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EP1307193A1 - Agonists of follicle stimulating hormone activity - Google Patents

Agonists of follicle stimulating hormone activity

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Publication number
EP1307193A1
EP1307193A1 EP01955958A EP01955958A EP1307193A1 EP 1307193 A1 EP1307193 A1 EP 1307193A1 EP 01955958 A EP01955958 A EP 01955958A EP 01955958 A EP01955958 A EP 01955958A EP 1307193 A1 EP1307193 A1 EP 1307193A1
Authority
EP
European Patent Office
Prior art keywords
substituted
group
alkyl
mmol
member selected
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.)
Withdrawn
Application number
EP01955958A
Other languages
German (de)
French (fr)
Inventor
Randall A. Scheuerman
Stephen D. Yanofsky
Christopher P. Holmes
Derek Maclean
Beatrice Ruhland
Ronald W. Barrett
Jay E. Wrobel
Wenling Kao
Ariamala Gopalsamy
Fuk-Wah Sum
Baihua Hu
John F. Rogers
James W. Jetter
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.)
SmithKline Beecham Corp
Original Assignee
Affymax Researh Institute Inc
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Filing date
Publication date
Application filed by Affymax Researh Institute Inc filed Critical Affymax Researh Institute Inc
Publication of EP1307193A1 publication Critical patent/EP1307193A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • 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
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • 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
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • A61P5/10—Drugs for disorders of the endocrine system of the posterior pituitary hormones, e.g. oxytocin, ADH
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/08—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D277/12—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/14—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • This invention relates broadly to novel thiazolidinones. More specifically, the invention relates to thiazolidinones which modulate Follicle Stimulating Hormone (FSH) activity.
  • FSH Follicle Stimulating Hormone
  • germ cells are stored in the ovaries of the human female at the time of puberty. No further germ cells are made. Beginning at the time of puberty and ending at menopause, there are approximately 400 ovulatory rnenstrual cycles which consume essentially all of the germ cells in the human ovary. About 1,000 germ cells are consumed in each menstrual period. However, in any one menstrual cycle, only one germ cell, developed in what becomes the dominant follicle, is ovulated and available for pregnancy.
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • hCG chorionic gonadotropin
  • GRF somatotrophin releasing factor
  • Thiazolidinones are a class of small molecule organic compounds which have found limited pharmaceutical use. For example, thiazolidinones have been found to have central nervous system activity. See, for example, Tripathi, et al, "Thiazolidinone congeners as central nervous system active agents.” Arzneistoffforschung 43:632-5 (1993). CNS activities which have been identified include, for example, antipsychotic properties.
  • Thiazolidinones have also been used as antimicrobial agents. See, for example, Ley, et al, "Inhibition of multiplication of Mycobacterium leprae by several antithyroid drugs.” Am. Rev. Respir. Dis. 111:651-5 (1975).
  • novel thiazolidinones offer the promise for discovering new pharmaceutical agents with applications in areas as diverse as, for example, antimicrobial therapy and the treatment of strokes with CNS antuschemic agents.
  • novel thiazolidinones are regulators of mammalian fertility.
  • the present invention provides a class of novel thiazolidinones possessing a range of pharmaceutical applications and activities.
  • the present invention provides novel thiazolidinones having the formula:
  • R 1 is a member selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocylic groups;
  • R 2 is a member selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocylic groups;
  • R 5 and R 6 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups;
  • Y is a member selected from the group consisting of O, S, and NH;
  • Y is a member selected from the group consisting of CH 2 , O, S, and NR ;
  • Y is a member selected from the group consisting of O and NR ;
  • R 7 is a member selected from the group consisting of hydrogen and lower alkyl
  • X 2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, phenyl, substituted phenyl, heterocychc, substituted heterocychc, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups; m is an integer from 0 to 3; n is 0 or 1 ; and s is 1 or 2.
  • the present invention provides novel thiazolidinones having the structure:
  • R 1 is a member selected from the group consisting of aryl, substituted aryl, arylalkyl and substituted arylalkyl groups;
  • R 2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocychc groups;
  • R 3 and R 4 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups;
  • the invention provides a class of FSH receptor agonists, wherein the receptor agonists are noncompetitive with FSH for the receptor FSH binding site.
  • the invention provides a class of compounds that modulate FSH hormone activity, the compounds having: (a) a molecular weight of from about 50 daltons to about 1000 daltons; and (b) an FSH agonist activity corresponding to an EC 50 standard of no more than 50 ⁇ M, preferably no more than 2 ⁇ M; wherein the agonist activity of this class of compounds to the FSH receptor is competitively inhibited by a compound having the formula: wherein,
  • R 1 is a member selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocylic groups;
  • R 2 is a member selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocylic groups;
  • R 5 and R 6 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups;
  • Y is a member selected from the group consisting of O, S, and NH;
  • Y 2 is a member selected from the group consisting of CH 2 , O, S, and NR 5 ;
  • Y 3 is a member selected from the group consisting of O and NR 6 R 7 ;
  • R is a member selected from the group consisting of hydrogen and lower alkyl
  • X 2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, phenyl, substituted phenyl, heterocychc, substituted heterocychc, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups; m is an integer from 0 to 3; n is O or 1; and s is 1 or 2.
  • the invention provides a class of compounds that modulate FSH hormone activity, the compounds having: (a) a molecular weight of from about 200 daltons to about 1000 daltons; and (b) an FSH agonist activity corresponding to an EC50 standard of no more than 50 ⁇ M, preferably no more than 2 ⁇ M; wherein the agonist activity of this class of compoimds to the FSH receptor is competitively inhibited by a compound having the formula:
  • R 1 is a member selected from the group consisting of aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups
  • R 2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocychc groups
  • R 3 and R 4 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups
  • this class of compounds has a molecular weight of about 300 daltons to about 800 daltons. In another preferred embodiment, this class of compounds has an FSH receptor agonist activity, as expressed by an EC 5 o standard, of no more than 1 ⁇ M and, more preferably, of no more than 500 nM.
  • the invention provides methods of using the compounds, i.e., thiazolidinones, for diverse pharmaceutical applications including, for example, CNS antuschemic agents, agents with antipsychotic or other psychoactive properties, antimicrobial agents and mammalian fertility regulating agents.
  • the thiazolidinones are preferably agonists of the FSH receptor.
  • the present invention provides pharmaceutical compositions which contain one or more of the compounds of the invention in conjunction with pharmaceutically acceptable excipients, carriers, diluents, etc.
  • the pharmaceutical compositions can also contain agents which are themselves pharmacologically active and which serve to enhance, supplement, decrease or otherwise regulate the pharmacological effect of the pharmaceutical compositions.
  • HATU [O-(7-Azabenzotriazol-l-yl)-l, 1,3,3- tetramethyluromumhexafluorophosphate]
  • DIEA diisopropylethylamine
  • FMOC fluorenylmethoxycarbonyl
  • DECP diethyl cyanophosphonate
  • DCM dichloromethane
  • DBU l,8-diazabicyclo[5.4.0]undec-7-ene
  • CHO Chinese hamster ovary
  • RBF round- bottomed flask.
  • R groups e.g., R 1 , R 2 , R 4 and R 5
  • R 1 , R 2 and R 3 may all be substituted alkyls or R 1 and R 2 may be a substituted alkyl and R 3 may be an aryl, etc.
  • R group will generally have the structure which is recognized in the art as corresponding to R groups having that name.
  • representative R groups as enumerated above are defined herein. These definitions are intended to supplement and illustrate, not preclude, the definitions known to those of skill in the art.
  • alkyl is used herein to refer to a branched or unbranched, saturated or unsaturated, monovalent hydrocarbon radical having from 1-12 carbons and preferably, from 1-6 carbons. When the alkyl group has from 1-6 carbons, it is referred to as a "lower alkyl.”
  • Suitable alkyl radicals include, for example, methyl, ethyl, n-propyl, i-propyl, 2-propenyl (or allyl), n-butyl, t-butyl (or 2-methylpropyl), etc.
  • Substituted alkyl refers to alkyl as just described including one or more functional groups such as lower alkyl, aryl, acyl, halogen, (i.e., alkylhalos, e.g., CF 3 ), hydroxy, nitro, cyano, amino, alkoxy, alkylamino, acylamino, acyloxy, aryloxy, arloxyalkyl, mercapto, carboxylic acid, carboxylic acid derivatives, sulfonic acids, sulfonic acid derivatives, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like. These groups may be attached to any which are fused to the arom heterocycles and the like. These groups may be attached to any carbon of the alkyl moiety.
  • aryl is used herein to refer to an aromatic substituent having a single aromatic ring or multiple aromatic rings which are fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety.
  • the common linking group may also be a carbonyl as in benzophenone.
  • the aromatic ring(s) may include phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone among others.
  • Substituted aryl refers to aryl as just described including one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF 3 ), hydroxy, nitro, cyano, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, carboxylic acid amide, sulfonic acid amide and both saturated and unsaturated cyclic hydrocarbons which are fused to the aromatic ring(s), linked covalently or linked to a common group such as a methylene or ethylene moiety.
  • the linking group may also be a carbonyl such as in cyclohexyl phenyl ketone.
  • arylalkyl is used herein to refer to a subset of “aryl” in which the aryl group is attached through an alkyl group as defined herein. Examples include, but are not limited to, benzyl, phenylethyl and phenylpropyl groups.
  • Substituted arylalkyl defines a subset of “arylalkyl” wherein the aryl moiety of the arylalkyl group is substituted as defined herein for aryl groups.
  • halogen is used herein to refer to fluorine, bromine, chlorine and iodine atoms.
  • hydroxy is used herein to refer to the group -OH.
  • amino is used herein to refer to the group-NRR', where R and R' may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl or acyl.
  • alkoxy is used herein to refer to the -OR group, where R is a lower alkyl or substituted lower alkyl, wherein the alkyl and substituted lower alkyl groups are as described herein.
  • Suitable alkoxy radicals include, for example, methoxy, ethoxy, t-butoxy, etc.
  • aryloxy is used herein to refer to the -OR group, wherein R is an aryl, substituted aryl, arylalkyl or substituted arylalkyl as described above. Examples include phenoxy, benzyloxy, phenethyloxy and substituted derivatives thereof.
  • alkylamino denotes secondary and tertiary amines wherein the alkyl groups may be either the same or different and may consist of straight or branches, saturated or unsaturated hydrocarbons.
  • heterocyclychc is used herein to describe a monovalent group having a single ring or multiple condensed rings from 1-12 carbon atoms and from 1-4 heteroatoms selected from nitrogen, sulfur or oxygen within the ring.
  • heterocycles include, for example, tetrahydrofuran, morpholine, piperidine, pyrrolidine, thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan, benzo-fused analogs of these rings, etc.
  • substituted heterocychc as used herein describes a subset of “heterocychc” wherein the heterocycle nucleus is substituted with one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF 3 ), hydroxy, amino, alkoxy, alkylmino, acylamino, acyloxy, mercapto, etc.
  • functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF 3 ), hydroxy, amino, alkoxy, alkylmino, acylamino, acyloxy, mercapto, etc.
  • heterocyclicalkyl is used herein to refer to a subset of “heterocylic” in which the heterocylcic group is attached through an alkyl group as defined herein.
  • Substituted heterocyclicalkyl defines a subset of “heterocyclicalkyl” wherein the heterocychc moiety of the heterocyclicalkyl group is substituted as defined herein for heterocychc groups.
  • pharmaceutically acceptable salt refers to those salts of compounds which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and organic acids such as, for example, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • Pharmaceutically acceptable salts include, for example, alkali metal salts, such as sodium and potassium, alkaline earth salts and ammonium salts.
  • contacting is used herein interchangeably with the following: combined with, added to, mixed with, passed over, incubated with, flowed over, etc.
  • the thiazolidinone compounds of present invention can be "administered" to a subject by any conventional method such as, for example, parenteral, oral, topical and inhalation routes as described herein.
  • An amount sufficient” or “an effective amount” is that amount of a given thiazolidinone analog which exhibits the binding/activity of interest or, which provides an improvement in gamete recruitment.
  • EC50 is the effective concentration, i.e., the concentration of a compound at which 50% of the maximal response of that obtained with FSH would be achieved.
  • Non-competitive refers to the nature of the agonist activity exhibited by the compounds of the invention, wherein the compounds act as agonists of and activate the FSH receptor without substantially reducing the magnitude of binding of FSH to the receptor.
  • Magnitude of binding refers to the amount of FSH bound by a receptor population and/or the strength of the binding interaction between FSH and the FSH receptor.
  • the present invention is directed to novel thiazolidinone compounds which exhibit a range of pharmaceutical activities.
  • the novel compounds are small molecule FSH receptor agonists. These compounds offer numerous advantages over the current state of the art (i.e., gonadotrophins of urinary origin and recombinant FSH).
  • the compounds of the instant invention are inexpensive and both easily prepared and purified.
  • the compounds exhibit a range of activity regarding the FSH receptor. Such a manifold of compounds of differing activity provides an opportunity to the clinician to modulate the desired level of fertility induction by judicious choice of the fertility-inducing agent.
  • novel thiazolidinones exhibit a pharmacokinetic profile which is distinct from that of conventional peptidic hormone preparations.
  • the pharmacokinetic profile can be further modified by judicious choice of the route of administration and manipulating the nature of the substituents on the thiazolidinone nucleus.
  • the present invention provides a compound having a formula:
  • R 1 is a member selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocylic groups;
  • R 2 is a member selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocylic groups;
  • R 5 and R 6 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups;
  • Y is a member selected from the group consisting of O, S, and NH;
  • Y 2 is a member selected from the group consisting of CH 2 , O, S, and NR 5 ;
  • Y is a member selected from the group consisting of O and NR R ;
  • R 7 is a member selected from the group consisting of hydrogen and lower alkyl
  • X 2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, phenyl, substituted phenyl, heterocychc, substituted heterocychc, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups; m is an integer from 0 to 3; n is O or 1; and s is 1 or 2.
  • the present invention provides a compound wherein,
  • R 5 and R 6 are independently members selected from the group consisting of hydrogen, -(CH 2 ) q X 2 , and wherein p is an integer from 1 to 2; q is an integer from 0 to 5;
  • X is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, phenyl, substituted phenyl, heterocychc, substituted heterocychc, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups; and
  • R 1 is a member selected from the group consisting of
  • R > 8 and ⁇ R9 are independently members selected form the group consisting of H, halogen, ketone, alkyl, substituted alkyl, phenyl, substituted phenyl, lower alkoxy, aryloxy, substituted aryloxy, carboxylic acid, carboxylic acid amide, sulfonic acid, sulfonic acid amide, alkynyl, substituted alkynyl and -CONR ⁇ CH ⁇ t Z wherein t is an integer from one to four;
  • R 10 is a member selected from the group consisting of H and lower alkyl
  • A is a member selected from the group consisting of -C alkyl and substituted -C 4 alkyl, wherein each carbon atom is independently substituted with members selected from the group consisting of H, amino, alkyl, substituted alkyl, spirocyclic alkyl, substituted spirocyclic alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc, and substituted heterocychc groups;
  • X 3 is a member selected from the group consisting of O, S, SO, SO 2 and NR 10 ;
  • Z is a member selected from the group consisting of alkylamino, dialkylamino,
  • w is an integer from 1 to 3;
  • Y is a member selected from the group consisting of O, S and NR 10 ;.
  • R 2 is a member selected from the group consisting of
  • R » ⁇ and R , 12 are independently members selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, phenyl, substituted phenyl, aryloxy, substituted aryloxy, alkynyl, substituted alkynyl, nitro, cyano, aminoarylalkyl and substituted aminoarylalkyl; u is an integer from 1 and 4;
  • X 4 is a member selected from the group consisting of O, S, NR 13 , and CR 13 ;
  • X 5 is a member selected from the group consisting of O, S, NH, and CH;
  • R is a member selected from the group consisting of H, and lower alkyl.
  • R and R are independently members selected form the group consisting of H, halogen, ketone, alkyl, substituted alkyl, carboxylic acid amide, sulfonic acid amide and alkynyl and -CONR 1 (CH 2 ) t Z wherein t is an integer from one and four; wherein
  • Z is a member selected from the group consisting of alkylamino, dialkylamino,
  • w is an integer from 1 to 5;
  • Y is a member selected from the group consisting of O, S, and NR 10 ;
  • R 1 is a member selected from the group consisting of H, and lower alkyl.
  • the present invention provides a compound wherein,
  • R and R are independently members selected from the group consisting of hydrogen, halogen, alkoxy, substituted alkoxy, aryloxy, -----R 14 , and -X 6 -( CH 2 ) V R 14 wherein
  • R 14 is a member selected from the group consisting of phenyl, substituted phenyl, alkyl, alkenyl, cyloalkyl, cycloalkenyl, CH 2 (X 7 ) Z R 15 , CONHR 15 , COR 15 , pyridine, thiophene, furan, pyrrole and phenylsulfonyl;
  • X 6 is a member selected from the group consisting of O, S, NH, and CH 2 O;
  • X 7 is.a member selected from the group consisting of O, S, and R 14 ;
  • R 15 is a member selected from the group consisting of H, alkyl and phenyl; v is an integer from 0 to 3; and z is O or 1.
  • the present invention provides a compound wherein, when substituent R 4 , on C-5, is H, and a second substituent at C-5 (R ) is not H, said substituent R on C-5 and substituent R on C-2 are oriented in a cis manner.
  • the present invention provides novel thiazolidinones having the formula:
  • R 1 is a member selected from the group consisting of aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups;
  • R 2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocychc groups;
  • R 3 and R 4 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups; and
  • R 1 is aryl or substituted aryl. In another preferred embodiment, R 1 is phenyl or substituted phenyl. In a further preferred embodiment, R 1 is substituted phenyl as in Formula (IN)
  • R 11 , R 12 , R 13 , R 14 and R 15 are independently members selected from the group consisting of H, halogen, lower alkyl, substituted lower alkyl, phenyl, lower alkoxy, aryloxy, substituted aryloxy, carboxyl, ester and amide groups.
  • R 1 is a substituted phenyl according to Formula (IN) and R 11 , R 12 , R 13 , R 14 and R 15 are independently members selected from the group consisting of H, halogen, substituted alkyl, ketone, ester, amide and nitro groups.
  • R 1 is substituted phenyl according to Formula (IN) and R 11 , R 12 , R 13 , R 14 and R 15 are independently members selected from the group consisting of H, halogen and amide groups.
  • R is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocychc groups.
  • R 2 is an aryl group.
  • a preferred aryl group is the phenyl group and a preferred substituted aryl group is a substituted phenyl group.
  • R 2 is a substituted phenyl according to Formula (N) or a five-membered ring according to Formula (VI)
  • R 21 , R 22 , R 23 , R 24 , R 25 are members independently selected from the group consisting of H, halogen, lower alkyl, substituted lower alkyl, lower alkoxy, substituted lower alkoxy, phenyl, substituted phenyl, aryloxy, substituted aryloxy, alkynyl, substituted alkynyl and nitro groups.
  • Preferred aryloxy groups are phenoxy and benzyloxy and preferred substituted aryloxy groups are substituted phenoxy and substituted benzyloxy.
  • Y is a member selected from the group consisting of-CH 2 -, -O-, -S- and NR 26 wherein R 26 is H or lower alkyl.
  • R 21 , R 22 , R 23 , R 24 and R 25 are independently members selected from the group consisting of H, halogen, lower alkoxy and substituted aryl groups according to Formula (Nil)
  • R 31 , R 32 , R 33 , R 34 and R 35 are members independently selected from the group consisting of hydrogen, halogen, nitro and trifluoromethyl, alkyl, substituted alkyl, alkoxy and hydroxy.
  • X 1 is a member selected from the group consisting of O, ⁇ R 36 , S, C, CH and CH 2 ; R is H or lower alkyl; m is an integer from 0 to 5; n is an integer from 0 to 3, p is an integer from 0 to 3 and q is an integer from 0 to 2. When q ⁇ 2, a multiple bond exists between CH q and X 1 .
  • R 21 , R 22 , R 23 , R 24 and.R 25 are independently chosen from hydrogen and the groups according to Formulae (NIII) and (IX):
  • R 41 , R 42 , R 43 , R 44 and R 45 are members independently selected from the group consisting of hydrogen, halogen, nitro and trifluoromethyl.
  • n is an integer from 0 to 3 and X 1 is O or ⁇ H.
  • R 3 and R 4 are the same or different and are members independently selected from the group consisting of H and structures according to Formula (X):
  • X is an integer from 0 to 5, preferably from 1 to 5.
  • X 2 is a member selected from the group consisting of aryl, substituted aryl, heterocychc and substituted heterocychc. In a further preferred embodiment, X is heterocychc or substituted aryl. In still further preferred embodiments, X 2 is phenyl, substituted phenyl, indole or substituted indole.
  • the compounds of the invention can exist in a number of different isomeric and stereoisomeric forms.
  • the configuration of C-2 and C-5 can be such that their substituents are in either a cis or trans configuration. In preferred embodiments, the compounds exist in the cis configuration. Additionally, the combination of absolute configurations available to C-2 and C-5 can take any one of four permutations.
  • the thiazolidinone nucleus can be 2S, 5S; 2R, 5R; 2S, 5R; or 2R, 5S. Presently preferred embodiments are those in which the configuration at C- 2 and C-5 are 2S, 5R.
  • the compounds of the present invention can be used for diverse pharmaceutical applications including, for example, CNS antuschemic agents, agents with antipsychotic or other psychoactive properties, antimicrobial agents and mammalian fertility regulating agents.
  • the thiazolidinones are preferably agonists of the FSH receptor.
  • Tables I-NI Examples of presently preferred thiazolidinones having FSH agonist activity are displayed in Tables I-NI.
  • the EC 5 o values of the compounds displayed in Tables I-NI are less than about 500 riM. Table I.
  • the invention provides a class of FSH receptor agonists, wherein the receptor agonist activity is noncompetitve with FSH.
  • the non-competitive FSH agonists are organic molecules with a molecular weight of from about 50 daltons to about 1000 daltons, more preferably from about 200 daltons to about 1000 daltons.
  • the inventiorf provides for pharmaceutical formulations containing a FSH receptor agonist which is non- competitive with FSH.
  • the invention provides regulators of mammalian fertility which are useful in the diverse applications described herein for the thiazolidinones of the invention.
  • the present invention provides pharmaceutical compositions which contain one or more of the compounds of the invention in conjunction with pharmaceutically acceptable excipients, carriers, diluents, etc.
  • the pharmaceutical compositions can also contain other agents which are themselves pharmacologically active and which serve to enhance, supplement, decrease or otherwise regulate the pharmacological effect of the pharmaceutical compositions.
  • the compounds, i.e., thiazolidinones, of the present invention can be administered to a mammal, e.g., a human patient, alone, in the form of a pharmaceutically acceptable salt, or in the form of a pharmaceutical composition where the compound is mixed with suitable carriers or excipient(s) in a therapeutically effective amount.
  • the compounds and compositions of the invention can be admimstered to induce greater fertility in the patient or they can be administered to stimulate the production of ova which will be removed, fertilized in vitro and implanted in the patient or a surrogate.
  • the compounds and compositions of the present invention can be used for the treatment of male, as well as female, infertility. See, for example, Reichert, et al., "The follicle stimulating hormone (FSH) receptor in testis: interaction with FSH, mechanism of signal transduction, and properties of the purified receptor," Biol. Reprod. 40:13-26 (1989), the disclosure of which is incorporated herein by reference.
  • FSH follicle stimulating hormone
  • the compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration.
  • the compound can be administered in a local rather than systemic manner, for example, via injection of the compound directly into an ovary, often in a depot or sustained release formulation.
  • the compounds can be administered in a targeted drug delivery system, for example, in a liposome coated with an organ surface receptor-specific antibody. Such liposomes will be targeted to and taken up selectively by the organ.
  • the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • the discussion which follows is based on the use of the compounds of the invention as fertility-inducing agents. That pharmaceutical compositions containing the novel thiazolidinones are useful in other applications, and are not limited to use as fertility-inducing agents will be apparent to those of skill in the art.
  • adjuncts which serve a purpose analogous to those discussed below (i.e., enhance or supplement the thiazolidinone therapeutic activity) can be included within the formulation.
  • the thiazolidinone analogs of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g., fertility-inducing agent, such as FSH, LH and hMG).
  • Other agents which can be included in the pharmaceutical compositions include ovulation adjuncts such as, for example, cytokines (e.g., IGF-1 and TGF- ⁇ ) and narrow action oligopeptides (e.g., activins, irihibins and follistatins). See, for example, Gast, "Evolution of clinical agents for ovulation induction.” Am. J.
  • compositions described herein can be manufactured in a manner that is known to those of skill in the art, i.e., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the compounds can be formulated into preparations by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining with pharmaceutically acceptable carriers that are well known in the art.
  • Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained by mixing the compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form ofan aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, or from propellant-free, dry-powder inhalers.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, or from propellant-free, dry-powder inhalers.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas
  • propellant-free, dry-powder inhalers e.g.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, carbowaxes, polyethylene glycols or other glycerides, all of which melt at body temperature, yet are solidified at room temperature.
  • rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, carbowaxes, polyethylene glycols or other glycerides, all of which melt at body temperature, yet are solidified at room temperature.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, " as a sparingly soluble salt.
  • hydrophobic pharmaceutical compounds may be employed.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in a therapeutically effective amount.
  • the amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. Determination of an effective amounUs well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the EC 5 o as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vitro or in vivo data.
  • Initial dosages can also be formulated by comparing the effectiveness of the compounds described herein in cell culture assays with the effectiveness of known drugs. For instance, when used as fertility agents, initial dosages can be formulated by comparing the effectiveness of the compounds described herein in cell culture assays with the effectiveness of known fertility agents such as hMG or FSH. In this method, an initial dosage can be obtained by multiplying the ratio of effective concentrations obtained in cell culture assay for the compound of the present invention and a known fertility drug by the effective dosage of the known fertility drug.
  • a compound of the present invention is twice as effective in cell culture assay as hMG (i.e., the EC 5 o of that compound is equal to one-half the EC 50 of hMG in the same assay)
  • an initial effective dosage of the compound of the present invention would be one-half the known dosage for hMG.
  • toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 5 o (the dose required to cause death in 50% of the subjects tested) and the ED 5 o (the dose that produces a defined effect in 50% of the subjects tested).
  • the dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LD 5 o and ED 50 .
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is appropriate for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 5 o with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, for example, Fingl, et al, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1 (1975).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain therapeutic effect.
  • Usual patient dosages for oral administration range from about 50-2000 mg/kg/day, commonly from about 100-1000 mg/kg/day, preferably from about 150-700 mg/kg/day and most preferably from about 250-500 mg/kg/day.
  • therapeutically effective serum levels will be achieved by administering multiple doses each day.
  • the effective local concentration of the drug may not be related to plasma concentration.
  • One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
  • compositions of the invention When used as fertility-inducing agents in the female, the compositions of the invention can be evaluated for their effectiveness by any of a number of art accepted parameters including number of follicles, number of oocytes, number of transferrable embryos, number of pregnancies, the total dose administered and the treatment length. Similarly accepted criteria are available for evaluating the safety of a fertility-inducing agent including incidence of ovarian hyperstimulation and incidence of multiple gestation. When used to enhance fertility in the male, effectiveness can be adduced by increased sperm count, sperm motility and the like. Additional criteria and methods for assessing the efficacy of a thiazolidinone-containing pharmaceutical composition, when used as a fertility-inducing agent or for another purpose, will be apparent to those of skill in the art.
  • the thiazolidinones can be incorporated into the pharmaceutical formulation as mixtures of diastereomers, mixtures of enantiomers or as stereochemically distinct compounds.
  • the origin of the isomerism is the chirality of the carbons at positions 2 and 5 of the thiazolidinone ring structure (Formula I).
  • the thiazolidinone component of the pharmaceutical composition is a mixture of cis and trans isomers.
  • the mixture of cis and trans isomers is enriched in the cis isomer relative to the trans isomer.
  • the thiazolidinone is present as the substantially pure cis isomer.
  • the stereochemistry of the carbon atoms at positions 2 and 5 of the ring is yet another feature of the thiazolidinone constituent which can be varied.
  • the thiazolidinone constituent is a mixture of the 2S, 5R and 5S, 2R isomers.
  • the thiazolidinone constituent is enriched in the 2S, 5R isomer.
  • the thiazolidinone constituent is substantially pure 2S, 5R.
  • the compounds of the invention are useful in vitro as unique tools for understanding the biological role of FSH, including the evaluation of the many factors thought to influence, and be influenced by, the production of FSH and the interaction of FSH with the FSH-R (e.g., the mechanism of FSH signal transduction/receptor activation).
  • the present compounds are also useful in the development of other compounds that interact with the FSH-R, because the present compounds provide important structure-activity relationship (SAR) information that facilitate that development.
  • SAR structure-activity relationship
  • Compounds of the present invention that bind to the FSH receptor can be used as reagents for detecting FSH receptors on living cells, fixed cells, in biological fluids, in tissue homogenates, in purified, natural biological materials, etc. For example, by labelling such compounds, one can identify cells having FSH-R on their surfaces.
  • compounds of the present invention can be used in in situ staining, FACS (fluorescence-activated cell sorting), western blotting, ELISA (enzyme-linked immunoadsorptive assay), etc.
