, [M+H]+). LRMS (FAB-): 357.2(100, [M-H]'). HRMS (FAB+) 359.202332 C2sH27O2 requires 359.201105
3-Hydroϊcy-13-methyl-16-(3,4,5-trimethoxy-benzylidene)-6, 7, 8, 9, 11, 12, 13, 14, 15, 1@-d@cahydro-cyclop@nta[a]ph@nanthren-17-on@ (CAB01124, STX328).
Figure imgf000238_0001
GΛB01124
Potassium hydroxide (1.0 g) was added to a solution of estrone (1.350 g, 2.0 mmol) and 3,4,5-trimethoxybenzaldehyde (1.00 g, 5.10 mmol) in ethanol (50 ml) at room temperature. The resulting dark brown solution was stirred at this temperature for 4h, then glacial acetic acid (ca. 2.5 ml) was added with stirring. The colour changed to yellow and a white solid precipitated. The solid was filtered off and washed with water (50 ml), ethanol (20 ml), diethyl ether (50 ml) and hexane and recrystallised from ethanol. Yield: 2.063 g (92%) light yellow crystals. 1H-NMR (CDCl3, 400 MHz) δ: 1.00 (s, 3H), 1.40-1.78 (m,5H), 2.00-2.11 (m, 2H), 2.26-2.34 (m, 1 H), 2.38-2.56 (m, 2H), 2.84- 3.04 (m, 3H), 3.90 (s, 3H), 3.91 (s, 6H), 4.93 (s, 1 H, -OH), 6.60 (d, J = 2.3 Hz, 1 H), 6.66 (dd, J = 8.2, 2.3 Hz, 1 H), 6.80 (s, 2H), 7.17 (d, J = 8.2 Hz, 1 H), 7.40 (s, 1H). 3C-NMR (CDCI3, 100 MHz) δ : 15.0, 26.4, 27.2, 29.4, 32.1 , 38.4, 44.4, 48.2, 49.0, 56.6, 61.3, 108.2, 113.2, 115.6, 126.6, 131.3, 131.9, 133.9, 135.2, 137.9, 139.7, 153.4, 154.0, 209.9. LRMS (FAB+): 449.3 (100, [M+H]+). LRMS (FAB-): 447.3 (100, [M-H]'). HRMS (FAB+): 448.223763. C28H32O5 requires 448.224974 C28H32O5 (448.6) calculated: C 74.97% H 7.19% found: C 75.05% H 7.18%
13-Methyl-16-(3,4,5-trimethoxy-benzylidene)-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthrene-3, 17-diol (CAB02001, STX329).
Figure imgf000238_0002
GAB02C91 A solution of 3-Hydroxy-13-methyl-16-(3,4,5-trimethoxy-benzylidene)-6, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta[a]phenanthren-17-one (448 mg, 1.0 mmol, CAB01124) in THF/methanol (1 : 1, 40 ml) was cooled to 0°C (ice bath) and sodium borohydride (100 mg,..2.64 mmol) was added. The reaction mixture was stirred for 2h at this temperature, while it was turning from slightly yellow to colourless. The solution was transferred into a separation funnel and ethyl acetate (100 ml) and water (100 ml) were added. The organic layer was separated, washed with water (50 ml) and brine (50 ml), dried over sodium sulphate and concentrated under reduced pressure. Yield 445 mg (99%) white solid. ^H-NMR (CDCI3, 400 MHz) δ: 0.74 (s, 3H), 1.20-1.60 (m, 6H), 1.90- 1.98 (m, 1H), 2.01-2.08 (m, 1H), 2.21-2.41 (m, 3H), 2.74-2.90 (m, 3H), 3.86 (s, 3H), 3.89 (s, 6H), 4.15 (d, J = 9.4 Hz, 1H, H-17), 4.62 (s, 1H, -OH), 6.49-6.51 (m, 1H), 6.59 (d, J = 2.3 Hz, 1H), 6.63-6.68 (m, 3H), 7.18 (d, J = 8.2Hz, 1H). LRMS (FAB+): 451.3 (100, [M+H]+).
16-Benzylidene-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-3,17-diol (CAB01128, STX313).
Figure imgf000239_0001
CAB01128
16-Benzylidene-3-hydroxy-13-methyl-6,7, 8,9,11,12,13,14,15,16-decahydro- cyclopenta[a]phenanthren-17-one (179 mg, 0.50 mmol, CAB01122) was dissolved in ethanol (10 ml) and THF (10 ml). The resulting light yellow solution was cooled to 0°C (ice bath) and sodium borohydride (100 mg, 2.64 mmol) was added. The reaction mixture was stirred overnight at room temperature, the solvents were removed under reduced pressure and the residue was dissolved in ethyl acetate (40 ml). The organic solution was washed with water (2 x 25 ml) and brine (25 ml), dried over sodium sulphate and concentrated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate and precipitated by addition of hexane. The white solid was filtered off and dried under high vacuum. Yield 175 mg (97%). 1H-NMR (d6-DMSO, 270 MHz) δ: 0.61 (s, 3H), 1.20-1.50 (m, 5H), 1.82-.98 (m, 2H), 2.20-2.35 (m, 3H), 2.58- 2.82 (m, 3H), 3.95-4.05 (m, 1H, H-17), 5.15 (d, J = 6.2 Hz, 1H), 6.43 (s, 1H), 6.46 (d, J = 2.3 Hz, 1H), 6.52 (dd, J = 8.2, 2.3 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 7.15-7.20 (m, 1H), 7,31-7.41 (m, 4H). LRMS (FAB+): 343.2 (80), 360.2 (100, [M]+). LRMS (FAB-): 359.3 (100, [M-H]'). HRMS (FAB+) 360.209709 C25H28O2 requires 360.208930
Sulfamic acid 16-b@n2ylid©n©-13-methyl-17-øx©-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- d©cahydro-@H-cyclop©nta[a]phenan hren-3-yl ester (CAB01148, ST2C314).
Figure imgf000240_0001
CAB01148
5 ml of a 0.7 M sulphamoyl chloride solution in toluene (3.5 mmol) were concentrated under reduced pressure (30°C water bath temperature). The residue was cooled to 0°C
(ice bath) and DMA (10 ml) and 16-Benzylidene-3-hydroxy-13-methyl-6, 7, 8, 9, 11, 12,
13, 14, 15, 16-decahydro-cyc1openta[a]phenanthren-17-one (717 mg, 2.0 mmol,CAB01122) were added. The resulting clear solution was stirred for 1h at 0°C, then allowed to warm up to room temperature and stirred overnight. Ethyl acetate (50 ml) was added and the solution was washed with water (2 x 30 ml) and brine. The organic layer was dried over sodium sulphate and concentrated under reduced pressure.
The resulting off-white solid was dissolved in a small amount of ethyl acetate and precipitated by addition of hexane. Yield: 683 mg (78%) off-white solid. 1H-NMR (CDCI3,
400 MHz) δ: 0.96 (s, 3H), 1.20-2.60 (m, 11H), 2.80-3.02 (m, 3H), 5.38 (s, 2H, -NH2), 7.06 (d, J = 2.3 Hz, 1H), 7.11 (dd, J = 8.0, 2.3 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.34-
7.47 (m, 2H), 7.52-7.65 (m, 2H). LRMS (FAB+): 438.2 (100, [M]+).
Sulfamic acid 16-benzylidene-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthren-3-yl ester (CAB01152, STX3 5).
Figure imgf000240_0002
Sodium borohydride (100 mg, 2.64 mmol) was added to a solution of sulfamic acid 16- benzylidene-13 methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthren-3-yl ester (438 mg, 1.0 mmol, CABΘ1148) in THF (20 ml) and ethanol (20 ml) at 0°C. The mixture was stirred for 1h at this temperature (TLC-control) and ethyl acetate (100 ml) and water (50 ml) were added. The organic layer was separated and washed with water (50 ml) and brine (50 ml), dried over sodium sulphate and concentrated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate and precipitated by addition of diethyl ether. Yield: 440 mg (quant.) white powder. 1H-NMR (CDCI3, 400 MHz) δ: 0.67 (s, 3H), 1.36-1.62 (m, 6H), 2.00-2.10 (m, 2H), 2.22-2.41 (m, 3H), 2.78 (dd, J = 17.4, 7.2 Hz, 1H), 2.90-2.96 (m, 2H), 4.15 (d, J = 9.4 Hz, 1H, H-17), 4.86 (s, 2H, -NH2), 6.55 (d, J = 2.3 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 7.10 (dd, J = 8.2, 2.3 Hz,1H), 7.19-7.42 (m, 6H). LRMS (FAB+): 439.2 (100, [M]+).
Sulfamic acid 13-methyl-17-oxo-16-(3,4,5-trimethoxy-benzylidene)-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthren-3-yl ester(CAB02069, STX692).
Figure imgf000241_0001
CAB02069
5 ml of a 0.59 M sulphamoyl chloride solution in toluene (2.95 mmol) were concentrated under reduced pressure (30°C water bath temperature). The residue was cooled to 0°C
(ice bath) and DMA (10 ml) and 3-Hydroxy-13-methyl-16-(3,4,5-trimethoxy-benzylidene)-
6, 7, 8, 9, 11 , 12, 13, 14, 15, 16-decahydro-cyclopenta[a]phenanthren-17-one (899 mg,
2.0 mmol,CAB01124) were added. The resulting clear solution was stirred for 1h at 0°C, then allowed to warm up to room temperature and stirred overnight. Ethyl acetate (50 ml) was added and the solution was washed with water (2 x 30 ml) and brine. The organic layer was dried over sodium sulphate and concentrated under reduced pressure.
The resulting off-white solid was dissolved in a small amount of acetone and precipitated by addition of hexane. Yield: 913 mg (86%) light yellow solid. 1H-NMR (d6-DMSO, 400
MHz) δ: 0.91 (s, 3H), 1.35-1.74 (m, 4H), 1.86-1.92 (m, 1H), 2.02-2.10 (m, 1H), 2.26-2.34 (m, 1 H), 2.40-2.46 (m,1H) 2.60-2.70 (m, 1H), 2.84-2.96 ( , 3H), 3.32 (s, 9H), 6.95 (s,
2H), 6.98 (d, J = 2.3 Hz, 1H), 7.02 (dd, J = 8.2, 2.3 Hz, 1H), 7.29 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.90 (s, 2H, -NH2). LRMS (FAB+): 528.2 (100, [M+H]+). LRMS (FAB-): 526.2 (100, [M-H]').
Sulfamic acid 17-hydro2-y-13-me hyl-16-(3>4,5-trim©thθHy-ben2ylϊdene)-7> 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclop©nta[aJph®nanthren-3-yl
©st©r(CAB02084, ST 693).
Figure imgf000242_0001
CAB02084
