Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/08—Drugs for disorders of the urinary system of the prostate
• • 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
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • 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
  • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
• • 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
  • A61P9/12—Antihypertensives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero 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
  • C07D417/04—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
  • C07D417/14—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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
  • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
  • C07D513/04—Ortho-condensed systems

Definitions

• Steroid biosynthesis begins in cells of the adrenal gland where the initial product in sterol biosynthesis, cholesterol, is converted into the adrenal steroid hormones aldosterone, hydrocortisone, and corticosterone by a series of P 45 o -mediated hydroxylation steps.
• the cholesterol side-chain cleavage activity that represents the first step in steroid hormone biosynthesis is a P 45 o -mediated oxidation and cleavage of a pair of adjacent methylene groups to two carbonyl fragments, pregnenolone and isocaprylaldehyde (see Walsh (1979) Enzymatic Reaction Mechanisms: W.H. Freeman and Company, pp. 474-77).
• CYP 17, P 450 17 Another critical set of enzymatic conversions in steroid metabolism is facilitated by 17-alpha- hydroxylase-17,20-lyase (CYP 17, P 450 17).
• CYP 17 is a bifunctional enzyme which possesses both a C17,20-lyase activity and a C17-hydroxylase activity.
• these two alternative enzymatic activities of CYP 17 result in the formation of critically different intermediates in steroid biosynthesis and each activity appear to be differentially and developmentally regulated (see e.g. l'AUemand et al. (2000) Eur. J. Clin. Invest. 30: 28-33).
• DHEA dehydroepiandrosterone
• DHT dihydrotestosterone
• Estradiol and estrone are key intermediates in the synthesis of not only the androgens testosterone and dihydrotestosterone (DHT), but also the estrogens 17-beta-estradiol and estrone.
• DHT dihydrotestosterone
• adrenal and ovarian estrogens are the main sources of estrogens in postmenopausal women (see e.g. Harris et al. (1988) Br. J.
• the C17-hydroxylase activity of CYP 17 catalyzes the conversion of the common intermediate progesterone to 17- hydroxyprogesterone, a precursor of cortisol. Therefore the first activity of CYP 17, the C17-hydroxylase activity, promotes the formation of glucocorticoids while the second activity of CYP 17, the C17,20-lyase activity, promotes the formation of sex hormones - particularly androgens including testosterone as well as estrogens.
• Prostate cancer is currently one of the most frequently diagnosed forms of cancer in men in the U.S. and Europe.
• Prostate cancer is typically androgen-dependent and, accordingly, the reduction in androgen production via surgical or pharmacological castration remains the major treatment option for this indication.
• complete rather than partial withdrawal of androgens may be more effective in treating prostate cancer (Labrie, F. et al, Prostate, 1983, 4, 579 and Crawford, E.D. et al, N. Engl. J. Med, 1989, 321, 419).
• Pharmacological inhibition of CYP 17 may be a promising alternative treatment to antiandrogens and LHRH agonists in that testicular, adrenal, and peripheral androgen biosynthesis would be reduced rather than only testicular androgen production (Njar V, et al, J. Med. Chem., 1998, 41, 902).
• CYP17 inhibitor the fungicide ketoconazole
• this drug is a relatively non-selective inhibitor of cytochrome P450 (CYP) enzymes, has weak CYP 17 activity, and has a number of notable side effects associated with it including liver damage (De Coster, R. et al, J. Steroid Biochem. Mol Bio , 1996, 56, 133 and Lake-Bakaar, G. et al, Br. Med. J., 1987, 294, 419).
• ketoconazole In postmenopausal patients with advanced breast cancer, treatment with high doses of ketoconazole resulted in suppression of both testosterone and estradiol levels, implicating CYP 17 as a potential target for hormone therapy (Harris, A. L. et al, Br. J. Cancer, 1988, 58, 493).
• Chemotherapy is usually not highly effective, and is not a practical option for most patients with prostate cancer because of the adverse side effects which are particularly detrimental in older patients.
• Current treatment by orchidectomy or administration of gonadotropin- releasing hormone (GnRH) agonists results in reduced androgen production by the testis, but does not interfere with androgen synthesis by the adrenals.
• total androgen blockade as first line therapy may be more effective than conventional androgen deprivation by achieving maximum suppression of androgen concentrations which may also prevent AR amplification. It is presently unclear whether sequential treatment with different agents can prolong the benefits of the initial therapy. This strategy has been found effective in breast cancer treatment. New agents which act by different mechanisms could produce second responses in a portion of relapsed patients. Although the percentage of patients who respond to second-line hormonal therapy may be relatively low, a substantial number of patients may benefit because of the high incidence of prostate cancer. Furthermore, there is the potential for developing more potent agents than current therapies, none of which are completely effective in blocking androgen effects.
• the invention provides substituted 3-pyridyl heterocyclic compounds which inhibit the lyase activity of enzymes, e.g., 17 ⁇ -hydroxylase-C 17,20 lyase.
• enzymes e.g., 17 ⁇ -hydroxylase-C 17,20 lyase.