  • compounds of the present invention can be used in receptor purification, or in purifying cells expressing FSH receptors on the cell surface (or inside permeabilized cells).
  • the compounds of the invention can also be utilized as commercial research reagents for various medical research and diagnostic uses. Such uses can include but are not limited to: (1) use as a calibration standard for quantitating the activities of candidate FSH agonists in a variety of functional assays; (2) use as blocking reagents in random compound screening, i.e., in looking for new families of FSH receptor ligands, the compounds can be used to block recovery of the presently claimed FSH compounds; (3) use in the co-crystallization with FSH receptor, i.e., the compounds of the present invention will allow formation of crystals of the compound bound to the FSH receptor, enabling the determination of receptor/compound structure by x-ray crystallography; (4) other research and diagnostic applications wherein the FSH-receptor is preferably activated or such activation is conveniently calibrated against a known quantity of an FSH agonist, and the like; (5) use in assays as probes for determining the expression of FSH receptors on the surface of cells; and (6) developing assays
  • Example 1 details the assembly of a library in which the amino acid component is varied.
  • Example 2 the synthesis of a library in which the aldehyde component is varied is set forth.
  • Example 3 illustrates a library, wherein the amine component is varied.
  • Example 4 illustrates a scaled-up synthesis of one exemplary novel thiazolidinone.
  • Example 5 sets forth the synthetic route to enantiomerically pure thiazolidinones from an enantiomerically pure mercaptosuccinic acid precursor.
  • Example 6 details the synthesis of thiophene compounds of the invention.
  • Example 7 sets forth the synthesis of phenethylamine compounds of the invention.
  • Example 8 details the synthesis of a benzyl ether derivative of the invention.
  • Example 9 details the preparation of iodobenzyl derivatives of compounds of the invention.
  • Example 10 illustrates the exchange of acetylene for iodine in the compound of Example 9.
  • Example 11 illustrates the coupling of pyridine to the acetyline moiety of the compound of Example 10.
  • Example 12 illustrates the derivatization of the carboxylic acid moiety of the compound of Example 11 with an amine.
  • Example 13 illustrates the oxidation of the ring sulfur of a thiazolidinone compound of the invention.
  • Example 14 details the experimental protocol for assaying the compounds of the invention for their ability to act as FSH antagonists.
  • Example 15 illustrates an assay for determining whether the compounds of the invention compete with FSH for the FSH binding site.
  • Example 1
  • This example details the synthesis of a library of thiazolidinones in which the amino acid component is varied.
  • a 96 well parallel synthesis apparatus was added 50 mg of Argogel-Rink Amide-FMOC (0.33 mmol/g loading) to 30 of the wells.
  • the resin was washed with dichloromethane (100 mL) and N,N-dimethylformamide (100 mL).
  • the resin was then deprotected with 20% piperidine in N,N-dimethylformamide (1 mL) for 30 minutes.
  • the resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide. To each well was added 5 eq.
  • the resin was then treated with 20 eq. of 4-(phenethynyl)benzaldehyde and 40 eq. of mercaptosuccinic acid in THF (1 mL) and heated at 60°C for 16 hours. The apparatus was cooled and washed with hot THF. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide.
  • This example illustrates the assembly of a library of the compounds of the novel thiazolidinones of the invention in which the structure of the aldehyde component is varied.
  • the resin was distributed into 96 wells on a parallel synthesis apparatus (-50 mg/well). To each well was added 20 eq. one of 96 different aldehydes (see below) and 40 eq mercaptosuccinic acid. THF (1 mL) was added to each well and heated at 70°C for 24 hours. The apparatus was cooled and each well was washed exhaustively with THF, DCM, DMF, DCM, MeOH, DCM, DMF in sequence.
  • This example illustrates the assembly of a thiazolidinone library, wherein the amine component of the thiazolidinone is varied over the library.
  • the resin was transferred to a 250 mL RBF and treated with 20 eq of 4- (phenethynyl)benzaldehyde and 40 eq of mercaptosuccinic acid in THF and heated at 60°C for 16 hours.
  • the vessels were cooled and transferred with THF to a 250 mL peptide vessel and washed with hot THF (250 mL).
  • the resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide.
  • the resin was distributed into 96 wells on a parallel, synthesis apparatus (-50 mg/well). To each well was added and reacted with 20 eq. of one of 96 different amines, 20 eq.
  • Example 4 sets forth a preparative synthetic route to a representative thiazolidinone of the invention, 3-[5- ⁇ [-(3,4-dichlorophenyl)ethylcarbamoyl]methyl ⁇ -4- oxo-2-(4-phenylethynylphenyl)-thiazolidin-3-yl]benz--mide.
  • the material was purified by preparative HPLC (1.25 mL injection, 8 column, isocratic 60% AcCN H 2 0, 30 mL/min), collected in 50 mL centrifuge tubes, and lyophilized to give the cis enantiomers of 3-[5- ⁇ [2-(3 ,4-dichlorophenyl)ethylcarbamoyl]methyl ⁇ -4-oxo-2-(4-phenylethynylphenyl)- thiazolidin-3-yl]benzamide as a white powder (35.0 mg, 0.056 mmol, 56%).
  • the enantiomers can be resolved by chiral chromatography to obtain optically pure compounds. Conditions for separation varies with each compound. A preparative Chiracel OD column was used to resolve the enantiomers of AF17102 and AF17439.
  • the individual enantiomers can be prepared synthetically by employing optically pure mercaptosuccinic acid in the thiazolidinone synthesis.
  • Example 5
  • L-aspartic acid ((S)-aspartic acid, 25 g, 188 mmol) and 245 mL of 5 N HBr.
  • the reaction was cooled in an ice bath to 0-50°C, followed by the dropwise addition of sodium nitrite (20.7 g, 301 mmol) in 75 mL of water over five hours. The temperature was maintained below 50°C during the addition. After the addition was complete, the reaction was allowed to stir for 12 hours at 23-25°C.
  • the reaction was diluted with diethyl ether (120 mL). The aqueous layer was removed and the organic phase was washed with 1 N HCI (100 mL).
  • the resin was split into 2 equal portions and each portion was treated with 10 eq of 4-benzyloxybenzaldehyde and 20 eq. of either R (70% EE) or S (75% EE) mercaptosuccinic.
  • Acetonitrile (5 mL) was added and the reaction was left at RT for 48 hours, then 55 °C for 48 hours.
  • the vessels were cooled and their contents were transferred with THF to a peptide vessel, and washed with hot THF.
  • the resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide.
  • the trans isomer was transferred to a 500 mL RBF, with 200 mL THF, and 10 equivalents of DBU.
  • the reaction was heated under reflux, followed by the addition of -20 mL of methanol.
  • the reaction was reflux for 24 hours, cooled, and the solvent removed under reduced pressure.
  • the residue was dissolved in EtOAc (250 mL) and washed with 1 N HCI (2 x 300 mL).
  • the organic layer was filtered to remove the trans isomer, and then concentrated under reduced pressure.
  • the remaining material was triturated with acetonitrile, filtered, and process repeated to achieve material with 95:5 cistrans ratio. This material was then recrystallized from acetonitrile to afford the cis isomer (>97:3).
  • This example illustrates the synthesis of a benzyl ether derivative of the invention.
  • This example illustrates the preparation of iodobenzyl derivatives of compounds of the invention.
  • This example illustrates the exchange of acetylene for iodine in the compound of Example 9.
  • This example illustrates the coupling of pyridine to the acetyline moiety of the compound of example 10.
  • This example illustrates the derivatization of the carboxylic acid moiety of the compound of Example 11 with an amine.
  • This example details the protocols utilized to assay the thiazolidinone library compounds for FSH agonist activity. a. Testing compounds for agonist activity using a cell-based reporter assay
  • Binding of FSH to the FSH receptor results in an increase in intracellular cAMP.
  • Increases in cAMP can be monitored by the use of reporter constructs (George, SE, Bungay, PJ and Naylor, LH, "Functional coupling of endogenous serotonin and calcitonin receptor in CHO cells to a cAMP responsive lucif erase reporter gene," J. Neurochem. 69 1278-1285 (1997), the teachings of which are incorporated herein by reference), in which the firefly luciferase gene is placed under CRE control.
  • the human FSH receptor gene was cotransfected into CHO cells with a CRE-luciferase vector and cells expressing the FSH receptor were sorted by FACS as described above for the binding assay. Individual clones were examined for their ability to produce luciferase in response to stimulation with 1 ⁇ M FSH. Clone 1D7, which produces a response of 20-40,000 cps against a basal level of about 1,000 cps, was chosen for testing of compounds. This clone responded to FSH with an EC50 of about 80 pM.
  • the human FSH receptor was cloned into the ⁇ -T8-12CA5-KH expression vector (Koller, et alJ, "A generic method for the production of cell lines expressing high levels of transmembrane receptors," Analytical Biochem. 250:51-60 (1997), which is incorporated herein by reference), and transfected into CHO cells. After G418 selection, cells were stained with FITC-labeled 12CA5 antibody and those expressing the FSH receptor were collected by FACS. Individual clones were expanded and examined for binding of 125 I labeled FSH. CHO FSH-R clone 1H6 was expanded in a 15 liter spinner and membranes were prepared as described (Koller, et al, Analytical Biochem. 250 51-60 (1997)).
  • Membranes were prepared from Chinese hamster ovary (CHO) cells which expressed FSH-R as described above. These cells specifically bind 125 I-labeled FSH. When a binding assay was performed in the presence of 100 ⁇ M thiazolidinone, no inhibition of the radiolabeled FSH was observed. Thus, although the thiazolidinones are able to bind to the FSH receptor and to elicit a response, they do not block the interaction between FSH and its receptor.
  • the resin prepared above was distributed into a number of scintillation vials according to the number of different aldehydes to be used.
  • To each amino acid on Rink Amide Resin (0.424 mmol) was added a solution of lOeq. of aldehyde (4.24mmol) and 20eq. of mercaptosuccinic acid (1.27g, 8.48mmol) in anhydrous THF (10 ml).
  • the resulting reaction mixture was heated at 60°C on the J-KEM block for 48 hr, the mixture was filtered, washed with THF (3 x 10ml), MeOH (3 x 10ml), and CH 2 C1 2 (3 x 10ml).
  • the products were cleaved from the solid support for characterization according to the following procedure. To each well was added 95% TFA DCM (2 ml). The resin was left standing for 1 h, and the solution were filtered into a 48 or 96 wells Robbins microtiter plate. The resin in each well was washed with dichloromethane (2 ml). The solutions were concentrated under a nitrogen stream and dried in Savant under vacuum. The compounds were purified by Gilson prep HPLC and the required fractions were concentrated in Savant. The final product was characterized by LC/MS. The structure for the libraries generated are shown in the following Table.
  • the ethyl acetate fraction was washed two additional times with acid.
  • the organic layer was separated, washed with brine and dried using MgSO 4 .
  • the solvent was removed under reduced pressure, yielding a mixture of cis and trans epimers as a white solid.
  • This material was dissolved in DMF (10 ml) and K 2 CO 3 (280 mg, 2 mmol) and benzyl bromide (340 mg, 2 mmol) were added. After 3h, the reaction was complete.
  • the DMF was partitioned between ethyl acetate and brine twice.
  • the organic layer was washed with 3N HCI twice.
  • the organic layer was washed with brine, dried over MgSO4 and concentrated under vacuum to yield an oil.
  • the DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer dried using magnesium sulfate. The solvent was removed under vacuum. The crude material was purified using silica gel flash chromotography (3% methanol:DCM), yielding the title compound (32 mg, 60%) as a yellow oil.
  • HATU (380 mg, 1.0 mmol) was added and the solution was allowed to stir at room temperature for 15 hours.
  • the DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer was washed twice with 3N HCI. The organic layer was washed again with brine and dried using MgSO 4 . The solvent was removed under vacuum, yielding the crude products as a yellow oil.
  • HATU (380 mg, 1.0 mmol) was added and the solution was allowed to stir at room temperature for 15 hours.
  • the DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer was washed twice with 3N HCI. The organic layer was washed again with brine and dried using MgSO 4 . The solvent was removed under vacuum, yielding the crude products as a yellow oil.
  • N-[2-(3-ethoxy-4-methoxyphenyl)ethyl]-2,2,2,-trifluoroacetamide 500 mg, 1.7 mmol was dissolved in MeOH (16 mL). Iodine (765 mg, 3.0 mmol) was added. This solution was treated with HIO 3 (264 mg, 1.5 mmol, dissolved inl7 mL of water). The reaction mixture was protected from light, and it was allowed to stir under nitrogen for 20 hours. The reaction mixture was partitioned between ethyl acetate and a weak sodium bisulfite solution. The ethyl acetate layer was washed one additional time with bisulfite, and then it was washed with bicarbonate.
  • N-[2-(5-Ethoxy-2-iodo-4-methoxyphenyl)ethyl]-2,2,2,-trifluroacetamide (2.0 g, 4.8 mmol) was stirred in MeOH (60 mL) and water (20 mL). Lithium hydroxide monohydrate (1.05 g, 25 mmol) was added and the mixture was heated to 60°C and allowed to stir for 1 hour. The reaction mixture was diluted with ethyl acetate, and washed three times with water. It was dried using brine and magnesium sulfate, and concentrated under vacuum to yield the title compound (1.4 g, 89%).
  • the DMF solution was diluted with ethyl acetate, washed twice with brine, and twice with 3N HCI.
  • the ethyl acetate was dried over MgSO4, and concentrated by rotary evaporation.
  • This crude was purified using silica gel column chromatography (6% MeOH/DCM) to yield the product (1 lOmg, 25%) as yellow solid.
  • the DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer dried using magnesium sulfate. The solvent was removed under vacuum. The crude material was purified using silica gel flash chromotography (4 % methanol:DCM), yielding the title compound (20 mg, 28%) as a yellow oil.
  • a CH 2 Ph 0 a) CH 3 CN, mole, sieves 4A, 80°C; b) BOP, DIEA, THF for 6 hrs then reduced with 1.15 eq NaBH 4 ; c) LiCl, LiTMS 2 , RX, -78°C to -40°C; d) NH4OH, CH 3 OH; e) Jones oxidation, 0°C; f) RNH 2 , HATU, DIAE, DMF, g) MCPBA, NMP, 60°C.
  • this compound (2.51 g) was dissolved in THF (15 ml) and treated with 1M diborane in THF (36 ml, 36 mmol). The solution was heated under reflux for 2 hours, then cooled to 0°, treated with water (10 ml) and 20% KOH aqueous solution (10 ml).
  • Example 85 Example 85
  • Example 86 Using the procedure of Example 83 and substituting 3- chlorophenethylamine, the title compound was obtained as a white solid; 1 HNMR (DMSO-d 6 ) ⁇ 6.80-8.00 (m, 17 H), 6.55 (s, IH), 4.99 (s, 2H), 4.17 (m, 2H), 3.23 (m, 2H), 2.71 (t, 2H), 2.17 (m, 2H); MS (ES-positive): [M+ H] + 644. HPLC determined that this compound 90.8 % purity.
  • Example 86 Example 86
  • Example 87 Using the procedure of Example 83 and substituting 3- triflouromethoxyphenethylamine, the title compound was obtained as a white solid; 1 HNMR (DMSO-d 6 ) ⁇ 6.80-8.00 (m, 17 H), 6.55 (s, IH), 5.00 (s, 2H), 4.25 (m, 4H), 2.20 (m, 2H), 1.65 (s, 3H); MS (ES-positive): [M+ H] + 680. HPLC determined that this compound 93.9 % purity.
  • Example 87
  • Example 89 Using the procedure of Example 83 and substituting 3-iodophenethylamine, the title compound was obtained as a white solid; 1 HNMR (DMSO-d 6 ) ⁇ 6.70-8.15 (m, 17 H), 6.55 (s, IH), 4.98 (s, 2H), 4.17 (m, 4H), 2.25 (m, 2H), 1.65 (s, 3H); MS (ES-positive): [M+ H] + 722; HPLC determined that this compound 91.3 % purity.
  • Example 89 Example 89

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Abstract

In one aspect, the present invention provides novel compounds. In addition, the invention provides a FSH receptor agonist, wherein the agonist binds to a FSH receptor having a FSH binding site and, wherein the agonist is noncompetitve with FSH for the FSH binding site. In another aspect, the invention provides methods of using the compounds of the present invention for diverse pharmaceutical applications including, for example, CNS antiischemic agents, agents with antipsychotic or other psychoactive properties, antimicrobial agents and mammalian fertility regulating agent.

Description

AGONISTS OF FOLLICLE STIMULATING HORMONE ACTIVITY
FIELD OF THE INVENTION
This invention relates broadly to novel thiazolidinones. More specifically, the invention relates to thiazolidinones which modulate Follicle Stimulating Hormone (FSH) activity.
BACKGROUND OF THE INVENTION
Approximately 400,000 germ cells are stored in the ovaries of the human female at the time of puberty. No further germ cells are made. Beginning at the time of puberty and ending at menopause, there are approximately 400 ovulatory rnenstrual cycles which consume essentially all of the germ cells in the human ovary. About 1,000 germ cells are consumed in each menstrual period. However, in any one menstrual cycle, only one germ cell, developed in what becomes the dominant follicle, is ovulated and available for pregnancy.
Although the details are not accurately known, the mechanism by which a single egg is selected each month to become the dominant egg is dependent upon a complex interaction between one or more hormones from the ovary, hypothalamus and the pituitary. Three glycoprotein hormones (luteinizing hormone (LH), follicle stimulating hormone (FSH) and chorionic gonadotropin (hCG)) act on the ovary to stimulate steroid synthesis and secretion. LH and FSH are secreted by the pituitary and together play a central role in regulating the menstrual cycle and ovulation. hCG is secreted by the developing placenta from the early stages of pregnancy and its role is to maintain steroid secretion by the corpus luteum, which is necessary to prevent ovulation during pregnancy.
In the normal cycle, there is a mid-cycle surge in LH concentration which is followed by ovulation. An elevated estrogen level, which is brought about by the endogenous secretion of LH and FSH, is required for the LH surge to occur. The estrogen mediates a positive feedback mechanism which results in the increased LH secretion.
For more than twenty years, it has been possible to induce ovulation and menstruation, and sometimes pregnancy, in patients whose ovulatory mechanism is deranged so that normal cyclic ovulation and menstruation does not occur, by the administration of suitable amounts of a 1:1 mixture of FSH and LH known as human menopausal gonadotropin (hMG). In the field of in vitro fertilization, exogenous hormonal stimulation is employed by administering hMG. Women treated with hMG, however, often fail to demonstrate a timely LH surge despite serum estradiol levels sufficient to elicit positive feedback of LH secretion.
It is well established that the appropriate application of mixed exogenous gonadotrophins is efficacious for ovulation induction or for multiple egg retrieval during in vitro fertilization therapy in women. However, ovarian stimulation through exogenous gonadotrophins for in vivo and in vitro fertilization therapy is notoriously difficult to manage and the lack of uniform success with conventional hMG medications is widely appreciated. Individual response to hMG varies markedly, thereby complicating patient management even when the most flexible (individualized) protocols are used.
Attempts have been made to vary therapeutic hormone regimens in order to provide improved methods of inducing ovulation which increase the likelihood of more uniform follicular maturation or ovulation. One such attempt involves the induction of follicular maturation or ovulation by the administration of FSH in the absence of exogenous LH. Hodgen, U.S. Patent No. 4,854,077. To produce FSH which is uncontaminated by LH, post-menopausal urinary gonadotropin has been purified using immunoaffinity chromatography and reverse-phase HPLC. Arpaia, et al., U.S. Patent No. 5,128,453. Additional advances in this area have been realized by the use of hMG preparations which contain a markedly increased ratio of FSH to LH. Jones, Jr., et al, U.S. Patent No. 4,725,579.
Other hormone preparations which have been used to treat infertility include somatotrophin releasing factor (GRF) either alone or in combination with FSH. Fabbri, et al, U.S. Patent No. 5,017,557. Studies carried out in normal subjects during the menstrual cycle have demonstrated that intravenous administration of GRF produces an increase in serum levels of somatomedin C, but not of LH or FSH. Evans, et al "Effects of human pancreatic growth hormone releasing factor 40 on serum growth hormone, prolactin, luteinizing hormone, follicle stimulating hormone and somatomedin C concentrations in normal women throughout the menstrual cycle." J. Clin. Endoc. Metab. 59:1006 (1984). Two synthetic compounds, buserelin and triptorelin, have been used as gonadotrophin-releasing hormone agonists. See, for example, Out, et al, "A prospective, randomized, assessor-blind, multicentre study comparing recombinant FSH (Puregon) either given intramuscularly or subcutaneously in subjects undergoing INF." Hum. Reprod. 10 (Abstract Book 1):6 (1995), and Hedon, et al, "Efficacy and safety of recombinant FSH (Puregon) in infertile women pituitary-suppressed with triptorelin undergoing in vitro fertilization: a prospective, randomized, assessor-blind, multicentre trial." Hum. Reprod. 10: 3102-3106 (1995). Currently, there are no small molecule FSH receptor agonists available for clinical use.
Thiazolidinones are a class of small molecule organic compounds which have found limited pharmaceutical use. For example, thiazolidinones have been found to have central nervous system activity. See, for example, Tripathi, et al, "Thiazolidinone congeners as central nervous system active agents." Arzneimittelforschung 43:632-5 (1993). CNS activities which have been identified include, for example, antipsychotic properties. See, Mutlib, et al, "Metabolism of an atypical antipsychotic agent, 3-[4-[4-(6- fluoroberιzo[b]thien-3-yl)-l-piperazinyl]butyl]-2,5,5-trimethyl-4-thiazolidinone (HP236)." Drug Metab. Dispos. 24: 1139-50 (1996). Other thiazolidinones have been found to be CNS antuschemic agents. See, Ruterbories, et al, "Pharmacokinetics of a novel butylated hydroxytoluene-thiazolidinone CNS antuschemic agent LY256548 in rats, mice, dogs and monkeys." DrugMetab. Dispos. 18:674-9 (1990). Thiazolidinones have also been used as antimicrobial agents. See, for example, Ley, et al, "Inhibition of multiplication of Mycobacterium leprae by several antithyroid drugs." Am. Rev. Respir. Dis. 111:651-5 (1975).
The synthesis of novel thiazolidinones offers the promise for discovering new pharmaceutical agents with applications in areas as diverse as, for example, antimicrobial therapy and the treatment of strokes with CNS antuschemic agents. Of particular interest is the use of novel thiazolidinones as regulators of mammalian fertility.
Although the recent introduction of recombinant FSH has eliminated many of the difficulties associated with the use of gonadotrophins of urinary origin (e.g., cumbersome collection of urine, contamination of FSH by LH and low specific activity), there remains still a need for new fertility-regulating agents which are useful for both in vivo and in vitro applications. A class of small molecule FSH receptor agonist compounds which are inexpensive to prepare, easily purified, easily administered and which exhibit a broad range of activities would represent a significant advance in the field of human fertility medicine. Quite surprisingly, the present invention provides such small molecule thiazolidinone FSH receptor agonists. SUMMARY OF THE INVENTION
The present invention provides a class of novel thiazolidinones possessing a range of pharmaceutical applications and activities. Thus, in one aspect, the present invention provides novel thiazolidinones having the formula:
wherein,
R1 is a member selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocylic groups;
R2 is a member selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocylic groups;
R3 and R4 are independently members selected from the group including hydrogen, alkyl, -(CH2)mCONR5R6, -(CH2)mOCONR5R6, -(CH2)mCH2Y2R6, - (CH2)mCH=CHR6,
and -(CH2)mCH2NR5CO(Y3)nR6;
R5 and R6 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups;
X is a member selected from the group consisting of S, S=O, and O=S=O;
Y is a member selected from the group consisting of O, S, and NH;
Y is a member selected from the group consisting of CH2, O, S, and NR ;
Y is a member selected from the group consisting of O and NR ;
R7 is a member selected from the group consisting of hydrogen and lower alkyl;
X2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, phenyl, substituted phenyl, heterocychc, substituted heterocychc, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups; m is an integer from 0 to 3; n is 0 or 1 ; and s is 1 or 2.
In a second aspect, the present invention provides novel thiazolidinones having the structure:
cm) wherein,
R1 is a member selected from the group consisting of aryl, substituted aryl, arylalkyl and substituted arylalkyl groups;
R2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocychc groups;
R3 and R4 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups; and
X is a member selected from the group consisting of S, S=O, and O=S=O.
In another aspect, the invention provides a class of FSH receptor agonists, wherein the receptor agonists are noncompetitive with FSH for the receptor FSH binding site.
In yet another aspect, the invention provides a class of compounds that modulate FSH hormone activity, the compounds having: (a) a molecular weight of from about 50 daltons to about 1000 daltons; and (b) an FSH agonist activity corresponding to an EC50 standard of no more than 50 μM, preferably no more than 2 μM; wherein the agonist activity of this class of compounds to the FSH receptor is competitively inhibited by a compound having the formula: wherein,
R1 is a member selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocylic groups;
R2 is a member selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocylic groups;
R3 and R4 are independently members selected from the group including hydrogen, alkyl, -(CH2)mCONR5R6, -(CH2)mOCONR5R6, -(CH2)mCH2Y2R6, - (CH2)mCH=CHR6, -(CH2)mCH2NR5CO(Y3)nR6,
R5 and R6 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups;
X is a member selected from the group consisting of S, S=O, and O=S=O;
Y is a member selected from the group consisting of O, S, and NH;
Y2 is a member selected from the group consisting of CH2, O, S, and NR5;
Y3 is a member selected from the group consisting of O and NR6R7;
R is a member selected from the group consisting of hydrogen and lower alkyl;
X2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, phenyl, substituted phenyl, heterocychc, substituted heterocychc, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups; m is an integer from 0 to 3; n is O or 1; and s is 1 or 2. In yet a further aspect, the invention provides a class of compounds that modulate FSH hormone activity, the compounds having: (a) a molecular weight of from about 200 daltons to about 1000 daltons; and (b) an FSH agonist activity corresponding to an EC50 standard of no more than 50 μM, preferably no more than 2 μM; wherein the agonist activity of this class of compoimds to the FSH receptor is competitively inhibited by a compound having the formula:
wherein: R1 is a member selected from the group consisting of aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups; R2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocychc groups; R3 and R4 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups; and X is a member selected from the group consisting of S, SO, and O=S=O.
In a preferred embodiment, this class of compounds has a molecular weight of about 300 daltons to about 800 daltons. In another preferred embodiment, this class of compounds has an FSH receptor agonist activity, as expressed by an EC5o standard, of no more than 1 μM and, more preferably, of no more than 500 nM.
In still another aspect, the invention provides methods of using the compounds, i.e., thiazolidinones, for diverse pharmaceutical applications including, for example, CNS antuschemic agents, agents with antipsychotic or other psychoactive properties, antimicrobial agents and mammalian fertility regulating agents. When used as mammalian fertility regulating agents, the thiazolidinones are preferably agonists of the FSH receptor.
As such, in another aspect, the present invention provides pharmaceutical compositions which contain one or more of the compounds of the invention in conjunction with pharmaceutically acceptable excipients, carriers, diluents, etc. The pharmaceutical compositions can also contain agents which are themselves pharmacologically active and which serve to enhance, supplement, decrease or otherwise regulate the pharmacological effect of the pharmaceutical compositions.
Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description which follows.
DETAILED DESCRIPTION OF THE INVENTION A. Abbreviations and Definitions
HATU, [O-(7-Azabenzotriazol-l-yl)-l, 1,3,3- tetramethyluromumhexafluorophosphate]; DIEA, diisopropylethylamine; FMOC, fluorenylmethoxycarbonyl; DECP, diethyl cyanophosphonate; DCM, dichloromethane; DBU, l,8-diazabicyclo[5.4.0]undec-7-ene; CHO, Chinese hamster ovary; RBF, round- bottomed flask.
The term "independently selected" is used herein to indicate that the R groups, e.g., R1, R2, R4 and R5, can be identical or different (e.g., R1, R2 and R3 may all be substituted alkyls or R1 and R2 may be a substituted alkyl and R3 may be an aryl, etc.).
A named R group will generally have the structure which is recognized in the art as corresponding to R groups having that name. For the purposes of illustration, representative R groups as enumerated above are defined herein. These definitions are intended to supplement and illustrate, not preclude, the definitions known to those of skill in the art.
The term "alkyl" is used herein to refer to a branched or unbranched, saturated or unsaturated, monovalent hydrocarbon radical having from 1-12 carbons and preferably, from 1-6 carbons. When the alkyl group has from 1-6 carbons, it is referred to as a "lower alkyl." Suitable alkyl radicals include, for example, methyl, ethyl, n-propyl, i-propyl, 2-propenyl (or allyl), n-butyl, t-butyl (or 2-methylpropyl), etc.
"Substituted alkyl" refers to alkyl as just described including one or more functional groups such as lower alkyl, aryl, acyl, halogen, (i.e., alkylhalos, e.g., CF3), hydroxy, nitro, cyano, amino, alkoxy, alkylamino, acylamino, acyloxy, aryloxy, arloxyalkyl, mercapto, carboxylic acid, carboxylic acid derivatives, sulfonic acids, sulfonic acid derivatives, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like. These groups may be attached to any which are fused to the arom heterocycles and the like. These groups may be attached to any carbon of the alkyl moiety.