Sodium borohydride (100 mg, 2.64 mmol) was added to a solution of sacid 13-methyl- 17-0X0-16-(3,4,5-trimethoxy-benzylidene)-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro- 6H-cyclopenta[a]phenanthren-3-yl ester (528 mg, .0 mmol, CAB02069) in THF (20 ml) and ethanol (20 ml) at 0°C. The mixture was stirred for 1h at this temperature (TLC- control) and ethyl acetate (100 ml) ahd water (50 ml) were added. The organic layer was separated and washed with water (50 ml) and brine (50 ml), dried over sodium sulphate and concentrated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate and precipitated by addition of diethyl ether. Yield: 527 mg (99%) white powder. 1H-NMR (CDCI3, 400 MHz) δ: 0.72 (s, 3H), 1.20-1.60 (m, 4H), 1.64-1.72 (m, 2H), 1.88-1.96 (m, 1H), 2.02-2.10 (m, 1H), 2.21-2.40 (m, 3H), 2.71-2.94 (m, 3H), 3.83 (s, 3H), 3.86 (s, 6H), 4.13 (d, J = 9.4 Hz, 1H, H-17), 5.17 (s, 2H, -NH2), 6.48 (d, J = 2.3 Hz, 1H), 6.61 (s, 2H), 7.02 (d, J = 2.3 Hz, 1H), 7.09 (dd, J = 8.2, 2.3 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H). LRMS (FAB+): 529.2 (100, [M]+). LRMS (FAB-): 528.2 (100, [M-H]').
SECTION 6
General procedure for debenzylation of protected phenol intermediates using a Radleys GreenHouse synthesiser.
Starting materials were dissolved/suspended in THF (1cm3) and to each suspension was added ethanol (1cm3). Each suspension was degassed by bubbling nitrogen through for a few minutes before Pd/C (5% wt., catalytic) was added and each solution/suspension was degassed for a further few minutes. Hydrogen gas (balloon) was then passed over the reactions and they were stirred under a hydrogen atmosphere at room temperature for 24h. 1H NMR of one of the reactions at this stage showed that the debenzylation had not gone to completion therefore all were degassed again and more Pd added, followed by stirring under hydrogen for a further 21 h. Each reaction was then filtered through celite and the celite washed with ethyl acetate and methanol. The solutions were concentrated in vacuo and the products purified by flash chromatography.
2-Hydroxy-6a-m®thyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydr©-7, S-diasa- pentaleno[2,1 -a]phenanthrene-9-carfoossylic acid (tetrahydro-furan-2-ylm©thyl)- amide STX 892 (GMA02058-2)
Figure imgf000243_0001
Rf 0.36 (EtOAc). 1H NMR (270MHz, CD3OD) δ: 0.92 (s, 3H), 1.25-1.95 (multiplets, 9H), 2.01-2.58 (m, 5H), 2.72-2.82 (m, 3H), 3.25-3.45 (m, 2H), 3.59-3.71 (m, 1H), 3.74-3.85 (m, 1H), 3.89-4.00 (m, 1H), 6.40 (d, J=2.5Hz, 1 H), 6.45 (dd, J=8.4, 2.7Hz, 1H), 6.99 (d, J=8.4Hz, 1 H); HPLC 100% (Rt 1.70, 4:96 H2O:MeOH); LCMS (APCI) 422.27 (M+H+)
2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid methylamide STX 893 (GMA02058-4)
Yield 57%. Rf 0.40 (EtOAc); 1H NMR (270MHz, CD3OD) δ: 1.03 (s, 3H), 1.39-2.45 (multiplets, 10H), 2.83-2.88 (m, 3H), 2.89 (s, 3H), 6.50 (d, J=2.5Hz, 1 H), 6.54 (appdd, J=8.4Hz, 1 H), 7.09 (d, J=8.2Hz, 1 H); HPLC 99% (Rt= 1.66, 4:96 H2O:MeOH); LCMS (APCI), 350.21 (M-H+)
2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- p©ntaleno[2,1-a3phenanthrene-9-carbojcylic cid ethyl-methyl-amid®
ST 894 (GfiflA02058-5)
Figure imgf000244_0001
Yield 76%. Rf 0.33 (EtOAc); 1H NMR (270MHz, CD3OD) δ: 0.96 (s, 3H), 1.12 (t, J=7.2H∑, 3H), 1.25-2.36 (multiplets, 10H), 2.60-2.79 (m, 3H), 2.95 and 3.09 (singlets, comb. 3H), 3.44-3.54 (m, 2H), 6.40 (d, J=2.5H∑, 1 H), 6.46 (dd, J=8.4, 2.7Hz, 1 H), 7.00 (d, J=8.4Hz, 1 H); HPLC 93% (Rt=1.69, 4:96 H2O:MeOH); Ace. Mass (FAB+) calcd 380.233803, obsd. 380.232979 (M+H+
2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (1-methyl-1 H-pyrrol-2-ylmethyl)- amide '
STX 895 (GMA02058-8)
Figure imgf000244_0002
Yield 36%. Rf 0.75 (EtOAc); 1H NMR (270MHz, CD3OD) δ: 0.94 (s, 3H), 1.05-1.90 (multiplets, 6H), 2.15-2.36 (m, 4H), 2.72-2.79 (m, 3H), 3.52 (s, 3H), 4.43 (s, 2H), 5.85- 5.86 (m, 1 H), 5.96-5.98 (m, 1 H), 6.40 (d, J=2.7Hz, 1 H), 6.45 (dd, J=8.4, 2.7Hz, 1H), 6.51-6.53 (m, 1H), 7.01 (d, J=8.4Hz, 1H); HPLC 100% (Rt=2.11 , 4:96 H2O:MeOH); Ace. Mass (FAB+) calcd. 431.244702, obsd. 431.244164 (M+H+)
2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)- amide STX 896 (GMA02058-9)
Figure imgf000244_0003
Yield 82%. Rf 0.26 (EtOAc); 1H NMR (400MHz, CD3OD) δ: 1.04 (s, 3H), 1.06-2.50 (multiplets, 10H), 2.54 (s, 3H), 2.78-2.90 (m, 3H), 4.66 (s, 2H), 6.50 (d, J=2.3Hz, 1 H), 6,55 (dd, J=8.2, 2.7Hz, 1H), 7.09 (d, J=8.6Hz, 1H), 8.48 (s, 1H), 8.49 (s, 1H); HPLC 89% (Rt=1.67, 4:96 H2O:MeOH); LCMS (APCI) 442.30 (M-H+)
(2-Hydroxy-6a-m©thyl-4fø, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-d@cahydr©-7, 8-diaza- pentaleno[2,1-a]phenanthr©n-9-yl)-(4-m©thyl-pip©ra2in-1-yl)-methanon© ST2C 897 (GRUA02058-12)
Figure imgf000245_0001
Yield 96%. Rf 0.1 (10%MeOH in DCM); 1H NMR (270MHz, CD3OD) δ: 1.05 (s, 3H), 1.28- 2.02 (m, 5H), 2.09-2.50 (m, 8H), 2.33 (s, 3H), 2.67-2.94 (m, 3H), 3.52-3.90 (m, 4H), 6.50 (d, J=2.5Hz, 1H), 6.55 (dd, J=8.3, 2.6Hz, 1H), 7.10 (d, J=8.7Hz, 1H); HPLC 94% (Rt=1.68, 4:96 H2O:MeOH); LCMS (APCI) 421.36 (M+H+)
2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (pyridin-2-ylmethyl)-amide STX 915 (GMA02070-1)
Figure imgf000245_0002
To a suspension of the acid starting material (0.095g, 0.28mmol) in dry DCM (6cm3) was added DMAP (catalytic), EDC (0.065g, 0.3mmol) and triethylamine (0.04cm3, 0.37mmol). The starting material did not dissolve therefore dry DMF (2cm3) was added and the resulting solution stirred at rt for 15min. To this solution was added 2- (aminomethyl) pyridine and the reaction was stirred at rt for 4d. The solution was washed with sat. aq. NaHCO3 and the organic layer separated and concentrated in vacuo. Flash chromatography using DCM followed by 10% MeOH in DCM as eluents gave a solution of the product in DMF. Addition of hexane to this followed by a small amount of DCM gave a white powder. H NMR (270MHz, DMSO-d6) δ: 0.94 (s, 3H), 1.72-1.95 (m, 5H), 2.00-2.49 (m, 4H), 2.55-2.97 (m, 4H), 4.45-4.55 (m, 2H), 6.45 (d, 1H), 6.51 (dd, J=8.4Hz, 1H), 7.05 (d, J=8.9Hz, 1H), 7.22-7.33 (m, 1H), 7.74 (m, 1H), 8.28 (m, NH), 8.52 (apps, 1 H), 9.03 (s, 1 H); HPLC 99.8% (Rt=1.67, 4:96 H2O:MeOH)
2-Hydro∑_y-6a-m©thyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-d©cahydr©-7, 8-diasa- pen£aleno[2,1-a]phenanthr ne-9-carbo--ylic acid (2-pyridin-3-yl-©thyl)-amid© STX 91β (CsRi.A02070-2)
Figure imgf000246_0001
Procedure as for STX 915. Precipitation gave a cream-coloured powder. 1H NMR (270MHz, DMSO-d6) δ: 0.93 (s, 3H), 1.36-3.5 (multiplets, 17H), 6.47 (appd, 1H), 6.52 (appdd, 1H), 7.07 (d, J=8.2Hz, 1H), 7.31-7.36 (m, 1H), 7.67 (appd, J~.7Hz, 1H), 8.45 (m, 2H), 9.07 (s, 1 H); LCMS (APCI) 441.48 (M-H+)
Alkylation Reactions
2-Benzyloxy-6a-methyl-7-phenethyl-4b, 5, 6, 6a, 7, 10, 10a, 10b, 11, 12-decahydro- 7, 8-diaza-pentaleno[2,1-a]phenanthren-9-ol, GMA02076
Figure imgf000246_0002
To a stirred, cooled (ice bath) solution of the benzyl protected hydroxypyrazole starting material (0.050g, 0.12mmol) in dry DMF (3cm3) was added sodium hydride (0.006g of 60% dispersion, 0.14mmol) and the reaction was stirred at 0°C for 15min. To this was added 2-bromoethyl benzene (0.024cm3, 0.18mmol) and the reaction was allowed to warm to room temperature and stirred overnight. Water (5cm3) was added and the product extracted with ethyl acetate. Flash chromatography using hexane followed by ethyl acetate as eluents gave an alkylated product as the second fraction, Rf 0.8 (EtOAc). 1H NMR (270MHz, CDCI3) δ: 1.01 (s, 3H), 1.29-2.41 (multiplets, 10H), 2.60 (dd, J=12.6, 5.9Hz, 1H), 2.82-2.95 (m, 2H), 3.07 (t, J=2.7Hz, 2H), 4,36 (t, J=7.2Hz, 2H), 5.04 (s, 2H), 6.73 (d, J=2.7Hz, 1H), 6.79 (appdd, J=8.5, 2.6Hz, 1H), 7.18-7.45 ( , 11H); LCMS (APCI) 505.37 (M+H+)
A: 2-Ben2ylo2sy-S-metho2.y-6a, 7-dim@thyl-4b, 5, 6, 6a, 7, 10, 10a, i, 11, 12- d©cahydr©-7, 8-diaza-pentaleno[2,1-a]phenanthrene
B: 2-Bensyloxy-6a,7, 8-trimethyl-5, 6, 6a, 7, 8, 10, 10a, 10b, 11, 12 decahydro- 7, 8-diaza-pentalenof2, 1-a]phenanthren-9-on®, GMAQ2107
Figure imgf000247_0001
B