• Compounds of the invention have the formula
• R 1 ⁇ N ⁇ (CH 2 ) n — R 2 in which n is 1 or 2; R 1 represents
• R is C 1-4 alkyl or C 3-5 cycloalkyl; and p is 0, 1, or
• R 2 is other than a pyridyl group
• R 2 represents
• R represents C 1-4 alkyl, C 1-4 alkoxy, C 3-6 cycloalkyl, halogen, NO 2 , or CN; and q is 0, 1, or 2;
• the invention also provides pharmaceutical compositions for inhibiting lyase activity, comprising a compound of the invention and a pharmaceutically acceptable carrier.
• the invention also provides methods for inhibiting lyases, comprising contacting the lyase with a compound of the invention. More particularly, the invention provides a method of inhibiting a 17 ⁇ -hydroxylase-C 17,20 lyase, comprising contacting a 17 ⁇ -hydroxylase- C 17,20 lyase with a compound of the invention.
• the invention further provides methods for treating diseases which can benefit from an inhibition of a lyase enzyme.
• diseases are lyase-associated diseases, e.g., diseases resulting from an excess of androgens or estrogens.
• the invention provides a method for treating cancer in a subject, comprising administering to the subject a pharmaceutically effective amount of a compound of the invention, such that the cancer is treated.
• the method of treatment may be applied where the subject is equine, canine, feline, or a primate, in particular, a human.
• the cancer may, for example, be prostate or breast cancer.
• a method for treating prostate cancer in a subject comprises administering to the subject a therapeutically effective amount of a compound of the invention, such that the prostate cancer in the subject is treated.
• a method for treating breast cancer in a subject comprises administering to the subject a therapeutically effective amount of a compound of the invention, such that the breast cancer in the subject is treated.
• the invention is based at least in part on the discovery that substituted 3-pyridyl heterocyclic compounds inhibit the enzyme 17 ⁇ -hydroxylase-C 17,20 lyase.
• R is C alkyl or C 3-5 cycloalkyl; and p is 0, 1, or
• R 2 represents
• R represents C ⁇ alkyl, C 1-4 alkoxy, halogen, NO 2 , or CN; and q is 0, 1, or 2.
• R 1 represents
• R • 3 J is C 1-4 alkyl or C 3-5 cycloalkyl; and p is 0, 1, or 2; or
• R 2 represents
• R represents C 1- alkoxy, halogen, or NO 2; and q is
• agonist of an enzyme refers to a compound that binds to the enzyme and stimulates the action of the naturally occurring enzyme, or a compound which mimics the activity of the naturally occurring enzyme.
• antagonist of an enzyme refers to a compound that binds to the enzyme and inhibits the action of the naturally occurring enzyme.
• analog of a compound refers to a compound having a some structural similarity to a particular compound and having essentially the same type of biological activity as the compound.
• CYP 17 substrate includes any of the various steroid hormones acted upon by a CYP 17 or a CYP 17-like P 45 o enzyme. Examples include pregnenolone, progesterone and their 17 ⁇ -hydroxylated forms. Pregnenolone is converted to DHEA via a CYP 17 C17,20-lyase reaction, but is also subject to C17 ⁇ -hydroxylation via the C 17,20- lyase activity.
• Progesterone is converted to delta 4- androstenedione via a CYP 17 C 17,20- lyase reaction, but is also subject to C17 alpha-hydroxylation via the C17-hydroxylase activity to form 17-hydroxyl-progesterone, a precursor to hydrocortisone (i.e. cortisol).
• CYP 17 metabolite refers to any of the steroid hormones that are synthesized from a cholesterol precursor via a CYP17-mediated reaction, such as a C17- hydroxylase reaction or a C17,20-lyase reaction.
• CYP 17 metabolites include the androgens, such as testosterone, which are synthesized via a CYP 17 C17,20-lyase reaction from CYP 17 substrate precursors such as pregnenolone (converted to DHEA by the CYP 17 C17,20-lyase activity), and progesterone (converted to delta 4- androstenedione by the CYP 17 C17,20-lyase activity).
• Progestagens such as progesterone are primarily synthesized in the corpus luteum.
• the androgens are responsible for, among other things, development of male secondary sex characteristics and are primarily synthesized in the testis.
• Other examples include the estrogens, which are also synthesized from a cholesterol precursor via a CYP 17-mediated reaction.
• the estrogens are responsible for, among other things, the development of female secondary sex characteristics and they also participate in the ovarian cycle and are primarily synthesized in the ovary.
• Another group of CYP 17 metabolites are the glucocorticoids, such as hydrocortisone (i.e. cortisol), which is synthesized from progesterone via a CYP 17-mediated reaction.
• the glucocorticoids among other functions, promote gluconeogenesis and the formation of glycogen and also enhance the degradation of fat.
• the glucocorticoids are primarily synthesized in the adrenal cortex.
• CYP 17 metabolite is further meant to include other steroid hormones which, although not necessarily synthesized by a CYP 17-mediated reaction, may nonetheless be understood by the skilled artisan to be readily affected by an alteration in a CYP 17-mediated activity.
