Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
  • C07D209/04—Indoles; Hydrogenated indoles
  • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
  • C07D209/32—Oxygen atoms
  • C07D209/34—Oxygen atoms in position 2
• • 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
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/18—Feminine contraceptives
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/56—Ring systems containing three or more rings
  • C07D209/96—Spiro-condensed ring systems
• • 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
  • C07D401/02—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 two hetero rings
  • C07D401/04—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 two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur 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
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/10—Spiro-condensed systems

Definitions

• Intracellular receptors form a class of structurally related gene regulators known as "ligand dependent transcription factors” (R. M. Evans, Science, 240, 889,
• the steroid receptor family is a subset of the IR family, including progesterone receptor (PR), estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR).
• PR progesterone receptor
• ER estrogen receptor
• AR antigen receptor
• GR glucocorticoid receptor
• MR mineralocorticoid receptor
• the natural hormone, or ligand, for the PR is the steroid progesterone, but synthetic compounds, such as medroxyprogesterone acetate or levonorgestrel, have been made which also serve as ligands.
• a ligand Once a ligand is present in the fluid surrounding a cell, it passes through the membrane via passive diffusion, and binds to the IR to create a receptor/ligand complex. This complex binds to specific gene promoters present in the cell's DNA. Once bound to the DNA the complex modulates the production of mRNA and protein encoded by that gene.
• a compound that binds to an IR and mimics the action of the natural hormone is termed an agonist, whilst a compound which inhibits the effect of the hormone is an antagonist.
• PR antagonists may also be useful for the treatment of hormone dependent breast cancers (Horwitz, et al, Horm. Cancer, 283, pub: Birkhaeuser, Boston, Mass., ed. Vedeckis) as well as uterine and ovarian cancers. PR antagonists may also be useful for the treatment of non-malignant chronic conditions such as fibroids (Mu ⁇ hy, et al, J. Clin. Endo. Metab., 76, 513, 1993) and endometriosis (Kettel, et al, Fertility and Sterility, 56, 402, 1991).
• hormone dependent breast cancers Horm. Cancer, 283, pub: Birkhaeuser, Boston, Mass., ed. Vedeckis
• PR antagonists may also be useful for the treatment of non-malignant chronic conditions such as fibroids (Mu ⁇ hy, et al, J. Clin. Endo. Metab., 76, 513, 1993) and endometriosis (Kettel, et al, Fer
• PR antagonists may also be useful in hormone replacement therapy for post menopausal patients in combination with a partial ER antagonist such as tamoxifen (U.S. Patent No. 5,719,136).
• PR antagonists such as mifepristone and onapristone
• PR antagonists have been shown to be effective in a model of hormone dependent prostate cancer, which may indicate their utility in the treatment of this condition in men (Michna, et al, Ann. N.Y. Acad. Sci., 761, 224, 1995).
• Rl F, Cl, Br, alkyl, NH 2
• R2 alkyl, alkoxy, F, Cl, NH2, CF 3
• Bohm, et al include the generic structure U (WO 91/04974).
• U JP 63112584 A contains the generic structure V:
• Boar, et al described the dioxoiane W as an intermediate for preparation of acetyl- cholinesterase inhibitors (WO 93/12085 Al).
• Kende, et al described methodology for preparing 3, 3 -substituted oxindoles, e.g. X, that was utilized in the present invention (Synth. Commun., 12, 1, 1982).