To a stirred solution of the benzyl protected hydroxypyrazole starting material (0.200g, O.δmmol) in dry THF (2.3cm3) was added sodium hydride (0.06g of 60% dispersion, 1.5mmol) and the reaction was stirred at room temperature for 30min. To this was added iodomethane (0.1cm3, 1.5mmol) and stirring under nitrogen at room temperature was continued for 6h. Water was added and the products extracted into ethyl acetate. The combined extracts were concentrated in vacuo. Chromatography on a 20g flash column (flashmaster) using a hexane to ethyl acetate gradient followed by a methanol wash gave the title compounds as the second fraction, Rf 0.7 (EtOAc) and fifth fraction, Rf -baseline (EtOAc) and 0.65 (10%MeOH in DCM), respectively. Yield of A: 0.046g, 21%; Η NMR (270MHz, CDCI3) δ: 1.01 (s, 3H), 1.38-1.54 (m, 1H), 1.56-1.75 (m, 2H), 1.83- 1.97 (m, 2H), 2.15-2.44 (m, 5H), 2.50-2.62 (m, 1H), 2.82-2.93 (m, 2H), 3.65 (s, 3H), 3.87 (s, 3H), 5.03 (s, 2H), 6.74 (d, J=2.5Hz, 1H), 6.79 (dd, J=8.4, 2.7Hz, 1H), 7.19 (d, J=8.4Hz, 1 H), 7.29-7.45 (m, 5H); NOESY (400MHz, CDCI3) showed interaction between methyl peaks at 1.01 and 3.65ppm; HPLC 98% (Rt=4.70, 4:96 H2O:MeOH); LCMS (ES') 427.48 (M-H+); Aec. Mass (FAB+) calcd. 429.254204, obsd. 429.254517 (M+H+) Yield of B: 0.031 g, 14%; 1H NMR (270MHz, CDCI3) δ: 1.02 (s, 3H), 1.36-1.52 (m, 1H), 1.55-1.74 (m, 2H), 1.82-1.96 (m, 2H), 2.03-2.20 (m, 3H), 2.28-2.43 (m, 2H), 2.47-2.57 (m, 1H), 2.83-2.91 (m, 2H), 3.24 (s, 3H), 3.28 (s, 3H), 5.01 (s, 2H), 6.72 (d, J=2.5Hz, 1H), 6.77 (dd, J=8.5, 2.6Hz, 1H), 7.16 (d, J=8.4Hz, 1H), 7.27-7.69 (m, 5H); LCMS (ES") 427.42 (M-H+)
®-Rfl®t θ2sy-6sι, 7-dim®ϊhyl-4b, 5, 6, 6a, 7, 10, 10a, 10b, 11, 12-d©cahydrø-7, 8-diaκa- pentalen©[2, 1-a]ph®nan r®n-2-ol, QRflA 02114
Figure imgf000248_0001
To a stirred solution of the benzylated starting material (0.02δg, 0.06mmol) in dry DCM
(2cm3) under nitrogen, cooled to -78°C (dry ice/acetone) was added boron tribromide (0.065cm3 of a 1M solution in DCM, 0.065mmol) and stirring at -78°C was continued for
2.5h. Water was added, the reaction was allowed to warm to room temperature and the resulting white precipitate was collected by filtration. This was shown by 1H NMR and
LCMS to consist of a 3:1 mixture of dimethylated and monomethylated products. The dichloromethane layer of the filtrate was found to contain starting material and this was redissolved in DCM and reacted further with boron tribromide at -78°C for 1.5h before quenching with water as before. The white precipitate formed in this case was solely the dimethylated product. 1H NMR (270MHz, CD3OD) δ: 1.09 (s, 3H), 1.40-1.77 (m, 3H),
1.87-2.01 (m, 2H), 2.23-2.61 (m, 5H), 2.69-2.89 (m, 3H), 3.71 (s, 3H), 3.94 (s, 3H), 6.60
(d, J=2.5Hz, 1H), 6.55 (appdd, J-8.3, 2.6Hz, 1H), 7.09 (d, J=8.2Hz, 1H); LCMS (ES ) 337.35 (M-H+)
2-Hydroxy-6a, 7, 8-trimethyl-5, 6, 6a, 7, 8, 10, 10a, 10b, 11, 12-decahydro-4bH-7, 8- diaza-pentaleno[2, 1-a]phenanthren-9-one, STX942, GMA02116
Figure imgf000248_0002
To a stirred solution of the benzylated starting material (0.031 g, 0.07mmol) in dry DCM (3cm3) under nitrogen, cooled to -78°C, was added boron tribromide (0.15cm3 of a 1M solution in DCM (O.lδmmol) and the reaction was stirred at -78°C for 2h. The reaction was then quenched with water and the resulting white precipitate was collected by filtration and washed with water and DCM. Recrystallisafion from methanol gave a pale orange powder which was collected by filtration and washed with DCM and hexane, yield 0.0125g, 53%; H NMR (270MH∑, CD3OD) δ: 1.15 (s, 3H), 1.40-1.80 (m, 3H), 1.93-2.04 (m, 2H), 2.21-2.53 (m, 5H), 2.70 (dd, J=13.1 , 5.7H∑, 1H), 2.80-2.95 (m, 2H), 3.34 (s, 1H), 3.71 (s, 3H), 3.84 (s, 3H), 6.51 (s, 1H), 6.5,6 (dd, J=8.4, 2.7Hz, 1H), 7.09 (d, J=8.4Hz, 1H); LCMS (ES-): 337.16 (M-H+); HPLC 99.6% (Rt=2.46, 10:90 H2O:MeOH)
BIOLOGICAL DATA
The following assay for determining 17B-HSD Type I and Type II activities was performed.
17B-HSD TYPE I AND TYPE II ASSAY
♦ PURPOSE
To determine 17β-hydroxysteroid dehydrogenase activity (types I and II) in human breast cancer cells (T-47D and MDA) in the absence or presence of regulatory agents.
• SAFETY NOTES
• Wear gloves and a lab coat when working with human cell lines. • "H-EL 3H-E2, 14C-E.,, and 14C-E2 are radioactive.
• Assume E1f E2 and inhibitors to be carcinogenic and/or teratogenic.
• DMSO promotes absorption through the skin and so when used as a solvent the solute may be easily absorbed - avoid bodily contact.
• THF can form potentially explosive peroxides upon long standing in air. © Diethyl ether has extremely flammable liquid and vapour. It is harmful when swallowed, inhaled or absorbed through skin. Causes irritation to skin, eyes and respiratory tract and affects the central nervous system. o Solid CO2 - contact with the product may cause frostbite or freeze burns in exposed tissues. • Methanol is highly flammable. • Dichloromethane must be handled with caution - causes respiratory tract, skin and eye irritation and may be a carcinogen. o Ethyl acetate - flammable liquid and vapour which is harmful if swallowed or inhaled.
Causes irritation to skin, eyes and respiratory tract and affects the central nervous system.
« Ecoscint A - contact hazard - will irritate skin and eyes.
0 PROCEDURE
Figure imgf000250_0001
Equipment o Beckman LS6000SC Scintillation Spectrometer. o SMI Multi-tube Vortexer - model 2601.
• Fume Hood. o TLC Aluminium Sheets Silica Gel 60 F254. ERCK
θ Purification of 3H-E1/3H-E2. © Set up a TLC plate with a lane at either end and a large lane in the middle for streaking.
• Streak 100 μCi (100 μl or rather 5 x 20μl) 3H-E1/3H-E2 in the middle lane.
• Spot 20 μl cold E.,/E2 in the outer lanes.
• Leave the plate in the TLC tank for 1 - 1 1/2 hours. • Visualise the spots of E^E., (in the outer lanes) under UV light and circle.
• Cut out the area where the labelled E-,/E2 should be (according to the position of the cold E-,/E2 on the TLC plate) and place in a glass vial.
• Elute in 5.0 ml of diethyl ether.
• Vortex mix the contents of the vial and leave for 12 hours at 4°C. • Vortex the vial again and allow the contents to settle down.
• Add 1 ml of distilled water (to cover all the bits of plate) and freeze the aqueous phase in a solid carbon dioxide/methanol mixture.
• Decant the diethyl ether phase into another glass vial and evaporate to dryness under an airstream at 40°C. • Dissolve the residue in 1 ml ethanol and count a 5 μl aliquot for radioactivity using a liquid scintillation counter.
• Calculations
Use Specific Activity of 3H-E1/3H-E2 to calculate how much 3H-E1/3H-E2 needs to be added to each flask for physiological concentration (6 pmol) per flask. Also calculate how much needs to be added to each well for physiological concentration (3 pmol) per well when carrying out the assay in 24-well plate format.
• Assay of 17β-hydroxysteroid dehydrogenase activity in T-47D cells (E-, → E2). • Remove the growth medium from all flasks. • Add 2.5 ml of substrate [(assay medium + 3H-Eι) + appropriate inhibitor] to each flask. β Incubate the flasks at 37°C for 0.5 hours. o Take 2 ml from each flask and add directly to tubes containing 14C-oestradiol (5000 cpm). o Extract the steroids from the medium with 4 ml diethyl ether.
® Mechanically shake the tubes for 3 minutes to get a good partition. o Separate the ether phase by freezing the aqueous phase in a solid carbon dioxide/methanol mixture and decant the ether into tubes containing 50 μg (20 μl of 10 mM solution) unlabelled product to help visualise the labelled product upon TLC. • Evaporate the ether phase to dryness under an airstream at 40°C.
• Dissolve the residue in 6-8 drops of diethyl ether and spot onto a TLC plate containing a fluorescent indicator.
• Separate the oestrone and oestradiol by TLC using dichloromethane/ethyl acetate (4:1 v/v) as solvents. • Visualise the spots of product steroid (3H-E2) under UV light, cut them out, and place them in scintillation vials.
• Add 0.5 ml methanol to the vials to elute the pieces of TLC.
• Add 0.5 ml assay medium to each vial to correct for volume.
• Add 10 ml Ecoscint A to each vial. • Measure the total activity of the 3H isotope by counting 0.5 ml substrate solution containing the 3H isotope (3H-E-,), 0.5 ml methanol and 10 ml Ecoscint A. Measure the total activity of the 14C isotope by counting 14C-oestradiol (5000 cpm), 0.5 mi assay medium, 0.5 ml methanol and 10 ml Ecoscint A.
• Count product and recovery radioactivity in a liquid scintillation counter using a program for dual [3H/14C] isotopes.
• Calculations
Overall, four corrections are applied to the raw data: Crossover Correction. Recovery Correction. Blank Correction. Dilution Correction. Use Microsoft Excel.