• the mineralocorticoids such as aldosterone
• progesterone is also converted to the glucocorticoids and sex steroids via CYP 17-mediated reactions
• an alteration of a CYP 17 activity can alter the amount of progesterone available for conversion to aldosterone.
• inhibition of CYP 17 activity can increase the amount of progesterone available for conversion into aldosterone.
• the mineralocorticoids function, among other things, to increase reabsorption of sodium ions, chloride ions, and bicarbonate ions by the kidney, which leads to an increase in blood volume and blood pressure.
• the mineralocorticoids are primarily synthesized in the adrenal cortex.
• CYP 17 metabolite-associated disease or disorder refers to a disease or disorder which may be treated by alteration of the level of one or more CYP 17 metabolites. Examples include a hormone dependent cancer, such as an androgen-dependent prostate cancer, which may be treated by inhibiting CYP 17-mediated androgen synthesis, and an estrogen-dependent breast cancer or ovarian cancer, which may be treated by inhibiting CYP 17-mediated estrogen synthesis.
• hormone dependent cancer such as an androgen-dependent prostate cancer
• an estrogen-dependent breast cancer or ovarian cancer which may be treated by inhibiting CYP 17-mediated estrogen synthesis.
• Other examples of "CYP 17 metabolite-associated diseases or disorders” are Cushing's disease, hypertension, prostatic hyperplasia, and glucocorticoid deficiency.
• derivative of a compound refers to another compound which can be derived, e.g., by chemical synthesis, from the original compound.
• a derivative of a compound has certain structural similarities with the original compound.
• Disease associated with an abnormal activity or level of a lyase refers to diseases in which an abnormal activity or protein level of a lyase is present in certain cells, and in which the abnormal activity or protein level of the lyase is at least partly responsible for the disease.
• a “disease associated with a lyase” refers to a disease that can be treated with a lyase inhibitor, such as the compounds disclosed herein.
• a “lyase” refers to an enzyme having a lyase activity.
• Lyase activity refers to the activity of an enzyme to catalyze the cleavage of the bond C17-C20 in 17 ⁇ -hydroxy-pregnenolone and 17 ⁇ -hydroxy-progesterone to form dehydroepiandrosterone (DHEA) and delta4-androstenedione, respectively. Lyase activity also refers to the cleavage of a similar bond in related compounds.
• a “lyase inhibitor” is a compound which inhibits at least part of the activity of a lyase in a cell.
• the inhibition can be at least about 20%, preferably at least about 40%, even more preferably at least about 50%, 70%, 80%, 90%, 95%, and most preferably at least about 98% of the activity of the lyase.
• a "patient” or “subject” to be treated by the subject method can mean either a human or non-human animal.
• Treating refers to preventing, curing or improving at least one symptom of a disease.
• heteroatom as used herein means an atom of nitrogen, oxygen, or sulfur.
• alkyl refers to the radicals of saturated aliphatic groups, including straight-chain alkyl groups and branched-chain alkyl groups.
• cycloalkyl refers to radicals of cycloalkyl compounds, examples being cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
• aralkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
• alkenyl and alkynyl refer to unsaturated aliphatic groups that contain at least one double or triple bond respectively.
• lower alkyl as used herein means an alkyl group but having from one to six carbons, preferably from one to four carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Preferred alkyl groups are lower alkyls.
• aryl as used herein means an aromatic group of 6 to 14 carbon atoms in the ring(s), for example, phenyl and naphthyl. As indicated, the term “aryl” includes poly cyclic ring systems having two or more rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic.
• heteroaryl as used herein means an aromatic group which contains at least one heteroatom in at least one ring. Typical examples include 5-, 6- and 7-membered single-ring aromatic groups that may include from one to four heteroatoms. Examples include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. These aryl groups may also be referred to as “aryl heterocycles" or "heteroaromatics.”
• alkoxyl or "alkoxy” as used herein refer to moiety in which an alkyl group is bonded to an oxygen atom, which is in turn bonded to the rest of the molecule. Examples are methoxy, ethoxy, propyloxy, tert-butoxy, etc.
• nitro means -NO2; the term “halogen” designates -F, -CI, -
• triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, r»-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively.
• triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, / ?-tomenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.
• Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, -toluenesulfonyl and methanesulfonyl, respectively.
• a more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list are hereby incorporated by reference.
• each expression e.g. alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
• substitution or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
• the term "substituted" is contemplated to include all permissible substituents of organic compounds.
• the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
• Illustrative substituents include, for example, those described herein above.
• the permissible substituents can be one or more and the same or different for appropriate organic compounds.
• the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
• protecting group means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
• protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
• the field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 3 rd ed.; Wiley: New York, 1999).
• Exemplary compounds of the invention are set forth in Table 1 below.
• the compounds of Table 1 are producible from known compounds (or from starting materials which, in turn, are producible from known compounds), through the general preparative methods described in the General Methods or Examples.
• Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms.