• This invention comprises compounds of the Formula 1:
• R, and R 2 are chosen independently from H, alkyl, substituted alkyl; OH; O(alkyl); O(substituted alkyl); OAc; aryl; optionally substituted aryl; heteroaryl; optionally substituted heteroaryl; alkylaryl; alkylheteroaryl; 1-propynyl; or 3- propynyl; or R, and R, are joined to form a ring comprising one of the following: -CH-(CH 2 ) ⁇ CH 2 -; -CH 2 CH 2 CMe 2 CH 2 CH 2 -; -O(CH 2 ) m CH 2 -; 0(CH 2 ) p O; -CH 2 CH 2 OCH 2 CH 2 -; or -CH 2 CH,N(H or alkyl)CH 2 CH 2 -; or R, and R- comprise a double bond to CMe 2 , C(cycloalkyl), O, or C(cyloether); n is
• R 3 is selected from H, OH, NH,, C, to alkyl, substituted C, to C 6 alkyl, C 3 to C 6 alkenyl, alkynyl or substituted alkynyl, or COR A ;
• R A is selected from H, C, to C 3 alkyl, substittited C, to C 3 alkyl, C, to C 3 alkoxy, substituted C, to C 3 alkoxy, C, to C 3 aminoalkyl, or substituted C, to C 3 aminoalkyl;
• R 4 is selected from H, halogen, CN, NH,, C, to C 6 alkyl, substituted C, to C 6 alkyl, C, to C 6 alkoxy, substituted C, to C 6 alkoxy, C, to C 6 aminoalkyl. or substituted C, to C 6 aminoalkyl;
• R 5 is selected from the groups a), b) or c): a) R 5 is a trisubstituted benzene ring containing the substituents X, Y and Z as shown below:
• X is selected from the group of halogen, OH, CN, C, to C 3 alkyl, substituted C, to C 3 alkyl, C, to C 3 alkoxy, substituted C, to C 3 alkoxy, C, to C 3 thioalkyl, substituted C, to C 3 thioalkyl, S(O)alkyl, S(O) 2 alkyl, C, to C 3 aminoalkyl, substituted C, to C 3 aminoalkyl, NO,, C, to C 3 perfluoroalkyl, 5 or 6 membered heterocyclic ring containing 1 to 3 heteroatoms, COR B , OCOR B , or NR c COR B ;
• R B is H, C, to C 3 alkyl. substituted C, to C 3 alkyl, aryl, substituted aryl, C, to C 3 alkoxy, substituted C, to C 3 alkoxy, C, to C 3 aminoalkyl, or substituted C, to C 3 aminoalkyl;
• R c is H, C, to C 3 alkyl, or substituted C, to C 3 alkyl; Y and Z are independently selected from H, halogen, CN, N0 2 , C, to C 3 alkoxy, C, to C 3 alkyl, or C, to C 3 thioalkyl; or b) R 5 is a five or six membered heterocyclic ring with 1, 2, or 3 heteroatoms selected from O, S, SO, SO, or NR 6 and containing one or two independent substituents from the group of H. halogen, CN, NO, and C, to C 3 alkyl, C, to C 3 alkoxy, C, to C 3 aminoalkyl. COR D , or NR E COR D ;
• R D is H, C, to C 3 alkyl, substituted C, to C 3 alkyl, aryl, substituted aryl, C, to C 3 alkoxy, substituted C, to C 3 alkoxy, C, to C 3 aminoalkyl, or substituted C, to C 3 aminoalkyl;
• R E is H, C, to C 3 alkyl, or substituted C, to C 3 alkyl
• R 5 is a disubstituted benzene ring containing the substiments X and Y as shown below:
• Another preferred group of this invention comprises compounds of formulas 2 and 2a wherein R 5 is a five membered ring with the structure shown below:
• R 6 is H, or C, to C 3 alkyl, C, to C 4 CO,alkyl, X' is selected from the group of halogen, CN, NO,, C, to C 3 alkyl or C, to C 3 alkoxy; with a proviso that, when X' is CN, U is not NR 6 ;
• Y' is selected from H, F, CN, NO, or C, to C 4 alkyl; or a pharmaceutically acceptable salt thereof.
