Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D473/00—Heterocyclic compounds containing purine ring systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D473/00—Heterocyclic compounds containing purine ring systems
  • C07D473/40—Heterocyclic compounds containing purine ring systems with halogen atoms or perhalogeno-alkyl radicals directly attached in position 2 or 6

Definitions

• the present invention relates to 2-substituted-6-trifluoromethyl purine derivatives as selective adenosine antagonists, in particular adenosine -A 3 receptor antagonists, to methods for the preparation of these compounds and to novel intermediates useful for the synthesis of said purine derivatives.
• the invention also relates to the use of a compound disclosed herein for the manufacture of a medicament giving a beneficial effect. A beneficial effect is disclosed herein or apparent to a person skilled in the art from the specification and general knowledge in the art.
• the invention also relates to the use of a compound of the invention for the manufacture of a medicament for treating or preventing a disease or condition.
• the invention relates to a new use for the treatment of a disease or condition disclosed herein or apparent to a person skilled in the art from the specification and general kno wledge in the art.
• specific compounds disclosed herein are used for the manufacture of a medicament useful in the treatment of disorders in which adenosine-A 3 receptors are involved, or that can be treated via manipulation of those receptors.
• Adenosine receptors At present, four types of adenosine receptors have been identified and designated A 1 , A 2A , A 2B and A 3 respectively. All four belong to the super-family of seven transmembrane G-protein coupled receptors. Adenosine receptors are ubiquito us and involved in a great variety of biological processes. Thus, during the past decades the therapeutic potential of adenosine receptor ligands has resulted in a substantial research interest.
• adenosine A 3 receptors may play a basic role in different pathologies such as inflammation and neurodegeneration, ischemic brain damage, cardiac ischemia, hypotension, ischemic heart pre-conditioning, asthma and cancer. (P.G. Baraldi et al., European Journal of Medicinal Chemistry, 38, 367-382, 2003, and references cited therein).
• adenosine A 3 receptor antagonists have been claimed to be useful for the treatment of acute and chronic pain, inflammatory diseases including, arthritis, multiple sclerosis, asthma and psoriasis; gastro-intestinal disorders such as ulcers, inflammatory bowel disease (Crohn's disease) and ulcerative colitis; allergic responses such as eczema, atopic dermatitis and rhinitis; cardio-vascular disorders such as myocardial infarction, arrhythmias, hypertension, thrombosis, anaemia, arteriosclerosis, angina pectoris, cutaneous diseases such as urticaria, lupus erythematosus and pruritus; opthalmological disorders like glaucoma; respiratory disorders including chronic obstructive pulmonary disease, bronchitis and cystic fibrosis; central nervous system disorders including various forms of epilepsy, stroke, depression, sleep apnoea; disorders characterized by impairment of cognition and memory such
• the available literature does not provide incentives towards the design as selective adenosine A 3 receptor antagonists of the uniquely substituted purine derivatives of the present invention , in particular with respect to the trifluoromethyl moiety at position 6 of the purine core.
• novel compounds obtained by this new synthetic route turned out to be adenosine receptor ligands (in contrast to previo usly described 6- CF 3 derivatives), more specifically: adenosine-A 3 receptor selective antagonists.
• the invention relates to compounds of the general formula ( 1 ):
• - Ri represents -tX-(CH 2 )n]m-Y, in which formula X is either NH or oxygen, n is 0, 1 , 2 or 3, m is 0 or 1 , and Y is an optionally substituted 5 - or 6-membered aromatic or non-aromatic ring, containing 0, 1 or 2 hetero -atoms selected from N, O and S, or Ri represents a nitro goup,
• R 2 is hydrogen, d -3 -alkyl, ribose, -CH 2 -O-CO-CM-butyl or aryl-(Ci -3 )alkyl,
• R 3 represents hydrogen, halogen, NH 2 , -NH-(Ci -6 )alkyl, -N-di(Ci -6 )alkyl, -NH-cyclo(C 3-8 )alkyl, C 2-4 -alkenyl(hetero)aryl or C 2-4 -alkynyl(hetero)aryl, of which the hetero-atoms are selected from N, O, and S, and which (hetero)aryl groups may be optionally substituted, or R 3 represents an optionally substituted 5- or 6- membered aromatic or non -aromatic ring, containing 0, 1 or 2 hetero-atoms selected from nitrogen, oxygen and sulphur,
• Such compounds are new and are adenosine-A 3 receptor antagonists.