The term "aryl" is used herein to refer to an aromatic substituent having a single aromatic ring or multiple aromatic rings which are fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety. The common linking group may also be a carbonyl as in benzophenone. The aromatic ring(s) may include phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone among others.
"Substituted aryl" refers to aryl as just described including one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF3), hydroxy, nitro, cyano, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, carboxylic acid amide, sulfonic acid amide and both saturated and unsaturated cyclic hydrocarbons which are fused to the aromatic ring(s), linked covalently or linked to a common group such as a methylene or ethylene moiety. The linking group may also be a carbonyl such as in cyclohexyl phenyl ketone.
The term "arylalkyl" is used herein to refer to a subset of "aryl" in which the aryl group is attached through an alkyl group as defined herein. Examples include, but are not limited to, benzyl, phenylethyl and phenylpropyl groups.
"Substituted arylalkyl" defines a subset of "arylalkyl" wherein the aryl moiety of the arylalkyl group is substituted as defined herein for aryl groups.
The term "halogen" is used herein to refer to fluorine, bromine, chlorine and iodine atoms.
The term "hydroxy" is used herein to refer to the group -OH.
The term "amino" is used herein to refer to the group-NRR', where R and R' may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl or acyl.
The term "alkoxy" is used herein to refer to the -OR group, where R is a lower alkyl or substituted lower alkyl, wherein the alkyl and substituted lower alkyl groups are as described herein. Suitable alkoxy radicals include, for example, methoxy, ethoxy, t-butoxy, etc.
The term "aryloxy" is used herein to refer to the -OR group, wherein R is an aryl, substituted aryl, arylalkyl or substituted arylalkyl as described above. Examples include phenoxy, benzyloxy, phenethyloxy and substituted derivatives thereof. The term "alkylamino" denotes secondary and tertiary amines wherein the alkyl groups may be either the same or different and may consist of straight or branches, saturated or unsaturated hydrocarbons.
The term "heterocychc" is used herein to describe a monovalent group having a single ring or multiple condensed rings from 1-12 carbon atoms and from 1-4 heteroatoms selected from nitrogen, sulfur or oxygen within the ring. Such heterocycles include, for example, tetrahydrofuran, morpholine, piperidine, pyrrolidine, thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan, benzo-fused analogs of these rings, etc.
The term "substituted heterocychc" as used herein describes a subset of "heterocychc" wherein the heterocycle nucleus is substituted with one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF3), hydroxy, amino, alkoxy, alkylmino, acylamino, acyloxy, mercapto, etc.
The term "heterocyclicalkyl" is used herein to refer to a subset of "heterocylic" in which the heterocylcic group is attached through an alkyl group as defined herein.
"Substituted heterocyclicalkyl" defines a subset of "heterocyclicalkyl" wherein the heterocychc moiety of the heterocyclicalkyl group is substituted as defined herein for heterocychc groups.
The term "pharmaceutically acceptable salt" refers to those salts of compounds which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and organic acids such as, for example, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutically acceptable salts include, for example, alkali metal salts, such as sodium and potassium, alkaline earth salts and ammonium salts.
The term "contacting" is used herein interchangeably with the following: combined with, added to, mixed with, passed over, incubated with, flowed over, etc. Moreover, the thiazolidinone compounds of present invention can be "administered" to a subject by any conventional method such as, for example, parenteral, oral, topical and inhalation routes as described herein. "An amount sufficient" or "an effective amount" is that amount of a given thiazolidinone analog which exhibits the binding/activity of interest or, which provides an improvement in gamete recruitment.
"EC50" is the effective concentration, i.e., the concentration of a compound at which 50% of the maximal response of that obtained with FSH would be achieved.
"Non-competitive" refers to the nature of the agonist activity exhibited by the compounds of the invention, wherein the compounds act as agonists of and activate the FSH receptor without substantially reducing the magnitude of binding of FSH to the receptor. "Magnitude of binding" refers to the amount of FSH bound by a receptor population and/or the strength of the binding interaction between FSH and the FSH receptor.
The present invention is directed to novel thiazolidinone compounds which exhibit a range of pharmaceutical activities. In a presently preferred embodiment, the novel compounds are small molecule FSH receptor agonists. These compounds offer numerous advantages over the current state of the art (i.e., gonadotrophins of urinary origin and recombinant FSH). For example, the compounds of the instant invention are inexpensive and both easily prepared and purified. Further, the compounds exhibit a range of activity regarding the FSH receptor. Such a manifold of compounds of differing activity provides an opportunity to the clinician to modulate the desired level of fertility induction by judicious choice of the fertility-inducing agent. In addition, the novel thiazolidinones, as small molecules, exhibit a pharmacokinetic profile which is distinct from that of conventional peptidic hormone preparations. The pharmacokinetic profile can be further modified by judicious choice of the route of administration and manipulating the nature of the substituents on the thiazolidinone nucleus.
In a first aspect, the present invention provides a compound having a formula:
wherein, R1 is a member selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocylic groups;
R2 is a member selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocylic groups;
R3 and R4 are independently members selected from the group including hydrogen, alkyl, -(CH2)mCONR5R6, -(CH2)mOCONR5R6,-(CH2)mCH2Y2R6, - (CH2)mCH=CHR6,
and -(CH2)mCH2NR:>CO(Y nRo;
R5 and R6 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups;
X is a member selected from the group consisting of S, S=O, and O=S=O;
Y is a member selected from the group consisting of O, S, and NH;
Y2 is a member selected from the group consisting of CH2, O, S, and NR5;
Y is a member selected from the group consisting of O and NR R ;
R7 is a member selected from the group consisting of hydrogen and lower alkyl;
X2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, phenyl, substituted phenyl, heterocychc, substituted heterocychc, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups; m is an integer from 0 to 3; n is O or 1; and s is 1 or 2.
In a presently preferred embodiment, the present invention provides a compound wherein,
R5 and R6 are independently members selected from the group consisting of hydrogen, -(CH2)qX2, and wherein p is an integer from 1 to 2; q is an integer from 0 to 5;
X is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, phenyl, substituted phenyl, heterocychc, substituted heterocychc, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups; and
X is a member selected from the group consisting of S, S=O, and O=S=O.
More preferably, R1 is a member selected from the group consisting of
R > 8 and τ R9 are independently members selected form the group consisting of H, halogen, ketone, alkyl, substituted alkyl, phenyl, substituted phenyl, lower alkoxy, aryloxy, substituted aryloxy, carboxylic acid, carboxylic acid amide, sulfonic acid, sulfonic acid amide, alkynyl, substituted alkynyl and -CONR^CH^ tZ wherein t is an integer from one to four;
R10 is a member selected from the group consisting of H and lower alkyl;
A is a member selected from the group consisting of -C alkyl and substituted -C4 alkyl, wherein each carbon atom is independently substituted with members selected from the group consisting of H, amino, alkyl, substituted alkyl, spirocyclic alkyl, substituted spirocyclic alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc, and substituted heterocychc groups;
X3 is a member selected from the group consisting of O, S, SO, SO2 and NR10; Z is a member selected from the group consisting of alkylamino, dialkylamino,
w is an integer from 1 to 3;
Y is a member selected from the group consisting of O, S and NR 10 ;. R2 is a member selected from the group consisting of
ICBύa
wherein,
R »π and R , 12 are independently members selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, phenyl, substituted phenyl, aryloxy, substituted aryloxy, alkynyl, substituted alkynyl, nitro, cyano, aminoarylalkyl and substituted aminoarylalkyl; u is an integer from 1 and 4;
X4 is a member selected from the group consisting of O, S, NR13, and CR13;
X5 is a member selected from the group consisting of O, S, NH, and CH;
R is a member selected from the group consisting of H, and lower alkyl. o n *
More preferably, R and R are independently members selected form the group consisting of H, halogen, ketone, alkyl, substituted alkyl, carboxylic acid amide, sulfonic acid amide and alkynyl and -CONR1(CH2)tZ wherein t is an integer from one and four; wherein
Z is a member selected from the group consisting of alkylamino, dialkylamino,
w is an integer from 1 to 5; Y is a member selected from the group consisting of O, S, and NR10;
R1 is a member selected from the group consisting of H, and lower alkyl.
In another preferred embodiment, the present invention provides a compound wherein,
R and R are independently members selected from the group consisting of hydrogen, halogen, alkoxy, substituted alkoxy, aryloxy, -----R14, and -X6-( CH2)VR14 wherein
R14 is a member selected from the group consisting of phenyl, substituted phenyl, alkyl, alkenyl, cyloalkyl, cycloalkenyl, CH2(X7)ZR15, CONHR15, COR15, pyridine, thiophene, furan, pyrrole and phenylsulfonyl;
X6 is a member selected from the group consisting of O, S, NH, and CH2O;
X7 is.a member selected from the group consisting of O, S, and R14;
R15 is a member selected from the group consisting of H, alkyl and phenyl; v is an integer from 0 to 3; and z is O or 1.
In yet a further preferred embodiment, the present invention provides a compound wherein, when substituent R4, on C-5, is H, and a second substituent at C-5 (R ) is not H, said substituent R on C-5 and substituent R on C-2 are oriented in a cis manner.
In a second aspect, the present invention provides novel thiazolidinones having the formula:
(1TD wherein,
R1 is a member selected from the group consisting of aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups;
R2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocychc groups; R3 and R4 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups; and
X is a member selected from the group consisting of S, S=O and O=S=O.
In a presently preferred embodiment of this aspect of the invention, R1 is aryl or substituted aryl. In another preferred embodiment, R1 is phenyl or substituted phenyl. In a further preferred embodiment, R1 is substituted phenyl as in Formula (IN)
wherein R11, R12, R13, R14 and R15 are independently members selected from the group consisting of H, halogen, lower alkyl, substituted lower alkyl, phenyl, lower alkoxy, aryloxy, substituted aryloxy, carboxyl, ester and amide groups.
In other preferred embodiments, R1 is a substituted phenyl according to Formula (IN) and R11, R12, R13, R14 and R15 are independently members selected from the group consisting of H, halogen, substituted alkyl, ketone, ester, amide and nitro groups.
In still further preferred embodiments, R1 is substituted phenyl according to Formula (IN) and R11, R12, R13, R14 and R15 are independently members selected from the group consisting of H, halogen and amide groups.
In another presently preferred embodiment, R is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocychc groups. In another preferred embodiment, R2 is an aryl group. A preferred aryl group is the phenyl group and a preferred substituted aryl group is a substituted phenyl group.
In a further preferred embodiment, R2 is a substituted phenyl according to Formula (N) or a five-membered ring according to Formula (VI)
wherein R21, R22, R23, R24, R25 are members independently selected from the group consisting of H, halogen, lower alkyl, substituted lower alkyl, lower alkoxy, substituted lower alkoxy, phenyl, substituted phenyl, aryloxy, substituted aryloxy, alkynyl, substituted alkynyl and nitro groups. Preferred aryloxy groups are phenoxy and benzyloxy and preferred substituted aryloxy groups are substituted phenoxy and substituted benzyloxy. Y is a member selected from the group consisting of-CH2-, -O-, -S- and NR26 wherein R26 is H or lower alkyl.
In a further preferred embodiment, R21, R22, R23, R24 and R25 are independently members selected from the group consisting of H, halogen, lower alkoxy and substituted aryl groups according to Formula (Nil)
wherein R31, R32, R33, R34 and R35 are members independently selected from the group consisting of hydrogen, halogen, nitro and trifluoromethyl, alkyl, substituted alkyl, alkoxy and hydroxy. X1 is a member selected from the group consisting of O, ΝR36, S, C, CH and CH2; R is H or lower alkyl; m is an integer from 0 to 5; n is an integer from 0 to 3, p is an integer from 0 to 3 and q is an integer from 0 to 2. When q < 2, a multiple bond exists between CHq and X1.
In certain presently preferred embodiments, R21, R22, R23, R24 and.R25 are independently chosen from hydrogen and the groups according to Formulae (NIII) and (IX):
(VIII) (IX) wherein R41, R42, R43, R44 and R45 are members independently selected from the group consisting of hydrogen, halogen, nitro and trifluoromethyl. In Formula (NIII). n is an integer from 0 to 3 and X1 is O or ΝH. In yet a further preferred embodiment, R3 and R4 are the same or different and are members independently selected from the group consisting of H and structures according to Formula (X):
-(CH2)S-X2
(X) . wherein s is an integer from 0 to 5, preferably from 1 to 5. X2 is a member selected from the group consisting of aryl, substituted aryl, heterocychc and substituted heterocychc. In a further preferred embodiment, X is heterocychc or substituted aryl. In still further preferred embodiments, X2 is phenyl, substituted phenyl, indole or substituted indole.
Due to the chiral carbons at positions 2 and 5 (i.e., C-2 and C-5) of the thiazolidinone ring structure (see, for example, Formula III), the compounds of the invention can exist in a number of different isomeric and stereoisomeric forms. The configuration of C-2 and C-5 can be such that their substituents are in either a cis or trans configuration. In preferred embodiments, the compounds exist in the cis configuration. Additionally, the combination of absolute configurations available to C-2 and C-5 can take any one of four permutations. Thus, the thiazolidinone nucleus can be 2S, 5S; 2R, 5R; 2S, 5R; or 2R, 5S. Presently preferred embodiments are those in which the configuration at C- 2 and C-5 are 2S, 5R.
The compounds of the present invention can be used for diverse pharmaceutical applications including, for example, CNS antuschemic agents, agents with antipsychotic or other psychoactive properties, antimicrobial agents and mammalian fertility regulating agents. When used as mammalian fertility regulating agents, the thiazolidinones are preferably agonists of the FSH receptor.
Examples of presently preferred thiazolidinones having FSH agonist activity are displayed in Tables I-NI. The EC5o values of the compounds displayed in Tables I-NI are less than about 500 riM. Table I.
Table II.
R26 R 2-6
Table III.
Table TV.
Table V.
Table VI.
In another aspect, the invention provides a class of FSH receptor agonists, wherein the receptor agonist activity is noncompetitve with FSH. In a preferred embodiment, the non-competitive FSH agonists are organic molecules with a molecular weight of from about 50 daltons to about 1000 daltons, more preferably from about 200 daltons to about 1000 daltons. In another preferred embodiment, the inventiorf provides for pharmaceutical formulations containing a FSH receptor agonist which is non- competitive with FSH. In this aspect, the invention provides regulators of mammalian fertility which are useful in the diverse applications described herein for the thiazolidinones of the invention. B. Pharmaceutical Compositions and Uses
In another embodiment, the present invention provides pharmaceutical compositions which contain one or more of the compounds of the invention in conjunction with pharmaceutically acceptable excipients, carriers, diluents, etc. The pharmaceutical compositions can also contain other agents which are themselves pharmacologically active and which serve to enhance, supplement, decrease or otherwise regulate the pharmacological effect of the pharmaceutical compositions.
The compounds, i.e., thiazolidinones, of the present invention can be administered to a mammal, e.g., a human patient, alone, in the form of a pharmaceutically acceptable salt, or in the form of a pharmaceutical composition where the compound is mixed with suitable carriers or excipient(s) in a therapeutically effective amount. Further, the compounds and compositions of the invention can be admimstered to induce greater fertility in the patient or they can be administered to stimulate the production of ova which will be removed, fertilized in vitro and implanted in the patient or a surrogate. There are a number of art accepted techniques and technologies for accomplishing both of these goals. See, for example, Jennings, et al, "in vitro fertilization: A review of drug therapy and clinical management." Drugs 52:313-343 (1996), the disclosure of which is incorporated herein by reference.
By analogy to the demonstrated efficacy of gonadotrophins on the Sertoli cell, that is, the male equivalent of the ovarian granulosa cells, the compounds and compositions of the present invention can be used for the treatment of male, as well as female, infertility. See, for example, Reichert, et al., "The follicle stimulating hormone (FSH) receptor in testis: interaction with FSH, mechanism of signal transduction, and properties of the purified receptor," Biol. Reprod. 40:13-26 (1989), the disclosure of which is incorporated herein by reference.
The compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. Moreover, the compound can be administered in a local rather than systemic manner, for example, via injection of the compound directly into an ovary, often in a depot or sustained release formulation. In addition, the compounds can be administered in a targeted drug delivery system, for example, in a liposome coated with an organ surface receptor-specific antibody. Such liposomes will be targeted to and taken up selectively by the organ.
In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. In the interest of brevity, the discussion which follows is based on the use of the compounds of the invention as fertility-inducing agents. That pharmaceutical compositions containing the novel thiazolidinones are useful in other applications, and are not limited to use as fertility-inducing agents will be apparent to those of skill in the art. In these further applications, adjuncts which serve a purpose analogous to those discussed below (i.e., enhance or supplement the thiazolidinone therapeutic activity) can be included within the formulation.
The thiazolidinone analogs of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g., fertility-inducing agent, such as FSH, LH and hMG). Other agents which can be included in the pharmaceutical compositions include ovulation adjuncts such as, for example, cytokines (e.g., IGF-1 and TGF-β) and narrow action oligopeptides (e.g., activins, irihibins and follistatins). See, for example, Gast, "Evolution of clinical agents for ovulation induction." Am. J. Obstet Gynecol 172:753-59 (1995), Meldrum, "Ovarian stimulation for assisted reproduction," Curr. Opin. Obstet. Gynecol 8: 166-70 (1996), which are incorporated herein by reference. Other ovulation adjuncts are known to those of skill in the art and are useful with the compounds of the present invention. A number of suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences (Mack Publishing Company, Philadelphia, PA, 17th ed. (1985), which is incorporated herein by reference. Moreover, for a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990), which is incorporated herein by reference. The pharmaceutical compositions described herein can be manufactured in a manner that is known to those of skill in the art, i.e., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The following methods and excipients are merely exemplary and are in no way limiting.
For injection, the compounds can be formulated into preparations by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. Preferably, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining with pharmaceutically acceptable carriers that are well known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form ofan aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, or from propellant-free, dry-powder inhalers. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, carbowaxes, polyethylene glycols or other glycerides, all of which melt at body temperature, yet are solidified at room temperature.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example," as a sparingly soluble salt.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in a therapeutically effective amount. The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. Determination of an effective amounUs well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any compound used in the method of the invention, a therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the EC5o as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be estimated from in vitro or in vivo data.
Initial dosages can also be formulated by comparing the effectiveness of the compounds described herein in cell culture assays with the effectiveness of known drugs. For instance, when used as fertility agents, initial dosages can be formulated by comparing the effectiveness of the compounds described herein in cell culture assays with the effectiveness of known fertility agents such as hMG or FSH. In this method, an initial dosage can be obtained by multiplying the ratio of effective concentrations obtained in cell culture assay for the compound of the present invention and a known fertility drug by the effective dosage of the known fertility drug. For example, if a compound of the present invention is twice as effective in cell culture assay as hMG (i.e., the EC5o of that compound is equal to one-half the EC50 of hMG in the same assay), an initial effective dosage of the compound of the present invention would be one-half the known dosage for hMG. Using these initial guidelines one having ordinary skill in the art could determine an effective dosage in humans or other mammals. Moreover, toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD5o (the dose required to cause death in 50% of the subjects tested) and the ED5o (the dose that produces a defined effect in 50% of the subjects tested). The dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LD5o and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is appropriate for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED5o with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, for example, Fingl, et al, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1 (1975).
Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain therapeutic effect. Usual patient dosages for oral administration range from about 50-2000 mg/kg/day, commonly from about 100-1000 mg/kg/day, preferably from about 150-700 mg/kg/day and most preferably from about 250-500 mg/kg/day. Preferably, therapeutically effective serum levels will be achieved by administering multiple doses each day. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
When used as fertility-inducing agents in the female, the compositions of the invention can be evaluated for their effectiveness by any of a number of art accepted parameters including number of follicles, number of oocytes, number of transferrable embryos, number of pregnancies, the total dose administered and the treatment length. Similarly accepted criteria are available for evaluating the safety of a fertility-inducing agent including incidence of ovarian hyperstimulation and incidence of multiple gestation. When used to enhance fertility in the male, effectiveness can be adduced by increased sperm count, sperm motility and the like. Additional criteria and methods for assessing the efficacy of a thiazolidinone-containing pharmaceutical composition, when used as a fertility-inducing agent or for another purpose, will be apparent to those of skill in the art.
The thiazolidinones can be incorporated into the pharmaceutical formulation as mixtures of diastereomers, mixtures of enantiomers or as stereochemically distinct compounds. The origin of the isomerism is the chirality of the carbons at positions 2 and 5 of the thiazolidinone ring structure (Formula I). For example in one preferred embodiment, the thiazolidinone component of the pharmaceutical composition is a mixture of cis and trans isomers. In another preferred embodiment, the mixture of cis and trans isomers is enriched in the cis isomer relative to the trans isomer. In a further preferred embodiment, the thiazolidinone is present as the substantially pure cis isomer.
The stereochemistry of the carbon atoms at positions 2 and 5 of the ring is yet another feature of the thiazolidinone constituent which can be varied. In a preferred embodiment, the thiazolidinone constituent is a mixture of the 2S, 5R and 5S, 2R isomers. In a more preferred embodiment, the thiazolidinone constituent is enriched in the 2S, 5R isomer. In still further preferred embodiments, the thiazolidinone constituent is substantially pure 2S, 5R.
In addition to the foregoing, the compounds of the invention are useful in vitro as unique tools for understanding the biological role of FSH, including the evaluation of the many factors thought to influence, and be influenced by, the production of FSH and the interaction of FSH with the FSH-R (e.g., the mechanism of FSH signal transduction/receptor activation). The present compounds are also useful in the development of other compounds that interact with the FSH-R, because the present compounds provide important structure-activity relationship (SAR) information that facilitate that development.
Compounds of the present invention that bind to the FSH receptor can be used as reagents for detecting FSH receptors on living cells, fixed cells, in biological fluids, in tissue homogenates, in purified, natural biological materials, etc. For example, by labelling such compounds, one can identify cells having FSH-R on their surfaces. In addition, based on their ability to bind the FSH receptor, compounds of the present invention can be used in in situ staining, FACS (fluorescence-activated cell sorting), western blotting, ELISA (enzyme-linked immunoadsorptive assay), etc. In addition, based on their ability to bind to the FSH receptor, compounds of the present invention can be used in receptor purification, or in purifying cells expressing FSH receptors on the cell surface (or inside permeabilized cells).
The compounds of the invention can also be utilized as commercial research reagents for various medical research and diagnostic uses. Such uses can include but are not limited to: (1) use as a calibration standard for quantitating the activities of candidate FSH agonists in a variety of functional assays; (2) use as blocking reagents in random compound screening, i.e., in looking for new families of FSH receptor ligands, the compounds can be used to block recovery of the presently claimed FSH compounds; (3) use in the co-crystallization with FSH receptor, i.e., the compounds of the present invention will allow formation of crystals of the compound bound to the FSH receptor, enabling the determination of receptor/compound structure by x-ray crystallography; (4) other research and diagnostic applications wherein the FSH-receptor is preferably activated or such activation is conveniently calibrated against a known quantity of an FSH agonist, and the like; (5) use in assays as probes for determining the expression of FSH receptors on the surface of cells; and (6) developing assays for detecting compounds which bind to the same site as the FSH receptor binding ligands.
The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.
EXAMPLES
The following examples illustrate the preparation of three libraries of novel thiazolidinones. In brief, the thiazolidinones are constructed from three distinct components; an amino acid, an aldehyde and an amine. In each of the three libraries, the identity of one of these constituents is varied over the library. Example 1 details the assembly of a library in which the amino acid component is varied. In Example 2, the synthesis of a library in which the aldehyde component is varied is set forth. Example 3 illustrates a library, wherein the amine component is varied. Example 4 illustrates a scaled-up synthesis of one exemplary novel thiazolidinone. Example 5 sets forth the synthetic route to enantiomerically pure thiazolidinones from an enantiomerically pure mercaptosuccinic acid precursor. Example 6 details the synthesis of thiophene compounds of the invention. Example 7 sets forth the synthesis of phenethylamine compounds of the invention. Example 8 details the synthesis of a benzyl ether derivative of the invention. Example 9 details the preparation of iodobenzyl derivatives of compounds of the invention. Example 10 illustrates the exchange of acetylene for iodine in the compound of Example 9. Example 11 illustrates the coupling of pyridine to the acetyline moiety of the compound of Example 10. Example 12 illustrates the derivatization of the carboxylic acid moiety of the compound of Example 11 with an amine. Example 13 illustrates the oxidation of the ring sulfur of a thiazolidinone compound of the invention. Example 14 details the experimental protocol for assaying the compounds of the invention for their ability to act as FSH antagonists. Example 15 illustrates an assay for determining whether the compounds of the invention compete with FSH for the FSH binding site. Example 1
This example details the synthesis of a library of thiazolidinones in which the amino acid component is varied. To a 96 well parallel synthesis apparatus was added 50 mg of Argogel-Rink Amide-FMOC (0.33 mmol/g loading) to 30 of the wells. The resin was washed with dichloromethane (100 mL) and N,N-dimethylformamide (100 mL). The resin was then deprotected with 20% piperidine in N,N-dimethylformamide (1 mL) for 30 minutes. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide. To each well was added 5 eq. of 30 different N-Fmoc protected amino acids (see, Table VI), 10 eq. of HATU, and 10 eq. of DIEA in DMP (1 mL) for 16 hours. The resin was then washed exhaustively with dichloromethane and N,N-dimethylfoιmamide. The resin was then deprotected with 20% piperidine in N,N- dimefhylformamide (1 mL) for 30 minutes. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide.
The resin was then treated with 20 eq. of 4-(phenethynyl)benzaldehyde and 40 eq. of mercaptosuccinic acid in THF (1 mL) and heated at 60°C for 16 hours. The apparatus was cooled and washed with hot THF. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide.
Each well was reacted with 20 eq. 3,4-dimethoxyphenethylamine (60 μL/well), 20 eq. DIEA (60 μL/well), and 20 eq. diethylcyanophosphate (60 μL/well) in DCM (1 mL) for 3 hours. The resin was washed exhaustively with THF, DMF, DCM, MeOH, DCM in sequence. The products were cleaved with 95% TFA/DCM for 1 hour, drained and collected into a 96 well plate. The solvent was removed under reduced pressure on a speed vac overnight. Acetonitrile (1 mL/well) was added and removed by speed vac. Methanol (1 mL/well) was added and removed by Gene Vac. Submitted for biological assay at an estimated concentration of 8.25 μmol/well.
TABLE VI.
Example 2
This example illustrates the assembly of a library of the compounds of the novel thiazolidinones of the invention in which the structure of the aldehyde component is varied.
To a 250 mL peptide vessel was added 5.0 g of Argogel-Rink Amide- FMOC (0.33 mmol/g loading). The resin was washed with dichloromethane (100 mi) and N,N-dimethylformamide (100 mL). The resin was then deprotected with 20% piperidine in N,N-dimethylformamide (50 mL) for 30 minutes. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide. 3-Aminobenzoic acid (N-Fmoc protected, 2,0 g, 5.6 mmol) was coupled to the resin with HATU (2.327 g, 6.1 mmol) and DIEA (1.07 mL, 6.1 mmol) in DMF (12 mL) for 16 hours. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide. The resin was then deprotected with 20% piperidine in N,N-dimethylformamide (50 mL) for 30 minutes. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide.
The resin was distributed into 96 wells on a parallel synthesis apparatus (-50 mg/well). To each well was added 20 eq. one of 96 different aldehydes (see below) and 40 eq mercaptosuccinic acid. THF (1 mL) was added to each well and heated at 70°C for 24 hours. The apparatus was cooled and each well was washed exhaustively with THF, DCM, DMF, DCM, MeOH, DCM, DMF in sequence.
Each.. well was reacted with 20 eq. 3,4-dimethoxyphenethylamine (60 μL/well), 20 eq. DIEA (60 μL/well), and 20 eq. diethylcyanophosphate (60 μL/well) in DCM (1 mL) for 3 hours. The resin was washed exhaustively with THF, DMF, DCM, MeOH, DCM in sequence. The products were cleaved with 95% TFA/DCM for 1 hour, drained and collected into a 96 well plate. The solvent was removed under reduced pressure on a speed vac overnight. Acetonitrile (1 mL/well) was added and removed by speed vac. Methanol (1 mL/well) was added and removed by Gene Vac. Submitted for biological assay at an estimated concentration of 8.25 μmol/well.
The manifold of structures which were derived from the various aldehyde constituents and the point at which these structures were attached to the thiazolidinone ring are displayed in Table VII.
TABLE VII.
Example 3
This example illustrates the assembly of a thiazolidinone library, wherein the amine component of the thiazolidinone is varied over the library.