• the present invention contemplates all such compounds, including cis- and tra/w-isomers, R- and S-enantiomers, diastereomers, ( ⁇ )-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
• Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
• a particular enantiomer of a compound of the present invention may be prepared by asymmetric synthesis, or by derivatizaton with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
• the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
• Compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids.
• pharmaceutically acceptable salts refers to the relatively nontoxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
• Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J Pharm. Sci. 66:1-19).
• compositions of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids.
• such conventional nontoxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
• the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
• These salts can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically- acceptable organic primary, secondary or tertiary amine.
• a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically- acceptable organic primary, secondary or tertiary amine.
• Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
• Organic amines useful for the formation of base addition salts include efhylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra).
• Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof (e.g., functioning as 17 ⁇ -hydroxylase-C17,20-lyase inhibitors), wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound in binding to 17 ⁇ -hydroxylase-C17,20-lyase receptors.
• the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.
• the present invention provides a method of inhibiting a lyase, e.g., 17 ⁇ -hydroxylase- C 17,20 lyase, comprising contacting a lyase with a compound of the invention.
• the activity can be inhibited by at least 20%, preferably at least about 50%, more preferably at least about 60%, 70%, 80%, 90%, 95%, and most preferably at least about 98%.
• the invention provides a method for inhibiting a lyase in vitro.
• the lyase is in vivo or ex vivo.
• the invention provides methods for inhibiting a lyase in a cell, comprising contacting the cell with a compound of the invention, such that the activity of the lyase is inhibited.
• the cell may further be contacted with a composition stimulating the uptake of the compound into the cell, e.g., liposomes.
• the invention provides a method for inhibiting a lyase in a cell of a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present invention, or a formulation comprising a compound of the present invention, such that the lyase is inhibited in a cell of the subject.
• the subject can be one having a disease associated with a lyase, e.g., cancer.
• a disease associated with a lyase e.g., cancer.
• Preferred types of cancer that can be treated according to the invention include prostate cancer and breast cancer.
• Other diseases that can be treated include diseases in which it is desired to prevent or inhibit the formation of a hormone selected from the group consisting of the androgens testosterone and dihydrotestosterone (DHT) and the estrogens 17 ⁇ -estradiol and estrone.
• DHT dihydrotestosterone
• any disease that can be treated by inhibiting the activity of a lyase e.g., 17 ⁇ -hydroxylase- C17,20-lyase, can be treated with the compounds of the invention.
• the invention provides methods and compositions for the treatment of CYP 17 metabolite-associated diseases and disorders.
• CYP 17 metabolite-associated diseases and disorders include particularly sex steroid hormone dependent cancers, such as androgen-dependent prostate cancer, which may be treated by inhibiting CYP 17-mediated androgen synthesis, and estrogen-dependent breast cancer or ovarian cancer, which may be treated by inhibiting CYP 17-mediated estrogen synthesis.
• adenocarcinoma of the prostate is a common disease that causes significant morbidity and mortality in the adult male population (see Han and Nelson (2000) Expert Opin. Pharmacother. 1 : 443-9).
• Hormonal therapy for prostate cancer is considered when a patient fails with initial curative therapy, such as radical prostatectomy or definitive radiation therapy, or if he is found with an advanced disease. Hormonal agents have been developed to exploit the fact that prostate cancer growth is dependent on androgen. Non- steroidal anti-androgens (NSAAs) block androgen at the cellular level. Castration is another, albeit drastic means of decreasing androgens levels in order to treat or prevent prostate cancer.
• the methods and compositions of the invention are useful in inhibiting the CI 7,20- lyase activity of CYP 17 and thereby decreasing levels of androgen production and the associated growth of androgen-dependent cancers such as prostate cancer.
• breast cancer in postmenopausal women, can be treated by administration of a C 17,20-lyase inhibitor of the invention because adrenal and ovarian androgens are the main precursors of the estrogens which stimulate the growth of hormone dependent breast cancer.
• breast cancer can be treated with inhibitors of aromatase that prevent interconversion of estrogens and adrenal and ovarian androgens (see Harris et al. (1983) Eur. J. Cancer Clin. Oncol. 19: 11). Patients failing to respond to aromatase inhibitors show elevated levels of androgens in response to aromatase inhibitor treatment (see Harris et al. (1988) Br. J. Cancer 58: 493-6).
• inhibitors of the invention may be used alone or in combination with other drugs to treat or prevent hormone-dependent cancers such as breast and prostate cancer.
• compositions of the invention are particularly suited to treating or preventing hormone-dependent cancers in individuals genetically predisposed to such cancers, particularly those predisposed due to an alteration in the CYP 17 gene.
• CYP 17 metabolite-associated diseases or disorders amenable to treatment with the compositions and methods of the invention include those associated with mineralocorticoid excess such as hypertension caused by sodium retention at renal tubules. Such a mechanism operates in hypertension such as primary hyperaldosteronism and some forms of congenital adrenal hyperplasia. Recently, deficient cortisol metabolism in the aldosterone target organ has been recognized as a novel form of hypertension known as apparent mineralocorticoid excess.