B & C represent columns of 3H & 14C raw data respectively: D. C x 0.14 = Crossover
E. B-D = Crossover Corrected
F. C / Mean Total Activity 1 C = Recovery
G. E / F = Recovery Corrected
H. G - Blank (mean) = Blank Corrected
I. H x Constant (see below) = fmol/flask/O.Shrs
J- K(Xι + ^2) x 272) / 1 ,000,000] = cells/flask (millions)
K. I / J = fmol/0.5hrs/million cells
L K x 2 = fmol/hr/million cells
M. Mean of each triplicate = Mean Activity
N. Standard Deviation of each triplicate = Standard Deviation Activity
O. [(100 / Mean adtivity of control) x L] = % Activity
P. 100 - O = % Inhibition
Q. Mean % Inhibition of each triplicate = Mean % Inhibition
R. SD % Inhibition of each triplicate = Standard Deviation % Inhibition
S. Label Treatments and Concentrations = Inhibitor Codes & Concentrations
Graphs and statistics as required.
Dilution (2.5/2)
Constant = [1.25 x (pmol 3H-E1 / total cpm 3H-E1/flask)]
• Assay of 17β-hydroxysteroid dehydrogenase activity in MDA-MB-231 cells (E2 → E.,) • Remove the growth medium from all flasks.
• Add 2.5 ml substrate medium [(assay medium + 3H-E2) + appropriate inhibitor] to each flask.
• Incubate the flasks at 37°C for 3 hours.
» Take 2 ml from each flask and add directly to tubes containing 14C-oestrone (5000 cpm). e Extract the steroids from the medium with 4 ml diethyl ether. o Mechanically shake the tubes for 3 minutes to get a good partition. • Separate the ether phase by freezing the aqueous phase in a solid carbon dioxide/methanol mixture and decant the ether into tubes containing 50 μg (20 μl of 10 mM solution) unlabelled product to help visualise the labelled product upon TLC. o Evaporate the ether phase to dryness under and airstream at 40°C. o Dissolve the residue in 6-8 drops of diethyl ether and spot onto a TLC plate containing a fluorescent indicator. o Separate the oestrone and oestradiol by TLC using dichloromethane/ethyl acetate
(4:1 v/v) as solvents. β Visualise the spots of product steroid (3H-E.,) under UV light, cut them out, and place into scintillation vials.
• Add 0.5 ml methanol to the vials to elute the pieces of TLC.
• Add 0.5 ml assay medium to each vial to correct for volume.
• Add 10 ml Ecoscint A to each vial.
• Measure the total activity of the 3H isotope by counting O.δ ml substrate solution containing the 3H isotope (3H-E2), 0.5 ml methanol and 10 ml Ecoscint A. Measure the total activity of the 14C isotope by counting 4C-oestrone (5000 cpm), 0.5 ml assay medium, 0.5 ml methanol and 10 ml Ecoscint A.
• Count product and recovery radioactivity in a liquid scintillation counter using a program for dual [3H/14C] isotopes.
• Calculations
Overall, four corrections are applied to the raw data: Crossover Correction. Recovery Correction. Blank Correction. Dilution Correction, Use Microsoft Excel.
B & C represent columns of 3H and 14C raw data respectively.
D. C x 0.14 = Crossover
E. B - D = Crossover Corrected
F. C / Mean Total Activity 14C = Recovery
G. E / F = Recovery Corrected H. G - Blank (Mean) = Blank Corrected I. H x Constant = fmol/flask/3 hrs
J. [((Xi + x2) x 272) / 1 ,000,000] = cells / flask (millions)
K. I / J = fmol/3 hrs/million cells
L K / 3 = fmol/hr/million cells
Mean of each triplicate = Mean Activity N Standard Deviation' of each triplicate = Standard Deviation Activity O (100 x Mean Activity of Control) x L = % Activity P 100 - O = % Inhibition
Q Mean % Inhibition of each triplicate = Mean % Inhibition R, SD % Inhibition of each triplicate = SD % Inhibition
S Label Treatments and Concentrations = Inhibitor Codes & Concentrations
Graphs and statistics as required.
THER NOTES:
Dilution (2.5/2)
Constant = [1.25 x (pmol 3H-E1 / total cpm 3H-E1/flask)] ■■
• Account for all radioactive isotope usage.
• The assay of 17B-HSD Type I activity in T-47D cells is often carried out in 24-well plate format (rather than T25 flasks). Under these circumstances only 1.5 ml substrate solution (± inhibitor) is used and the incubation time is increased to 3 hours. After incubation only 1 ml is taken from each well.
♦ EXAMPLES OF EXPECTED MEAN % INHIBITION VALUES FOR THE POSITIVE CONTROL:
ET → E2 Typical % inhibitions obtained with 10 μM E1 are between 97% and 99% inhibition.
E2 — > E1 Typical % inhibitions obtained with 10 μM E2 are between 37% and 43% inhibition. RESULTS
Figure imgf000256_0001
Figure imgf000257_0001
Figure imgf000258_0001
Figure imgf000259_0001
258
Figure imgf000260_0001
Figure imgf000261_0001
260
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Figure imgf000265_0001
264
Figure imgf000266_0001
Figure imgf000267_0001
Figure imgf000268_0001
Figure imgf000269_0001
All publications and patents and patent applications mentioned in the above specification are herein incorporated by reference.
Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.
References
(I) Saunders, C. M.; Baum, M. Management of early breast cancer. Oncol, in Pract. 1994, 3, 4-8 (2) Nicholls P. J. Breast cancer management: science and care together. Pharm. J. 1997, 259, 459-470 '
(3) Miller, B. A.; Kolonel, L. N.; Bernstein, L.; Young, Jr. J. L.; Swanson, G. M.; West, D.; Key, C. R.; Lift, J. M.; Glover, C. S.; Alexander, G. A.; et al. (eds). Racial/Ethnic patterns of cancer in the united states 1988-1992. National Cancer Institute 1996 (4) (a) Kaae, S. and Johansen, H. Does simple mastectomy followed by irradiation offer the survival comparable to radical procedures? International Journal of Radiation Oncology, Biology, Physics. 1977, 2, 1163-1166 (b) Holli, K.; Saaristo, R.; Isola, J.; Joensuu, H. and Hakama, M. Lumpectomy with or without postoperative radiotherapy for breast cancer with favourable prognostic features: results of a randomised study. Br. J. Cancer 2001, 84(2), 164-169
(5) Early Breast Cancer Trialists' Collaborative Group. Effects of adjuvant Tamoxifen and of cytotoxic therapy on mortality in early breast cancer. N. Eng. J. Med. 1988, 319, 1681-1692
(6) Gorski, J.; Toft, D.; Shyamala, G.; Smith, D.; Notides, A. Hormones receptors: studies on the interaction of estrogens with the uterus. Recent Prog. Horm. Res.
1968, 24, 46-80
(7) Gorski, J. and Gannon F. Current models of steroid hormone action: a critique. Ann. Rev. Physiol. 1976, 38, 425-460
(8) Coulson, C. J. Steroid biosynthesis and action, 2nd edition. Molecular Mechanism of Drug Action. 1994, 95-122
(9) (a) Horwitz, K. B. and McGuire, W. L. Nuclear mechanism of estrogen action: effects of oestradiol and anti-estrogens on estrogens receptors and nuclear receptor processing. J. Biol. Chem. 1978, 253, 8185-8191 (b) Horwitz, K. B.; Koseki, Y. and McGuire, W. L. Oestrogen control of progesterone receptor in human breat cancer: role of oestradiol and antiestrogen. Endocrinology 1978, 103, 1742-1751
(10) (a) Jordan, V. C. The strategic use of antiestrogens to control the development and growth of breast cancer. Cancer. 1992, 70, 977-982 (b) Powles, T. J. Breast cancer prevention Breast Cancer Res. 2000, 2, 10-12
(I I) Wakeling, A. E.; Bowler, J. Steroidal pure antiestrogens. J. Endocrinol. 1987, 112, R7-R10
(12) Sexton, M. J.; Gherman, R. B. Selective estrogen receptor modulators : the ideal estrogen replacement? Prim. Care. Update Ob/Gyns 2001, 8(1), 25-30
(13) Agnusdei, D.; Liu-Leage, S.; Augendre-Ferrante, B. Ann. Endocrinol. 1999, 60(3), 242-246
(14) John Smith, H.; Nicholls, P. J.; Simons, O; Le Lain, R. Inhibitors of steroidogenesis as agents for the treatment of hormone-dependent breast cancer. Exp.
Opin. Ther. Patents 2001, 7 f, 789-824
(15) (a) Castiglione-Gertsch, M. New aromatase inhibitors: more selectivity, less toxicity, unfortunately, the same activity. Eur. J. Cancer 1996, 32A, 393-395 (b) Miller, W. R. Aromatase inhibitors - where are we now? Br. J. Cancer 1996, 73, 415-417 (16) Santner, S. J.; Fell, P. D and Santen, R. J. In situ estrogen production via the oestrone sulphatase pathway in breast tumour: relative importance vs the aromatase pathway. J. Clin. Endocrin. Metab. 1984, 59, 29-33
(17) Purohit, A. Williams, G. J.; Howarth, N. M.; Potter, B. V. L. and Reed, M. J. Inactivation of steroid sulphatase by an active site-directed inhibitor, estrone-3-O- sulfamate. Biochem. 1995, 34, 11508-11514
(18) Purohit, A.; Williams, G. J.; Roberts, C. J.; Potter, B. V. L; Reed, M. J. In vivo inhibition of oestrone sulphatase and dehydroepiandrosterone sulphatase by estrone-3- O-sulfamate. Int. J. Cancer 1995, 62, 106-111