To a 250 mL peptide vessel was added 5.0 g of Argogel-Rink Amide- FMOC (0.33 mmol/g loading). The resin was washed with dichloromethane (100 mL) and N,N-dimethylformamide (100 mL). The resin was then deprotected with 20% piperidine in N,N-dimethylformamide (50 mL) for 30 minutes. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide. 3-Aminobenzoic acid (N-Fmoc protected, 2.0 g, 5.6 mmol) was coupled to the resin with HATU (2.327 g, 6.1 mmol) and DMA (1.07 mL, 6.1 mmol) in DMP (12 mL) for 16 hours. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide. The resin was then deprotected with 20 % piperidine in N,N-dimethylformamide (50 mL) for 30 minutes. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide,
The resin was transferred to a 250 mL RBF and treated with 20 eq of 4- (phenethynyl)benzaldehyde and 40 eq of mercaptosuccinic acid in THF and heated at 60°C for 16 hours. The vessels were cooled and transferred with THF to a 250 mL peptide vessel and washed with hot THF (250 mL). The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide. The resin was distributed into 96 wells on a parallel, synthesis apparatus (-50 mg/well). To each well was added and reacted with 20 eq. of one of 96 different amines, 20 eq. DIEA (60 μL/well), and 20 eq. diethylcyanophosphate (60 μL/well) in DCM (1 mL). The reaction was allowed to continue for 3 hours. The resin was washed exhaustively with THF, DMF, DCM, MeOH, DCM in sequence. The products were cleaved with 95% TFA/DCM for 1 hour, drained and collected into a 96 well plate. The solvent was removed under reduced pressure on a speed vac overnight. Acetonitrile (1 mL/well) was added and removed by speed vac. Ethanol (1 mL/well) was added and removed by Gene Nac. Submitted for biological assay at an estimated concentration of 8.25 μmol well.
The various amines which were incorporated into the thiazolidinone library are displayed in Table NIII, below.
TABLE mi .
Example 4
Example 4 sets forth a preparative synthetic route to a representative thiazolidinone of the invention, 3-[5-{[-(3,4-dichlorophenyl)ethylcarbamoyl]methyl}-4- oxo-2-(4-phenylethynylphenyl)-thiazolidin-3-yl]benz--mide. 4.1 Synthesis
Into a 1000 mL RBF was added 3-aminobenzamide (3.4 g, 25 mmol), 4- phenethynylbenzaldehyde (5.2 g, 25 mmol), mercaptosuccinic acid (7.5 g, 50 mmol) and toluene (500 mL). The reaction was heated under reflux with azeotropic removal of water via a Dean-Stark trap for 8 hours. The reaction was cooled to RT, concentrated under reduced pressure, and transferred to a 2 L separatory funnel with EtOAc (500 mL). The organic phase was washed with water (2 x 1 L) and extracted with 1 N NaOH (3 x 300 mL). The combined basic extracts were washed with EtOAc (500 mL) and acidified with con. HCI. The product was then extracted with EtOAc (500 mL) and washed with saturated sodium chloride solution (2 x 500 mL). The organic layer was dried (MgSO4), filtered, and concentrated under reduced pressure to leave a yellow solid. The solid was triturated with hot DCM and the resulting off-white solid was collected by filtration. The solid proved to be predominately the trans isomer (7 g, 15.3 mmol, 61%).
Into a 500 mL RBF was added the predominately trans acid (7 g, 15.3 mmol), THF (250 mL), DBU (4.5 mL, 3 eq.), and MeOH (15 mL). The reaction was heated under reflux for 24 hours. HPLC indicated equilibration to 1:5 ratio of cis:trans, an additional 2 mL of DBU was added along with 15 mL of MeOH and refluxed an additional 24 hours. HPLC indicated equilibration to 1:2 ratio of cis:trans. The reaction was cooled to RT and the solvent was removed under reduced pressure leaving a yellow syrup. The syrup was dissolved in EtOAc (125 mL) and washed with 1 N HCI (3 x 100 mL). The organic layer was concentrated under reduced pressure to leave a yellow syrup.
4.2 Purification and Resolution of Enantiomers
The syrup from 4.1, above, was dissolved in DMF (20 mL) and purified by preparative HPLC (1 mL injections, C18 column, isocratic 47% AcCN/H2O, 30 mL/min) to give the cis enantiomers (2.6 g, 5.6 mmol, 38%), and the trans enantiomers (4 g, 8.8 mmol, 58%).
Into a 4 mL vial was added the cis enantiomers (46 mg, 0.1 mmol), 3,4- dichlorophenethylamine (57 mg, 0.3 mmol) in DMF (1 mL), DECP (60 μL, 0.3 mmol), and DIEA (65 μL, 0.3 mmol). The reaction was stirred at room temperature for 3 hours. HPLC analysis showed the reaction to be complete. The material was purified by preparative HPLC (1.25 mL injection, 8 column, isocratic 60% AcCN H20, 30 mL/min), collected in 50 mL centrifuge tubes, and lyophilized to give the cis enantiomers of 3-[5- { [2-(3 ,4-dichlorophenyl)ethylcarbamoyl]methyl} -4-oxo-2-(4-phenylethynylphenyl)- thiazolidin-3-yl]benzamide as a white powder (35.0 mg, 0.056 mmol, 56%).
The enantiomers can be resolved by chiral chromatography to obtain optically pure compounds. Conditions for separation varies with each compound. A preparative Chiracel OD column was used to resolve the enantiomers of AF17102 and AF17439.
Alternatively, the individual enantiomers can be prepared synthetically by employing optically pure mercaptosuccinic acid in the thiazolidinone synthesis. Example 5
This example details the preparation of optically pure mercaptosuccinic acid and the synthesis of optically pure thiazolidinones from this precursor. 5.1 Synthesis of optically pure mercaptosuccinic acid a. Preparation of (R)-Bromosuccinic acid
5 N HBr
NH2 NaN02 Br ooc. 0-5°C HOOC- X.
^-^ X. OOH ^^ OOH
C4H7N04 C4H5Br04 Mol. Wt: 133.1036 Mol. Wt.: 196.9851 To a 500 mL round bottom flask was added D-aspartic acid ((R)-aspartic acid, 25 g, 188 mmol) and 245 mL of 5 N HBr. The reaction was cooled in an ice bath to 0-5°C, followed by the dropwise addition of sodium nitrite (20.7 g, 301 mmol) in 75 mL of water over five hours. The temperature was maintained below 5°C during the addition. After the addition was complete, the reaction was allowed to stir for 12 hours at 23-25°C. The reaction was diluted with diethyl ether (120 mL). The aqueous layer was removed and the organic phase was washed with 1 N HCI (100 mL). The combined aqueous phases were washed with EtOAc (100 mL). The combined organic extracts were dried (MgSO4), filtered and concentrated under reduced pressure to leave the product as a slightly yellow solid. The solid was recrystallized from EtOAc (-100 mL) and hexanes (-10 mL) to obtain the product (16.58 g, 84 mmol, 45%) as a white crystalline solid. b. - Preparation of (S)-Bromosuccinic acid
Mol. Wt.: 133.1036 Mol. Wt.: 196.9851
To a 500 mL round bottom flask was added L-aspartic acid ((S)-aspartic acid, 25 g, 188 mmol) and 245 mL of 5 N HBr. The reaction was cooled in an ice bath to 0-50°C, followed by the dropwise addition of sodium nitrite (20.7 g, 301 mmol) in 75 mL of water over five hours. The temperature was maintained below 50°C during the addition. After the addition was complete, the reaction was allowed to stir for 12 hours at 23-25°C. The reaction was diluted with diethyl ether (120 mL). The aqueous layer was removed and the organic phase was washed with 1 N HCI (100 mL). The combined aqueous phases were washed with EtOAc (100 mi). The combined organic extracts were dried (MgSO4), filtered and concentrated under reduced pressure to leave the product as a slightly yellow solid. The solid was recrystallized from EtOAc (-100 mL) and hexanes (-10 mL) to obtain the product (19.03 g, 97 mmol, 51%) as a white crystalline solid. c. Preparation of (S)-Mercaptosuccinic acid. HOO
MoL Wt: 196.9851 Form. Wt: 396.1837 MoL Wt: 150.1490
To a suspension of sodium thiophosphate dodecahydrate (6 g, 15 mmol) in toluene (50 mL) in an oil bath at 60°C was added (R)-bromosuccinic acid (0.5 g, 2.5 mmol). The reaction was stirred at 60°C for 3.5 hours (as the reaction temperature approaches 60°C, the sodium thiophosphate melts forming a biphasic reaction medium). The toluene was then removed under reduced pressure and the resulting white solid was diluted with water (25 mL) and 1 N hydrochloric acid (30 mL), a pH of 1-1.5. The reaction was stirred-at 23-25°C for 1-2 hours, then extracted with EtOAc (3x50 mL). The combined organic extracts were dried over MgSO4, filtered and concentrated to dryness under reduced pressure. The resulting white solid was dissolved in water (3.0 mL) and filtered through a 0.2 μm nylon filter. The filtrate was purified by preparative HPLC (a single injection of 3.0 mL, a Waters PrepPak cartridge Delta-Pak C18 compression column, 15 μm 25x100 mm, 95/5 water/acetonitrile at 12.0 mL/min). The product was collected and lyophilized to afford the product (276 mg, 18.4 mmol, 72.5%) as a white solid. d. Preparation of (R)-Mercaptosuccinic acid
C4H5B-04 HMNajOw-PS (W)4S
Mol. Wt: 196.9851 FOOT. Wt: 396.1837 Mol. Wt: 150.1490
To a suspension of sodium thiophosphate dodecahydrate (6 g, 15 mmol) in toluene (50 mL) in an oil bath at 60°C was added (S)-bromosuccinic acid (0.5 g, 2.5 mmol). The reaction was stirred at 60°C for 3.5 hours (as the reaction temperature approaches 60°C, the sodium thiophosphate melts forming a biphasic reaction medium). The toluene was then removed under reduced pressure and the resulting white solid was diluted with water (25 mL) and 1 N hydrochloric acid (30 mL), a pH of 1-1.5. The reaction was stirred at 23-25° C for 1-2 hours, then extracted with EtOAc (3x50 mL). The combined organic extracts were dried over MgSO4, filtered and concentrated to dryness under reduced pressure. The resulting white solid was dissolved in water (3.0 mL) and filtered through a 0.2 μm nylon filter. The filtrate was purified by preparative HPLC (a single injection of 3.0 mL, a Waters PrepPak cartridge Delta-Pak C18 compression column, 15 μm 25x100 mm, 95/5 water/acetonitrile at 12.0 ml min). The product was collected and lyophilized to afford the product (280 mg, 18.7 mmol, 73.5%) as a white solid. e. Determination of Enantiomeric Excess (%ee)
To a 1 wt% solution of Nα-(2,4-dinitrofluorophenyl)-L-valinamide in acetone (2.0 ml) is added mercaptosuccinic acid (2.0 mg) and 0.5 M NaHCO3 (1.0 ml). The reaction mixture is heated to 57°C for 45 minutes. The mixture is removed and diluted with 0.5 N NaHCO3 (5.0 mL), and washed with ethyl acetate (10 mL). The aqueous phase is acidified with 1 N HCI and extracted with ethyl acetate (5 mL). The adduct is then analyzed by HPLC. 5.2 Synthesis of optically pure thiazolidinones
The synthesis of optically pure thiazolidinones from the optically pure precursor, mercaptosuccinic acid proceeds as outlined in Scheme 1.
To a 100 mL peptide vessel was added 2.0 g of Argogel-Rink Amide- FMOC (0.33 mmol g loading). The resin was washed with dichloromethane (50 mL) and N,N-dimethylformamide (50 mL). The resin was then deprotected with 20% piperidine in N,N-dimethylformamide (50 mL) for 30 minutes. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide. 3-Aminobenzoic acid (N-Fmoc protected, 1.0 g, 2.8 mmol) was coupled to the resin with HATU (1.16 g, 3.0 mmol) and DIEA (0.53 mL, 6.0 mmol) in DMF (12 mL) for 16 hours. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide. The resin was then deprotected with 20% piperidine in N,N-dimethylformamide (50 mL) for 30 minutes. The resin was then washed exhaustively with dichloromethane and N,N- dimethylformamide.
The resin was split into 2 equal portions and each portion was treated with 10 eq of 4-benzyloxybenzaldehyde and 20 eq. of either R (70% EE) or S (75% EE) mercaptosuccinic. Acetonitrile (5 mL) was added and the reaction was left at RT for 48 hours, then 55 °C for 48 hours. The vessels were cooled and their contents were transferred with THF to a peptide vessel, and washed with hot THF. The resin was then washed exhaustively with dichloromethane and N,N-dimethylformamide.
Each portion was further reacted with 20 eq. of 3,4- dimethoxyphenethylamine, 20 eq. DIEA, and 20 eq. diethylcyanophosphate in DCM for three hours. The resin was washed exhaustively with THF, DMF, DCM, MeOH, DCM in sequence. The products were cleaved with 95% TFA/DCM for 1 hour, drained and washed with DCM (2 x 2 mL). The solvent was removed under reduced pressure leaving a yellow solid which was purified by preparative HPLC. R-Mercaptosuccinic acid afforded the cis isomer (18 mg, as a 96:4 mixture of 2S,5R:2S,5S). S-Mercaptosuccinic acid afforded the cis isomer (4 mg, as a 55:45 mixture of 2R,5S:2S,5R) and trans isomer (20 mg, as a 55:45 mixture of 2S,5S:2R,5R). The enantiomeric purity was determined on a Pirkle Leucine column employing 65% THF/35% hexane as the eluent at 0J ml/min. Example 6
This example details the synthesis of thiophene compounds of the invention.
Into a 250 mL RBF was added 3-aminobenzamide (1-6 g, 11.8 mmol), 5- (phenethynyl)thiophene-2-carboxaldehyde (2.5 g, 11.8 mmol), mercaptosuccinic acid (5.3 g, 35.4 mmol) and acetonitrile (200 mL). The reaction was heated under reflux for 3 days. A white solid had formed. The solid was collected by filtration, and washed with acetonitrile. The solid proved to be the trans isomer (4.0 g, 8.6 mmol, 73%). The filtrate was discarded. The trans isomer was transferred to a 500 mL RBF, with 200 mL THF, and 10 equivalents of DBU. The reaction was heated under reflux, followed by the addition of -20 mL of methanol. The reaction was reflux for 24 hours, cooled, and the solvent removed under reduced pressure. The residue was dissolved in EtOAc (250 mL) and washed with 1 N HCI (2 x 300 mL). The organic layer was filtered to remove the trans isomer, and then concentrated under reduced pressure. The remaining material was triturated with acetonitrile, filtered, and process repeated to achieve material with 95:5 cistrans ratio. This material was then recrystallized from acetonitrile to afford the cis isomer (>97:3).
Into a 2 mL vial was added the carboxylic acid (25 mg, 0.054 mmol), tryptamine (25 mg) in DMF (0.5 mL), DECP (30 μL), and DIEA (50 μL). The reaction was stirred at room temperature for 24 hours. HPLC analysis showed the reaction to be complete. The material was purified by preparative HPLC (C18 column, 5-95% AcCN/H2O over 40 minutes, 30 mL/min) to give the cis enantiomers as a white solid (AF21639, 28.0 mg, 0.046 mtnol 86%). HPLC, MS confirm product. Example 7
This example details the synthesis of phenethylamine compounds of the invention.
(a) AcCN/reflux; (b) DECP/DIEA DMF/amine; (c) Lil/DMF; (d) Boc2O/DMF/pyridine/NH4CO3; (e) SnCl2/DMF; (f) benzaldehyde/DMF/NaBH3CN. Into a 500 mL RBF was added methyl 5-amino-2-chlorobenzoate (6.5 g, 35 mmol), 4-nitrobenzaldehyde (5.3 g, 35 mmol), mercaptosuccinic acid (15.75 g, 105 mmol), acetonitrile (250 mL). The reaction was heated under reflux for 2 days. The reaction was diluted in EtOAc (1000 mL) and washed with water (3 x 500 mL). The organic layer was separated, dried (MgSO4), and concentrated under reduced pressure to leave a yellow syrup. The syrup was dissolved in DMF (20 mL) and standard DECP coupling to 3,4-dimethoxyphenethylamine was employed. Reaction was washed, dried, and purified by flash chromatography. The resulting solid was dissolved in DMF with 3 equivalents of lithium iodide and heated at 120 °C for three days. The reaction was complete, and was washed, dried, and used directly for the Ungashe reaction. After workup, the nitro group was reduced with 3 equivalents of tindichloride in DMF. The material was washed, and reductively alkylated with benzaldehyde and sodium cyanoborohydride. The product was worked up as usual, and purified by preparative HPLC to give the product as a white powder. Example 8
This example illustrates the synthesis of a benzyl ether derivative of the invention.
(a) AcCN, reflux; (b) DECP/DIES/DMF/a ine; (c) Lil/DMF; BOC2O/DMF/pyridine/-NH4CO3.
Into a 500 mL RBF was added methyl 5-amino-2-chlorobenzoate (6.5 g, 35 mmol), 4-benzyloxybenzaldehyde (7.43 g, 35 mmol), mercaptosuccinic acid (15.75 g, 105 mmol), acetonitrile (250 mL). The reaction was heated under reflux for 3 days. The reaction was diluted in EtOAc (1000 mL) and washed with water (3 x 500 mL). The organic layer was separated, dried (MgSO4), and concentrated under reduced pressure to leave a yellow syrup. The syrup was dissolved in DMF (20 mL) and standard DECP coupling to 3,4-dimethoxyphenethylamine was employed. Reaction was washed, dried, and purified by flash chromatography. The resulting solid was dissolved in DMF with 3 equivalents of lithium iodide and heated at 120 °C for three days. The reaction was complete, and was washed, dried, and used directly for the Ungashe reaction. The product was worked up as usual, and purified by preparative HPLC to give the product as a white powder. Example 9
This example illustrates the preparation of iodobenzyl derivatives of compounds of the invention.
Into a 500 mL was added sulfanilamide (3.71 g, 21.6 mmol) 4- iodobenzaldehyde (5 g, 21.6 mmol), mercaptosuccinic acid (10 g, 64.8 mmol) and acetonitrile (300 mL). The reaction was heated under reflux for 3 days. The reaction was cooled and concentrated under reduced pressure to leave a yellow solid. The solid was dissolved in EtOAc (250 mL) and washed with IN HCI (2 x 250 mL), water (3 x 250 mL), and saturated sodium chloride solution (1 x 100 mL). The organic layer was separated, dried (MgSO4), filtered, and concentrated under reduced pressure to leave a yellow solid. The solid was refluxed in chloroform (300 mL), filtered and dried to afford the product (10.5 g, 20.2 mmol, 93%) as a 1:8 ratio of cis:trans enantiomers. HPLC, MS, 1H NMR, and 13C NMR all confirm product.
Into a 500 mL RBF was added the predominately trans acid (5.2 g, 10 mmol). THF (200 mL), DBU (15 mL), and MeOH (50 mL). The reaction was heated under reflux for 48 hours. The reaction was cooled to RT and the solvent was removed under reduced pressure leaving a yellow syrup. The syrup was dissolved in EtOAc (250 mL) and washed with IN HCI (3 x 250 mL). The organic layer was concentrated under reduced pregsure to leave a yellow solid. The solid was refluxed in chloroform (300 mL), filtered and dried to afford the product (2.25 g, 4.3 mmol, 43%) as a 2;3 ratio of cis:trans enantiomers. HPLC, MS, 1H NMR, and 13C NMR all confirm product. Example 10
This example illustrates the exchange of acetylene for iodine in the compound of Example 9.
Into a 100 mL 3-necked RBF was added the predominately trans acid (5.2 g, 10 mmol), NMP (75 mL), DIEA (51 mmol), and trimethylsilylacetylene (51 mmol). The reaction was deoxygenated by alternating application of vacuum and nitrogen. Tetrakis(triphenylphosphine)palladium(0) (1.156 g, 1 mmol) and copper(I)iodide (760 mg, 4 mmol) were added and the reaction was again deoxygenated. The reaction was stirred under nitrogen at RT for 20 hours. The reaction was diluted with EtOAc (250 mL) and washed with IN HCI (3 x 250 mL). The organic layer was concentrated under reduced pressure to leave an orange syrup. The syrup was triturated with CHC13 (100 mL) to precipitate the product (3.4 g, 7.0 mmol, 70%) as an off-white solid. HPLC, MS, 1H NMR, and I3C NMR all confirm product.
Into a 200 mL RBF containing the carboxylic acid (3.4 g, 7 mmol) was added methanol (100 mL) and potassium carbonate (10 g, 70 mmol). The reaction mixture was stirred at RT for 4 hours. The reaction mixture was diluted with EtOAc (200 mL) and washed with :N HCI (3 x 500 mL). The organic phase was separated, dried (MgSO ), filtered, and concentrated under reduced pressure to leave a yellow syrup. The syrup was dissolved in DMF (6 mL) and purified by preparative HPLC (2 mL injection, C18 column 50% AcCN/H2O, 30 mL/min) to give the product (2.4 g, 5.8 mmol, 82%) as a 1 :2 mixture of cis:trans isomers. HPLC, MS both confirm product. Example 11
This example illustrates the coupling of pyridine to the acetyline moiety of the compound of example 10.
Into an 8.mL vial was added the carboxylic acid (250 mg, 0.6 mmol), 3- iodopyridine (200 mg), dichlorobis (triphertylphosphine)palladium(II) (45 mg, 0.06 mmol), copper(I) iodide (49 mg, 0.26 mmol), NMP (4 mL) and DIEA (0.44 mL). The reaction was stirred at RT for 24 hours. The reaction mixture was filtered through a 0.2 micron PTFE filter and purified by preparative HPLC (2 mL injections, C18 column, 5-95% AcCN H20 over 60 minutes, 30 mL/min) to give the product (225 mg, 0.46 mmol, 76%) as a white solid. HPLC, MS, 1H NMR, 13C NMR all confirm product. Example 12
This example illustrates the derivatization of the carboxylic acid moiety of the compound of Example 11 with an amine.
Into a 20 mL vial was added the carboxylic acid (225 mg, 0.46 mmol), 3- ethoxy-4-methoxyphenethylarnine (102 μL, 0.55 mmol) in DMF (4 mL), DECP (90 μL, 0.55 mmol), and DIEA (240 μL, 0.55 mmol). The reaction was stirred at room temperature for 24 hours. HPLC analysis showed the reaction to be complete. The material was purified by preparative HPLC (2.25 mL injections, C18 column, isocratic 35% AcCN/H2O, 30 mL/min) to give the cis enantiomers as a slightly yellow powder (AP20645, 61.9 mg, 0.092 mmol, 20%). HPLC, MS confirm product. Example 13
This example illustrates the oxidation of the ring sulfur of a thiazolidinone compound of the invention. HiNoc α
/ V 0M
Into a 4 mL vial was added the carboxylic acid (25 mg, 0.038 mmol), NMP (1 mL), and meta-chloroperbenzoic acid (46 mg). The reaction was heated at 60°C for 24 hours. The material was purified by preparative HPLC (C18 column, 5% to 95% AcCN/H2O over 40 minutes, 30 mL/min) to give the cis:trans enantiomersas a 1:1 mixture as a white powder (AF19470,12,2 mg, 0.0178 mmol, 47%). HPLC, MS confirm product. Example 14
This example details the protocols utilized to assay the thiazolidinone library compounds for FSH agonist activity. a. Testing compounds for agonist activity using a cell-based reporter assay
Binding of FSH to the FSH receptor results in an increase in intracellular cAMP. Increases in cAMP can be monitored by the use of reporter constructs (George, SE, Bungay, PJ and Naylor, LH, "Functional coupling of endogenous serotonin and calcitonin receptor in CHO cells to a cAMP responsive lucif erase reporter gene," J. Neurochem. 69 1278-1285 (1997), the teachings of which are incorporated herein by reference), in which the firefly luciferase gene is placed under CRE control. The human FSH receptor gene was cotransfected into CHO cells with a CRE-luciferase vector and cells expressing the FSH receptor were sorted by FACS as described above for the binding assay. Individual clones were examined for their ability to produce luciferase in response to stimulation with 1 μM FSH. Clone 1D7, which produces a response of 20-40,000 cps against a basal level of about 1,000 cps, was chosen for testing of compounds. This clone responded to FSH with an EC50 of about 80 pM. Compounds were mixed with CHO FSH-R CRE-luciferase cells (100,000 cells per well in a 96 well plate) in DMEM/F12 without phenol red and incubated at 37°C for 4-6 hours. An equal volume of LucLite (Packard) was added and the plates were counted in a TopCount (Packard). Example 15
This example illustrates the procedure utilized to test whether the thiazolidinone compounds of the invention competed with FSH for binding to the FSH receptor. a. ' Testing compounds for inhibition of binding of I FSH to the human FSH receptor expressed on the surface of CHO cells
The human FSH receptor was cloned into the α-T8-12CA5-KH expression vector (Koller, et alJ, "A generic method for the production of cell lines expressing high levels of transmembrane receptors," Analytical Biochem. 250:51-60 (1997), which is incorporated herein by reference), and transfected into CHO cells. After G418 selection, cells were stained with FITC-labeled 12CA5 antibody and those expressing the FSH receptor were collected by FACS. Individual clones were expanded and examined for binding of 125I labeled FSH. CHO FSH-R clone 1H6 was expanded in a 15 liter spinner and membranes were prepared as described (Koller, et al, Analytical Biochem. 250 51-60 (1997)).
Individual compounds were examined for their inhibition of 125I FSH binding to these membranes as follows: Mix:
• 50 μl membranes diluted in binding buffer (10 mM Tris pH 7.2, 1 mM MgCl2, 1 mM CaCl7 containing 0.1% BSA)-use amount of membranes to generate a 10:1 signa noise
25 μl sample or buffer containing 4 μM unlabeled FSH (Cortex
Biochem.)
25 μl 125I FSH (30,000 cpm per well) Incubate for 2 hours at room temperature and filter onto pretreated GF/B Unifilter plates (blocked with 0.1% PEI for 30 minutes). Dry filter at 37°C, add 40 μl of Microscent 20 (Packard) and count using Packard TopCount. b. Results
Membranes were prepared from Chinese hamster ovary (CHO) cells which expressed FSH-R as described above. These cells specifically bind 125I-labeled FSH. When a binding assay was performed in the presence of 100 μM thiazolidinone, no inhibition of the radiolabeled FSH was observed. Thus, although the thiazolidinones are able to bind to the FSH receptor and to elicit a response, they do not block the interaction between FSH and its receptor. Example 16
This example illustrates a general procedure for library production by parallel synthesis on Rink Amide resin. Step 1: Deprotection of Fmoc from Rink Amide Resin
Rink Amide Resin (loading: 0.53 mmol g; 2.4g, 1.272mmol) was treated with a solution of 20% piperidine in DMF (2 x 25 ml, 10 min for the first time and 20 min for the second time) to remove the Fmoc protecting group from the resin. The mixture was filtered and the resin was washed with DMF (3 x 25 ml), MeOH (3 x 25ml), and CH2C12 (3 x 25ml). Step 2: Attachment of Various Fmoc-Protected Amino Acids to the Resin
The resin (1.272 mmol) was swollen in anhydrous DMF (10 ml). A solution of Fmoc-protected amino acid (2eq., 2.544 mmol), HOBT (389.2mg, 2.544 mmol) and HBTU (964.2mg, 2.544 mmol) in anhydrous DMF (15 ml) was added to the resin followed by adding DIEA (886.3μl, 5.088 mol). The mixture was shaked at room temperature on an orbital shaker overnight. The mixture was filtered and the resin was washed with DMF (3 x 25 ml), MeOH (3 x 25ml), CH2C12 (3 x 25ml), and dried. Step 3: Deprotection of Fmoc Group
The resin (1.272 mmol), prepared as described in step 2 above, was again treated with a solution of 20% piperidine in DMF (2 x 25 ml, 10 min for the first time and 20 min for the second time) to remove the Fmoc protecting group. The mixture was filtered and the resin was washed with DMF (3 x 25 ml), MeOH (3 x 25ml), and CH2C12 (3 x 25ml). Step 4: Reaction with Various Aldehydes
The resin prepared above was distributed into a number of scintillation vials according to the number of different aldehydes to be used. To each amino acid on Rink Amide Resin (0.424 mmol) was added a solution of lOeq. of aldehyde (4.24mmol) and 20eq. of mercaptosuccinic acid (1.27g, 8.48mmol) in anhydrous THF (10 ml). The resulting reaction mixture was heated at 60°C on the J-KEM block for 48 hr, the mixture was filtered, washed with THF (3 x 10ml), MeOH (3 x 10ml), and CH2C12 (3 x 10ml). Step 5: Reaction with Various Amines
The resin again was distributed into 48 or 96 wells on a Robbins apparatus depending on the number of different amines to be used. To the resin-bound acid (0.027 mmol) was added a solution of HOBT (16.52mg, 0.108 mmol) and HBTU (41mg, 0.108 mmol) in anhydrous DMF (2 ml). DIEA(37.6μl, 0.216mmol) was then added into each well followed by lOeq. of amines(0.27mmol). The reaction mixture was rotated at room temperature on an orbital shaker overnight. The mixture was then filtered and the resin was washed with DMF .(3 x 2 ml), MeOH (3 x 2ml), CH2C12 (3 x 2ml), and dried. Step 6: Cleavage from the Solid Support
The products were cleaved from the solid support for characterization according to the following procedure. To each well was added 95% TFA DCM (2 ml). The resin was left standing for 1 h, and the solution were filtered into a 48 or 96 wells Robbins microtiter plate. The resin in each well was washed with dichloromethane (2 ml). The solutions were concentrated under a nitrogen stream and dried in Savant under vacuum. The compounds were purified by Gilson prep HPLC and the required fractions were concentrated in Savant. The final product was characterized by LC/MS. The structure for the libraries generated are shown in the following Table.