• Disorders associated with mineralocorticoid synthesis include abnormalities of mineralocorticoid synthesis and/or metabolism which profoundly affect the regulation of electrolyte and water balance and of blood pressure (see e.g. Connell et al.
• CYP 17 metabolite- associated diseases or disorders would include those associated with altered levels of aldosterone production (e.g. hypertension, primary adrenal hyperplasia).
• CYP 17 metabolite-associated diseases or disorders are Cushing's disease, prostatic hyperplasia, glucocorticoid deficiency, and endometrial cancer.
• the subject that can be treated according to the invention can be a mammal, e.g., a primate, equine, canine, bovine, ovine, porcine, or feline.
• the mammal is a human.
• the invention provides methods for inhibiting the lyase activity of enzymes that are present in organisms other than mammals, e.g., yeast and fungus, e.g., mildew. Certain compounds of the invention may function as antifungal compounds.
• the therapeutic methods of the invention generally comprise administering to a subject in need thereof, a pharmaceutically effective amount of a compound of the invention, or a salt, prodrug or composition thereof.
• the compounds of the invention can be O 03/027095
• the compounds of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice.
• the compounds can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
• Toxicity and therapeutic efficacy of the compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD o (the dose lethal to 50% of the population) and the ED 5 o (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 5 o.
• Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such reagents to the site of affected tissue in order to minimize potential damage to normal cells and, thereby, reduce side effects.
• Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage 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 therapeutically effective dose can be estimated initially from cell culture assays.
• a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of activity) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
• the compounds of the invention have an IC50 less than 10 ⁇ M as determined by the biochemical or cellular assay described herein. Some compounds of the invention are effective at concentrations of 10 nM, 100 nM, or 1 ⁇ M. Based on these numbers, it is possible to derive an appropriate dosage for administration to subjects.
• prodrugs are well known in the art in order to enhance the properties of the parent compound. Such properties include solubility, absorption, biostability and release time (see “Pharmaceutical Dosage Form and Drug Deliveiy Systems” (Sixth Edition), edited by Ansel et al, publ. by Williams & Wilkins, pgs. 27-29, (1995)). Commonly used prodrugs of the disclosed compounds can be designed to take advantage of the major drug biotransformation reactions and are also to be considered within the scope of the invention.
• compositions can be prepared so that they may be administered orally, dermally, parenterally, nasally, ophthalmically, otically, sublingually, rectally or vaginally.
• Dermal administration includes topical application or transdermal administration.
• Parenteral administration includes intravenous, intraarticular, intramuscular, intraperitoneal, and subcutaneous injections, as well as use of infusion techniques.
• One or more compounds of the invention may be present in association with one or more non-toxic pharmaceutically acceptable ingredients and optionally, other active anti-proliferative agents, to form the pharmaceutical composition.
• These compositions can be prepared by applying known techniques in the art such as those taught in Remington's Pharmaceutical Sciences (Fourteenth Edition), Managing Editor, John E. Hoover, Mack Publishing Co., (1970) or Pharmaceutical Dosage Form and Drug Delivery Systems (Sixth Edition), edited by Ansel et al, publ. by Williams & Wilkins, (1995).
• compositions containing a compound of the invention may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
• Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically acceptable preparations.
• Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
• excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, macrocrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia; and lubricating agents, for example, magnesium stearate, stearic acid or talc.
• the tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
• a water soluble taste masking material such as hydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed.
• Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
• an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
• water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
• Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions.
• excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl- cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacan h and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example lecithin; or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate; or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol; or condensation products of ethylene oxide with partial esters derived firom fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate; or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan mono
• the aqueous suspensions may also contain one or more preservatives, for example ethyl or n- propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
• preservatives for example ethyl or n- propyl p-hydroxybenzoate
• coloring agents for example ethyl or n- propyl p-hydroxybenzoate
• flavoring agents such as sucrose, saccharin or aspartame.
• sweetening agents such as sucrose, saccharin or aspartame.
• Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
• the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
• Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
• These compositions may be preserved by the addition of an anti-oxidant such as butylated liydroxyanisol or alpha-tocopherol.
• Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compound of the invention in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid. Pharmaceutical compositions of the invention may also be in the form of an oil-in- water emulsions.
• the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
• Suitable emulsifying agents may be naturally occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived firom fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
• the emulsions may also contain sweetening, flavouring agents, preservatives and antioxidants.
• Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
• sweetening agents for example glycerol, propylene glycol, sorbitol or sucrose.
• Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
• compositions may be in the form of a sterile injectable aqueous solutions.
• acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
• Sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the compound of the invention is dissolved in the oily phase.
• the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution is then introduced into a water and glycerol mixture and processed to form a microemulation.
• the injectable solutions or microemulsions may be introduced into a patient's blood stream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the active compound.
• a continuous intravenous delivery device may be utilized.
• An example of such a device is the Deltec CADD-PLUSTM model 5400 intravenous pump.
• the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration.
• This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid find use in the preparation of injectables.
• compositions of the invention may also be administered in the form of a suppository for rectal administration of the drug.