(19) Woo, L. W. L; Howarth, N. M.; Purohit, A.; Hejaz, H. A. M.; Reed, M. J. and Potter, B. V. L. Steroidal and nonsteroidal sulfamates as potent inhibitors of steroid sulphatase. J. Med. Chem. 1998, 47, 1068-1083
(20) Purohit, A.; Woo, L. W. L; Singh, A.; Winterbom, C. J.; Potter, B. V. L. and Reed, M. J. In vivo activity of 4-methylcoumarin-7-O-sulfamate, a non steroidal, non estrogenic steroid sulphatase inhibitor. Cancer Res. 1996, 56, 4950-4955 (21) (a) Woo, L. W. L; Purohit, A.; Malini, B.; Reed, M. J. and Potter, B. V. L. Potent active site-directed inhibition of steroid sulphatase by tricyclic coumarin-based sulfamates. Chemistry & Biology 2000, 7, 773-791 (b) Malini, B.; Purohit, A.; Ganeshapillai, D.; Woo, L. W. L.; Potter, B. V. L.; Reed, M. J. Inhibition of steroid sulphatase activity by tricyclic coumarin sulfamates. J. Steroid Biochem. Molec. Biol. 2000, 75, 253-25 (c) Purohit, A.; Woo, L. W. L; Barrow, D.; Hejaz, H. A. M.; Nicholson, R. I.; Potter, B. V. L.; Reed, M. J. Non-steroidal and steroidal sulfamates: new drugs for cancer therapy. Mol. Cell. Endocrinol. 2001, 777, 129-135
(22) Purohit, A.; Woo, L. W. L.; Potter, B. V. L; Reed, M. J. In vivo inhibition of oestrone sulphatase activity and growth of nitrosomethylurea-induced mammary tumours by 667 COUMATE. Cancer Res. 2000, 60, 3394-3396
(23) Claussner, A.; Nedelec, L; Nique, F.; Philibert, D.; Teush, G.; Van de Velde, P. 11 β-Amidoalkylestradiols as new series of pure anti-estrogens. J. Steroid. Biochem. 1992, 41, 609-6,14
(24) Li, P-K.; Chu, G-O; Guo, J. P.; Selcer, K. W. Development of potent non- estrogenic oestrone sulphatase inhibitors. Steroids 1098, 63, 425-432 (25) (a) Jin, J-Z.; Lin, S-X. Human estrogenic 17β-hydroxysteroid dehydrogenase: predominance of oestrone reduction and its induction by NADPH. Biochem. Biophys. Res. 1999, 259, 489-493 (b) Penning, T. M. Molecular endocrinology of hydroxysteroid dehydrogenases. Endocrine Reviews 1997, 18, 281-305
(26) (a) Labrie, F. At the cutting edge. Intracrinology. Mol. Cell. Endocrinol. 1991, 78, C113-C118 (b) Poulin, R.; Labrie, F. Stimulation of cell proliferation and estrogenic response by adrenal C19-?5-steroids in the ZR-75-1 Human Breast Cancer Cell Line. Cancer Res. 1986, 46, 4933-4937
(27) (a) Peltoketo, H.; Luu-The, V.; Simard, J.; Adamski, J. 17β-hydroxysteroid dehydrogenase (HSD) / 17-ketosteroid reductase (KSR) family; nomenclature and main characteristics of the 17 HSD / KSR enzymes. J. Mol. Endocrinol. 1999, 23, 1-11 (b) Peltoketo, H.; Isomaa, V.; Maentausta, O.; Vihko, R. Complete amino acid sequence of human placental 17β-hydroxysteroid dehydrogenase deduced from cDNA. FEBS Lett. 1988, 239, 73-77 (c) Wu, L; Einstein, M.; Geissler, W. M.; Chan, H. K.; Elliston, K. O.; Andersson, S. Expression cloning and characterization of human 17β-hydroxysteroid dehydrogenase type 2, a microsomal enzyme possessing 20 -hydroxysteroid dehydrogenase activity. J. Biol. Chem. 1993, 268, 12964-12969 (d) Geissler, W. M.; Davis, D. L; Wu, L; Bradshaw, K. D.; Patel, S.; Mendonca, B. B.; Elliston, K. O.; Wilston, J. D.; Russell, D. W.; Andersson, S. Male pseudohermaphroditism caused by mutation of testicular 17β-hydroxysteroid dehydrogenase 3. Nat. Genet. 1994, 7, 34- 39 (e) Adamski, J.; Normand, T; Leenders F.; Monte, D.; Begue, A.; Stehelin, D.; Jungblut, P. W.; de Launoit, Y. Molecular cloning of a novel widely expressed human 80 kDa 17β-hydroxysteroid dehydrogenase IV. Biochem. J. 1995, 377, 437-443 (f) Deyashiki, Y.; Ohshima, K.; Nakanishi, M.; Sato, K.; Matsuura, K.; Hara, A. Molecular cloning and characterization of mouse oestradiol 17β-dehydrogenase (A-specific), a member of the aldoketoreductase family. J. Biol. Chem. 1995, 270, 10461-10467
(28) Tremblay, M. R.; Auger, S. and Poirier, D. Synthesis of 16-(bromoalkyl)-estradiols having inhibitory effect on human placental oestradiol 17β-hydroxysteroid dehydrogenase (17β-HSD type 1). Bioorg. Med. Chem. 1995, 3, 506-523
(29) Tremblay, M. R.; Poirier, D. Overview of a rational approach to design type I 17β- hydroxysteroid dehydrogenase inhibitors without estrogenic activity: chemical synthesis and biological evaluation. J. Steroid. Biochem. 1998, 66, 179-191 (30) Collins, B. M.; Mac Lachlan, J. A.; Arnold, S. F. The estrogenic and anti- oestrogenic activities of phytochemicals with the human estrogen receptor expressed in yeast. Steroids 1997, 62, 365-372 (31 ) (a) Makela, S.; Poutanen, M.; Kostian, M. L.; Lehtimaki, N.; Strauss, L.; Santti, R.; Vihko, R. Inhibition of 17 befa-hydroxysteroid oxidoreductase by flavonoids in breast and prostate cancer cells. Proc. Soc. Exp. Biol. Med. 1998, 277, 310-316
(32) LeBail, J. C; Laroche, T.; Marre-Fournier, F.; Habrioux, G. Aromatase and 17β- hydroxysteroid dehydrogenase inhibition by flavonoids. Cancer Lett. 1998, 733, 101- 106
(33) Coldham, N. G.; James, V. H. T. A possible mechanism for increased breast cell proliferation by progestins through increased reductive 17β-hydroxysteroid dehydrogenase activity. Int. J. Cancer 1990, 45, 174-178
(34) Purohit, A.; Hejaz, H. A. M.; Walden, J.; MacCarthy-Marrogh, L.; Packam, G.; Potter, B. V. L.; Reed, M. J. The effect of 2-methoxyestrone-3-O-sulphamate on the growth of breast cancer cells and induced mammary tumours. Int. J. Cancer 2000, 85, 584-689
(35) Heer, J.; Miescher, K. ϋber Steroide. Marrianol- und Doisynolsaure. Uber oestrogene carbonsauren II. Helv. Chim. Acta 1945, 28, 156-165 (36) (a) Matkovics, B.; Tarόdi, B.; Balaspiri, L. Rearangement of steroids, VII. Schmidt reaction and Beckmann rearrangement of oestrone and its derivatives. Acta Chim. Acad. Scien. Hung. 1974, 80, 79-87 (b) Regan, B. M.; Newton Hayes, F. 17- and 17- Aza-D-homosteroids. J. Am. Chem. Soc. 1956, 78, 639-643
(37) Gupta, R. and Jindal, D. P. Synthesis and biological activity of some D-ring modified oestrone derivatives. Ind. J. Chem. 1999, 38B, 563-571
(38) Love, B. and Dawson, C. R. Alkylphenols related to the poison ivy principle. An improved method of synthesis involving the Na-Butanol cleavage of benzyl ethers. J. Am. Chem. Soc. 1956, 78, 6095-6101
(39) Okada, M.; Iwashita, S.; Koizumi, N. Efficient general method for sulfamoylation of a hydroxyl group. Tet. Lett. 2000, 47, 7047-7051.
(40) C. A. Hioruchi & J. Y. Satoh, Regioselective 2-lodination of Estradiol, Estriol & Oestrone, J. Chem. Soc, Chem. Commun., 1982, 671-672.
(41 ) M. Numazawa and Y. Ogura, J. Chem Soc, Chem. Commun. 1983, 9, 533.
(42) Williams, G. J.; Woo, L. W. L; Mahon, M. F.; Purohit, A.; Reed, M. J.; Potter, B. V. L. X-ray crystal structure and mechanism of action of oestrone 3-O-sulphamate, a synthetic active site-directed inhbitor of oestrone sulphatase. Pharm. Sci. 1996, 2, 11- 16
(43) Ghosh, D.; Pletnev, V. Z.; Zhu, D-W. et al. Structure of the human estrogenic 17 beta-hydroxysteroid dehydrogenase at 2.20 A resolution. Structure, 1995, 3, 503-613
(44) (a) Lin, S. X.; Han, Q.; A∑∑i, A.; Zhu, D-W.; Gongloff, A.; Campbell, R. L. 3D structure of human estrogenic 17β-HSD: binding with various steroids. J. Steroid
Biochem. Mol. Biol. ' 1999, 69, 425-429 (b) Puranen, T.; Poutanen, M.; Ghosh, D.;
Vihko, R. and Vihko, P. Origin of substrate specificity of human and rat 17β- hydroxysteroid dehydrogenase Type 1 , using chimeric enzymes and site-directed substitutions. Endocrinology 1997, 738, 3532-3539 (45) Breton, R.; Housset, D.; Maz∑a, O; Fontecilla-Camps, J. C. The structure of a complex of human 17β-hydroxysteroid dehydrogenase with oestradiol and NADP+ identifies two principal targets for the design of inhibitors. Structure (Lond) 1996, 4, 905-
915
(46) Apel, R.; Berger, G. Uber das hydrazidosulfamid Chem. Ber. 1958, 97, 1339-1341 (47) Woo, L. W. W.; Lightowler, M.; Purohit, A.; Reed, M. J.; Potter, B. V. L.
Heteroatom-substituted analogues of the active-site directed inhibitor estra-1 , 3, 5 (10)- trien-17-one-3-sulphamate inhibit oestrone sulphatase by different mechanism. J.
Steroid Biochem Mύl. Biol. 1996, 57, 79-88
(48) Duncan, L; Purohit, A.; Howarth, N. M.; Potter, B. V. L.; Reed, M. J. Inhibition of oestrone sulphatase activity by estrone-3-methyl-thiophosphonate : a potential therapeutic agent in breast cancer Cancer Res. 1993, 53, 298-303