• the invention relates to racemates, mixtures of diastereomers as well as the individual stereoisomers of the compounds having formula (1).
• the invention also relates to the E isomer, Z isomer and E/Z mixtures of compounds having formula (1).
• E isomer Z isomer
• E/Z mixtures of compounds having formula (1) Throughout this specification the generally accepted atom numbering system of the purine skeleton is used:
• Ci_ 3 -alkyl' means 'methyl, ethyl, n-propyl or isopropyl'.
• Optionally substituted' means that a group may or may not be further substituted by one or more groups selected from alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxyl, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, oxo, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5 - or 6-membered aromatic or non -aromatic ring containing 0, 1 or 2 heteroatoms selected from nitrogen,
• 'alkyl' means d -3 -alkyl
• 'alkenyl' means Ci -3 -alkenyl
• 'alkynyl' means Ci -3 -alkynyl
• 'acyl' means Ci -3 -acyl and 'aryl' means furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1 ,3,5-triazynyl, phenyl, indazolyl, indolyl, indolizinyl, isoindolyl, benzi[b]furanyl, benzo[b]thiophenyl, benz -imidazolyl, benzthiazoly
• Optional substituents may themselves bear additional optional substituents.
• Preferred optional substituents include Ci -3 alkyl such as for example methyl, ethyl, and trifluoromethyl, fluoro, chloro, bromo, hydroxyl, C i -3 alkyloxy such as for example methoxy, ethoxy and trifluoromethoxy, and amino.
• Prodrugs are therapeutic agents which are inactive per se but are transformed into one or more active metabolites.
• Prodrugs are bioreversible derivatives of drug molecules used to overcome some barriers to the utility of the parent drug molecule. These barriers include, but are not limited to, solubility, permeability, stability, presystemic metabolism and targeting limitations (Medicinal Chemistry: Principles and Practice, 1994, ISBN 0-85186-494-5, Ed.: F. D. King, p. 215; J. Stella, "Prodrugs as therapeutics", Expert Opin. Ther. Patents, 14(3), 277-280, 2004; P. Ettmayer et al., "Lessons learned from marketed and investigational prodrugs", J.Med.Chem., 47, 2393-2404, 2004).
• Pro-drugs i.e. compounds which when administered to humans by any known route, are metabolised to compounds having formula (1), belong to the invention.
• this relates to compounds with primary or secondary amino or hydroxy groups.
• Such compounds can be reacted with organic acids to yield compounds having formula (1) wherein an additional group is present which is easily removed after administration, for instance, but not limited to amidine, enamine, a Mannich base, a hydroxyl -methylene derivative, an O-(acyloxymethylene carbamate) derivative, carbamate, ester, amide or enami none.
• the invention particularly relates to compounds of formula (1) in which :
• - Ri represents -
• R 2 represents hydrogen, methyl, ribose or -CH 2 -O-CO-CM-butyl
• R 3 represents hydrogen, halogen, -NH-cyclopentyl, ethenylphenyl or ethynylphenyl, which phenyl groups may be optionally substituted with a CF 3 group, or R 3 represents an optionally substituted 5- or 6-membered aromatic or non-aromatic ring, containing 0, 1 or 2 hetero -atoms selected from nitrogen, oxygen and sulphur, and tautomers, stereoisomers, prodrugs and salts thereof.
• the invention relates to compounds of formula (1) in which
• - Ri represents -[X-(CH 2 ) n ] m -Y, in which formula X is either NH or oxygen, n is 1 or 2, m is 0 or 1 , and Y is an optionally hydroxylated 6-membered aromatic or non- aromatic ring, containing 0 or 1 N-atoms, - R 2 represents hydrogen, methyl, ribose or -CH 2 -O-CO-CM-butyl,
• R 3 represents hydrogen, halogen, -NH-cyclopentyl, ethenylphenyl or ethynylphenyl, which phenyl groups may be optionally substituted with a CF 3 group, or R 3 represents an optionally substituted 5- or 6-membered aromatic or non-aromatic ring, containing 0, 1 or 2 hetero -atoms selected from nitrogen, oxygen and sulphur, and tautomers, stereoisomers, prodrugs and salts thereof.