• These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
• suitable non-irritating excipient include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
• creams, ointments, jellies, solutions or suspensions, etc., containing the compound of the invention can be employed.
• topical application shall include mouth washes and gargles.
• the compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art.
• the dosage administration will preferably be continuous rather than intermittent throughout the dosage regimen.
• the compounds of the invention may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated.
• the compounds may be administered simultaneously or sequentially.
• the active compounds may be useful in combination with known anti-cancer and cytotoxic agents.
• the active compounds may be useful in combination with agents that are effective in the treatment and prevention of osteoporosis, inflammation, neurofibromatosis, restinosis, and viral infections.
• the active compounds may also be useful in combination with inhibitors of other components of signaling pathways of cell surface growth factor receptors.
• Drugs that can be co-administered to a subject being treated with a compound of the invention include antineoplastic agents selected from vinca alkaloids, epipodophyllotoxins, anthracycline antibiotics, actinomycin D, plicamycin, puromycin, gramicidin D, taxol, colchicine, cytochalasin B, emetine, maytansine, or amsacrine.
• antineoplastic agents selected from vinca alkaloids, epipodophyllotoxins, anthracycline antibiotics, actinomycin D, plicamycin, puromycin, gramicidin D, taxol, colchicine, cytochalasin B, emetine, maytansine, or amsacrine.
• Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described
• Radiation therapy including x-rays or gamma rays which are delivered from either an externally applied beam or by implantation of tiny radioactive sources, may also be used in combination with a compound of the invention to treat a disease, e.g., cancer.
• a composition according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient's symptoms.
• a compound of the invention, materials and/or reagents required for administering the compounds of the invention may be assembled together in a kit.
• the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.
• the kit may further comprise one or more other drugs, e.g., a chemo- or radiotherapeutic agent. These normally will be a separate formulation, but may be formulated into a single pharmaceutically acceptable composition.
• the container means may itself be geared for administration, such as an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, or injected into an animal, or even applied to and mixed with the other components of the kit.
• an inhalant such as syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, or injected into an animal, or even applied to and mixed with the other components of the kit.
• kits also may be provided in dried or lyophilized forms.
• reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means.
• the kits of the invention may also include an instruction sheet defining administration of the agent. Kits may also comprise a compound of the invention, labeled for detecting lyases.
• kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
• a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
• the kits of the invention also may comprise, or be packaged with a separate instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
• a separate instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
• Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
• Other instrumentation includes devices that permit the reading or monitoring of reactions or amounts of compounds or polypeptides.
• Formula I are prepared by the general method described below, according to methods described below, or according to methods commonly employed in the art.
• the 3-cyanopyridines II used to prepare compounds of Formula I are commercially available, are prepared according to methods described below to prepare Intermediates A-F or are prepared according methods described in the following references: Comins, D. L., Smith, R., Stroud, E., Heterocycles, Nol. 22, No. 2, 1984, 339; Leete, E.; Leete, S. A. S., J. Org. Chem. Vol. 43, No. 11, 1978, 2122; Kim, J. G.; Yu, D. S.; Moon, S. H.; Park, J.; Park, W. W. J. Korean Chem. Soc. Vol. 37, No. 9, 1993, 826. Other methods commonly employed in the art may also be used to prepare 3-cyanopyridines II.
• Treatment of II with sodium azide and ammonium chloride in a polar solvent such as DMF, DMA, DMSO, N-methylpyrrolidinone, or water at a temperature of about 80 - 180 °C provides 5-(3-pyridyl)tetrazoles III.
• a polar solvent such as DMF, DMA, DMSO, N-methylpyrrolidinone, or water
• the solvent is DMF and the temperature is about 120-140 °C.
• Other methods commonly employed in the art to prepare aryl tetrazoles from aryl nitriles maybe used to prepare 5-(3-pyridyl)tetrazoles III; representative references are: Musicki, B.; Verert, J.-P. Tetrahedron Lett. Nol 35, No 50, 1994, 9391; Wittenberger, S.
• Tetrazoles III are treated with alkylating agent IN using General Methods A or B to prepare 3-(pyridyl)tetrazoles of Formula I.
• Alkylating agents IN are commercially available or are prepared according to methods commonly employed in the art to prepare benzyl/phenethyl bromides, chlorides, tosylates and triflates.
• R 1 may be prepared by standard N-oxidation conditions from the corresponding Formula I compound where R 1 represents .
• An example of such conditions are hydrogen peroxide in acetic acid at a temperature of about 80 °C for about four hours.
• LC/MS mass spectral data were obtained using a Hewlett-Packard 1100 HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (2 x 23 mm, 120A), and a Finnigan LCQ ion trap mass spectrometer with electrospray ionization. Spectra were scanned from 120-1200 amu using a variable ion time according to the number of ions in the source.
• the eluents were A: 2% acetonitrile in water with 0.02% TFA and B: 2% water in acetonitrile with 0.018% TFA. Gradient elution from 10% B to 95% B over 3.5 minutes at a flowrate of 1.0 mL/min was used with an initial hold of 0.5 minutes and a final hold at 95% B of 0.5 minutes. Total run time was 6.5 minutes.