Claims

Formula
Figure imgf000276_0001
lerein (I) R1 is a selected from
(i) an alkyloxyalkyl group
(ii) a nitrile group, and wherein R2is capable of forming a hydrogen bond
(iii) alkylaryl group, wherein the aryl group is substituted by other than a C1-10 group
(iv) alkenylaryl group wherein the aryl group is substituted
(v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group
(vi) alkenylheteroaryl group
(vii) =N-O-alkyl or =N-O-H group
(viii) branched alkenyl
(ix) alkyl-alcohol group
(x) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or -
CH2CH2-, (b) the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
(xi) -CHO so that RT together with R3 provide the enol tautomer
Figure imgf000276_0002
OR R1 together with R3 form (xii) a pyrazole wherein (a) R4 is =N-O-alkyl or =N-O-H group, (b) the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl group, branched alkyl group, and an amide and/or (c) the 2 position is substituted with a group selected from -OH and -O-hydrocarbyl (xiii) a heteroaryl ring to provide a compound of the formula
Figure imgf000277_0001
(II) ' R2 is selected from groups capable of forming a hydrogen bond , a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and
(III) R3 is selected from
-OH, =O, or a -C(=O)- mimetic
2. Use of a compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with 17β-hydroxysteroid dehydrogenase (17β-HSD), wherein the compound is of Formula I
Formula I
Figure imgf000277_0002
wherein
(I) R1 is a selected from
(i) an alkyloxyalkyl group (ii) a nitrile group
(iii) alkylaryl group, wherein the aryl group is substituted by other than a methyl group
(iv) alkenylaryl group
(v) alkyiheteroaryl group, wherein when heteroaryl group comprises only C and N in the ring, the aryl group is substituted by other than a methyl group (vi) alkenylheteroaryl group (vii) =N-O-alkyl or =N-O-H group
(viii) carboxylic acid esters wherein (a) R3 is =O and/or (b) the ester is selected from -CO2X, -CH2CO2X, and -CH2CH2CO2X, wherein X is a hydrocarbyl group (ix) CO2H or alkyI-CO2H group, wherein alkyl is CH2 or CH2CH2 group
(x) branched alkenyl
(xi) alkyl-alcohol group or alkenyl-alcohol group
(xii) amide or alkylamide wherein (a) the alkyl of the alkylamide is -CH2- or - CH2CH2-, (bj the amide is di-substituted and/or (c) the amide is substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkyiheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkylamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group, (xiv) -CHO so that RT together with R3 provide the enol tautomer
Figure imgf000278_0001
OR R, together with R3 form
(xii) a pyrazole wherein (a) R4 is =N-O-alkyl or =N-O-H group, (b) the pyrazole is substituted and/or (c) the 2 position is substituted with a group selected from -OH and -O-hydrocarbyl (xiii) a heteroaryl ring to provide a compound of the formula
Figure imgf000278_0002
(II) R2 is selected from groups capable of forming a hydrogen bond , a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and (III) R3 is selected from
-OH, =O, or a -C(=O)- mimetic
3. Use according to claim 2 wherein the aryl group of the alkenylaryl group is substituted.
4. Use according to claim 2 or 3 wherein the carboxylic acid ester is selected from - CO2X, -CH2CO2X, and -CH2CH2CO2X, wherein X is a hydrocarbyl group.
5. Use according to claim 2 or 3 wherein X is a hydrocarbon group alkyl.
6. Use according to any one of claims 2 to 5 wherein the pyrazole is substituted with one of alkyl-OH group, alkyl ester group, alkyloxyalkyl group, branched alkyl group, and an amide.
7. Use of a compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with 17β-hydroxysteroid dehydrogenase (17β-HSD), wherein the compound is a compound as defined in claim 1.
8. The invention of any one of the preceding claims wherein when R1 is a nitrile group, R2is -OH.
9. The invention of any one of the preceding claims wherein the aryl group of the alkylaryl group is substituted by a group selected from N,N-dialkyl, alkoxy, nitro (-NO2 "), nitrile, and azaalkyl groups.
10. The invention of claim 9 wherein the N,N-dialkyl is NMe2.
11. The invention of claim 9 or 10 wherein the alkoxy group is methoxy.
12. The invention of any one of the preceding claims wherein the alkylaryl group is -CH2-Ph.
13. The invention of any one of the preceding claims wherein the aryl group of the alkenylaryl group is substituted by a group selected from N,N-dialkyl, alkoxy, nitro (-NO2 ~ ), and nitrile, groups
14. The invention of claim 13 wherein the N,N-dialkyl is NMe2.
15. The invention of claim 13 or 14 wherein the alkoxy group is methoxy.
16. The invention of any one of the preceding claims wherein the alkenylaryl group is 5 =CH-Ph.
17. The invention of any one of the preceding claims wherein the aryl group of the alkyiheteroaryl group is substituted by a group selected from N.N-dialkyl, alkoxy, nitro (-
NO2 "), and nitrile, groups 0
18. • The invention of claim 17 wherein the N,N-dialkyl is NMe2.
19. The invention of claim 17 or 18 wherein the alkoxy group is methoxy.
5 20. The invention of any one of the preceding claims wherein the alkyiheteroaryl • ■ group is selected from
Figure imgf000280_0001
21. The invention of any one of the preceding claims wherein the aryl group of the alkenylheteroaryl group is substituted. 0
22. The invention of any one of the preceding claims wherein the aryl group of the alkenylheteroaryl group is substituted by a group selected from N.N-dialkyl, alkoxy, nitro
(-NO2~), and nitrile, groups.
5 23. The invention of claim 22 wherein the N,N-dialkyl is NMe2.
24. The invention of claim 22 or 23 wherein the alkoxy group is methoxy.
25. The invention of any one of the preceding claims wherein the alkenylheteroaryl 0 group is selected from
Figure imgf000281_0001
26. The invention of any one of the preceding claims wherein the =N-O-alkyl or =N- O-H group is a =N-O-alkyl group
27. The invention of any one of the preceding claims wherein when R1 is selected from =N-O-alkyl and =N-O-H groups and R3 is a =N-O-alkyl group or =N-O-H group.
28. , The invention of any one of the preceding claims wherein when R1 and R3 form a pyrazole the 2 position is substituted with a group selected from -OH and -O- hydrocarbyl.
29. The invention of any one of the preceding claims wherein when R1 and R3 form a pyrazole the 2 position is substituted'with a group selected from -OH and -O-alkyl
30. The invention of any one of the preceding claims wherein the amide is of the formula -C(=O)NR5R6 or -N(CO-R7)R8, wherein R5, R6, R7 and R8 are independently selected from H and hydrocarbyl groups.
31. The invention of any one of the preceding claims wherein the amide or alkylamide is an alkylamide.
32. The invention of any one of the preceding claims wherein the substituents of the di-substituted amide together form a cyclic structure.
33. The invention of claim 32 wherein the substituents of the di-substituted amide together form an aryl ring.
34. The invention of claim 32 or 33 wherein the substituents of the di-substituted amide together forma heterocyclic ring.
35. The invention of any one of the preceding claims wherein R2 is selected from groups capable of forming a hydrogen bond and a sulphamate group.
36. The invention of claim 35 wherein the group capable of forming a hydrogen bond are selected from -OH
37. The invention of any one of the preceding claims wherein the -C(=O)- mimetic is selected from -CN, =N-O-alkyI group, =N-O-H group, pyridine, pyrimidine, and groups of the formula
Figure imgf000282_0001
38. The invention of any one of the preceding claims wherein R3 is selected from OH, and =O.
39. The invention of any one of the preceding claims wherein R3 is =O.
40. The invention of any one of the preceding claims wherein the compound is of Formula II
Formula II
Figure imgf000282_0002
41. The invention of any one of the preceding claims wherein the compound is of Formula III
Formula III
Figure imgf000282_0003
42. The invention of any one of the preceding claims wherein the compound is of Formula IV
Formula IV
Figure imgf000283_0001
43. The invention of any one of the preceding claims wherein the compound is of Formula IX
Formula IX
Figure imgf000283_0002
wherein R11 is an alkoxy group or an alkyl group.
44. The invention of claim 43 wherein R11 is an alkoxy group.
45. The invention of claim 44 wherein R11 is methoxy.
46. The invention of claim 43 wherein R11 is an alkyl group.
47. The invention of claim 46 wherein R11 is a C|-C10 alkyl group, preferably a G,-C6 alkyl group, preferably a C,-C3 alkyl group.
48. The invention of claim 46 or 47 wherein R11 is -CH3 or -CH 2Cv-.Hi ι3.
49. The invention of any one of the preceding claims wherein the sulphamate group is of the formula
Figure imgf000283_0003
wherein R9 and R10 are independently selected from H, alkyl, cycloalkyl, alkenyl and aryl, or combinations thereof, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
50. The invention of claim 49 wherein at least one of R9 and R10 is H.
51. The invention of claim 49 wherein R9 and R10 is H.
52. A pharmaceutical composition comprising a compound according to any one of claims 1 and 8 to 51 optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
53. A compound according to any one of claims 1 and 8 to 61 for use in medicine.
54. A compound as substantially hereinbefore described with reference to any one of the Examples.
56. A composition as substantially hereinbefore described with reference to any one of the Examples.
56. A use as substantially hereinbefore described with reference to any one of the Examples.
PCT/GB2004/001234 2003-03-24 2004-03-22 17beta-hydroxysteroid dehydrogenase inhibitors Ceased WO2004085457A2 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP2006505995A JP5000294B2 (en) 2003-03-24 2004-03-22 Compound
CA2519956A CA2519956C (en) 2003-03-24 2004-03-22 Compound
MXPA05010340A MXPA05010340A (en) 2003-03-24 2004-03-22 17beta-hydroxysteroid dehydrogenase inhibitors.
BRPI0408697-0A BRPI0408697A (en) 2003-03-24 2004-03-22 compound
AU2004224061A AU2004224061B2 (en) 2003-03-24 2004-03-22 17beta-hydroxysteroid dehydrogenase inhibitors
EP04722316A EP1631579A2 (en) 2003-03-24 2004-03-22 17beta-hydroxysteroid dehydrogenase inhibitors
US11/234,868 US8003783B2 (en) 2003-03-24 2005-09-23 17B-hydroxysteroid dehydrogenase inhibitors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0306718.8 2003-03-24
GBGB0306718.8A GB0306718D0 (en) 2003-03-24 2003-03-24 Compound