Scheme 1. For Examples 17-19
a) CH3CN, 80°C; b) RNH2, HATU, DIAE, DMF Example 17
Preparation of [3-[3-{aminocarbonyl}phenyl-2-{4-hex-l-ynyl}phenyl]-4- oxo-1, 3-thiazolidin-5-yl] acetic acid.
A solution of 3-aminobenzamide (5.4 g , 40 mmol) ,4-hex-l-yn-l-yl- benzaldehyde (7.3 g, 40 mmol ), and mercaptosuccinic acid (18 g, 120 mmol ) in acetonitrile (250 mL ) was heated to reflux for 48 hours. After cooling to room temperature, the mixture was concentrated in vacuo and the residue was crystallized from methanol to afford the title compound, m.p 210-212 °C.- H NMR (DMSO-d6) δ 0.94 (t, J = 7.1 Hz, 3H ),1.50 (m, 4 H), 2.44 (t , J = 6.7 Hz, 2 H), 2.96 (dd, J=17.3, 8.5 Hz, IH), 3.13 (dd, J = 17.3, 3.8 Hz, IH), 4.58 (dd, J = 8.2, 3.4 Hz,l H), 6.53 (s,l H), 7.33 (d, J=8.1 Hz, IH ), 7.45 (m , 5H ), 7.73 (d, J = 7.2 Hz, IH), 7.90 (s, IH), 8.01 (s , IH), 12.77 (broad s, IH). MS (Fl NEG) m/z 435 (M-Z)". Example 18 Preparation of 3-[(2S*, 5 R*)-5(2-{3-ethoxy-4-methoxyphenyl}- ethylamino-2-oxoethyl)-2-{4-hex-l-ynylphenyl}-4-oxo-l,-3-thiazolidin-3-yl]benzamide.
A solution of [3-[3-{aminocarbonyl}phenyl-2-{4-hex-l-ynyl}phenyl]-4- oxo-1, 3-thiazolidin-5-yl] acetic acid (1.8 g, 4.1 mmol ), and l,8-diazabicyclo[5.4.0]- undec-7-ene (0.57 g, 3.1 mmol) in methanol (225 mL) and dimethylformamide (25 mL) was stirred at room temperature for 60 hours. The solution was concentrated in vacuo , the residue was triturated with methylene chloride and 1 N hydrochloric acid, and the solid crude product, a mixture of trans and cis isomers in a ratio 2.3/1, was collected by filtration and dried. A solution of this crude product, 3-ethoxy-4-methoxyphenethyl- amine (0.9 g, 4.6 mmol), diisopropylethylamine (0.58 g, 4.6 mmol), and O-(7- azabenzotriazol-l-yl)-N,N,N',N-tetramethyluronium hexafluorophosphate (2J g, 5.8 mmol) in dimethylformamide (30 mL) was stirred at room temperature for 20 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The ethyl acetate solution was washed with brine, dried over magnesium sulfate, and concentrated. The residue was purified by column chromatography (Zorpax PRO C18, acetonitrile/water 60/40) to afford the title compound (0.46 g, 18%) m.p.l30-133°C.1H NMR (DMSO-d6) δ 0.87 (t, J = 7.2 Hz, 3H), 1.27 (t, J = 7.0 Hz, 3 H), 1.42 (m, 4 H), 2.39 (t, J = 6.8 Hz, 2 H), 2.63 (t, J = 7.1 Hz , 2 H), 2.71 (dd, J = 15.5, 9.7 Hz, IH), 3.06 (dd, J = 14.5, 3.6 Hz, IH), 3.26 (q, J = 6.4 Hz, 2H), 3.70 (s, 3H), 3.94 (q, J = 7.0 Hz, 2H), 4.39 (dd, J = 9.3, 3.5 HzlH), 6.49 (s, IH), 6.73 (m, 3H), 7.24 (d, J = 8.2 Hz, 2H), 7.40 (m, 5H), 7.64 (d, J = 7.5 Hz, IH), 7.81 (s, IH), 7.93 (s, IH), 8.15 (t, J = 5.5 Hz, IH), 7.81 (s, IH), 7.93 (s, IH), 8.15 (t, J = 5.5 Hz,lH); .MS (Fl POS) m z 614(M+H); Anal.Calc. for C24H24N2O4S. C: 66.04, H: 5.54, N: 6.42, found. C: 65.83, H: 5.67, N: 6.09. Example 19
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(4-ethoxy-3-methoxyphenyl)ethyl]- amino}-2-oxoethyl)-2)-4-hex-l-ynylphenyl)-4-oxo-l,3-thiazolidn-3-yl]benzamide.
Similar to the above example[3-[3-{aminocarbonyl}phenyl-2-{4-hex-l- ynyl}phenyl]-4-oxo-l, 3-thiazolidin-5-yl] acetic acid (3.1 g,8.2 mmol), 4-ethoxy-3- methoxyphenethylamine (2.12 g, 16.4 mmol), and O-(7-azabenzotriazol-l-yl)-N,N,N1,N1- tetramethyluronium-hexafluorophosphate (4.11 g, 10.8 mmol) were reacted to obtain the title compound (1.4 g, 28%); m.p. 154-157 °C. 1H NMR (DMSO-d6) δ 0.87 (t, J = 7.2 Hz, 3H), 1.29 (t, J = 7.0 Hz, 3H), 1.43 (m, 4H), 2.37 (t, J = 6.8 Hz, 2H), 2.64 (t, J = 7.2 Hz, 2H), 3.07 (dd, J = 15.6, 3.5 Hz, s, IH), 3.27 (q, J = 6.6 Hz, 2H), 3.69 (s, 3H), 3.93 (q, J = 7.0 Hz, 2H), 4.39 (dd, J = 9.7, 3.5 Hz, IH), 6.49 (s, IH), 6.73 (m, 3H), 7.24 (d, J - 8.2 Hz, 2H), 7.38 (m, 5H), 7.64 (d, J = 7.5 Hz, IH), 7.82 (s, IH), 7.93 (s, IH), 8.16 (t, J = 5.5 Hz, IH).; MS (FIPOS ) m/z 614 (M+H): Anal.Calc. for C35H39N3O5S C: 68.49, H: 6.40, N: 6.85, found. C: 68.15, H: 6.42, N: 6.78. Scheme 2. For Examples 20-37, 43-47.
a) CH3CN, mole, sieves 4A, 80°C; b) DBU, MeOH, THF, 70°C; c) PhCH2Br, K2CO3; d) recrystalize from EtOAc; e) TFA, thioanisole; f) RNH2, HATU, DIAE, DMF; g) R'X, K2CO3, DMF
Example 20
Preparation of {(2S*,5S*)-3-[3-(aminocarbonyl) phenyl]-2-[4-(benzyloxy) phenyl]-4-oxo-l,3-thiazolidin-5-yl} acetic acid.
A round-bottomed flask was fitted with a bump trap containing molecular sieves (4A). 3-aminobenzamide (5.4 g, 40 mmol) and 4-benzyloxybenzaldehyde (10.2 g, 48 mmol) were added to the flask and allowed to stir in acetonitrile (300 mL) at 80°C for 1 hour. Mercaptosuccinic acid (18.0 g, 120 mmol) was added to the reaction mixture. The solution was allowed to stir for 48 hours. The suspension was cooled in an ice-water bath for 1 hour and filtered, yielding the title compound (11.8g, 64%) as a white solid. 1H NMR (DMSO-d6):δ 12.49 (bs, IH), 7.93 (b, IH), 7.80 (s, IH), 7.64 (d, IH, 8Hz), 7.30-7.42 (m, 10H), 6.87 (d, IH, 8Hz), 6.45 (s, IH), 4.95 (s, 2H), 4.50 (dd, IH, 9Hz, 3Hz), 3.15 (dd, IH, 18Hz, 4Hz), 2.80 (dd, IH, 18Hz, 9Hz). MS (FI-NEG):[M-H]"= 461.Anal.Calc. for C25H22N2O5S: C, 64.92, H, 4J9, N, 6.06. Found: C, 61.52, H, 4.35, N, 8.89. Example 21
Preparation of {(2S*,5R*)-3-[3-(aminocarbonyl) phenyl] -2- [4-(benzyloxy) phenyl]-4-oxo-l,3-thiazolidin-5-yl} acetic acid.
{(2,5-Trans)-3-[3-(aminocarbonyl) phenyl]-2-[4-(benzyloxy) phenyl]-4- oxo-l,3-thiazolidin-5-yl} acetic acid (3.0 g, 6.5 mmol) was dissolved in methanol (100 mL) and THF (100 mL) at 70°C. DBU (11.9 g, 78 mmol) was added, and the solution was allowed to stir for 48 hours. Analytical HPLC (isocratic, 40% acetonitrile/water) showed two components in a 60:40 ratio. The more abundant material proved upon co-injection to be the trans epimer. The solvent was removed under vacuum, and the oil remaining was partitioned between ethyl acetate and 3 N HCI. The ethyl acetate fraction was washed two additional times with acid. The organic layer was separated, washed with brine and dried using MgSO4. The solvent was removed under reduced pressure, yielding a mixture of cis and trans epimers as a white solid. Preparative HPLC (isocratic, 40% acetonitrile/water ) was performed on this material, purifying the title compound (830 mg, 28%). 1H NMR (DMSO-d6): δ 7.93 (b, IH), 7.80 (s, IH), 7.64(d, IH, 8Hz), 7.30-7.42 (m, 10H), 6.87 (d, IH, 8Hz), 6.45 (s, IH), 5.00 (s, 2H), 4.40 (dd,lH, 9Hz, 3Hz), 3.15 (dd, IH, 18Hz, 4Hz), 2.80 (dd, IH, 18Hz, 9Hz). MS (FI-NEG):[M-H]-= 461.Anal. Calc. for C25H22N2O5S: C, 64.92, H, 4.79, N, 6.06. Found: C, 61.30, H, 4.64, N, 5.71. Example 22
Preparation of 3-[(2S*,5R*)-5-(2-{[2-(3-ethoxy-4-methoxyphenyl)ethyl]- --mino}-2-oxoethyl)-2-(4-benzyloxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] berizamide.
{(2S*,5R*)-3-[3-(aminocarbonyl) phenyl]-2-[benzyloxyphenyl]-4-oxo-l,3- thiazolidin-5-yl} acetic acid (8.00 g, 17.3 mmol) was dissolved in methanol (150 mL) and THF (150 mL) at 80°C. DBU (31.6 g, 208 mmol) was added,, and the solution was allowed to stir for 15h. Analytical HPLC (isocratic, 45 % acetonitrile/water) showed two components in a 60:40 ratio. The solvent was removed under vacuum, and the oil remaining was partitioned between ethyl acetate and 3 N HCI. The ethyl acetate fraction was washed two additional times with acid. The organic layer was separated, washed with brine and dried using MgSO4. The solvent was removed under reduced pressure, yielding a mixture of CM and trans epimers as a white solid. This crude material was dissolved in DMF (100 mL) with DIEA (3.06 g, 23 mmol) and 3-ethoxy-4-methoxyphenethylamine (4.29 g, 23 mmol). HATU (8.7 g, 23 mmol) was added and the solution was allowed to stir at room temperature for 15 h. The DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer was washed twice with 3N HCI. The organic layer was washed again with brine and dried using MgSO4. The solvent was removed under vacuum, yielding the crude products as a yellow oil. A portion of this mixture (4 g) was purified using preparative HPLC (40 acetonitrile/water (.1% TFA)) to yield the title product (1.10 g, 37%). The cis epimer:1H NMR (DMSO-d6): δ 8.15 (m, IH), 7.92 (bs, IH), 7.72 (s, IH), 7.62 (d, IH, 8Hz), 7.30-7.42 (m, 9H), 6.70-6.82 (m, 4H), 6.43 (s, IH), 4.37 (dd, IH, 9Hz, 3Hz), 3.99 (q, 2H, 8 Hz), 3.64 (s, 3H), 3.26 (m, 2H), 3.15 (dd, IH, 18Hz, 4Hz), 2.80 (dd, IH, 18Hz, 9Hz), 2.62 (m, 2H), 1.32 (t, 3H, 8 Hz). MS (ESI-NEG):[M-H] = 639. Anal.Calc. for C36H37N3O6S: C, 67.59, H, 5.83, N, 6.57. Found: C, 67.16, H, 5.82, N, 6.39. Example 23
Preparation of 3-[(2S*,5S*)-5-(2- {[2-(3-ethoxy-4-methoxyphenyl)ethyl]- amino}-2-oxoethyl)-2-(4-benzyloxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide.
{(2S*, 5S*)-3-[3-(aminocarbonyl) phenyl]-2-[benzyloxyphenyl]-4-oxo-l ,3- thiazolidin-5-yl} acetic acid (8.00 g, 17.3 mmol) was dissolved in methanol (150 mL) and THF (150 mL) at 80°C. DBU (31.6 g, 208 mmol) was added, and the solution was allowed to stir for 15 hours. Analytical HPLC (isocratic, 45% acetonitrile/water) showed two components in a 60:40 ratio. The solvent was removed under vacuum, and the oil remaining was partitioned between ethyl acetate and 3 N HCI. The ethyl acetate fraction was washed two additional times with acid. The organic layer was separated, washed with brine and dried using MgSO4. The solvent was removed under reduced pressure, yielding a mixture of cis and trans epimers as a white solid. This crude material was dissolved in DMF (100 mL) with DIEA (3.06 g, 23 mmol) and 3-ethoxy-4-methoxyphenethylamine (4.29 g, 23 mmol). HATU (8.7g, 23 mmol) was added and the solution was allowed to stir at room temperature for 15 h. The DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer was washed twice with 3N HCI. The organic layer was washed again with brine and dried using MgSO4. The solvent was removed under vacuum, yielding the crude products as a yellow oil. A portion of this mixture (4 g) was purified using preparative HPLC (40 acetonitrile/water (.1% TFA)) to yield the trans title product (1.52 g, 51%). 1H NMR (DMSO-d6): δ 8.15 (m, IH), 7.92 (bs, IH), 7.72 (s, IH), 7.62 (d, IH, 8Hz), 7.30-7.42 (m, 9H), 6.70-6.82 (m, 4H), 6.39 (s, IH), 4.46 (m, IH), 3.99 (q, 2H, 8 Hz), 3.64 (s, 3H), 3.26 (m, 2H), 3.15 (dd, lH,18Hz, 4Hz), 2.80 (dd, IH, 18Hz, 9Hz), 2.62 (m, 2H), 1.32 (t, 3H, 8 Hz). MS (ESI-NEG):[M-H] = 639. Anal.Calc. for C36H37N3O6S: C, 67.59, H, 5.83, N, 6.57. Found: C, 65.68, H, 5.63, N, 6.22. Example 24
Preparation of benzyl {(2S*,5R*)-3-[3-(aminocarbonyl) phenyl]-2-[4- (benzyloxy) phenyl]-4-oxo-l,3-thiazolidin-5-yl} acetate.
{(2S*,5S*)-3-[3-(aminocarbonyl) phenyl]-2-[benzyloxyphenyl]-4-oxo-l,3- thiazolidin-5-yl} acetic acid (500 mg, 1.1 mmol) was dissolved in methanol (10 mL) and THF (10 tnL) at 80°C. DBU ( 1.87 g, 12.3 mmol) was added, and the solution was allowed to stir for 15 hours. Analytical HPLC (isocratic, 45% acetonitrile/water) showed two components in a 60:40 ratio. The solvent was removed under vacuum, and the oil remaining was partitioned between ethyl acetate and 3 N HCI. The ethyl acetate fraction was washed two additional times with acid. The organic layer was separated, washed with brine and dried using MgSO4. The solvent was removed under reduced pressure, yielding a mixture of cis and trans epimers as a white solid. This material was dissolved in DMF (10 ml) and K2CO3 (280 mg, 2 mmol) and benzyl bromide (340 mg, 2 mmol) were added. After 3h, the reaction was complete. The DMF was partitioned between ethyl acetate and brine twice. The organic layer was washed with 3N HCI twice. The organic layer was washed with brine, dried over MgSO4 and concentrated under vacuum to yield an oil. The oil was triturated in ether (180 mL) for 1 hour. This suspension was filtered, yielding a white powdery solid. This material was dissolved in ethyl acetate (20 ml) with heating. It was placed in a freezer for 2 hours and then filtered. The ratio of cis: trans was increased significantly. This recrystallization was repeated twice, yielding the title compound (60 mg, 10%) as the pure cis epimer. 1H NMR (DMSO-d6):δ 7.93 (IH, b), 7.80 (IH, s), 7.62 (d, IH, 8Hz), 7.30-7.42 (m, 15H), 6.87 (d, IH, 8 Hz), 6.45 (s, IH), 5.17 (d, 2H, 3 Hz), 4.99 (s, 2H), 4.45 (dd, IH, 9Hz, 3Hz), 3.15 (dd, IH, 18 Hz, 4Hz), 2.80 (dd, IH, 18Hz, 9Hz). MS (ESI-POS):[M+H]+= 553. Anal. Calc. for C32H28N2O5S: C, 69.55, H, 5.11, N, 5.07. Found: C, 68.54, H, 4.81, N, 4.95. Example 25
Preparation of [(2S*,5R*)-3-[3-(aminocarbonyl) phenyl]-2-(4- (hydroxyphenyl)-4-oxo-l,3-thiazolidin-5-yl] acetic acid. Benzyl {(2S*,5R*)-3-[3-(aminocarbonyl) phenyl]-2-[4-(benzyloxy) phenyl]-4-oxo-l, 3-thiazolidin-5-yl} acetate (1.0 g , 1.8 mmol) was dissolved in trifluoroacetic acid (15 mL) and thioanisole (1.8 g, 15 mmol) at room temperature. This solution was allowed to stir for 24 hours. The solvent was removed under vacuum, leaving a pale yellow oil. This oil was dripped into stirring diethyl ether, and this suspension was allowed to stir for 24 hours. This mixture was filtered, yielding the title compound (640 mg, 95%) as a yellow solid. 1H NMR (DMSO-d6): δ 12.32 (bs, IH), 9.64 (bs, IH), 7.93 (bs, IH), 7.78 (s, IH), 7.62 (d, IH, 8Hz), 7.30-7.42 (m, 4H), 7.23 (d, 2H, 9Hz), 6.62 (d, IH, 8Hz), 6.38 (s, IH), 4.45 (dd, IH, 9Hz, 3Hz), 3.15 (dd, lH,18Hz, 4Hz), 2.80 (dd, lH,18Hz, 9Hz). MS (ESI-POS):[M-H] = 371. Anal.Calc. for Cι8Hι6N2O5S: C, 58.06, H, 4.33, N, 7.52. Found: C, 57.90, H, 4.34, N, 5.20. Example 26
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4- methoxyphenyl)ethyl]amino}-2-oxoethyl)-2-(4-hydroxyphenyl)-4-oxo-l,3-thiazolidin-3- yl] benzamide.
[(2S*,5R*)-3-[3-(aminocarbonyl) ρhenyl]-2-(4-(hydroxyρhenyl)-4-oxo-l,3- thiazolidin-5-yl] acetic acid (650 mg, 1.8 mmol) was dissolved in DMF (15 mL) with DIEA (270 mg, 2.1 mmol) and 3-ethoxy-4-methoxyphenethylamine (270 mg, 2.1 mmol). HATU (811 mg, 2.1 mmol) was added and the solution was allowed to stir at room temperature for 15 hours. The DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer was washed twice with 3N HCI. The organic layer was washed again with brine and dried using MgSO4. The solvent was removed under vacuum, yielding the crude product as a yellow oil. The title product (317 mg, 32%) was purified using silica gel flash chromatography (3% methanol: DCM). 1H NMR (DMSO-de): δ 8.15 (m, IH), 7.92 (bs, IH), 7.72 (s, IH), 7.62 (d, IH, 8Hz), 7.30-7.42 (m, 3H), 7.23 (d, 2H, 9Hz), 6.70-6.82 (m, 3H), 6.69 (d, IH, 8Hz), 6.35 (s, IH), 4.44 (dd, IH, 9Hz, 3Hz), 3.99 (q, 2H, 8 Hz), 3.64 (s, 3H), 3.26 (m, 2H), 3.15 (dd, IH, 18Hz, 4Hz), 2.80 (dd, IH, 18Hz, 9Hz), 2.62 (m, 2H), 1.32 (t, 3H, 8 Hz). MS (ESI-NEG):[M-H] = 550. Anal. Calc. for C29H3ιN3O6S: C, 63.37, H, 5.68, N, 7.64. Found: C, 61.57, H, 6.19, N, 6.87. Example 27 Preparation of 3-[(2S*,5R*)-5-(2-{[2-(3-ethoxy-4- methoxyphenyl)ethyl]amino}-2-oxoethyl)-2-(4-methoxyphenyl)-4-oxo-l,3-thiazolidin-3- yl] benzamide.
3-[(2S*,5R*)-5-(2-{[2-(3-ethoxy-4-methoxyphenyl) ethyl]amino}-2- oxoethyl)-2-(4-hydroxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide (50 mg, .09 mmol) was dissolved in DMF (1 mL) with methyl iodide (28 mg, .2 mmol). Potassium carbonate (28 mg, .2 mmol, dissolved in .5 mL of water) was added. The reaction mixture was allowed to stir under nitrogen for 24 hours. The DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer dried using magnesium sulfate. The solvent was removed under vacuum. The crude material was purified using silica gel flash chromotography (3% methanol:DCM), yielding the title compound (32 mg, 60%) as a yellow oil. 1H NMR (DMSO-d6): δ 8.15 (m, IH), 7.92 (bs, IH), 7.72 (s, IH), 7.62 (d, IH, 8Hz), 7.30-7.42 (m, 3H), 7.23 (d, 2H, 9Hz), 6.70-6.82 (m, 3H), 6.69 (d, IH, 8Hz), 6.35 (s, IH), 4.44 (dd, IH, 9Hz, 3Hz), 3.99 (q, 2H, 8 Hz), 3.64 (s, 3H), 3.60 (s, 3H), 3.15 (dd, IH, 18Hz, 4Hz), 2.80 (dd, IH, 18Hz, 9Hz), 2.62 (m, 2H), 1.32 (t, 3H, 8 Hz). MS (ESI- POS): [M+H]+= 564.
The above procedure was used, varying the alkylating agents, to make the following examples: Example 28
Preparation of 3-[(2,5-cis)-5-(2- {[2-(3-ethoxy-4-methoxyphenyl) ethyl] amino}-2-oxoethyl)-2-(4-ethoxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide.
From ethyl 'iodide (71%): MS (ESI-POS): [M+H]+= 578 ' Example 29
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4-methoxyphenyl) ethyl] amino}-2-oxoethyl)-2-(4-allyloxyphenyl)-4-oxo-l, 3-thiazolidin-3-yl] benzamide.
From allyl bromide (47%): MS (ESI-POS): [M+H]+= 590 Example 30
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4-methoxyρhenyl) ethyl] amino}-2-oxoethyl)-2-(4-isopropoxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide.
From isopropyl iodide (38%): MS (ESI-POS): [M+H]+= 592 Example 31 Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4-methoxyphenyl) ethyl] amino}-2-oxoethyl)-2-(4-(2,2,2-trifluoroethoxy) phenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide.
From 2,2,2-trifluoroethyl Inflate (22%): MS (ESI-POS): [M+H]+= 632 Example 32
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4-methoxyρhenyl) ethyl]amino}-2-oxoethyl)-2-(4-prop-2-ynloxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] benza mide.
From propynyl bromide (48%): MS (ESI-POS): [M+H]+= 588 Example 33
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4-methoxyphenyl) ethyl] amino}-2-oxoethyl)-2-(4-but-2-ynloxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide.
From but-2-ynl bromide (18%): MS (ESI-POS): [M+H]+= 602 Example 34
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4-methoxyphenyl) ethyl] amino}-2-oxoethyl)-2-(4-[(2-methylprop-2-enyl)oxy]phenyl}-4-oxo-l,3-thiazolidin-3-yl] benzamide.
From 2-methyl-prop-2-enyl bromide (23%): MS (ESI-POS): [M+H]+= 604 Example 35
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4-methoxyphenyl) ethyl]amino}-2-oxoethyl)-2-(4-(cyclopropylmethoxy) phenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide.
From cyclopropyl methyl bromide (38%): MS (ESI-POS): [M+H]+= 604 Example 36
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4-methoxyphenyl) ethyl] amino}-2-oxoethyl)-2-(4-butoxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide.
From 2-iodobutane (30%): MS (ESI-POS): [M+H]+= 606 Example 37
Preparation of 3-[(2S*,5R*)-5-(2-{[2-(3-ethoxy-4-methoxyphenyl) ethyl] amino}-2-oxoethyl)-2-(4-isobutoxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide.
From isobutyl iodide (21%): MS (ESI-POS): [M+H]+= 606 Scheme 3. For Examples 38-42, 76-79 a) CH3CN, mole, sieves 4A, 80°C; b) DBU, MeOH, THF, 70°C; c) RNH2, HATU, DIAE,
DMF.
Example 38
Preparation of {(2S*,5S*)-3-[3-(aminocarbonyl) phenyl]-2-[4- propoxyphenyl]-4-oxo-l, 3-thiazolidin-5-yl} acetic acid.
A round-bottomed flask was fitted with a bump trap containing molecular sieves (4A). 3-aminobenzamide (1.09 g, 8 mmol) and 4-propoxy-benzaldehyde (1.57 g, 9.6 mmol) were allowed to stir in acetonitrile (60 mL) at 80°C for 1 hour. Mercaptosuccinic acid (3.6 g, 24 mmol) was added to the reaction mixture. The solution was allowed to stir for 24 hours. The suspension was cooled in an ice-water bath for 1 hour and filtered, yielding the title compound (1.90 g, 59%) as a white solid. 1H NMR (DMSO-d6): δ 12.46 (bs, IH), 7.98 (b, IH), 7.80 (s, IH), 7.64 (d, IH, 8Hz), 7.30-7.42 (m, 5H), 6.87 (d, IH, 8Hz), 6.45 (s,lH), 4.50 (dd, IH, 9Hz, 3Hz), 3.85 (t, 2H, 6, Hz), 3.15 (dd, IH, 18Hz, 4Hz), 2.80 (dd, IH, 18Hz, 9Hz), 1.75 (q, 2H, 6Hz), .92 (t, 3H, 6Hz). MS (FI- NEG): M-H]-= 413. Anal.Calc. for C2]H22N2O5S: C, 60.85, H, 5.35, N, 6.76. Found: C, 60.49, H, 5.31, N, 6.51. Example 39
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4-methoxyphenyl)ethyl]- amino}-2-oxoethyl)-2-(4-propόxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide.
{(2S*,5S*)-3-[3-(aminocarbonyl) phenyl] -2- [4-ρroρoxyphenyl]-4-oxo- 1 ,3- thiazolidin-5-yl} acetic acid (500 mg, 1.2 mmol) was dissolved in methanol (20 mL) and THF (20 mL) at 80°C. DBU (2.27 g, 15 mmol) was added, and the solution was allowed to stir for 15 hours. Analytical HPLC (isocratic, 45% acetonitrile/water) showed two components in a 65:35 ratio. The more abundant material proved upon co-injection to be the trans epimer. The solvent was removed under vacuum, and the oil remaining was partitioned between ethyl acetate and 3 N HCI. The ethyl acetate fraction was washed two additional times with acid. The organic layer was separated, washed with brine and dried using MgSO4. The solvent was removed under reduced pressure, yielding a mixture of cis and trans epimers as a white solid. A portion of this crude material (350 mg, .87 mmol) was dissolved in DMF (5 mL) with DIEA (120 mg, 1.0 mmol) and 3-ethoxy-4- methoxyphenethylamine (195 mg, 1.0 mmol). HATU (380 mg, 1.0 mmol) was added and the solution was allowed to stir at room temperature for 15 hours. The DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer was washed twice with 3N HCI. The organic layer was washed again with brine and dried using MgSO4. The solvent was removed under vacuum, yielding the crude products as a yellow oil. The mixture was purified using preparative HPLC (40 acetonitrile/water (.1% TFA)) to yield the cis title product (110 mg, 22%): 1H NMR (DMSO-d6): δ 8.15 (m, IH), 7.92 (bs, IH), 7.72 (s, IH), 7.62 (d, IH, 8Hz), 7.30-7.42 (m, 4H), 6J0-6.82 (m, 4H), 6.39 (s, IH), 5.85 (s, IH), 4.38 (dd, IH, 9Hz, 3Hz), 3.99 (q, 2H, 8 Hz), 3.83 (t, 2H), 3.64 (s, 3H), 3.26 (m, 2H), 3.15 (dd, IH, 18Hz, 4Hz), 2.80 (dd, IH, 18 Hz, 9Hz), 2.62 (m, 2H), 1.75 (q, 2H, 6Hz), 1.32 (t, 3H, 8 Hz) .92 (t, 3H, 6Hz).. MS (ESI-NEG):[M-H] = 592. Anal.Calc. for C32H37N3O6S: C, 64.95, H, 6.30, N, 7.10. Found: C, 62.56, H, 6.33, N, 6.76. Example 40
Preparation of 3-[(2S*,5R*)-5-(2- {[2-(3-ethoxy-4- methoxyphenyl)ethyl]amino}-2-oxoethyl)-2-(4-propoxyphenyl)-4-oxo-l,3-thiazolidin-3- yl] benzamide.