• Step 1 2,6-Dihydroxy-4-methyl-3-pyridinecarbonitrile (150 g, 1 mol) and phosphorus oxychloride (600 mL, 6.4 mol) were stirred under an argon atmosphere and triethylamine (300 mL, 2.1 mol) was added. After refmxing for 16 hours, the mixture was concentrated in vacuo, and the residue was partitioned between ice water (6 L) and dichloromethane (2 L). The organic phase was washed with aqueous sodium bicarbonate solution, then with brine, dried (Na 2 SO 4 ), and then filtered through a pad of silica gel (465 g) on a sintered glass funnel.
• Step 2 2,6-Dichloro-4-methyl-3-cyanopyridine (40.8 g, 0.22 mol) was dissolved in anhydrous ethanol (680 mL) and triethylamine (120 mL) by warming, and the solution hydrogenated over 5% palladium on carbon at 10 psi of hydrogen. Upon completion of the reaction, catalyst was removed by filtration. The filtrate was concentrated in vacuo.
• Step 1 mono-Ethyl malonate (35.0 g, 265 mmol) and THF (300 mL) is placed in a 500 mL round-bottomed flask and cooled to -70 °C under Ar. To this solution is added 330 mL of 1.6 M n-BuLi (2.0 equiv., 530 mmol) slowly and the solution allowed to stir for 10 min at - 70 °C. The acid chloride, where R 3 is as defined above, is added to the solution slowly, stirred for one more hour at -70 °C, and then the reaction temperature is allowed to go to rt overnight.
• R 3 is as defined above
• the solution is concentrated in vacuo and the residue is partitioned between HCl solution (IN, 200 mL) and Et 2 O (2 x 300 mL).
• the organic layer is washed sequentially with NaHCO 3 (saturated 200 mL) and H 2 O (200 mL), then dried over Na 2 SO 4 .
• the filtrate is concentrated and the crude product is purified by chromatography using hexanes-EtOAc (95:5).
• the average yield of the beta-ketoester is generally about 30-50%.
• Step 2 The beta-ketoester (347 mmol) and 2-cyanoacetamide (347 mmol) are placed in a 500 mL round-bottomed flask and dissolved in 100 mL of THF under Ar.
• Step 3 In a 500 mL round-bottomed flask are placed the 4-substituted-2,6-dihydroxy-3- cyanopyridine (314 mmol) and POCl 3 (3.3 equiv, 1035 mmol) under Ar. Triethylamine (471 mmol, 65.5 mL) is added very slowly using an ice bath for cooling. The reaction mixture is heated to 130 °C for 8 hours under Ar after the addition finished. After cooling to rt, the reaction mixture is concentrated in vacuo and poured into ice (150 g). The residue is partitioned between CH 2 C1 2 (3 x 200 mL) and ice water.
• the separated organic layer is washed sequentially with NaHCO 3 (saturated 200 mL) and H 2 O (200 mL), and dried over Na 2 SO 4 .
• the filtrate is concentrated and purified by chromatography using hexanes-EtOAc (80:20) as eluant.
• the average yield of the 4-substituted-2,6-dichloro-3-cyanopyridines is generally about 35-50%.
• Step 4 In a 500 mL round-bottomed flask are placed the 4-substituted-2,6-dichloro-3- cyanopyridines (232 mmol), 10% Pd/C (2.0 g), Et 3 N (927 mmol, 130 mL) and EtOH (300 mL). The mixture is hydrogenated at atmospheric pressure for 24 to 48 hours at rt. The catalyst is removed by filtration and the filtrate is concentrated. The residue is partitioned between CH 2 C1 2 (3 x 200 mL) and H 2 O (200 mL), and then the separated organic layer is dried over Na 2 SO 4 . Concentration and purification by chromatography using hexanes-EtOAc (95:5) affords the 4-substituted-3-cyanopyridines in an average yields of about 85-95%.
• Step 3 A dry round-bottomed flask was charged with 5 % palladium on carbon (0.38 g, cat.) and anhydrous ethanol (5 mL). In another flask was charged 2,6-dichloro-4- phenylnicotinonitrile (3.83 g, 0.0154 mol), triethylamine (8.57 mL, 0.0615 mol) and more ethanol (80 mL, anhydrous). This was transferred to the reaction flask and this flask was then purged with argon. The flask was evacuated and again purged with argon; this was repeated twice more.
• a second crop can normally be obtained by acidifying the combined filtrates with 2 N HCl to pH 5 to 6, concentrating to dryness, triturating the residue with water, and then filtering and drying.
• the average yield of Intermediates H-L is 20-50%. The products are confirmed NMR and MS.
• HPLC - electrospray mass spectra were obtained using a Hewlett-Packard 1100 HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (2 x 23 mm, 120A), and a Finnigan LCQ ion trap mass spectrometer with electrospray ionization. Spectra were scanned from 120- 1200 amu using a variable ion time according to the number of ions in the source. The eluents were A: 2% acetonitrile in water with 0.02% TFA and B: 2% water in acetonitrile with 0.018% TFA.