• - Ri represents -[X-(CH 2 ) n ] m -Y, in which formula X is NH, n is 1 or 2, m is 1 , and Y is an optionally hydroxylated phenyl group, - R 2 represents hydrogen, ribose or -CH 2 -O-CO-O-£-butyl,
• R 3 represents hydrogen, and tautomers, stereoisomers, prodrugs and salts thereof.
• BocOM -CH 2 -O °
• the N9 position can be functionalised with different alkyl or arylalkyl groups via standard alkylation or Mitsunobu conditions.
• these compounds can be further functionalised by adding nucleophiles.
• Palladium catalysed coupling procedures (Sonogashira, StNIe, Suzuki, Buchwald -Hartwig) allow for a broad range of carbon and heteroatom nude ophiles that can be introduced on the 8 position of the halo-purines.
• the present invention therefore also relates to a process for the preparation of compounds as described above, comprising the steps of:
• R 1 has the meanings as given in claim 1, and,
• the trifluoromethylation step (a) is preferably carried out by adding CF 3 TMS to a suspenstion of the compound of formula (3) and CsF in dry THF.
• the nucleophilic substitution reaction on compound (4) is preferably carried out by dissolving the compound with the chosen amine in the presence of triethylamine.
• the reaction (c2) is preferably performed with trifluoroacetic acid.
• the strong base in reaction (c3) is preferably MeONa in metha nol.
• the alkylation reaction (d) of the compound of formula (6) can be performed by standard alkylation procedures or under Mitsunobu conditions.
• the halogenation reaction (e) is preferably carried out by cooling the solution of the compound with formula (7) in THF followed by addition of n-BuLi and Br 2 or I 2 .
• reaction (f) is performed by standard procedures, in case of an alkylation the reaction is preferably performed by Pd coupling procedure.
• salts may be obtained using standard procedures well known in the art, for example by mixing a compound of the present invention with a suitable acid, for instance an inorganic acid such as hydrochloric acid, or with an organic acid.
• a suitable acid for instance an inorganic acid such as hydrochloric acid, or with an organic acid.
• the compounds of the invention can be brought into forms suitable for administration by means of usual processes using auxiliary substances such as liquid or solid carrier material.
• the pharmaceutical compositions of the invention may be administered enterally, orally, parenterally (intramuscularly or intravenously), rectally or locally (topically). They can be administered in the form of solutions, powders, tablets, capsules (including microcapsules), ointments (creams or gel) or suppositories.
• Suitable excipients for such formulations are the pharmaceutically customary liquid or solid fillers and extenders, solvents, emulsifiers, lubricants, flavorings, colorings and/or buffer substances.
• auxiliary substances which may be mentioned are magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars or sugar alcohols, talc, lactoprotein, gelatin, starch, cellulose and its derivatives, animal and vegetable oils such as fish liver oil, sunflower, groundnut or sesame oil, polyethylene glycol and solvents such as, for example, sterile water and mono- or polyhydric alcohols such as glycerol.
• Compounds of the present invention are generally administered as pharmaceutical compositions which are important and novel embodiments of the invention because of the presence of the compounds, more particularly specific compounds disclosed herein.
• Types of pharmaceutical compositions that may be used include but are not limited to tablets, chewable tablets, capsules, solutions, parenteral solutions, suppositories, suspensions, and other types disclosed herein or apparent to a person skilled in the art from the specification and general knowledge in the art.
• the invention also includes the preparation or manufacture of said pharmaceutical compositions.
• a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of a pharmaceutical composition of the invention.
• Associated with such container(s) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals products, which notice reflects approval by the agency of manufacture, use, or sale for human or veterinary administration.
• adenosine-A 3 receptor antagonistic properties of the compounds of the invention were determined using the methods outlined below.
• Affinity of the compounds for human adenosine-A 3 receptors was determined using the receptor binding assay described by CA. Salvatore et al.: "Molecular cloning and characterization of the human A 3 adenosine receptor", Proc. Natl. Acad. Sci. USA, 90, 10365-10369, 1993. Briefly, membrane preparations were obtained from human recombinant (HEK 293) cells in which the human adenosine-A 3 receptor was stably expressed. Membranes were incubated at 22 0 C for 90 minutes with [ 125 I]-AB-MECA in the absence or presence of testcompounds in a concentration range from 10 ⁇ M down to 0.1 nM, diluted in a suitable buffer.