{(2S*,5S*)-3-[3-(aminocarbonyl) phenyl]-2-[4-proρoxyphenyl]-4-oxo- 1, 3- thiazolidin-5-yl} acetic acid (500 mg, 1.2 mmol) was dissolved in methanol (20 mL) and THF (20 mL) at 80°C. DBU (2.27 g, 15 mmol) was added, and the solution was allowed to stir for 15 hours. Analytical HPLC (isocratic, 45% acetonitrile/water) showed two components in a 65:35 ratio. The more abundant material proved upon co-injection to be the trans epimer. The solvent was removed under vacuum, and the oil remaining was partitioned between ethyl acetate and 3 N HCI. The ethyl acetate fraction was washed two additional times with acid. The organic layer was separated, washed with brine and dried using MgSO . The solvent was removed under reduced pressure, yielding a mixture of cis and trans epimers as a white solid. A portion of this crude material (350 mg, .87 mmol) was dissolved in DMF (5 mL) with DIEA (120 mg, 1.0 mmol) and 3-ethoxy-4- methoxyphenethylamine (195 mg, 1.0 mmol). HATU (380 mg, 1.0 mmol) was added and the solution was allowed to stir at room temperature for 15 hours. The DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer was washed twice with 3N HCI. The organic layer was washed again with brine and dried using MgSO4. The solvent was removed under vacuum, yielding the crude products as a yellow oil. The mixture was purified using preparative HPLC (40 acetonitrile/water (.1% TFA)) to yield the trans title product (180 mg, 40%): Η NMR (DMSO-d6): δ 8.15 (m, IH), 7.92 (bs, IH), 7.72 (s, IH), 7.62 (d, IH, 8Hz), 7.30-7.42 (m, 4H), 6J0-6.82 (m, 4H), 6.39 (s, IH), 5.88 (s, IH), 4.45 (m, IH), 3.99 (q, 2H, 8 Hz), 3.83 (t, 2H), 3.64 (s, 3H), 3.26 (m, 2H), 3.15 (dd, IH, 18Hz, 4Hz), 2.80 (dd, IH, 18Hz, 9Hz), 2.62 (m, 2H), 1.75 (q, 2H, 6Hz), 1.32 (t, 3H, 8 Hz) 0.92 (t, 3H, 6Hz). MS (ESI-NEG): [M-H] = 592. Anal.Calc. for C32H37N3O6S: C, 64.95, H, 6.30, N, 7.10. Found: C, 63.82, H, 6.21, N, 7.06. Example 41
Preparation of {(2S*,5S*)-3-[5-(aminocarbonyl) pyridin-2-yl]-2-[4- (benzyloxy) phenyl]-4-oxo-l,3-thiazolidin-5-yl} acetic acid.
A round-bottomed flask was fitted with a bump trap containing molecular sieves (8g, 4A). 6-Aminonicotimide (l.OOg, 7.3 mmol) and 4-benzyloxybenzaldehyde (1.87g, 8.8 mmol). were allowed to stir in acetonitrile (60 mL) at 80°C for 1 hour. Mercaptosuccinic acid (3.3g, 22 mmol) was added to the reaction mixture. The solution was allowed to stir for 15 hours. The precipitate was filtered, yielding the predominantly cis title product (2.44g, 72%) as a white solid. 1H NMR (DMSO-d6): δ 8.85 (m, \Η), 8.29 (dd, IH, 8 Hz, 2Hz), 8.15 (m, IH), 8.03 (bs, IH), 7.89 (d, IH, 8 Hz), 7.58 (bs, IH), 7.25- 7.42 (m, 4H), 6.70-6.98 (m, 4H), 5.01 (s, 2H), 4.44 (dd, IH, 9 Hz, 3Hz), 3.64 (s, 3H), 3.15 (dd, IH, 18 Hz, 4Hz), 2.80 (dd, IH, 18 Hz, 9Hz). MS (ESI-POS): [M-H] = 462. Anal.Calc.for C35H36N4O6S: C, 65.61, H, 5.66, N, 8.74. Found: C, 59.29, H, 5.13, N, 7.79. Example 42
Preparation of 6-[(2S*,5S*ns)-2-[4-(benzyloxy)phenyl]-5-(2-{[2-(3- ethoxy-4-methoxyphenyl)ethyl] amino)-2-oxoethyl)-4-oxo-l,3-thiazolidin-3-yl} nicotinamide.
{(2S*,5S*)-3-[5-(aminocarbonyl) ρyridin-2-yl]-2-[4-(benzyloxy) phenyl]- 4-oxo-l, 3-thiazolidin-5-yl} acetic acid (1.0 g, 2.2 mmol) was dissolved in DMF (20 mL) with HATU (1.25 g, 3.3 mmol), 3-methoxy-4-ethoxy-phenethylamine (644 mg, 3.3 mmol) and DIEA (430 mg, 3.3 mmol). The mixture was allowed to stir for 15 hours under nitrogen, when it was found to be complete by tic. The DMF solution was diluted with ethyl acetate, washed twice with brine and twice with 3N HCI solution. The organic layer . was filtered, yielding the title product (560 mg, 40%) as a white solid. 1H NMR (DMSO- d6): δ 8.85 (m, IH), 8.29 (dd, IH, dd, 8 Hz, 2 Hz), 8.15 (m, IH), 8.03 (bs, IH), 7.89 (d, IH, 8 Hz), 7.58 (bs, IH), 7.25-7.42 (m, 6H), 6.70-6.98 (m, 5H), 5.01 (s, 2H), 4.44 (dd, IH, 9 Hz, 3Hz), 3.99 (q, 2H, 8 Hz), 3.64 (s, 3H), 3.60 (s, 3H), 3.26 (m, 2H), 3.15 (dd, IH, 18Hz, 4Hz), 2.80 (dd, IH, 18 Hz, 9Hz), 2.62 (m, 2H), 1.32 (t, 3H, 8 Hz). MS (ESI-POS): [M+H]+= 564. Anal.Calc. for C35H36N4O6S: C, 65.61, H, 5.66, N, 8.74. Found: C, 59.29, H, 5.13, N, 1.19. Example 43
Preparation of N-[2-(3-ethoxy-4-methoxyphenyl)ethyl]-2,2,2,- trifluoroacetamide.
3-Methoxy-4-ethoxy-phenethylamine (10.0 g, 51 mmol) was dissolved in toluene (200mL) with DIEA (7.1 g, 55 mmol). This solution was treated with trifluoroacetic acid anhydride (12.9 g, 55 mmol). The solution was allowed to stir at room temperature for 2 hours. The solvent was removed under vacuum, and the residue was partitioned between ethyl acetate and water. The organic layer was washed three times with 3N HCI. It was dried with brine and magnesium sulfate. The solvent was removed by rotary evaporation to yield the title compound (11.6 g, 78%) as a yellow solid. 1H NMR (DMSO-d6): δ 9.42 (m, IH), 6.83 (d, IH, 7 Hz), 6.78 (d, IH, 1 Hz), 6.65 (dd, IH, 7 Hz, 1Hz), 3.95 (q, 2H, 8 Hz), 3J2 (s, 3H), 3.38 (q, 2H, 6 Hz), 2.72 (t, 3H, 6 Hz), 1.34 (t, 3H, 8 Hz). MS (ESI-POS): [M+H]+ = 292. Anal.Calc. for Cι3Hι6F3NO3: C, 53.61, H, 5.54, N, 4.81. Found: C, 51.90, H, 5.05, N, 4.54. Example 44
Preparation of N-[2-(5-ethoxy-2-iodo-4-methoxyphenyl)ethyl]-2,2,2,- trifluroacetamide.
N-[2-(3-ethoxy-4-methoxyphenyl)ethyl]-2,2,2,-trifluoroacetamide (500 mg, 1.7 mmol) was dissolved in MeOH (16 mL). Iodine (765 mg, 3.0 mmol) was added. This solution was treated with HIO3 (264 mg, 1.5 mmol, dissolved inl7 mL of water). The reaction mixture was protected from light, and it was allowed to stir under nitrogen for 20 hours. The reaction mixture was partitioned between ethyl acetate and a weak sodium bisulfite solution. The ethyl acetate layer was washed one additional time with bisulfite, and then it was washed with bicarbonate. It was dried with brine and magnesium sulfate, and concentrated under vacuum yielding the title product (575 mg, 81%).1H NMR (DMSO-d6): δ 9.48 (m, IH), 7.24 (s, IH), 6.88 (s, IH), 3.99 (q, 2H, 8 Hz), 3.72 (s, 3H), 3.38 (q, 2H, 6 Hz), 2.72 (t, 3H, 6 Hz), 1.34 (t, 3H, 8 Hz). MS (ESI-NEG):[M-H] = 416. Anal.Calc. for Cι3Hι5F3INO3: C, 37.43, H, 3.62, N, 3.18. Found: C, 36.76, H, 3.43, N, 3.18. Example 45
Preparation of 2-(5-ethoxy-2-iodo-4-methoxy-phenyl)-ethylamine.
N-[2-(5-Ethoxy-2-iodo-4-methoxyphenyl)ethyl]-2,2,2,-trifluroacetamide (2.0 g, 4.8 mmol) was stirred in MeOH (60 mL) and water (20 mL). Lithium hydroxide monohydrate (1.05 g, 25 mmol) was added and the mixture was heated to 60°C and allowed to stir for 1 hour. The reaction mixture was diluted with ethyl acetate, and washed three times with water. It was dried using brine and magnesium sulfate, and concentrated under vacuum to yield the title compound (1.4 g, 89%). 1H NMR (DMSO-d6): δ 7.27 (s, IH), 6.92 (s, IH), 3.99 (q, 2H, 8 Hz), 3.72 (s, 3H), 3.38 (q, 2H, 6 Hz), 2.72 (t, 3H, 6 Hz), 1.34 (t, 3H, 8 Hz). MS (ESI-POS) :[M+H]+= 322. Anal.Calc. for CnHι6INO2: C, 41.14, H, 5.02, N, 4.36. Found: C, 40.36, H, 4.75, N, 3.75. Example 46
. Preparation of 3-[(2S*,5R*)-5-(2-{[2-(5-ethoxy-2-iodo-4- methoxyphenyl)ethyl]amino}-2-oxoethyl)-2-(4-hydroxyphenyl)-4-oxo-l,3-thiazolidin-3- yl] benzamide.
[(2S*,5R*)-3-[3-(Aminocarbonyl) phenyl]-2-(4-(hydroxyphenyl)-4-oxo- l,3-thiazolidin-5-yl] acetic acid (240 mg, .65 mmol) was dissolved in DMF (6 mL) with HATU (296 mg, .78 mmol), 2-(5-ethoxy-2-iodo-4-methoxy-phenyl)-ethylamine (250 mg, .78 mmol) and DIEA (100 mg, .78 mmol).The solution was allowed to stir for 2 hours. The DMF solution was diluted with ethyl acetate, washed twice with brine, and twice with 3N HCI. The ethyl acetate was dried over MgSO4, and concentrated by rotary evaporation. This crude was purified using silica gel column chromatography (6% MeOH/DCM) to yield the product (1 lOmg, 25%) as yellow solid. 1H NMR (DMSO-d6): δ 9.54 (s, IH), 8.15 (m, IH), 7.92 (bs, IH), 7.72 (s, IH), 7.62 (d, IH, 8 Hz), 7.30-7.42 (m, 3H), 7.25 (d, IH, 9 Hz), 6.70-6.82 (m, 3H), 6.69 (d, IH, 8 Hz), 6.35 (s, IH), 4.44 (dd, IH, 9 Hz, 3 Hz), 3.89 (q, 2H, 8 Hz), 3.64 (s, 3H), 3.26 (m, 2H), 3.15 (dd, IH, 18 Hz, 4 Hz), 2.80 (dd, IH, 18 Hz, 9 Hz), 2.62 (m, 2H),1.32 (t, 3H, 8 Hz). MS (ESI-POS) :[M+H]+ = 676. Anal.Calc. for C29H30IN3O6S: C, 63.37, H, 5.68, N, 7.64. Found: C, 61.57, H, 6.19, N, 6.87. Example 47
Preparation of 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2- {[2-(5-ethoxy- 2-iodo-4-methoxyphenyl) ethyl]amino}-2-oxoethyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide.
3-[(2S*,5R*)-5-(2-{[2-(5-Ethoxy-2-iodo-4-methoxyρhenyl)ethyl]amino}- 2-oxoethyl)-2-(4-hydroxyphenyl)-4-oxo-l,3-thiazolidin-3-yl] benzamide (63 mg, .090 mmol) was dissolved in DMF (1.5 mL) with benzyl bromide (34 mg, .2 mmol). Potassium carbonate (28 mg, .2 mmol, dissolved in .5 mL of water) was added. The reaction mixture was allowed to stir under nitrogen for 24 hours. The DMF solution was partitioned between ethyl acetate and brine twice, and the organic layer dried using magnesium sulfate. The solvent was removed under vacuum. The crude material was purified using silica gel flash chromotography (4 % methanol:DCM), yielding the title compound (20 mg, 28%) as a yellow oil. 1H NMR (DMSO-d6): δ 8.15 (m, IH), 7.92 (bs, IH), 7.72 (s, IH), 7.66 (d, IH, 8 Hz), 7.30-7.56 (m, 10H), 7.25 (s, IH), 6.80-6.92 (m, 3H), 6.69 (s, IH), 6.35 (s, IH), 5.01 (s, 2H), 4.44 (m, IH), 3.95 (m, 2H), 3J4 (s, 3H), 3.26 (m, 2H), 3.15 (dd, IH, 18 Hz, 4 Hz), 2.80 (dd, IH, 18 Hz, 9 Hz), 2.62 (m, 2H),1.32 (t, 3H, 8 Hz). MS (ESI- POS):[M+H]+= 766. Anal.Calc. for C36H36IN3O6S: C, 56.47, H, 4.74, N, 5.49. Found: C, 55.89, H, 4.95, N, 5.21. Scheme 4. For Examples 48-50, 61-63, 68-75.
CONH2 R,
&3XX
Λ - " o
A CH2Ph 0 a) CH3CN, mole, sieves 4A, 80°C; b) BOP, DIEA, THF for 6 hrs then reduced with 1.15 eq NaBH4; c) LiCl, LiTMS2, RX, -78°C to -40°C; d) NH4OH, CH3OH; e) Jones oxidation, 0°C; f) RNH2, HATU, DIAE, DMF, g) MCPBA, NMP, 60°C.
Example 48
Preparation of [(2S*,5S*)-3-[3-(methoxycarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-4-oxo-l,3-thiazolidin-5-yl]acetic acid.
At room temperature under N2, methyl 3-aminobenzoate (18.12 g, 119.865 mmol), 4-benzyloxybenzaldehyde (25.44 g, 119.865 mmol), and mercaptosuccinic acid (27.00 g, 179.798 mmol) in acetonitrile (200 ml) was heated at reflux for 4 days. The resulting brown solution was concentrated in vacuuo. The brown syrup was partitioned between water and CH2C1 (400 ml, ea), extraction, separation, drying over MgSO4 and concentration in vacuuo to a brown syrup. SiO gravitational chromatography elution with Hexane : EtOAc (3:1, 2L), (2:1, 2L), (1:1, 4L), (1:1.5, 2L) afforded an orange powder (48.57 g, 85% yield). 1H NMR (DMSO-d6) δ 2.93 (dd, J = 8.37, 8.38 Hz, IH), 3.04 (dd, J = 17.26, 3.95 Hz, IH), 3.83 (s, 3H), 4.51 (ddd, J = 5.42, 3.94, 1.48 Hz, lH), 5.00 (s, 2H), 6.47 (d, J = 1.48 Hz, IH), 6.90 (d, J = 8 87 Hz, 2H), 7.31 (d, J = 6.90 Hz, IH), 7.34 (m, 6H), 7.43 (t, J = 7.88 Hz, IH), 7.53 (dd, J = 7.89, 0.99 Hz, IH), 7.73 (d, J = 7.89 Hz, IH), 7.94 (t, J = 1.48 Hz, IH), 12.66 (s broad, IH). MS (ESI) [M+H]" 476. Example 49
Preparation of methyl 3-[(2S*,5S*)-2-[4-(benzyloxy)phenyl]-5-(2- hydroxyethyl)-4-oxo-l,3-thiazolidin-3-yl]benzoate.
At room temperature under N2 [(2S*,5S*)-3-[3-(methoxycarbonyl)phenyl]- 2-[4-(benzyloxy)phenyl]-4-oxo-l,3-thiazolidin-5-.yl]acetic acid. (4.90 g, 10.277 mmol) in THF (200 ml), benzo-riazol-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (5.0 g, 11.305 mmol) and diisopropylethylamine (2.33 ml, 13.360 mmol) were added and stirred 6 hours. After the reagents were added, the initial yellow solution turned brown. The brown solution was cooled to 0° C upon which NaBEU (450 mg, 11.819 mmol) was added. Gas evolution persisted for about lA hour, after which the slightly cloudy brown solution with gradual warming to room temperature was stirred for 60 hours. To the resulting dark brown solution, concentration in vacuo to a brown syrup. Partition between EtOAc and cold 2N HClaq (250 ml ea) extraction, separation. Extraction of the aqueous layer with EtOAc (2 X 100 ml). All organic layers combined extraction with cold saturated NaHCO3 aq (200 ml), water (150 ml), brine (100 ml), dried over MgSO , filtered, and concentration in vacuo to a brown syrup. Biotage SiO2 chromatography (40M cartage) , 3:1 / Hex:EtOAc (1 L), 2:1 / Hex:EtOAc (3 L) afforded the title compound as a light brown powder (2.81 g , 63% yield). 1H NMR (DMSO-d6) δ 1.17 (m, IH), 2.22 (m, IH), 3.53 (m, IH), 3.64 (m, IH), 3.83 (s, 3H), 4.32 (dd, J = 4.02, 0.72 Hz, IH), 4.72 (t, J = 5.13 Hz, IH), 5.01 (s, 2H), 6.55 (s, IH), 6.88 (q,d, J = 8.79, 1.83 Hz, 2H), 7.30 (m, 7H), 7.43 (t, J = 8.05 Hz, IH), 7.55 (dd, J = 6.95, 1.10 Hz, IH), 7.12 (d, J = 8.06 Hz, IH), 7.95 (t, J = 2.20 Hz, IH). MS (ESI) [M+H]+ 464. Anal. RP-HPLC 75%. Example 50
Preparation of 3-[(2S*,5S*)-2-[4-(benzyloxy)ρhenyl]-5-(2-hydroxyethyl)- 4-oxo-l,3-thiazolidin-3-yl]benzamide.
Prepared according to the procedure in the above example starting from {(2S*,5S*)-3-[3-(aminocarbonyl) phenyl]-2-[4-(benzyloxy) phenyl]-4-oxo-l,3- thiazolidin-5-yl} acetic acid (5.57 g, 11.664 mmol) afforded the title compound as a brown powder (1.78 g, 35% Yield). MS (ESI) [M+H]+ 449. Scheme 5. For Examples 51-60, 64-67.
a) NH4OH, CH3OH; b) R'SO2Cl, pyridine, 0°C; c) R"NH2, benzene, reflux; d) Jones oxidition, 0°C; e) R,7NH2, NaBH(OAc)3, HOAc, CH2C12, rt; f) AcCl or CH3SO2Cl, Cs2CO2, DMF, rt; g) (R"S)2, NaBH4, DMF, 55°C; h) H2O2, H2O, HOAc, 0°C for n = 1, H2O2, acetone, 45°C for n = 2.
Example 51
Preparation of 2-{(2S*,5S*)-3-[3-(aminocarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-4-oxo-l ,3-thiazolidin-5-yl} ethyl 4-methylbenzenesulfonate.
3-[(2S*,5S*)-2-[4-(Benzyloxy)phenyl]-5-(2-hydroxyethyl)-4-oxo-l,3- thiazolidin-3-yl]benzamide (500 mg, 1.1 mmol) in pyridine (20 ml) was cooled to 0° C/N2 and treated with tosyl chloride (381 mg, 2 mmol). The resulting solution was stirred at rt/N for 18 hours. The solvent was evaporated at 40° under reduced pressure, the residue was treated with water (25- ml) and acidified with aqueous 2N HCI. The acidic solution was extracted with ethyl acetate (5 x 40 ml). The ethyl acetate solution was washed with water, dried with MgSO4 and evaporated. Chromatography of the crude product on silica gel and elution with 2 % methanol in CH2C12 afforded 175 mg (26 % yield) of the title compound as a white solid; 1HNMR (DMSO-d6) δ 8.0-6.8 (m, 17 H), 6.44 (s, 1 H), 5.00 (s, 2H), 4.20 (m, 3H), 2.42 ( s, 3H), 2.40, 2.13 (mm, 2H); MS (ES-positive): [M+H]+ 603. Example 52
Preparation of 2-{(2S*,5S*)-3-[3-(amino-carbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-4-oxo-l,3-thiazolidin-5-yl}ethyl methane-sulfonate. Using the same procedure as in the above example except using 690 mg 3- [(2S*,5S*)-2-[4-(benzyloxy)ρhenyl]-5-(2-hydroxyethyl)-4-oxo-l,3-thiazolidin-3- yl]benzamide, 408 mg methanesulfonyl chloride, 15 ml pyridine and all the other reagents scaled to this proportion afforded 500 mg (63 % yield) of the title compound. MS (ES- positive): [M+H 527. Example 53
Preparation of 3-[(2S*,5S*)-2-[4-(benzyloxy)phenyl]-5-(2- {[2-(3-ethoxy-4- methoxy-phenyl)ethyl]amino}ethyl)-4-oxo-l,3-thiazolidin-3-yl]benzamide.
At rt/N2, {(2S*,5S*)-3-[3-(aminocarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-4-oxo-l,3-thiazolidin-5-yl}ethyl 4-methylbenzenesulfonate (410 mg,0.68 mmol) was dissolved in benzene (10 ml) and treated with 3-ethoxy-4- methoxyphenethylamine (288 mg, 2.72 mmol). After refluxing under N2 for 9 hours, the solution was evaporated and the residue was further dried at 70° C in vacuum for 8 hours. The gummy residue was dissolved in CH2C12 (200 ml), washed with water (2 x 25 ml), dried over anhydrous K2CO3 and evaporated. Chromatography of the crude product on silica gel and elution with 2.5 % methanol in CH2C12 afforded 170 mg (35 % yield) of the title compound as a white foam; 'HNMR: (DMSO-d6) δ 8.0-6.6 (m, 16 H), 6.46 (s, 1 H), 5.01 (s, 2H), 4.34 (m, 1 H), 3.96 (m, 2H), 3.70 (s, 3H), 1.29 (m, 3H); MS (ES-positive): [M+H]+ 626. Example 54
Preparation of 3-[2-[4-(benzyloxy)phenyl]-5-(2-{[2-(3,4- dimethoxyphenyl)ethyl]amino}-ethyl)-4-oxo-1.3-thiazolidin-3-yl]benzamide.
(2-{(2S*,5S*)-3-[3-(aminocarbonyl)phenyl]-2-[4-(benzyloxy)ρhenyl]-4- oxo-l,3-thiazolidin-5-yl} ethyl ethanesulfonate (5.0 g, 9.5 mmol) was suspended in benzene (180 ml) and was treated with 3,4-dimethoxyphenethylamine (5.5 g, 28.5 mmol). The mixture was heated under reflux /N2 for 6.5 hours. Benzene was removed by evaporation, the residue was diluted with CH2C12 (900 ml), washed with water (7 x 120 ml), dried and evaporated to give an oil (8.5 g). This oil was dissolved in THF (150 ml), treated with l,8-diazabicyclo[5.4.0]undec-7-ene (20 ml, 118 mmol) and heated at reflux/N2 for 20 hours. The reaction mixture was diluted with CH2C12 (1 1), washed with water (10 x 120 ml), dried with MgSO4 and evaporated. Chromatography of the crude product on silica gel and elution with 4.5 % methanol in CH2C1 afforded 1.7 g (30 % yield for the two-step reaction) of the title compound as a pale white solid; 1HNMR (DMSO-d6) δ 7.9-6.6 (m, 16 H), 6.47, 6.46 (ss, IH, cis/trans isomeric ratio about 1/1), 5.01, 4.99 (2s, 2H, cis/trans isomeric ratio about 1/1), 4.34, 4.23 (2m, IH, cis/trans isomeric ratio about 1/1), 3.71, 3.70 (2s, 3H each); MS (ES-positive): [M+H]+ 612; Anal. Calc. For C35H37N3O5S 1/2 H2O: C.67.71. H,6.17. N.6.76. Found: Q67.40. H,6.454. N,6.75.; Analytical HPLC determined that this compound 99 % purity consisted cis/trans isomeric ratio 46/54.
This compound (0.9 g, 1.45 mmol) was dissolved in a solution of ethyl acetate (10 ml)/ethyl ether (2 ml) and cooled to 0° C/ N2. To the solution 1 M HCl(gas) in ether (1.5 ml) was added at 0° C/N2 and a crystalline material was formed immediately. The resulting suspension was stirred at 0° C/N2 for 2 hours. The crystalline material was collected by filtration, dried at in vacuum to afford 0.89 g (90 % yield) of the hydrochloride salt of the title compound as a pale white form; 1HNMR (DMSO-d6) δ 8.0- 6.1 (m, 16 H), 6.56, 6.53 (2s, IH, cis/trans isomeric ratio about 1/1), 5.01, 5.00 (2s, 2H, cis/trans isomeric ratio about 1/1), 4.51, 4.38 (mm, IH, cis/trans isomeric ratio about 1/1), 3.75, 3.72 (2s, 3H each); MS (ES-positive): [M+H]+ 612; Analytical HPLC determined that this compound, 99 % purity consisted cis/trans isomeric ratio 46/54. Example 55
Preparation of 3-[2-[4-(benzyloxy)phenyl]-5-(2- {[2-(4-ethoxy-3- methoxyphenyl)ethyl]amino}ethyl)-4-oxo-l,3-thiazolidin-3-yl]benzamide.
Using the procedure of the above example except using 700 mg (2- {(2S*,5S*)-3-[3-(aminocarbonyl)phenyl]-2-[4-(benzyloxy)phenyl]-4-oxo-l,3-thiazolidin- 5-yl} ethyl methahesulfonate), 780 mg 4-ethoxy-3-methoxy-phenethylamine, and 25 ml of benzene afforded 170 mg (30 % yield) of the title compound; MS (ES- positive): [M+H]+ 626. Example 56
Preparation of 3-{5-(2-{acetyl [2-(3,4-dimethoxyρhenyl)ethyl]- amino)ethyl)-2-[4-(benzyloxy)phenyl]-4-oxo-l,3-thiazolindin-3-yl)benzamide.
At rt/N2 [2-[4-(benzyloxy)phenyl]-5-(2- {[2-(3,4-dimethoxyphenyl)ethyl]- amino}ethyl)-4-oxo-1.3-thiazolidin-3-yl]benzamide (100 mg, 0.16 mmol) in DMF (2 ml) was treated with cesium carbonate (212 mg, 0.64 mmol). Acetyl chloride (50 mg, 0.64 mmol) in DMF (0.5 ml) was added and the suspension was stirred at rt/N2 for 40 hours. It was diluted with CH2C12 (200 ml), washed with water, dried with MgSO4 and evaporated. Chromatography of the crude product on silica gel and elution with 2 % methanol in CH2C12 afforded 62 mg (58 % yield) of the title compound as a pale white form; 1HNMR (DMSO-d6): δ 7.9-6.6 (m, 16H), 6.45 (2s, IH, cis/trans isomeric ratio about 1/1), 5.00, 4.98 (2s, 2H, cis/trans isomeric ratio about 1/1) 4.35-4.22 (m,lH), 3.70 (s, 6H), 1.86, 1.84 (2s, 3 H).; MS (ES-positive): [M+H]+ 654; Analytical HPLC determined that this compound 96 % purity consisted cis/trans isomeric ratio 46/50. Example 57
Preparation of 3-(2-[4-(benzyloxy)phenyl]-5- {2-[[2-(3,4- dimethoxyphenyl)ethyl]-(methylsulfonyl)amino]ethyl}-4-oxo-l,3-thiazolindin-3- yl)benzamide.
At rt/N2 3-[2-[4-(benzyloxy)phenyl]-5-(2-{[2-(3,4- dimethoxyphenyl)ethyl]amino}ethyl)-4-oxo-1.3-thiazolidin-3-yl]benzamide (100 mg, 0.16 mmol) in DMF (2 ml) was treated with cesium carbonate (212 mg, 0.64 mmol). To the suspension at 0° C/N2 methanesulfonyl chloride (73 mg, 0.64 mmol) in DMF (0.5 ml) was added and stirred at rt/N2 for 20 hours. It was diluted with CH2C12 (200 ml), washed with water, dried with MgSO4 and evaporated. Chromatography of the crude product on silica gel and elution with 2.5 % methanol in CH2C12 afforded the title compound 56 mg (50 % yield) as a pale white form; 1HNMR (DMSO-d6): δ 8.0-6J (m, 16H), 6.50, 6.47 (2s, IH, cis/trans isomeric ratio about 1/1), 5.00, 4.98 (2s, 2H, cis/trans isomeric ratio about 1/1) 4.30, 4.27 ( mm,lH, cis/trans isomeric ratio about 1/1), 3J3, 3J2, 3.71, (3s, 3H each); MS (ES-positive): [M+H]+ 690; Analytical HPLC determined that this compound 99 % purity consisted cis/trans isomeric ratio 48/52. Example 58
Preparation of 3-[2-[4-(benzyloxy)phenyl]-5-(2-{[2-(3,4- dimethoxyphenyl)ethyl] thio} ethyl)-4-oxo- 1 ,3-thiazolidin-3-yl]benzamide.