• R 5 is a six membered ring with the structure shown
• X I is N or CX 2 , X 2 is halogen, CN or NO 2 ; or pharmaceutically acceptable salt thereof
• the compounds of this invention may contain an asymmetric carbon atom and some of the compounds of this invention may contain one or more asymmetric centers and may thus give rise to optical isomers and diastereomers. While shown without respect to stereochemistry in Formula 1 and 2 the present invention includes such optical isomers and diastereomers; as well as the racemic and resolved, enantiomerically pure R and S stereoisomers; as well as other mixtures of the R and S stereoisomers and pharmaceutically acceptable salts thereof.
• alkyl is used herein to refer to both straight- and branched-chain saturated aliphatic hydrocarbon groups having 1 to 8 carbon atoms; "alkenyl” is intended to include both straight- and branched-chain alkyl group with 1 or 2 carbon- carbon double bonds and containing 2 to 8 carbon atoms; “alkynyl” group is intended to cover both straight- and branched-chain alkyl group with at least 1 or 2 carbon-carbon triple bonds and containing 2 to 8 carbon atoms.
• substituted alkyl refers to alkyl, alkenyl, and alkynyl as just described having one or more substituents from the group including halogen, CN, OH, NO,, amino, aryl, heterocyclic, substituted aryl, substituted heterocyclic, alkoxy, aryloxy, substituted alkyloxy, alkylcarbonyl, alkylcarboxy, alkylamino, arylthio.
• substituents may be attached to any carbon of alkyl, alkenyl, or alkynyl group provided that the attachment constitutes a stable chemical moiety.
• aryl is used herein to refers to an aromatic system which may be a single ring or multiple aromatic rings fused or linked together as such that at least one part of the fused or linked rings forms the conjugated aromatic system.
• the aryl groups include, but are not limited to, phenyl, naphthyl, biphenyl, anthryl, tetrohydronaphthyl, phenanthryl.
• substituted aryl refers to aryl as just defined having 1 to 4 substituents from the group including halogen, CN, OH, NO,, amino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryloxy, substituted alkyloxy, alkylcarbonyl, alkylcarboxy, alkylamino, or arylthio.
• heterocyclic groups include, but are not limited to, tetrahydrofuran, piperidinyl, piperazinyl, 2-oxopiperidinyl, azepinyl, pyrrolidinyl, imidazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, morpholinyl, indolyl, quinolinyl, thienyl, furyl, benzofuranyl, benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide, and isoquinolinyl.
• substituted heterocyclic is used herein to describe the heterocyclic just defined having 1 to 4 substiments selected from the group which includes halogen, CN, OH, N0 2 , amino, alkyl, substituted alkyl, cycloalkyl, alkenyl, substituted alkenyl, alkynyl, alkoxy, aryloxy, substituted alkyloxy, alkylcarbonyl, alkylcarboxy, alkylamino, or arylthio.
• thioalkyl is used herein to refer to the SR group, where R is alkyl or substituted alkyl, containing 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms.
• alkylcarboxy is used herein to refer to the COOR group, where R is alkyl or substituted alkyl, containing 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms.
• aminoalkyl refers to both secondary and tertiary amines wherein the alkyl or substituted alkyl groups, containing 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms, which may be either the same or different and the point of attachment is on the nitrogen atom.
• halogen refers to Cl, Br, F, or I.
• the compounds of this invention may be prepared according to the methods described below.
• oxindole 5 is treated with mixture a strong organo-metallic base (e.g. butyl lithium, lithium dusopropylamide, potassium hexamethyldisilazide) in an inert solvent (e.g. THF, diethyl ether) under nitrogen at reduce temperature (ca. -20 °C) (Kende, et al, Synth. Commun., 12, 1, 1982).
• a strong organo-metallic base e.g. butyl lithium, lithium dusopropylamide, potassium hexamethyldisilazide
• an inert solvent e.g. THF, diethyl ether
• excess electrophile such as an alkyl halide, preferably the iodide. If R, and R, are to be joined such as the product 6 contains a spirocycle at position 3, then the electrophile should be bifunctional, i.e.