• a stable monoclonal hA3-Aequorin cell line was provided by Euroscreen.
• the coding region encoding the human Adenosine A3 receptor was amplified by polymerase chain reaction (PCR), using human lung cDNA as template.
• the PCR product was ligated in the expression vector pEFIN3 (Invitrogen), and the complete sequence of the insert was then established. Sequencing revealed a complete identity with the sequence published by Salvatore, CA. et al. (Ace. Number GenBank: L22607).
• the expression plasmid containing the coding sequence of the human Adenosine A3 receptor was transfected in CHO-K1 cells stably expressing mitochondrially targetted Aequorin and G ⁇ 16.
• Resistant clones were selected in the presence of 400 ⁇ g/ml G418 and isolated by limiting dilution. The clone with the best response to 2 -CI-IB- MECA was selected for further work.
• the hA3 -Aequorin cells express mitochondrially targetted apo-Aequorin. Cells have to be loaded with coelenterazine, in order to reconstitute active Aequorin. After binding of agonists to the hA3 receptor, the intracellular calcium concentration increases, which leads to a luminescent response.
• Binding of calcium to the apo-Aequorin/coelenterazine complex leads to an oxidation reaction of coelenterazine, which results in the production of apo -Aequorin, coelenteramide, CO 2 and light ( ⁇ ma ⁇ 469 nm). This luminescent response is dependent on the agonist concentration.
• Adenosine receptor subtype selectivity of the compounds of the invention was determined using the methods described below In vitro affinity for human adenosine-Ai receptors
• Affinity of the compounds for human adenosine ⁇ receptors was determined using the receptor binding assay described by A. Townsend-Nicholson and P. R. Schofield (Biol. Chem., 269, 2373, 1994), using human recombinant receptors expressed in CHO cells, and [ 3 H]DPCPX as radioligand.
• Affinity of the compounds for human adenosine-A 2A receptors was determined using the receptor binding assay described by D. R. Luthin et al., (MoI. Pharmacol., 47, 307, 1995), using human recombinant receptors expressed in HEK-293 cells, and [ 3 H]CGS 21680 as radioligand.
• Affinity of the compounds for human adenosine-A 2 B receptors was determined using the receptor binding assay described by J. H. Stehle et al. (Mo/. Endocrinol., 6, 384, 1992), using human recombinant receptors expressed in HEK-293 cells, and [ 3 H]MRS 1754 as radioligand.
• the compounds of the invention have affinity for adenosine -A 3 receptors in the binding assay described above, and were shown to be antagonists in the functional assay, also described above. These properties make them useful in the treatment of disorders in which adenosine-A 3 receptors are involved, or that can be treated via manipulation of these receptors.
• the affinity of the compounds of the invention for adenosine A 3 receptors was determined as described above. From the binding affinity measured for a given compound of formula (1), one can estimate a theoretical lowest effective dose. At a concentration of the compound equal to twice the measured Kj-value, nearly 100% of the adenosine A 3 receptors likely will be occupied by the compound. Converting that concentration to mg of compound per kg of patient yields a theoretical lowest effective dose, assuming ideal bioavailability. Pharmacokinetic, pharmacodynamic, and other considerations may alter the dose actually administere d to a higher or lower value. The dosage expediently administered is 0.001 - 1000 mg/kg, preferably 0.1 -100 mg/kg of patient's bodyweight.
• treatment refers to any treatment of a mammalian, preferably human, condition or disease, and includes: (1) preventing the disease or condition from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it, (2) inhibiting the disease or condition, i.e., arresting its development, (3) relieving the disease or condition, i.e., causing regression of the condition, or (4) relieving the conditions caused by the disease, i.e., stopping the symptoms of the disease.
• Nuclear magnetic resonance spectra ( 1 H NMR and 13 C NMR, APT) were determined in the indicated solvent using a Bruker ARX 400 ( 1 H: 400 MHz, 13 C: 100 MHz) at 300 K, unless indicated otherwise.