At room temperature under N2, 3,4-dimethoxyphenethyl alcohol (2.00g, 10.976 mmol) in CHC13 (30 ml) was added pyridine (1.74g, 21.951 mmol) and methanesulfonyl chloride (1.63g, 14.269 mmol). The resulting yellow solution was stirred at room temperature for 63 hrs. The yellow solution was partitioned with 2N HCI aq (3 X 50 ml), extraction, separation, drying over MgSO , filtration and concentration in vacuuo provided, as a pale syrup, 3,4-dimethoxyphenethyl methanesulfonate (2.46g, 86% yield). 1H NMR (DMSO-d6) δ 2.90 (t, J = 6.90 Hz, 2H), 3.11 (s, 3H), 3.72 (s, 3H), 3.75 (s, 3H), 4.36 (t, J = 6.90 Hz, 2H), 6.78 (dd, J = 8.13, 1.78 Hz, IH), 6.87 (d, J = 8.13 Hz, IH), 6.91 (d, J = 1.78 Hz, IH). MS (ESI) [M+Naf 283.
At room temperature under N2, a solution of this compound (1.78g, 6.838 mmol) in DMF (25 ml) was degassed three times in vacuuo and vacuum replaced with N2. Upon which, sodium hydrosulfide mono-hydrate (460 mg, 8.206 mmol) was added producing a blue mixture. The blue mixture was stirred at room temperature for 63 hrs. The resulting white mixture was quenched with 2N HCI aq (20 ml). Partition between water (20 ml) and EtOAc (30 ml), extraction, separation. Extraction of the organic layer with water (3 x 15 ml), brine (15 ml), dried over MgSO4, filtration and concentration in vacuuo to a brown syrup. Gravitional SiO2 chromatography (6:1 / Hex: EtOAc) afforded a yellow syrup (780 mg, 55% yield) as 3,4-dimethoxyphenethyl thiol. 1H NMR (DMSO-d6) δ 1.55 (t, J = 7.65 Hz, IH), 2.70 (t, J = 7.65 Hz, 2H), 2.71 (t, J = 7.65 Hz, 2H), 3.75 (s, 3H), 3.77 (s, 3H), 6.71 (dd, J = 8.13, 1.78 Hz, IH), 6.80 (d, J = 8.13 Hz, IH), 6.83 (d, J = 1.78 Hz, IH). MS (ESI) [M+dimer-2H]+ 394.
At room temperature under N2, a solution of this compound (370 mg, 1.866 mmol) in DMF (10 ml) was degassed three times in vacuuo and the vacuum replaced with N2. To the yellow solution, sodium hydride (81 mg, 2.022 mmol) was added producing an initial gray mixture, which was stirred 30 min that eventually turned into a yellow solution. 2-{3-[3-(Amino-carbonyl)phenyl]-2-[4-(benzyloxy)phenyl]-4-oxo-l,3- thiazolidin-5-yl} ethyl methane-sulfonate (818 mg, 1.555 mmol) in DMF (5 ml) was heated at 55°C upon which sodium 3,4-dimethoxyphenethyl thiol in DMF (10.5 ml) was added dropwise over 20 min period. The resulting brown solution was heated at 55°C for 6 hrs. Cooling of the dark brown solution to room temperature and quenching with 2N HCI aq (20 ml). Partition of the aqueaous solution with EtOAc (20 ml) extraction, separation. Extraction and separation of the aqueous layer with EtOAc (10 ml). All organic layers were combined, extraction with water (2 X 15 ml), brine (15 ml), dried over MgSO4, filtration, and concentration in vacuuo to a brown syrup. Gravitational SiO2 chromatography 4% MeOH : CH2C12 afforded the title compound as a yellow foam (460 mg, 47% yield) of trans 3-[2-[4-(benzyloxy)phenyl]-5-(2-{[2-(3,4-dimethoxyρhenyl)ethyl] thio}ethyl)-4-oxo-l,3-thiazolidin-3-yl]benzamide. 1H NMR (DMSO-d6) δ 2.07 (m, IH), 2.27 (m, IH), 2.67 (m, 6H), 3.69 (s, 3H), 3.70 (s, 3H), 4.40 (dd, J = 7.79, 4.13 Hz, IH), 5.01 (s, 2H), 6.51 (s, IH), 6.71 (dd, J = 8.12, 1.83 Hz, IH), 6.82 (m, 3H), 7.30 (m, 9H), 7.64 (d, J = 7.52 Hz, IH), 7.86 (s, IH), 7.94 (d, J = 3.53 Hz, IH). MS (ESI) [M+H}+ 629. This compound was epimerized using DBU in refluxing THF to afford the title compound as a pale white powder (130 mg, 76% yield). MS (ESI) [M+H]+ 629. Anal. RP-HPLC 90%) purity, 80%) trans isomer, 20% cis isomer. Example 59
Preparation of 3-[2-[4-(benzyloxy)phenyl]-5-(2- {[2-(3,4-dimethoxyphenyl) ethyl]sulfinyl}ethyl)-4-oxo-l,3-thiazolidin-3-yl]benzamide.
At 0°C under N2, 3-[2-[4-(benzyloxy)phenyl]-5-(2-{[2-(3,4- dimethoxyphenyl) ethyl] thio}ethyl)-4-oxo-l,3-thiazolidin-3-yl]benzamide (300 mg, 0.476 mmol) in water (2 ml), acetic acid (5 ml) followed by hydrogen peroxide (30% aqueous solution, 0.5 ml) were added. The resulting yellow solution was stirred at 0°C for 3 hrs. Quenching with DMSO (0.25 ml) and partition between CH2C12 and water ( 50 ml ea), extraction and separation. The organic phase was dried over MgSO4, concentration in vacuuo to a yellow syrup. Gravitational SiO2 chromatography 3% MeOH: CH2C12 (500 ml), 5% MeOH : CH C12 (500 ml) afforded the title compound as a white foam (307 mg, 46% yield). 1H NMR (DMSO-d6) δ 2.14 (m, IH), 2.34 (m, IH), 2.71 (m, 7H), 3.67 (m, IH), 3.71 (s, 3H), 3.74 (s, 3H), 4.43 (m, IH), 5.01 (s, 2H), 6.55 (s, IH), 6.76 (dd, J = 8.10, 1.79 Hz, IH), 6.86 (m, 3H), 7.29 (m, 8H), 7.63 (d, J = 7.48 Hz, IH), 7.86 (s, IH), 7.97 (s broad, 2H). MS (ESI) [M+H]+ 645. This compound was epimerized using DBU in refluxing THF to afford the title compound as a yellow foam (107 mg, 76% yield). MS (ESI) [M+H]+ 645. Anal. RP-HPLC 75% purity, trans isomer 46%, cis isomer 54%. Example 60
Preparation of 3-[2-[4-(benzyloxy)phenyl]-5-(2- {[2-(3,4- dimethoxyphenyl)ethyl] sulfonyl}ethyl)-4-oxo-l,3-thiazolidin-3-yl]benzamide.
At room temperature under N2, 3-[2-[4-(benzyloxy)phenyl]-5-(2-{[2-(3,4- dimethoxyphenyl) ethyl] thio}ethyl)-4-oxo-l,3-thiazolidin-3-yl]benzamide (130 mg, 0.207 mmol) in acetone (15 ml), hydrogen peroxide (30% aqueous solution, 15ml) was added. The resulting yellow solution was heated at 45°C for 48 hrs. Cooling to room temperature and quenching with DMSO (4 ml) followed by partition between water and CH2C12 (50 ml ea). Extraction, separation of the organic layer, drying over MgSO4, filtration and concentration in vacuuo to a brown syrup. Gravitational SiO2 chromatography 4% MeOH: CH2C12 afforded the title compound as a yellow powder (40 mg, 29% yield). 1H NMR (DMSO-d6) δ 2.09 (m, IH), 2.83 (m, 7H), 3.71 (s, 3H), 3.73 (s, 3H), 4.28 (m, IH),
5.09 (s, 2H), 6.58 (s, IH), 6.73 (d, J = 8.17 Hz, IH), 6.85 (m, 2H), 7.06 (d, J = 8.71 Hz, 2H), 7.32 ( , 9H), 7.61 (dd, J = 7.90, 1.47 Hz, IH), 7.72 (d, J = 7.73 Hz, IH), 7.99 (m broad, 2H). MS (ESI) [M+H]" 659. This compound was epimerized using DBU in refluxing THF to afford the title compound as a yellow foam (20.4 mg, 50% yield), cis: trans mixture 20: 80 ratio. MS (ESI) [M+H]+ 659. Anal. RP-HPLC 84%, 85% trans isomer, 8% cis isomer.
Example 61
Preparation of methyl 3-[(2S*,5R*)-2-[4-(benzyloxy)ρhenyl]-5-(2- hydroxy ethyl)-5 -methy 1-4-oxo- 1 ,3 -thiazolidin-3 -yl]benzoate.
To THF (100 ml) at 0°C under N2, n-butyl lithium (48.37 ml, 120.917 mmol, 2.0 M solution in hexane) was added by syringe and stirred 5 min.. To the pale yellow solution was added 1,1,1,3,3,3-hexamethyl disilazane (32.00 g, 115.66 mmol) over 3 min. producing gas evolution. The resulting colorless solution was stirred at 0°C for 25 min. To methyl 3-[(2S*,5S*)-2-[4-(benzyloxy)ρhenyl]-5-(2-hydroxyethyl)-4-oxo-l,3- thiazolidin-3-yl]benzoate (24.27 g, 52.573 mmol), lithium chloride (8.91 g, 210.290 mmol) in THF (350 ml) at -45°C, was added lithium 1,1,1,3,3,3,-hexamethyldisilazane in THF (182 ml solution) at a rate where the reaction temperature was -45°C +/- 3°C (about 4 min.). The resulting dark brown mixture was stirred at -78°C for 3.5 hrs. To the dark brown solution, was added iodomethane (7.18 g, 262.863 mmol) and stirred at -78°C for 2.5 hrs. The resulting dark purple solution was warmed to -40°C upon which was quenched with saturated NH C1 aq (500 ml). The resulting brown solution was partitioned with EtOAc (500 ml), extraction, and separation. The aqueous layer was extracted with EtOAc (500 ml). All organic layers were combined, washed with brine (300 ml), dried over MgSO , filtration and concentration in vacuuo to a dark brown syrup. Biotage Flash- 75 (75-L cartridge) elution schedule: hexane : EtOAc / 3:1 (16L), 2:1 (12L), 1:1 (16L), 0:1 (8L). Collection of the clean fractions afforded a yellow foam (9.71 g, 39% yield) of the title compound and recovered starting material (12.18 g). 1H NMR (DMSO-d6) δ 1.63 (s, 3H), 1.99 (dd, J = 7.53, 2.38 Hz, IH), 2.19 (qd, J = 2.78, 0.50 Hz, IH), 3.65 (m, IH), 3.73 (m, IH), 3.83 (s, 3H), 4.69 (s broad, IH), 4.99 (s, 2H), 6.60 (s, IH), 6.87 (qd, J = 8.73,
1.10 Hz, 2H), 7.31 (m, 7H), 7.41 (t, J = 7.94 Hz, IH), 7.54 (dq, J = 7.93, 1.20 Hz, IH), 7.70 (dt, J = 7.93, 1.20 Hz, IH), 7.90 (t, J = 1.99 Hz, IH). MS (ESI) [M+H]+ 478. Anal. RP-HPLC 92%. Example 62
Preparation of methyl 3-[(2S*,5R*)-5-allyl-2-[4-(benzyloxy)phenyl]-5-(2- hydroxy ethyl)-4-oxo- 1 ,3-thiazolidin-3-yl]benzoate.
Using the procedure of the above example except using allyl bromide as the alkylating agent afforded the title compound as a yellow powder (790 mg, 36% yield). MS (ESI) [M+H]+ 504. Example 63
Preparation of 3-[(2S*,5R*)-2-[4-(benzyloxy)ρhenyl]-5-(2-hydroxyethyl)- 5-methyl-4-oxo- 1 ,3-thiazolidin-3-yl]benzamide.
At room temperature in a coated flask opened to the atmosphere, methyl 3- [(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2-hydroxyethyl)-5-methyl-4-oxo-l,3-thiazolidin- 3-yl]benzoate (1.00 g, 2.094 mmol) in methyl alcohol (15 ml) was added ammonium hydroxide (20 ml, 30% aqueous solution). The resulting brown cloudy mixture was sealed with a teflon screw capped and stirred 4 days. To the yellow solution, concentration in vacuuo to a brown syrup. Biotage SiO2 chromatography (5% MeOH: CH2C12) afforded the title compound as a yellow foam (640 mg, 66% yield). 1H NMR (DMSO-d6) δ 1.62 (s, 3H), 2.00 (qd, J = 8.02, 2.82 Hz, IH), 2.16 (q, J = 8.02 Hz, IH), 3.62 (m, IH), 3.71 (m, IH), 4.66 (t, J = 5.31 Hz, IH), 4.99 (s, 2H), 6.51 (s, 2H), 6.87 (d, J = 8.85 Hz, 2H), 7.30 (m, 7H), 7.40 (dq, J = 7.78, 0.05 Hz, 2H), 7.63 (dt, J = 7.78, 1.41 Hz, IH), 7.80 (t, J = 1.77 Hz, IH), 7.93 (s broad, 2H). MS (ESI) [M+H]+ 463. Anal. RP-HPLC 99% purity. Example 64
Preparation of methanesulfonic acid 2-[(2S*,5R*)-2-(4-benzyloxy-phenyl)- 3-(3-carbamoyl-phenyl)-5-methyl-4-oxo-thiazolidin-5-yl]-ethyl ester.
At 0°C under N2, 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2- hydroxyethyl)-5-methyl-4-oxo-l,3-thiazolidin-3-yl]benzamide (200 mg, 0.433 mmol) in pyridine (5 ml) was added methanesulfonyl chloride ( 148 mg, 1.29 mmol) was added. The resulting red solution was stirred at 0°C for 2 hrs. The red solution was contated in vacuuo to a red syrup. Biotage SiO chromatography 3% MeOH: CH2C12 afforded a yellow powder (130 mg, 56% yield). 1H NMR (DMSO-d6) δ 1.66 (s, 3H), 2.29 (m, IH), 2.45 (m, IH), 2.50 (s, 3H), 4.44 (m, 2H), 5.00 (s, 2H), 6.58 (s, IH), 6.86 (d, J = 8.65 Hz, 2H), 7.32 (m, 6H), 7.44 (m, 2H), 7.63 (d, J = 7.68 Hz, IH), 7.82 (s, IH), 7.89 (s, IH), 8.57 (s broad, 2H). MS (ESI) [M+H]+ 541. Example 65
Preparation of 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2-{[2-(3,4- dimethoxy phenyl)ethyl]amino}ethyl)-5-methyl-4-oxo-l,3-thiazolidin-3-yl]benzamide.
At room temperature under N2, methanesulfonic acid 2-[(2S*,5R*)-2-(4- benzyloxy-phenyl)-3-(3-carbamoyl-phenyl)-5-methyl-4-oxo-thiazolidin-5-yl]-ethyl ester (110 mg, 0.204 mmol) in DMF (10 ml), 3,4-dimethoxy phenethylamine (200 mg, 1.104 mmol) was added. The resulting yellow solution was heated at 75°C for 6 hrs. Concentration in vacuo to a brown syrup. Gravitational SiO2 chromatography 4% MeOH: CH2C12 afforded the title compound as a yellow powder (80 mg, 63% yield). 1H NMR (DMSO-d6) δ 0.24 (s broad, IH), 1.64 (s, 3H), 2.04 (m, IH), 2.18 (m, IH), 2.72 (m, 6H), 3.70 (s, 3H), 3.73 (s, 3H), 4.99 (s, 2H), 6.57 (s, IH), 6.74 (dd, J = 8.11, 1.53 Hz, IH), 6.84 (m, 4H), 7.28 (m, 9H), 7.63 (d, J = 7.56 Hz, IH), 7.86 (s, IH), 7.97 (s broad, 2H). MS (ESI) [M+H]+ 626. Anal. RP-HPLC 89% purity. Example 66
Preparation of 3-[(2S*,5R*)-2-[4-(benzyloxy)ρhenyl]-5-(2-{[2-(3,4- dimethoxy phenyl)ethyl]thio} ethyl)-5-methyl-4-oxo-l ,3-thiazolidin-3-yl]benzamide.
At room temperature under N2, 3,4-dimethoxyphenethane thiol (97 mg, 0.489 mmol) in DMF (4 ml) was added sodium hydride (20 mg, 0.509 mmol) and stirred for 30 min.. At room temperature under N2, methanesulfonic acid 2-[(2S*,5R*)-2-(4- benzyloxy-phenyl)-3-(3-carbamoyl-phenyl)-5-methyl-4-oxo-thiazolidin-5-yl]-ethyl ester (110 mg, 0.205 mmol) in DMF (30 ml) was added the sodium salt of 3,4- - dimethoxyphenethane thiol in DMF (4 ml) and heated at 55°C for 3 hrs. Cooling of the resultant yellow solution to room temperature and quenching with 2N HClaq (50 ml). Partition with CH2C12 (40 ml) extraction, separation, drying over MgSO4, filtration and concentration in vacuo. Biotage SiO2 chromatography 2% MeOH: CH2C12 afforded the title compound as a yellow powder (410 mg, 19% yield). 1H NMR (DMSO-d6) δ 1.63 (s, 3H), 2.00 (m, IH), 2.21 (m, IH), 2.54 (m, IH), 2.73 (m, 5H), 3.69 (s, 3H), 3.70 (s, 3H), 4.98 (s, 2H), 6.56 (s, IH), 6.73 (dd, J = 8.18, 1.47 Hz, IH), 6.83 (t, J = 9.93 Hz, IH), MS (ESI) [M+H]+ 643. Example 67 Preparation of 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2- {[2-(3-ethoxy- 4-methoxy henyl)ethyl]thio}ethyl)-5-methyl-4-oxo-l,3-thiazolidin-3-yl]benzamide.
Using the procedure of the above example and substituting 4-ethoxy-3- methoxyphenethane thiol for 3, 4-dimethoxyphenylethane thiol afforded the title compound as a yellow powder (220 mg, 62% yield). MS (ESI) [M+H]+ 657. Example 68
Preparation of 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2-{[2-(3-ethoxy- 4-methoxy phenyl)ethyl]amino}ethyl)-5-methyl-4-oxo-l,3-thiazolidin-3-yl]benzamide.
At 0 °C under N2, CH C12 (100 ml) was added pyridinium chlorochromate (4.51 g, 20.941 mmol). To the orange mixture was added methyl 3-[(2S*,5R*)-2-[4- (benzyloxy)phenyl]-5-(2-hydroxyethyl)-5-methyl-4-oxo-l,3-thiazolidin-3-yl]benzoate (1.00 g, 2.094) in CH2C12 (60 ml) dropwise over 30 min.. The resulting dark brown mixture was stirred at 0°C 2 hrs. Quenching with water (150 ml), dilution with CH2C12 (100 ml), filtration through celite, extraction and separation. Extraction of the organic layer with water (2 X 150 ml), drying over MgSO4, filtration and concentration in vacuuo to a dark brown syrup. Biotage SiO2 chromatography (40s cartridge) 1:1 / Hex: EtOAc afforded methyl 3-[(2S*,5R*)-2-[4-(benzyloxy) phenyl]-5-(2-aldehylethyl)-5-methyl-4- oxo-l,3-thiazolidin-3-yl]benzoate as a yellow powder (617 mg, 62% yield). 1H NMR (DMSO-d6) δ 1.74 (s, 3H), 3.26 (s, 2H), 3.88 (s, 3H), 5.03 (s, 2H), 6.70 (s, IH), 6.90 (d, J = 8.06, 2H), 7.35 (m, 7H), 7.46 (t, J = 8.06 Hz, IH), 7.62 (d, J = 7.87 Hz, IH), 7.76 (dd, J = 7.63, 0.78 Hz, IH), 7.98 (s, IH), 9.82 (s, IH). MS (ESI) [M+H]+ 476.
At room temperature under N2, a solution of this aldehyde (100 mg, 0.210 mmol) in dichloroethane (5 ml) was added sodium triacetoxyborohydride (67 mg, 0.315 mmol), 3-ethoxy-4-methoxy phenethylamine (45 mg, 0.231 mmol) and glacial acetic acid (12 μL). The resulting yellow mixture was stirred at room temperature for 90 min. Quenching with saturated NaHCO3 aq (10 ml), extraction, separation of the organic layer, dried over MgSO4, filtration and concentration in vacuuo to a brown syrup. Biotage SiO2 (40s) chromatography 3.5% MeOH : CH2C12 afforded methyl 3-[(2S*,5R*)-2-[4- (benzyloxy)phenyl]-5-(2-{[2-(3-ethoxy-4-methoxyphenyl)ethyl]amino}ethyl)-5-methyl-4- oxo-1, 3-thiazolidin-3-yl]benzoate as a brown powder (68 mg, 58% yield). 1H NMR (DMSO-d6) δ 1.26 (t, J = 6.95 Hz, 3H), 1.61 (s, 3H), 1.90 (m, IH), 2.11 (m, IH), 2.63 (m, 6H), 3.35 (s broad, IH), 3.70 (s, 3H), 3.81 (s, 3H), 3.92 (q, J = 6.95 Hz, 2H), 4.98 (s, 2H), 6.59 (s, IH), 6.69 (d, J = 8.09 Hz, IH), 6.80 (m, 4H), 7.18 (m, 8H), 7.54 (d, J = 8.18 Hz, IH), 7.69 (d, J = 7.69 Hz, IH), 7.91 (s, IH). MS (ESI) [M+H]+ 655. This compound (250 mg, 0.382 mmol) was reacted with ammonia in methanol to afford the title compound as a yellow powder (110 mg, 45% yield). MS (ESI) [M+H]+ 640. Anal. RP-HPLC 94% purity. Example 69
Preparation of {(2S*,5R*)-2-[4-(benzyloxy)phenyl]-3-[3- (methoxycarbonyl)phenyl] -5 -methy 1-4-oxo-l ,3-thiazolidin-5-yl} acetic acid.
Methyl 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2-hydroxyethyl)-5- methyl-4-oxo-l,3-thiazolidin-3-yl]benzoate (160 mg, 0.32 mmol) was dissolved in acetone (16 ml) and cooled to -10° C/N2. To the clear solution 1.4 M Jones reagent (0.4 ml) was added. The resulting mixture was stirred at -10° for 1.5 hours. It was treated with methanol (1ml), and 3 minutes later with saturated aqueous solution of sodium bicarbonate (2 ml) and 3 minutes later with acetic acid (1 ml) and filtered. After evaporation, the residue was diluted with water (15 ml) and extracted with CH2C12 (5x40 ml). The CH2C12 solution was washed with water, dried with MgSO4 and evaporated. Chromatography of the crude product on silica gel and elution with 1 % methanol in CH2C12 afforded 70 mg (44 % yield) of the title compound as a foam; 1HNMR (CDC13): δ 9.7 (broad, IH), 7.9- 6.7(m, 13H), 6.20 (s, IH) 4.83 (s, 2H), 3.82 (s, 3H), 3.30, 3.00 (dd, J=16 Hz, 2H), 1.79 (s, 3H); MS (ES-positive): [M+H]+ 492. Example 70
{(2S*,5R*)-5-allyl-2-[4-(benzyloxy)phenyl]-3-[3-(methoxycarbonyl) phenyl]-4-oxo-l,3-thiazolidin-5-yl}acetic acid.
Using the procedure from the above example, methyl 3-[(2S*,5R*)-5-allyl- 2-[4-(benzyloxy)phenyl]-5-(2-hydroxy ethyl)-4-oxo-l,3-thiazolidin-3-yl]benzoate (400 mg, 0.794 mmol) was converted to the title compound: yellow foam (275 mg, 67% yield). MS (ESI) [M+H]+ 518. Example 71
Preparation of methyl 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2- {[2-(3- ethoxy-4-methoxy-phenyl)ethyl]amino}-2-oxoethyl)-5-methyl-4-oxo-l,3-thiazolidin-3- yl]benzoate. {(2S*,5R*)-2-[4-(Benzyloxy)ρhenyl]-3-[3-(methoxycarbonyl)phenyl]-5- methyl-4-oxo-l,3-thiazolidin-5-yl} acetic acid (240 mg, 0.49 mmol) was dissolved in DMF (15 ml) and was treated at rt/N with diisopropylethylamine (76 mg, 0.59 mmol) and 3- ethoxy-4-methoxyphenethylamine (114 mg, 0.59 mmol) in DMF (0.5 ml). To the solution at rt/N2 O-(7-azabenzotriazole-l-yl)- N, N, N',N'-tetra-methyl uronium hexafluorphosphate (233 mg, 0.59 mmol) was added. The resulting solution was stirred at rt/N2 for 20 hours. It was diluted with ethyl acetate (220 ml), washed with brine (3x20 ml), dried with MgSO4 and evaporated. Chromatography of the crude product on silica gel and elution with 1.5 % methanol in CH2C12 afforded 250 mg (76 % yield) of the title compound as a white solid; HNMR (CDC13): δ 7.9-6.6 (m, 16H), 6.17 (s, IH) 4.95 (s, 2H), 4.04 (m, 2H), 3.87, 3.84 (ss, 3H each), 3.58, 3.50 (mm, 2H); MS (ES-positive): [M+H]+ 669. Example 72
Preparation of 3-[(2S*,5R*)-5-allyl-2-[4-(benzyloxy)phenyl]-5-(2- { [2-(3- ethoxy-4-methoxyphenyl)ethyl]amino}-2-oxoethyl)-4-oxo-l,3-thiazolidin-3- yl]benzamide.
Using the procedure from the above example, {(2S*,5R*)-5-allyl-2-[4- (benzyloxy)phenyl]-3-[3-(methoxycarbonyl) phenyl]-4-oxo-l,3-thiazolidin-5-yl}acetic acid (230 mg, 0.444 mmol) afforded methyl 3-[(2S*,5R*)-5-allyl-2-[4- (benzyloxy)phenyl]-5-(2-{[2-(3-ethoxy-4-methoxyphenyl)ethyl]amino}-2-oxoethyl)-4- oxo-1, 3-thiazolidin-3-yl]benzoate as a yellow powder (290 mg, 94%) yield). MS (ESI) [M+H]+ 695.
This compound (230 mg, 0.331 mmol) was stirred with ammonia in methanol to afford the title compound as a yellow syrup (190 mg, 84% yield). MS (ESI) [M+H]+ 695. Example 73
Preparation of {(2S*,5R*)-3-[3-(aminocarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-5-methyl-4-oxo-l,3-thiazolidin-5-yl}acetic acid.
3-[(2S*,5R*)-2-[4-(Benzyloxy)ρhenyl]-5-(2-hydroxyethyl)-5-methyl-4- oxo-l,3-thiazolidin-3-yl]benzamide (200 mg, 0.43 mmol) was dissolved in acetone (25 ml) and cooled to -10°/N2. To the solution 1.4 M Jones reagent (0.5 ml) was added over a period of 5 minutes. The orange mixture was stirred at -10° C for 2 hours. The mixture was treated successionally with methanol (1 ml) and 3 minutes later with saturated aqueous solution of sodium bicarbonate (2 ml) and 3 minutes later with acetic acid (1 ml) and filtered. After evaporation the residue was diluted with water (15 ml) and extracted with CH2C12 (4x40 ml). The CH2C12 solution was washed with water, dried with MgSO4 and evaporated. Chromatography of the crude product on silica gel and elution with 4 % methanol in CH2C12 afforded 100 mg (40 % yield) of the title compound as a white solid; 1HNMR (DMSO-d6): δ 8.0-6.8 (m, 13H), 6.51 (s, IH) 4.99 (s, 2H), 3.07, 2.87 (dd, J=16 Hz, 2H), 1.67 (s, 3H, CH3); MS (ES-positive): [M+H]+ 477. Anal. Calc. For C26H24N2O5S. Vi H2O: C,64.29. H,5.18. N.5.77. Found: C, 64.28. H, 5.27. N.5.58. Example 74
Preparation of 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2- {[2-(3-ethoxy- 4-methoxyphenyl)-ethyl] amino } -2-oxoethyl)-5-methyl-4-oxo- 1 ,3 -thiazolidin-3 - yl]benzamide.