• bromide 7 is reacted with a palladium salt (e.g. tetrakis(rriphenylphoshine)palladium(0)), in a suitable solvent (e.g. THF, dimethoxyethane, ethanol, toluene) at room temperamre under an inert atmosphere (argon, nitrogen).
• a palladium salt e.g. tetrakis(rriphenylphoshine)palladium(0)
• a suitable solvent e.g. THF, dimethoxyethane, ethanol, toluene
• the resultant mono-alkylated compound may be then isolated and re-subjected to the reaction conditions using R 2 -X, or alternatively used in-situ for the second alkylation with R,-X.
• This invention mcludes pharmaceutical compositions and treatments which comprise administering to a mammal a pharmaceutically effective amount of one or more compounds as described above as antagonists of the progesterone receptor.
• the progesterone receptor antagonists of this invention used alone or in combination, can be utilized in methods of contraception and the treatment and/or prevention of benign and malignant neoplastic disease.
• Specific uses of the compounds and pharmaceutical compositions of invention include the treatment and or prevention of uterine myometrial fibroids, endometriosis, benign prostatic hypertrophy; carcinomas and adenocarcinomas of the endometrium, ovary, breast, colon, prostate, pituitary, meningioma and other hormone-dependent tumors.
• active compounds may be administered orally as well as by intravenous, intramuscular, or subcutaneous routes.
• Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while liquid carriers include sterile water, polyethylene glycols, non-ionic surfactants and edible oils such as corn, peanut and sesame oils, as are appropriate to the nature of the active ingredient and the particular form of administration desired.
• Adjuvents customarily employed in the preparation of pharmaceutical compositions may be advantageously included, such as flavoring agents, coloring agents, preserving agents, and antioxidants, for example, vitamin E, ascorbic acid, BHT and BHA.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringe ability exits. It must be stable under conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacterial and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oil.
• the present invention may be further understood by the following non- limiting examples.
• di-/_. ⁇ -propylborate 25 cm 3 , 0.1 1 mol was added and the mixmre was allowed to warm to room temperamre. After 2 nrs. hydrochloric acid (IN, 500 cm 3 ) and ethyiacetate (500 cm 3 ) were added. The aqueous phase was extracted with ethyiacetate, then the combined organic layers were washed with water, brine, dried (MgSO 4 ) and evaporated.
• reaction mixmre was treated with 20 mL of sodium bicarbonate solution and was then extracted with ethyl acetate (2x50mL). The combined organic phases were washed with samrated brine, dried (MgSO 4 ). Recrystallization from ethanol gave 0.13 g of pure product, mp 227 - 228 °C.
• the title compound was prepared from (2'-oxo-2,3-dihydrospiro[cyclohexane- l,3'-[3H]indol]-5'-yl)boronic acid (3.2 g, 12.5 mmol) and 4-bromo-2-fluoro- nitrobenzene (3 g, 13.6 mmol) as described for example 18 (0.7 g, 16%) as a yellow solid: mp.
• the title compound was prepared from (2'-oxo-2,3-dihydrospiro[cyclohexane- l,3'-[3H]indol]-5'-yl)boronic acid (2.5 g, 10 mmol) and 5-bromo-2-fluoro- trifluoromethylbenzene (2 g, 8 mmol) as described for example 18, to afford the title compound (0.87 g, 30%) as a solid: mp.
• the title compound was prepared from (2'-oxo-2,3-dihydrospiro[cyclohexane- l,3'-[3H]indol]-5'-yl)boronic acid (2.8 g, 11 mmol) and 5-bromo-2-fluoro- nitrobenzene (2.7 g, 12.2 mmol) as described for example 18, to afford the title compound (2.5 g, 66%) as a solid: mp.
• the reaction solution was sti ⁇ ed at -78 °C for 30 minutes and treated with anhydrous DMF (100 mL).
• the mixmre was allowed to warm to ambient temperamre and was quenched with IN aqueous hydrochloride solution (IL).