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/234,868 Continuation-In-Part US8003783B2 (en) 2003-03-24 2005-09-23 17B-hydroxysteroid dehydrogenase inhibitors

Publications (2)

Publication Number Publication Date
WO2004085457A2 true WO2004085457A2 (en) 2004-10-07
WO2004085457A3 WO2004085457A3 (en) 2005-02-03

Family

ID=9955403

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2004/001234 Ceased WO2004085457A2 (en) 2003-03-24 2004-03-22 17beta-hydroxysteroid dehydrogenase inhibitors

Country Status (10)

Country Link
US (1) US8003783B2 (en)
EP (2) EP2336146A1 (en)
JP (1) JP5000294B2 (en)
CN (1) CN100526328C (en)
AU (1) AU2004224061B2 (en)
BR (1) BRPI0408697A (en)
CA (1) CA2519956C (en)
GB (1) GB0306718D0 (en)
MX (1) MXPA05010340A (en)
WO (1) WO2004085457A2 (en)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008545678A (en) * 2005-05-26 2008-12-18 ゾルファイ ファーマスーティカルズ ゲゼルシャフト ミット ベシュレンクテル ハフツング 17β-HSD1 and STS inhibitors
JP2009506089A (en) * 2005-08-31 2009-02-12 エフ．ホフマン−ラ ロシュ アーゲー Pyrazolone derivatives as 11-βHSD1 inhibitors
JP2009508839A (en) * 2005-09-15 2009-03-05 カウンシル・オブ・サイエンティフィック・アンド・インダストリアル・リサーチ Novel steroid derivatives and indan-1-one derivatives substituted with imidazolyl groups
RU2369614C2 (en) * 2003-11-12 2009-10-10 Зольвай Фармасьютиклз Гмбх NEW INHIBITORS OF 17 β-HYDROXYSTEROID-DEHYDROGENASE TYPE I
US8030298B2 (en) 2005-05-26 2011-10-04 Abbott Products Gmbh 17β-HSD1 and STS inhibitors
US8080540B2 (en) 2006-09-19 2011-12-20 Abbott Products Gmbh Therapeutically active triazoles and their use
US8088758B2 (en) 2003-11-12 2012-01-03 Abbott Products Gmbh 17β-hydroxysteroid dehydrogenase type I inhibitors
US8288367B2 (en) 2006-11-30 2012-10-16 Solvay Pharmaceuticals Gmbh Substituted estratriene derivatives as 17BETA HSD inhibitors
EP2147014A4 (en) * 2007-04-12 2012-11-07 Endorech Inc 17-ALPHA-SUBSTITUTED STEROIDS AS SYSTEMIC ANTI-ANDROGEN AND ANDROGEN RECEPTOR SELECTIVE MODULATORS
WO2014207309A1 (en) 2013-06-25 2014-12-31 Forendo Pharma Ltd Therapeutically active estratrienthiazole derivatives as inhibitors of 17 b-hydroxysteroid dehydrogenase, type 1
WO2014207310A1 (en) 2013-06-25 2014-12-31 Forendo Pharma Ltd Therapeutically active 17-nitrogen substituted estratrienthiazole derivatives as inhibitors of 17.beta.-hydroxysteroid dehydrogenase
WO2014207311A1 (en) 2013-06-25 2014-12-31 Forendo Pharma Ltd Therapeutically active estratrienthiazole derivatives as inhibitors of 17.beta.-hydroxy-steroid dehydrogenase, type 1
WO2016102775A1 (en) 2014-12-23 2016-06-30 Forendo Pharma Ltd PRODRUGS OF 17β-HSD1 -INHIBITORS
WO2016102776A1 (en) 2014-12-23 2016-06-30 Forendo Pharma Ltd Prodrugs of 17.beta.-hsd1 -inhibitors
WO2017211330A1 (en) 2016-06-07 2017-12-14 Ustav Organicke Chemie A Biochemie Av Cr, V.V.I. 15β-SUBSTITUTED ESTRONE DERIVATIVES AS SELECTIVE INHIBITORS OF 17β-HYDROXYSTEOID-DEHYDROGENASES, METHOD OF PREPARATION AND USE THEREOF
WO2018224736A2 (en) 2017-06-08 2018-12-13 Forendo Pharma Ltd Therapeutically active steroidal derivatives
WO2020115371A1 (en) 2018-12-05 2020-06-11 Forendo Pharma Ltd Estra-1,3,5(10)-triene compounds condensed in position 16(17) with a pyrazole ring as inhibitors of 17-hsd1
US11396525B2 (en) * 2016-07-11 2022-07-26 Sage Therapeutics, Inc. C17, C20, and C21 substituted neuroactive steroids and their methods of use

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040127473A1 (en) * 1996-12-05 2004-07-01 Reed Michael John Compound
AU2012207381B2 (en) 2011-01-18 2016-05-19 General Atomics Hydrolase enzyme substrates and uses thereof
HUP1100453A2 (en) * 2011-08-19 2013-02-28 Richter Gedeon Nyrt Carboxamides with sterane skeleton as h3 antagonists
CN102952168A (en) * 2012-12-12 2013-03-06 中国药科大学 Estrogen derivative used as vasoinhibitor, as well as preparation method and medical application thereof
PL3500265T3 (en) * 2016-08-22 2024-03-11 Or-Genix Therapeutics, Inc. Estrogen receptor beta selective ligands
CN114085216B (en) * 2021-11-15 2025-06-24 兰州大学 A method for synthesizing alkenyl indole derivatives

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3398140A (en) * 1959-08-24 1968-08-20 Sterling Drug Inc Steroido [17. 16-c] pyrozoles and preparation thereof
US3047568A (en) * 1960-11-23 1962-07-31 American Cyanamid Co 16-cyano steroids, intermediates and method of preparing same
US3243432A (en) * 1961-04-28 1966-03-29 Ormonoterapia Richter Spa Steroidal pyrazoles and related intermediate products
DE1176131B (en) 1961-07-25 1964-08-20 Merck Ag E Process for the preparation of 16-cyano and 16-cyano-16, 17-seco-steroids
GB1033520A (en) * 1962-03-30 1966-06-22 Sterling Drug Inc 3-amino steroids
US3159656A (en) * 1962-11-06 1964-12-01 Lasdon Foundation Inc 16, 17-bis-oximinoestraen-3-ols and ethers thereof
US3163641A (en) * 1962-11-15 1964-12-29 Sterling Drug Inc 16-substituted androstanes and androstenes
NL6402049A (en) 1964-02-29 1965-08-30
US5364847A (en) * 1989-03-10 1994-11-15 Endorecherche Inhibitors of sex steroid biosynthesis and methods for their production and use
US5880115A (en) * 1997-07-18 1999-03-09 Duquesne University Of The Holy Ghost Steroid sulfatase inhibitors and methods for making and using the same
GB2331987B (en) * 1997-12-04 2002-11-27 Imperial College Polycyclic sulphamate inhibitors of oestrone sulphatase
US6046186A (en) * 1997-12-24 2000-04-04 Sri International Estrone sulfamate inhibitors of estrone sulfatase, and associated pharmaceutical compositions and methods of use
GB0020498D0 (en) 2000-08-18 2000-10-11 Sterix Ltd Compound
GB0025788D0 (en) * 2000-10-20 2000-12-06 Sterix Ltd Use
AU2002243643A1 (en) 2001-01-25 2002-08-06 Yale University Estradiol-16alpha-carboxylic acid esters as locally active estrogens