• C 17,20 Lyase inhibitory activity of compounds can be determined using, e.g., the biochemical or the cellular assays set forth in the Examples. A person of skill in the art will recognize that variants of these assays can also be used.
• the compounds of the invention can also be tested in animal models, e.g., animal models of prostate or breast cancer.
• Each of the compounds of the invention was subjected to a biochemical assay and a cellular assay for determining its C 17,20 lyase inhibitory activity.
• Recombinant human C 17,20 lyase (hLyase) was expressed in (Sf9) cells, and hLyase enriched microsomes were prepared from cultures as described in the following reference: Baculo virus Expression of Bovine P 45 o in Sf9 Cells and Comparison with Expression in Yeast, Mammalian Cells, and E. Coli. Barnes H. J.; Jenkins, C. M.; Waterman, M. R., Archives of Biochemistry and Biophysics (1994) 315(2) 489-494. Recombinant murine C 17,20 lyase (mLyase) was prepared in a similar manner.
• hLyase and mLyase preparations were titrated using assay conditions to determine protein concentrations to be used for assays. Both mLyase and hLyase assays were run in an identical manner except that cytochrome b5 was omitted in the murine assays.
• Test compounds were diluted 1 :4, serially in six steps, with 100% DMSO starting from 800 ⁇ M going to 51.2 nM reserving the first 2 columns for the generation of a standard curve. Each of these compound solutions in 100% DMSO was further diluted twenty fold in H 2 O to obtain compound concentrations ranging from 40 ⁇ M to 2.56 nM in 5% DMSO.
• Dehydroepiandrosterone (DHEA) standards were serially diluted in 100% DMSO from 400 ⁇ M down to 120 nM in half-log dilutions. Each dilution was further diluted twenty fold in H 2 O to obtain 20 ⁇ M to 6 nM solutions in 5% DMSO using the first 2 columns. Five ⁇ l of these 5% DMSO dilutions were used in the assay.
• Enzymatic reactions were allowed to run for 2 hours at room temperature with gentle agitation. Reactions were terminated with the addition of 50 ⁇ M (final concentration) YMl 16, a potent C 17,20 lyase inhibitor.
• concentration of DHEA generated by hLyase was determined by radioimmunoassay (RIA) as described below.
• CPM CPM
• lyase inhibitory activity of each compound was determined as the concentration of DHEA generated in the presence of test compounds, expressed as a percent inhibition compared to the DHEA concentration generated in the absence of test compounds (l-(nM DHEA fo ⁇ ned in the presence of test compound/nM DHEA formed in the absence of test compounds) x 100).
• Human 293 lyase cells were prepared as described above for the Sf9 cells [Baculovirus Expression of Bovine Cytochrome P 45 o in Sf9 Cells and Comparison with Expression in Yeast, Mammalian Cells, and E.Coli. Barnes, H. J.; Jenkins, C. M.; Waterman, M. R. Archives of Biochemistry and Biophysics (1994) 315 (2) 489-494].
• the cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) /10% FBS/ l%S/P/l%L-Glu/0.8mg/mLG418/HEPES.
• DMEM Dulbecco's Modified Eagle's Medium
• human 293 lyase cells were plated at 10,000 cells/well/lOO ⁇ L in columns 2-12 of a 96-well tissue culture plate (Falcon 3075), and allowed to attach overnight (each mother plate needs two cell plates).
• DHEA standard was diluted with RPMI (4.5 ⁇ L of 500 ⁇ M into 3 mL RPMI, then 1:3 serial dilutions). The media from columns 2-12 of the cell plate was removed and replaced with 100 ⁇ L RPMI without phenol red. Diluted DHEA standards (100 ⁇ L) at a concentration of 750, 250, 83.3, 27.7, 9.2, 3, 1 and 0.3 nM were added to column 1 of the cell plate. 50 ⁇ L of 100% DMSO was added to columns 1 and 2 of the mother plate.
• the raw data was converted to a concentration of DHEA formed (nM) by use of the standard curve.
• the lyase inhibitory activity of the compounds was determined as the amount of DHEA formed in the presence of compound compared to the amount formed in the absence of compound in the form of a percent inhibition (1- (nM DHEA formed with compound/nM DHEA formed without compound) x 100).
• test compound was considered to be active if the IC 5 o in the human C 17,20 biochemical assay or in the human C 17,20 cellular assay was less than 10 ⁇ M. All the compounds tested have IC 5 o in the human C 17,20 biochemical assay or the human C 17,20 cellular assay of less than 10 ⁇ M.

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  • 2002-09-26 CA CA002461363A patent/CA2461363A1/en not_active Abandoned
  • 2002-09-26 WO PCT/US2002/030924 patent/WO2003027100A1/en not_active Ceased
  • 2002-09-26 JP JP2003530682A patent/JP2005528325A/ja not_active Withdrawn
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  • 2002-09-26 WO PCT/US2002/030483 patent/WO2003027085A2/en not_active Ceased

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