• the solution was extracted with ethyl acetate(3x450 mL). The extracts were washed with water, brine and dried (MgSO 4 ). After removal of solvent in vacuo, the subtitled compound was obtained as a white solid (46g, 88.3%).
• a sample of the product was crystallized from hexane: mp 63-65 °C; IR (KBr) 1654 cm 1 .
• EXAMPLE 80 5 -(3-Cyano-5-fluorophenyl)-spiro[cyclopentane-l,3 , -[3H]indoll-2'(l'H)-one
• 3-cyano-5-fluoro-bromobenzene 0.5 g, 2.6 mmol
• tetrakis(triphenylphosphine)palladium(0) 0.2 g
• In-vitro potencies lie in the range 0.01 nM - 10,000 nM, and in-vivo potencies in the range 1 ⁇ g/kg to 100 mg/kg.
• A. In-vitro biology The in-vitro biology is determined by (1 ) competitive Radioligand Binding: using the A-form of the human progesterone receptor with progesterone as the radioligand; (2) co-transfection assay, which provides functional activity expressed as agonist EC50 and Antagonist IC50 values; (3) a T47D cell proliferation, which is a further functional assay which also provides agonist and antagonist data; and (4) T47D cell alkaline phosphatase assay, which is a further functional assay which also provides agonist and antagonist data.
• the object of this assay is to determine a compound's progestational or antiprogestational potency based on its effect on PRE-luciferase reporter activity in CV-1 cells co-transfected with human PR and PRE-luciferase plasmids.
• the materials methods used in the assay are as follows.
• a. Medium The growth medium was as follows: DMEM (BioWhittaker) containing 10% (v/v) fetal bovine serum (heat inactivated), 0.1 mM MEM non-essential amino acids, lOOU/ml penicillin, lOOmg/ml streptomycin, and 2 mM GlutaMax (GIBCO, BRL).
• the experimental medium was as follows: DMEM (BioWhittaker), phenol red-free, containing 10% (v/v) charcoal- stripped fetal bovine serum (heat-inactivated), 0.1 mM MEM non-essential amino acids, lOOU/ml penicillin, lOOmg/ml streptomycin, and 2 mM GlutaMax (GIBCO, BRL).
• DMEM BioWhittaker
• phenol red-free containing 10% (v/v) charcoal- stripped fetal bovine serum (heat-inactivated)
• 0.1 mM MEM non-essential amino acids lOOU/ml penicillin
• lOOmg/ml streptomycin lOOmg/ml streptomycin
• 2 mM GlutaMax mM GlutaMax
• Stock CV-1 cells are maintained in growth medium. Co-transfection is done using 1.2xl0 7 cells, 5 mg pLEM plasmid with hPR-B inserted at Sphl and BamHl sites, 10 mg pGL3 plasmid with two PREs upstream of the luciferase sequence, and 50 mg sonicated calf thymus DNA as carrier DNA in 250 ml. Electroporation is carried out at 260 V and 1,000 mF in a Biorad Gene Pulser II. After electroporation, cells are resuspended in growth medium and plated in 96-well plate at 40,000 cells/well in 200 ⁇ l. Following overnight incubation, the medium is changed to experimental medium.
• Progesterone and trimegestone are reference progestins and RU486 is the reference antiprogestin. All reference compounds are mn in full dose- response curves and the EC S0 or IC 50 values are calculated.
• Progestational activity Compounds that increase PRE-luciferase activity significantly (p ⁇ 0.05) compared to vehicle control are considered active.
• IC 50 Concentration of a compound that gives half-maximal decrease in 3 nM progesterone induced PRE-luciferase activity (default-nM) with SE. 3. T47D cell proliferation assay
• the objective of this assay is the determination of progestational and antiprogestational potency by using a cell proliferation assay in T47D cells.
• a compound's effect on DNA synthesis in T47D cells is measured.