{(2S*,5R*)-3-[3-(Aminocarbonyl)phenyl]-2-[4-(benzyloxy)phenyl]-5- methyl-4-oxo-l,3-thiazolidin-5-yl} acetic acid (90 mg, 0.19 mmol) in DMF (7 ml) was treated at rt/N2 with diisopropylethylamine (29.3 mg, 0.23 mmol), 3-ethoxy-4- methoxyphen-ethylamine (44.2 mg, 0.23 mmol) in DMF (0.5 ml) and O-(7- azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (86.2 mg, 0.23 mmol). After stirring at rt/N2 for 20 hours, it was diluted with ethyl acetate (200 ml), washed with brine (3x 18 ml), dried with MgSO4 and evaporated. Chromatography of the crude product on silica gel and elution with 3 % methanol in CH2C12 afforded 74 mg (60 % yield) of the title compound as a white powder; 'HNMR (DMSO-d6): δ 8.2-6.7 (m, 16H .6.46 (s, IH), 4.96 (s, 2H), 3.82 (m, 2H), 3.69 (s, 3H), 2.81(s, 2H), 2.63 (q, 2H), 1.57 (s, 3H); MS (ES-positive): [M+H]+ 654. Example 75
Preparation of 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2- {[2-(3-ethoxy- 4-methoxy-phenyl)ethyl]amino}-2-oxoethyl)-5-methyl-l,l-dioxido-4-oxo-l,3-thiazolidin- 3-yl]benzamide.
3-[(2S*,5R*)-2-[4-(Benzyloxy)phenyl]-5-(2- { [2-(3-ethoxy-4- methoxyphenyl)-ethyl]--mino-2-oxoethyl)-5-methyl-4-oxo-l,3-thiazolidin-3-yl]benzamide (770 mg, 1.18 mmol) was dissolved in l-methyl-2-pyrrolidinone (20 ml) and treated with 3-chloroperoxy-benzoic acid ( 2 g, 60 %purity, 7 mmol). The resulting solution was stirred at 60° C/N2 for 2 days. All the solvent was removed at 60° under reduced pressure. The residue was dissolved in ethyl acetate (400 ml), washed with 10 % aqueous sodium sulfite solution (45 ml), saturated aqueous sodium bicarbonate solution (45 ml), brine and dried with MgSO . After evaporation of the solvent, the crude product was chromatographed on silica gel. Elution with 3 % methanol in CH2C12 afforded 695 mg (83 % yield) of the title compound as a white solid; 1HNMR (DMSO-d6): δ 8.24-6.71 (m, 16H), 6.80 (s, IH) 5.02 (s, 2H), 3.97 (q, 2H), 3.71 (s, 3H), 3.30 (m, 2H), 2.97 (m, 2H), 2.64 (q, 2H), 1.67 (s, 3H), 1.29 (t, 3H); MS (ES-positive): [M+H]+ 686. Example 76
Preparation of 2-{(2S*, 5R*)-3-(3-acetylphenyl)-2-[4-(benzyloxy)phenyl]- 4-oxo-l,3-thiazolidin-5-yl}-N-[2-(3-ethoxy-4-methoxyphenyl]ethyl]acetamide.
A solution of 3-aminoacetophenone (13.5 g, 100 mmol), 4- benzyloxybenzaldehyde (21.2 g, 100 mmol), and mercaptosuccinic acid (45 g, 300 mmol in acetonitrile (300 ml) was heated to reflux for 48 hours. The mixture was concentrated in vacuo. The residue was dissolved in methylene chloride, and the solution was washed with water, dried over magnesium sulfate, and concentrated. The residue was 2x triturated with ether, the combined ether extracts were dried and concentrated to afford crude product (8.5 g, 18 mmol). A solution of this product, and l,8-diazabicyclo[5.4.0]undec-7- ene (5.6 g, 36 mmol) in acetonitrile (200 ml) was heated to reflux for 20 hours. The solution was concentrated in vacuo, and the residue was triturated with ethylacetate/lN hydrochloric acid. The solid crude product, a mixture of trans and cis isomer in a ratio 55/45 was collected by filtration and dried to afford [3-(3-acetylphenyl)-2-(4- benzyloxyphenyl)-4-oxo-1.3-thiazolidin-5-yl]acetic acid. (5.2 g, 61%), which was used without further purification.
A solution of this product ( 1.2 g, 2.6 mmol), 3-ethoxy-4- methoxyphenylethylamine (0.5 g, 26 mmol), diisopropylethylamine (0.4 g, 31 mmol), and O-(7-azabenzotriazol-l-yl)-N,N,N,N-tetramethyluronium (1 g, 26 mmol) in dimethylformamide (30 ml) was stirred at room temperature for 20 hours. The reaction mixture was diluted with water, and extracted with ethylacetate. The ethylacetate solution was washed with brine, dried over magnesium sulfate, and concentrated. The residue was purified by column chromatography (Zorbax PRO 18, acetonitrile/water 60/40) to afford the title compound as a white solid, m.p.65-68 °C (0.13 g, 8%): 1H-NMR (DMSO-d6) δ 1.26 (t, J = 6.9 Hz, 3H), 2.51 (s, 3H), 2.64 (t, J = 7.1Hz, 2H), 2.74 (dd, J = 15.4..9.4 Hz, IH), 3.06 (dd, J = 15.5, 3.4 Hz, IH), 3.28 (m, 2H), 3.69 (s, 3H), 3.94 (q, J = 6.9 Hz, 2H), 4.38 (dd, J = 9.2, 3.5 Hz, IH), 4.98 (s, 2H), 6.50 (s, IH), 6.82 (m, 5H), 7.36 (m, 10H), 7.54 (d, J = 8.2 Hz, IH), 7.73 (d, J = 7.6 Hz, IH), 7.85 (s, IH), 8.16 (t, J = 5.5 Hz, IH); MS (Fl POS) m/z 639 (M+H); Anal. Calc. for C37H38N2O6S C: 69.57, H: 6.00, N; 3.77, found. C: 68.10, H: 5.83, N: 3.77. Example 77
Preparation of 2-{(2S*, 5S*)-3-(3-acetylphenyl)-2-[4-(benzyloxy)phenyl]- 4-oxo-l, 3-thiazolidin-5-yl}-N-[2-(3-ethoxy-4-methoxyphenyl]ethyl]acetamide.
From the HPLC chromatography of the above example, a second compound which was the title compound was obtained as a white solid (0.19 g, 11 %).: m.p. 65-70; Anal. Calc. for C37H38N2O6S C: 69.57, H: 6.00, N: 4.39, found C: 69.06, H: 5.96, N: 3.96; 1H-NMR (DMSO-d6) δ 1.29 (t, J = 6.9 Hz, 3H), 2.51 (s, 3H), 2.66 (t, J = 6.9 Hz, 2H), 2.74 (dd, J = 16.1, 6.4, IH), 2.96 (dd, J = 16.3, 3.1 Hz, IH), 3.30 (m, 2H), 3.70 (s, 3H), 4.00 (q, J = 6.9 Hz, 2H), 4.51 (dd, J = 4.1, 4.1, IH), 5.01 (s, 2H), 6.82 (m, 5H), 7.38 (m, 8H), 7.57 (d, J = 8 Hz, IH), 7.75 (d, J = 8 Hz, IH), 7.89 (s, IH), 8.15 (t, J = 5.3 Hz, IH), MS (Fl POS) m/z 639 (M+H). Example 78
Preparation of 2-{(2S*,5R*)-3-(3-benzoylphenyl)-2-[4- (benzyloxy)phenyl]-4-oxo-l,3-thiazolidin-5-yl}-N-[2-(3-ethoxy-4- methoxyphenyl]ethyl]acetamide.
Using the procedure from the previous two examples, 3- aminobenzophenone (25 g, 127 mmol), 4-benzyloxybenzaldehyde (26.9 g, 127mmol), and mercaptosuccinic acid (57.5 g, 381 mmol) were reacted to obtain [3-(3-benzoylphenyl)-2- (4-benzyloxyphenyl)-4-oxo-l,3-thiazolidin-5-yl]acetic acid (52 g, 78 %).
Using the procedure from the previous two examples a solution of this acid (0.53 g, 1 mmol), 3-ethoxy-4-methoxyphenylethylamine (0.2 g, 1.5 mmol), diisopropylethylamine (0.15 g, 1.2 mmol), and O-(7-azabenzotriazol-l-yl)-N,N,N,N- tetramethyluronium hexafluorophosphate (0.4 g, 1 mmol) in dimethylformamide (20 ml) was reacted to obtain the title compound as a white solid: m.p.70-72 °C (0.09 g, 13 %): Anal. Calc. for C42H40N2O6S C: 71.98, H: 5.75, N: 4.00, found: C: 72.34, H: 5.47, N: 3.77. 1H-NMR (DMSO-d6) δ 1.26 (t, J = 6.9 Hz, 3H), 2.64 (t, J = 7.1 Hz, 2H), 2.73 (dd, J = 15.5, 9.5 Hz, IH), 3.05 (dd, J = 15.5, 3.4, IH), 3.27 (m, 2H), 3.69 (s, 3H), 3.93 (q, J = 6.9 Hz, 2H), 4.38 (dd, J = 9.2, 3.4 Hz, IH), 5.02 (s, 2H), 6.47 (s, IH), 6.70 (d, J = 8.3 Hz, IH), 6.76 (s, IH), 6.82 (d, J = 8.3 Hz, IH), 6.93 (d, J = 8.7 Hz, 2H), 7.37 (m, 7H), 7.58 (m, 8H), 7.69 (t, J = 7.2 Hz, IH), 8.14 (t, J = 5.6 Hz, IH), MS (Fl POS) m z 701 (M+H). Example 79
Preparation of 2-{(2S*,5S*)-3-(3-benzoylphenyl)-2-[4-(benzyloxy)phenyl]- 4-oxo-l,3-thiazolidin-5-yl}-N-[2-(3-ethoxy-4-methoxyphenyl]ethyl]acetamide.
From the HPLC chromatography of the above example, a second compound which was the title compound was obtained as a white solid (0.25 g, 36 %): m.p. 70-72; Anal. Calc. for C42H40N2O6S C: 71.98, H: 5.75. N: 4.00, f. C: 71.93, H: 5.60, N: 3.78. 1H-NMR (DMSO-d6) δ 1.29 (t, J = 6.9 Hz, 3H), 2.64 (t, J = 7.2 Hz, 2H), 2.70 (dd, J = 5.8, 8.8 Hz, lH)t2.?l (dd, J = 15.9, 4.0, IH), 3.27 (q, J = 7.0 Hz, 2H), 3.70 (s, 3H), 3.98 (q, J = 6.9 Hz, 2H), 4.48 (dd, J = 8.5, 3.8 Hz, IH), 5.04 (s, 2H), 6.39 (s, IH), 6.70 (d, J = 8.3 Hz, IH), 6.79 (s, IH), 6.84 (d, J = 8.0, IH), 6.94 (d, J = 6.8, 2H), 7.36 (m, 7H), 7.58 (m, 8H), 7.68 (t, J = 7.3 Hz, IH), 8.10 (t, J = 5.6 Hz). Scheme 6. For Examples 80-91.
a) 4-nitrophenyl chloroformate, pyridine, CH2C12, 0 °C; b) R'"NH2, CH2C12, 0 °C.
Example 80
Preparation of 2-{(2S*,5R*)-3-[3-(aminocarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-5-methyl-4-oxo- 1 ,3-thiazolidin-5-yl} ethyl 4-nitrophenyl carbonate.
At 0 °C/N23-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2-hydroxyethyl)-5- methyl-4-oxo-l,3-thiazolidin-3-yl]benzamide (1.7 g, 3.8 mmol) in CH2C12 (50 mi pyridine (5 ml) was treated with 4-nitrophenyl chloroformate (1.1 g, 5.7 mmol). After stirring at rt/N2 for 1.5 hours, it was diluted with water (100 ml) and extracted with CH2C12 (4x 50 ml). The combined CH2C12 solution was washed with water, dried with MgSO4 and evaporated. Chromatography of the crude product on silica gel and elution with 2 % methanol in CH2C12 afforded 1.9 g (83 % yield) of the title compound as a white solid; 1HNMR (DMSO-d6): δ 8.40-6.80 (m, 17H, aromatic), 6.61 (s, IH) 4.99 (s, 2H), 4.59 (m, 2H), 2.62, 2.20 (mm, 2H), 1.69 (s, 3H); MS (ES-positive): [M+H]+ 628; Anal. Calc. For C33H29N3O8S: C.63.15. H.4.66. N.6.69. Found: C,62.86. H,4.55. N,6.47. Example 81
Preparation of methyl 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-methyl-5- (2-{[(4-nitrophenoxy)carbonyl]oxy}ethyl)-4-oxo-l,3-thiazolidin-3-yl]benzoate.
Using the procedure of the above example and substituting methyl 3- [(2S*,5R*)-2-[4-(benzyloxy)ρhenyl]-5-(2-hydroxyethyl)-5-methyl-4-oxo-l,3-thiazolidin- 3-yl]benzoate for 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-(2-hydroxyethyl)-5-methyl-4- oxo-1, 3-thiazolidin-3-yl]benzamide the title compound was obtained as a pale white powder: 2.83 g (84% yield). 1HNMR (DMSO-d6) δ 1.75 (s, 3H), 2.09 (dt, J = 15.04, 3.90 Hz, IH), 2.61 (m, IH), 3.62 (s, 3H), 4.51 (m, 2H), 4.99 (s, 2H), 6.69 (s, IH), 6.84 (d, J = 8.46 Hz, 2H), 7.28 (m, 8H), 7.53 (d, 8.82 Hz, 2H), 7.59 (dt, J = 7.2, 0.75 Hz, IH), 7.68 (d, J = 7.65 Hz, IH), 7.98 (s, IH), 8.32 (d, J = 8.81 Hz, 2H). MS ESI+ 643. Example 82
Preparation of 2- {(2S*,5R*)-3-[3-(aminocarbonyl)ρhenyl]-2-[4- (benzyloxy)phenyl]-5-methyl-4-oxo-l,3-thiazolidin-5-yl}ethyl 2-(3-ethoxy-4- methoxyphenyl)ethylcarbamate.
At -10 °C/N2 2-{(2S*,5R*)-3-[3-(aminocarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-5-methyl-4-oxo-l,3-thiazolidin-5-yl}ethyl 4-mtrophenyl carbonate (200 mg, 0.31 mmol) in dichloromethane (10 ml) was treated with 3-ethoxy-4- methoxyphenethyl-amine (200 mg, 1.03 mmol) in CH2C12 (2 ml). After stirring at 0°/ N2 for 5 hours, it was diluted with aqueous 1 N hydrochloric acid (50 ml) and extracted with CH2C12 (4x50 ml). The CH2C12 solution was washed with water, dried with MgSO4 and evaporated. Chromatography of the crude product on silica gel and elution with 2 % methanol in CH2C12 afforded 204 mg (90 % yield) of the title compound as a white solid; 1HNMR (DMSO-d6): δ 8.0-6.6 (m, 16H), 6.55 (s, IH) 4.98 (s, 2H), 4.20 (m, 2H), 3.96 (q, 2H), 3.69 (s, 3H), 3.18 (m, 2H), 2.62 (t, 2H), 2.20 (m, 2H), 1.64 (t, 3H), 1.29 (t, 3H); MS (ES-positive): [M+H]+ 684; Anal. Calc. For C38HnN3O7S: C,66.74. H,6.04. N.6.14. Found: C.66.75. H.6.23. N,6.07. Example 83 Preparation of 2-[2-(4-benzyloxy-phenyl)-3-(3-carbamoyl-phenyl)-5- methyl-4-oxo-thiazolidin-5-yl]-ethyl ester.
Using the procedure of the above example and substituting 3-phenyl- bezylamine in place of 3-ethoxy-4-methoxyphenethyl-amine, the title compound was obtained as a white solid: ΗNMR (DMSO-d6) δ 1.67 (s, 3H), 2.13 (m, 2H), 4.18 (m, 4H), 4.99 (s, 2H), 6.56 (s, IH), 6.82 (d, J = 8.85 Hz, 2H), 7.24 (m, 9H), 7.39 (m, 4H), 7.47 (t, J = 8.07 Hz, 2H), 7.52 (d, J = 6.27 Hz, IH), 7.60 (m, 3H), 7.74 )t, J = 6.07 Hz, IH), 7.81 (s, IH), 7.91 (s, IH). MS ESI+ 672. Example 84
Preparation of 2S*,5R*-(3-ethoxy-4-methoxybenzyl)-carbamic acid 2-[2- (4-benzyloxy-phenyl)-3-(3-carbamoyl-phenyl)-5-methyl-4-oxo-thiazolidin-5yl]-ethyl ester
At rt/N2 ethoxy-4-methoxybenzaldehyde (3.6 g, 20 mmol) and methyl hydroxylamine HCI salt (4.0 g, 48 mmol) were dissolved in dry pyridine (15 ml) and stirred for 5 hours. The resulting suspension was filtered and filtrate was evaporated at 40° under reduced pressure to give a light brown solid. The solid material was dissolved in methanol (25 ml), diluted with water (25 ml) and was cooled at 0° to induce crystallization. The white crystalline material was collected by filtration, rinsed with methanol, and dried in vacuum to give 2.75 g (66% yield) of 3-ethoxy-4- methoxybenzaldehyde methoxyoxime as white powder; '-HNMR ( DMSO-d6) δ 8.1 l(s, IH), 6.90-7.30 ( m, 3H), 4.00 (q, 2H), 3.85 (s, 3H), 3.78 (s, 3H),1.32 (t, 3H); MS (ES- positive): [M+ H]+ 210. HPLC determined that this compound 99 % purity.
At 0°/N2 this compound (2.51 g) was dissolved in THF (15 ml) and treated with 1M diborane in THF (36 ml, 36 mmol). The solution was heated under reflux for 2 hours, then cooled to 0°, treated with water (10 ml) and 20% KOH aqueous solution (10 ml). The mixture was heated under reflux for 1.5 hours and extracted with CH2C12 Evaporation of CH2C12 extract and chromatography of crude product on silica gel afforded 2.1 g (62 % yield) of 3-ethoxy-4-methoxybenzylamine as light brown oil; 1HNMR ( DMSO-d6) δ 6.70-7.00 (m, 3H), 4.28 (s, 2H),3.97 (q, 2H), 3.71 (s, 3H), 1.31 (t, 3H).
Using the procedure of Example 83 and substituting 3-ethoxy-4- methoxybenzylamine, the title compound was obtained as a white solid; 1HNMR ( DMSO-d6) δ 6.70-8.00 (m, 16 H), 6.55 (s, IH), 4.97 (s, 2H), 4.25 (m, 2H), 4.12 (d, J=7.5 Hz, 2H), 3.90 (q, 2H), 3.69 (s, 3H), 2.25 (m, 2H), 1.64 (s, 3H), 1.26 (t, 3H); MS (ES- positive): [M+ H]+ 670. HPLC determined that this compound 90.3 % purity. Example 85
Preparation of 2- {(2S*,5R*)-3-[3-(aminocarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-5-methyl-4-oxo-l,3-thiazolidin-5-yl}ethyl 2-(3- chlorophenyl)ethylcarbamate
Using the procedure of Example 83 and substituting 3- chlorophenethylamine, the title compound was obtained as a white solid; 1HNMR (DMSO-d6) δ 6.80-8.00 (m, 17 H), 6.55 (s, IH), 4.99 (s, 2H), 4.17 (m, 2H), 3.23 (m, 2H), 2.71 (t, 2H), 2.17 (m, 2H); MS (ES-positive): [M+ H]+ 644. HPLC determined that this compound 90.8 % purity. Example 86
Preparation of 2-{(2S*,5R*)-3-[3-(aminocarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-5-methyl-4-oxo- 1 ,3-thiazolidin-5-yl} ethyl 2-(3- trifluoromethoxyphenyl)ethylcarbamate
Using the procedure of Example 83 and substituting 3- triflouromethoxyphenethylamine, the title compound was obtained as a white solid; 1HNMR (DMSO-d6) δ 6.80-8.00 (m, 17 H), 6.55 (s, IH), 5.00 (s, 2H), 4.25 (m, 4H), 2.20 (m, 2H), 1.65 (s, 3H); MS (ES-positive): [M+ H]+ 680. HPLC determined that this compound 93.9 % purity. Example 87
Preparation of 2- {(2S*,5R*)-3-[3-(aminocarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-5-methyl-4-oxo-l,3-thiazolidin-5-yl}ethyl 2-(3- trifluoromethylphenyl)ethylcarbamate
Using the procedure of Example 83 and substituting 3- trifluoromethylphenethylamine, the title compound was obtained as a white solid; 1HNMR (DMSO-d6) δ 6.70-8.00 (m, 17 H), 6.55 (s, IH), 4.98 (s, 2H), 4.30 (m, 4H), 2.25 (m, 2H), 1.64 (s, 3H); MS (ES-positive): [M+ H]+664. HPLC determined that this compound 92.1 % purity. Example 88 Preparation of 2-{(2S*,5R*)-3-[3-(aminocarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-5-methyl-4-oxo-l,3-thiazolidin-5-yl}ethyl 2-(3- iodophenyl)ethylcarbamate,
Using the procedure of Example 83 and substituting 3-iodophenethylamine, the title compound was obtained as a white solid; 1HNMR (DMSO-d6) δ 6.70-8.15 (m, 17 H), 6.55 (s, IH), 4.98 (s, 2H), 4.17 (m, 4H), 2.25 (m, 2H), 1.65 (s, 3H); MS (ES-positive): [M+ H]+ 722; HPLC determined that this compound 91.3 % purity. Example 89
Preparation of 2-{(2S*,5R*)-3-[3-(aminocarbonyl)ρhenyl]-2-[4- (benzyloxy)phenyl]-5-methyl-4-oxo-l,3-thiazolidin-5-yl}ethyl 4-(l,2,3-thiadiazol-5- yl)benzylcarbamate
Using the procedure of Example 83 and substituting 4-(l,2,3-thiadiazol-5- yl)benzylamine, the title compound was obtained as a white solid; 1HNMR (DMSO-d6) δ 1.66 (s, 3H), 2.18 (m, IH), 2.32 (m, IH), 4.16 (m, IH), 4.27 (d, J = 6.18 Hz, 2H), 4.28 (m, IH), 4.96 (s, 2H), 6.57 (s, IH), 6.82 (d, J = 8.56 Hz, 2H), 7.25 (m, 8H), 7.38 (m, 4H), 7.61 (d, J = 7.65 Hz, IH), 7.78 (t, J = 6.18 Hz, IH), 7.81 (s, IH), 7.96 (s, IH), 8.20 (d, J = 8.08 Hz, 2H), 9.59 (s, IH). MS (ESI) M+ m/z =680. Example 90
Preparation of 2-{(2S,5R)-3-[3-(aminocarbonyl)phenyl]-2-[4- (benzyloxy)phenyl]-5-methyl-4-oxo-l,3-thiazolidin-5-yl}ethyl l,3-benzodioxol-5- ylmethylcarbamate
Using the procedure of Example 83 and substituting l,3-benzodioxol-5- ylmethylamine, the title compound was obtained as a white solid; IHNMR (DMSO-d6) δ 1.62 (s, IH), 2.11 (m, IH), 2.26 (m, IH), 4.06 (d, J = 5.65 Hz, 2H), 4.09 (m, IH), 4.23 (m, IH), 4.99 (s, 2H), 5.98 (s, 2H), 6.56 (s, IH), 6.69 (d, J = 8.08 Hz, IH), 6.79 (d, J = 8.06 Hz, 2H), 6.84 (d, J = 8.53 Hz, 2H),7.26 (m, 8H), 7.40 (m, 2H), 7.61 (d, J = 6.92 Hz, 2H), 7.81 (s, IH), 7.87 (s, IH). MS (ESI) M- m/z = 638. Example 91
General Procedure for carbamate containing library by parallel synthesis techniques.
In a vial open to the atmosphere at room temperature, was added carbonate resin (20 mg, 3.18 mmol / g , Argonout Tech. PIN 800289), the corresponding commercially available amine (450 μL, 0.1 M soln in THF), and 2-{(2S*,5R*)-3-[3- (aminocarbonyl)phenyl]-2-[4-(benzyloxy)phenyl]-5-methyl-4-oxo-l,3-thiazolidin-5- yl} ethyl 4-nitrophenyl carbonate or 3-[(2S*,5R*)-2-[4-(benzyloxy)phenyl]-5-methyl-5-(2- {[(4-nitrophenoxy)carbonyl]oxy}ethyl)-4-oxo-l,3-thiazolidin-3-yl]benzoate (300 μL, 0.1 M soln in THF). For each equivalent of hydrochloride contained in each commercial amine employed, an extra amount of carbonate resin was added (20 mg). The resulting yellow mixtures were capped and shaken on an orbital shaker for 5 hrs. To this yellow mixture was added isocyanate resin (20 mg, 1.49 mmol / g, Argonout Tech. PIN 800 262) re-capped and shaken 15 hrs at room temperature. Each yellow mixture was filtered. Each compound was purified by Gilson preparatory HPLC system and the required fractions were collected and concentrated in vacuuo. The final products were analyzed by LC / MS.
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It is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reading the above description. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent applications and publications, are incorporated herein by reference for all purposes.

Claims

WHAT IS CLAIMED:
1. A compound having a formula:
wherein,
R1 is a member selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocylic groups;
R2 is a member selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocylic groups;
R3 and R4 are independently members selected from the group including hydrogen, alkyl, -(CH2)mCONR5R6, -(CH2)mOCONR5R6, -(CH2)mCH2Y2R6, - (CH2)mCH=CHR6, -(CH2)mCH2NR5CO(Y3)nR6,
R5 and R6 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups;
X is a member selected from the group consisting of S, S=O, and O=S=O;
Y is a member selected from the group consisting of O, S, and NH;
Y is a member selected from the group consisting of CH2, O, S, and NR ; Y3 is a member selected from the group consisting of O, NR6R7;
R7 is a member selected from the group consisting of hydrogen and lower alkyl;
X2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, phenyl, substituted phenyl, heterocychc, substituted heterocychc, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups; m is an integer from 0 to 3; n is 0 or 1; and s is 1 or 2.
2. A compound according to claim 1, wherein said compound is a member selected from the group consisting of
144
145
157
3. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable excipient.
4. A method of activating a FSH receptor comprising contacting a cell comprising the FSH receptor with an effective amount of a compound according to claim 1.
5. A method of stimulating follicle maturation comprising contacting a follicle cell comprising a FSH receptor with an effective amount of a compound according to claim 1.
6. A method for inducing ovulation in a subject comprising administering to said subject an effective amount of a compound according to claim 1.
7. A method for in vitro fertilization comprising:
(a) treating a subject with a pharmaceutical formulation according to claim 3;
(b) collecting ova from said subject;
(c) fertilizing said ova; and
(d) implanting said fertilized ova into a host subject.
8. A compound having a formula:
(in) wherein,
R1 is a member selected from the group consisting of aryl, substituted aryl, arylalkyl and substituted arylalkyl groups;
R2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocychc groups;
R3 and R4 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocychc groups; and
X is a member selected from the group consisting of S, S=O, and O=S= .
9. A FSH receptor agonist, wherein said agonist stimulates the activity of a FSH receptor, wherein said agonist is noncompetitve with FSH for said FSH binding site.
10. An agonist according to claim 9, wherein said agonist is ah organic molecule having a molecular weight of from about 50 daltons to about 1000 daltons.
11. A pharmaceutical formulation comprising a non-competitive FSH receptor agonist according to claim 9.
12. A compound that modulates FSH receptor activity, said compound having: a molecular weight of from about 200 daltons to about 1000 daltons; and a FSH modulating activity as expressed by an EC5o standard of no more than 200 nM; wherein said FSH receptor modulating activity of said compound is competitively inhibited by a compound having the formula:
wherein,
R1 is a member selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocychc and substituted heterocylic groups;
R2 is a member selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocylic groups;
R3 and R4 are independently members selected from the group including hydrogen, -(CH2)mCONR5R6, -(CH2)mCH2Y2R6, -(CH2)mCH=CHR6, - (CH2)mCH2NR5CO(Y3)nR6,
R5 and R6 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups;
X is a member selected from the group consisting of S, S=O, and O=S=O;
Y is a member selected from the group consisting of O, S, and NH; '
Y2 is a member selected from the group consisting of CH2, O, S, and NR5;
Y3 is a member selected from the group consisting of O, and NR6R7;
R7 is a member selected from the group consisting of hydrogen, and lower alkyl;
X2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, phenyl, substituted phenyl, heterocychc, substituted heterocychc, arylalkyl, substituted arylalkyl, heterocyclicalkyl and substituted heterocyclicalkyl groups; m is an integer from 0 to 3; n is 0 or 1 ; and s is 1 or 2.
13. A compound that modulates FSH receptor activity, said compound having: a molecular weight of from about 200 daltons to about 1000 daltons; and a FSH modulating activity as expressed by an EC50 standard of no more than 200 nM; wherein said FSH receptor modulating activity of said compound is competitively inhibited by a compound having the formula:
wherein,
R1 is a member selected from the group consisting of aryl, substituted aryl, arylalkyl and substituted arylalkyl groups;
R2 is a member selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocychc and substituted heterocychc groups; and
R3 and R4 are independently members selected from the group consisting of hydrogen, alkyl, substituted alkyl, arylalkyl, substituted arylalkyl, heterocychc arid substituted heterocychc groups.
EP01955958A 2000-07-27 2001-07-24 Agonists of follicle stimulating hormone activity Withdrawn EP1307193A1 (en)

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