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2369614C2 (en) * 2003-11-12 2009-10-10 Зольвай Фармасьютиклз Гмбх NEW INHIBITORS OF 17 β-HYDROXYSTEROID-DEHYDROGENASE TYPE I
US8088758B2 (en) 2003-11-12 2012-01-03 Abbott Products Gmbh 17β-hydroxysteroid dehydrogenase type I inhibitors
US8030298B2 (en) 2005-05-26 2011-10-04 Abbott Products Gmbh 17β-HSD1 and STS inhibitors
US8435973B2 (en) 2005-05-26 2013-05-07 Abbott Products Gmbh 17-beta HSD1 and STS inhibitors
JP2008545678A (en) * 2005-05-26 2008-12-18 ゾルファイ ファーマスーティカルズ ゲゼルシャフト ミット ベシュレンクテル ハフツング 17β-HSD1 and STS inhibitors
JP2009506089A (en) * 2005-08-31 2009-02-12 エフ．ホフマン−ラ ロシュ アーゲー Pyrazolone derivatives as 11-βHSD1 inhibitors
JP2009508839A (en) * 2005-09-15 2009-03-05 カウンシル・オブ・サイエンティフィック・アンド・インダストリアル・リサーチ Novel steroid derivatives and indan-1-one derivatives substituted with imidazolyl groups
US8080540B2 (en) 2006-09-19 2011-12-20 Abbott Products Gmbh Therapeutically active triazoles and their use
US8288367B2 (en) 2006-11-30 2012-10-16 Solvay Pharmaceuticals Gmbh Substituted estratriene derivatives as 17BETA HSD inhibitors
US9284345B2 (en) 2007-04-12 2016-03-15 Endorecherche, Inc. 17alpha-substituted steroids as systemic antiandrogens and selective androgen receptor modulators
EP2147014A4 (en) * 2007-04-12 2012-11-07 Endorech Inc 17-ALPHA-SUBSTITUTED STEROIDS AS SYSTEMIC ANTI-ANDROGEN AND ANDROGEN RECEPTOR SELECTIVE MODULATORS
JP2016523886A (en) * 2013-06-25 2016-08-12 フォレンド ファーマ リミテッド Therapeutically active 17-nitrogen substituted estratrienthiazole derivatives as inhibitors of 17β-hydroxysteroid dehydrogenase
US9850272B2 (en) 2013-06-25 2017-12-26 Forendo Pharma Ltd. Therapeutically active estratrienthiazole derivatives as inhibitors of 17.beta-hydroxy-steroid dehydrogenase, type 1
WO2014207310A1 (en) 2013-06-25 2014-12-31 Forendo Pharma Ltd Therapeutically active 17-nitrogen substituted estratrienthiazole derivatives as inhibitors of 17.beta.-hydroxysteroid dehydrogenase
CN105518015A (en) * 2013-06-25 2016-04-20 佛恩多制药有限公司 Therapeutically active 17-nitrogen substituted estratrienthiazole derivatives as inhibitors of 17.beta.-hydroxysteroid dehydrogenase
WO2014207311A1 (en) 2013-06-25 2014-12-31 Forendo Pharma Ltd Therapeutically active estratrienthiazole derivatives as inhibitors of 17.beta.-hydroxy-steroid dehydrogenase, type 1
US10377791B2 (en) 2013-06-25 2019-08-13 Forendo Pharma Ltd. Therapeutically active estratrienthiazole derivatives as inhibitors of 17 B-hydroxysteroid dehydrogenase, type 1
JP2016523885A (en) * 2013-06-25 2016-08-12 フォレンド ファーマ リミテッド Therapeutically active estratrienthiazole derivatives as inhibitors of 17β-hydroxysteroid dehydrogenase, type I - Patents.com
WO2014207309A1 (en) 2013-06-25 2014-12-31 Forendo Pharma Ltd Therapeutically active estratrienthiazole derivatives as inhibitors of 17 b-hydroxysteroid dehydrogenase, type 1
JP2016527212A (en) * 2013-06-25 2016-09-08 フォレンド ファーマ リミテッド 17β-Hydroxysteroid dehydrogenase type 1 inhibitor with therapeutic activity as an estratrienthiazole derivative
US9663549B2 (en) 2013-06-25 2017-05-30 Forendo Pharma Ltd. Therapeutically active 17-nitrogen substituted estratreinthiazole derivatives as inhibitors of 17β-hydroxysteroid dehydrogenase
CN105518015B (en) * 2013-06-25 2017-09-15 佛恩多制药有限公司 17 nitrogen as 17 beta hydroxysteroid dehydrogenase inhibitors of therapeutic activity replace estratriene thiazole
CN107207561B (en) * 2014-12-23 2020-03-31 佛恩多制药有限公司 Prodrugs of 17 β -HSD 1-inhibitors
CN107207562A (en) * 2014-12-23 2017-09-26 佛恩多制药有限公司 Prodrugs of 17β‑HSD1 inhibitors
CN107207561A (en) * 2014-12-23 2017-09-26 佛恩多制药有限公司 Prodrug of 17β‑HSD1–inhibitor
WO2016102776A1 (en) 2014-12-23 2016-06-30 Forendo Pharma Ltd Prodrugs of 17.beta.-hsd1 -inhibitors
US10413557B2 (en) 2014-12-23 2019-09-17 Forendo Pharma Ltd. Prodrugs of 17.BETA.-HSD1-inhibitors
CN107207562B (en) * 2014-12-23 2020-03-06 佛恩多制药有限公司 Prodrugs of 17β-HSD1 Inhibitors
WO2016102775A1 (en) 2014-12-23 2016-06-30 Forendo Pharma Ltd PRODRUGS OF 17β-HSD1 -INHIBITORS
US10626140B2 (en) 2014-12-23 2020-04-21 Forendo Pharma Ltd Prodrugs of 17β-HSD1-inhibitors
US10759826B2 (en) 2016-06-07 2020-09-01 Ustav Organicke Chemie A Biochemie Av Cr, V.V.I. 15β-substituted estrone derivatives as selective inhibitors of 17β-hydroxysteroid-dehydrogenases, method of preparation and use thereof
WO2017211330A1 (en) 2016-06-07 2017-12-14 Ustav Organicke Chemie A Biochemie Av Cr, V.V.I. 15β-SUBSTITUTED ESTRONE DERIVATIVES AS SELECTIVE INHIBITORS OF 17β-HYDROXYSTEOID-DEHYDROGENASES, METHOD OF PREPARATION AND USE THEREOF
US11396525B2 (en) * 2016-07-11 2022-07-26 Sage Therapeutics, Inc. C17, C20, and C21 substituted neuroactive steroids and their methods of use
WO2018224736A2 (en) 2017-06-08 2018-12-13 Forendo Pharma Ltd Therapeutically active steroidal derivatives
US10717761B2 (en) 2017-06-08 2020-07-21 Forendo Pharma Ltd Therapeutically active steroidal derivatives
US11254703B2 (en) 2017-06-08 2022-02-22 Forendo Pharma Ltd Therapeutically active steroidal derivatives
WO2020115371A1 (en) 2018-12-05 2020-06-11 Forendo Pharma Ltd Estra-1,3,5(10)-triene compounds condensed in position 16(17) with a pyrazole ring as inhibitors of 17-hsd1

Also Published As

Publication number Publication date
JP5000294B2 (en) 2012-08-15
CA2519956A1 (en) 2004-10-07
AU2004224061A1 (en) 2004-10-07
BRPI0408697A (en) 2006-03-28
US8003783B2 (en) 2011-08-23
JP2006521338A (en) 2006-09-21
CA2519956C (en) 2012-12-18
WO2004085457A3 (en) 2005-02-03
CN100526328C (en) 2009-08-12
CN1791611A (en) 2006-06-21
EP2336146A1 (en) 2011-06-22
EP1631579A2 (en) 2006-03-08
GB0306718D0 (en) 2003-04-30
US20060074060A1 (en) 2006-04-06
MXPA05010340A (en) 2006-04-18
AU2004224061B2 (en) 2011-06-16

Similar Documents

Publication Publication Date Title
AU2004224061B2 (en) 17beta-hydroxysteroid dehydrogenase inhibitors
AU2004224053B2 (en) Oestrogen derivatives as inhibitors of steroid sulphatase
US20090186900A1 (en) Compound
US20030203855A1 (en) 17-Aryl linker derivatised estrogen 3-sulphamates as inhibitors of steroid sulphatase
US20040009959A1 (en) Thioether sulphamate steroids as steroid inhibtors and anti-cancer compounds
WO2002032409A2 (en) Use of compound in the manufacture of a pharmaceutical for inhibiting steroid sulphatase and steroid dehydrogenase activity
CA2636431C (en) Steroidal compounds as steroid sulphatase inhibitors
EP1448592B1 (en) Steroidal compounds for inhibiting steroid sulphatase
US8124614B2 (en) Steroidal compounds for inhibiting steroid sulphatase
CA2576614A1 (en) 2-substituted estrogen sulphamates for inhibition of steroid sulphatase
US20030134829A1 (en) Halogenated sulphamate-, phosphonate-, thiophosphonate-, sulphonate- and sulphonamide- compounds as inhibitors of steroid sulphatase
HK1126225B (en) Steroidal compounds as steroid sulphatase inhibitors

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2519956

Country of ref document: CA

Ref document number: 2006505995

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2004224061

Country of ref document: AU

Ref document number: 11234868

Country of ref document: US

Ref document number: 4307/DELNP/2005

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: PA/a/2005/010340

Country of ref document: MX

ENP Entry into the national phase

Ref document number: 2004224061

Country of ref document: AU

Date of ref document: 20040322

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2004722316

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2004224061

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 20048139150

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 2004722316

Country of ref document: EP

ENP Entry into the national phase

Ref document number: PI0408697

Country of ref document: BR

WWP Wipo information: published in national office

Ref document number: 11234868

Country of ref document: US