• 19 F NMR and 13 C NMR experiments were carried out on a Varian Inova 500 spectrometer o perating at 11.74 T (499.9 MHz for 1 H; 125.7 MHz for 13 C; 50.7 Mhz, 470.4 MHz for 19 F) using a 5 mm SW probe.
• the spectra were determined in deuterated chloroform or dichloromethane obtained from Cambridge Isotope Laboratories Ltd.
• Mass spectra and accurate mass measurements were performed using a JEOL JMS - SX/SX 102 A Tandem Mass Spectrometer using Fast Atom Bombardement (FAB). A resolving power of 10,000 (10% valley definition) for high resolution FAB mass spectrometry was used. Extinction coefficients were determined with a HP 8453 UV- Vis spectrophotometer.
• Trifluoroacetic acid anhydride (3.95 ml; 28 mmol) was added dropwise to a solution of commercially available 6-chloro-9-Me-purine (3 g; 17 mmol) and tetrabutylammoniumnitrate (TBAN) (8.65 g; 28 mmol) in dry DCM (50 ml) at 0 0 C under a nitrogen atmosphere. After stirring for 1.5 h the solution was poured into 100 ml of saturated aqueous NaHCCMce (1 :1) and Et 2 O (100 ml) was added. The aqueous layer was extracted with 3 portions of 60 ml Et 2 O-CH 2 CI 2 (3:1).
• TFAA (2.25 ml; 13 mmol) was added dropwise to a solution of 6 -chloro-9-BocOM- purine (2.84 g; 10 mmol, intermediate C) and TBAN (4.87 g; 13 mmol) in dry CH 2 CI 2 (25 ml) at 0 0 C under a nitrogen atmosphere. After stirring for 1.5 h the solution was poured into 75 ml of saturated aqueous NaHCO 3 -ice (1 :1) and Et 2 O (75 ml) was added. The aqueous layer was extracted with 3 portions of 50 ml Et 2 O-CH 2 CI 2 (3:1).
• 6-Trifluoromethyl-9-(Tri-O-acetate-ribose)-2-nitropurine (intermediate F, 0.100 g, 0.2 mmol) was treated with morpholine (0.175 ml, 2 mmol) and triethylamine (0.2 ml) in 1ml THF. After the reaction was complete, the product was purified by flash chromatography (EA) to yield compound 2 (30 mg, 37%).
• This compound was prepared from compound 12 (20 mg, 0.045 mmol), 2- thiopheneboronic acid (12 mg, 0.089 mmol), Pd(PPh 3 J 4 (13 mg, 0.011 mmol) and Na 2 CO 3 (51 mg, 0.179 mmol) in DME/H 2 O (6:1) (2.0 mL) by the procedure described for the preparation of 19 to give compound 14 (11 mg, 55%) as a yellow solid.
• This compound was prepared from compound 12 (40 mg, 0.089 mmol) and E-2- phenylvinylboronic acid (26 mg, 0.179 mmol) by the procedure described for the preparation of compound 19.
• Workup of the reaction mixture and flash chromatography (gradient: EA/PE (1 :5) - EA/PE (1 :2), 150 mL) gave, after evaporation, compound 21 (38 mg, 100%) as a red/orange oil. Yellow crystals (24 mg) were obtained after crystallization from methanol.
• This compound was prepared from compound 12 (40 mg, 0.089 mmol) and E-2-(4- trifluoromethylphenyl)vinylboronic acid (39 mg, 0.179 mmol) by the procedure described for the preparation of compound 19.
• flash chromatography gradient: EA/PE (1 :5) - EA/PE (1 :2), 150 mL
• compound 24 44 mg, 100%
• Adenosine receptor affinity data as well as functional adenosine -A 3 receptor data, obtained according to the protocols given above, are shown in the table below. All of the tested compounds have an affinity for adenosine A 3 receptors higher than that for the other adenosine receptor subpopulations. In all of the compounds investigated the affinity for adenosine A 3 receptors was shown to translate into adenosine A 3 receptor antagonism (pA 2 values ranging from 5.1 - 9.8). None of the investigated compounds showed adenosine A 3 receptor agonism in concentrations up to 10 "5 M (pEC 50 ⁇ 5.0).

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