• the materials and methods used in this assay are as follows. a. Growth medium: DMEM:F12 (1 :1) (GIBCO, BRL) supplemented with 10% (v/v) fetal bovine serum (not heat- inactivated), lOOU/ml penicillin, lOOmg/ml streptomycin, and 2 mM GlutaMax (GIBCO, BRL).
• Treatment medium Minimum Essential
• MEM Medium
• GIBCO GlutaMax
• c. Cell culture Stock T47 D cells are maintained in growth medium.
• BrdU incorporation assay cells are plated in 96-well plates (Falcon, Becton Dickinson Labware) at 10,000 cells/well in growth medium. After overnight incubation, the medium is changed to treatment medium and cells are cultured for an additional 24 hr before treatment.
• Trimegestone and medroxyprogesterone acetate (MPA) are reference progestins and RU486 is the reference antiprogestin. All reference compounds are run in full dose-response curves and the EC 50 or IC 50 values are calculated.
• EC 50 Concentration of a compound that gives half-maximal increase in BrdU incorporation with SE
• IC 50 Concentration of a compound that gives half-maximal decrease in 0.1 trimegestone induced BrdU incorporation with SE 4.
• This assay is to identify progestins or antiprogestins by determining a compound's effect on alkaline phosphatase activity in T47D cells.
• the materials and methods used in this assay are as follows. a. Culmre medium: DMEM:F 12 (1:1) (GIBCO, BRL) supplemented with 5% (v/v) charcoal stripped fetal bovine serum (not heat-inactivated), lOOU/ml penicillin, 100 ⁇ g/ml streptomycin, and 2 mM GlutaMax (GIBCO, BRL).
• Frozen T47D cells were thawed in a 37°C water bath and diluted to 280,000 cells/ml in culmre medium. To each well in a 96-well plate (Falcon, Becton Dickinson Labware), 180 ⁇ l of diluted cell suspension was added. Twenty ⁇ l of reference or test compounds diluted in the culture medium was then added to each well. When testing for progestin antagonist activity, reference antiprogestins or test compounds were added in the presence of 1 nM progesterone. The cells were incubated at 37 C C in a 5% CO,/humidified atmosphere for 24 hr. d.
• the primary in-vivo assay is the rat decidualization model which may be used to determine progestational effects of both agonists and antagonists.
• the secondary in-vivo assay is the rat ovulation inhibition model which is under development and hence the protocol is un-available.
• Rat decidualization assay The objective of this procedure is used to evaluate the effect of progestins and antiprogestins on rat uterine decidualization and compare the relative potencies of various test compounds.
• the materials and methods used in this assay are as follows. a. Methods: Test compounds are dissolved in 100% ethanol and mixed with corn oil (vehicle). Stock solutions of the test compounds in oil (MazolaTM) are then prepared by heating (-80 °C) the mixmre to evaporate ethanol. Test compounds are subsequently diluted with 100% corn oil or 10% ethanol in corn oil prior to the treatment of animals. No difference in decidual response was found when these two vehicles were compared. b.
• Rats are weighed and randomly assigned to groups of 4 or 5 before treatment.
• Test compounds in 0.2 ml vehicle are administered by subcutaneous injection in the nape of the neck or by gavage using 0.5 ml. The animals are treated once daily for seven days.
• animals are given the test compounds and a EC50 dose of progesterone (5.6 mg/kg) during the first three days of treatment. Following decidual stimulation, animals continue to receive progesterone until necropsy four days later.
• Dosing are prepared based upon mg/kg mean group body weight. In all studies, a control group receiving vehicle is included. Determination of dose-response curves is carried out using doses with half log increases (e.g.
• decidual induction Approximately 24 hr after the third injection, decidualization is induced in one of the uterine horns by scratching the antimesometrial luminal epithelium with a blunt 21 G needle. The contralateral hom is not scratched and serves as an unstimulated control. Approximately 24 hr following the final treatment, rats are sacrificed by CO2 asphyxiation and body weight measured. Uteri are removed and trimmed of fat. Decidualized (D-hom) and control (C-horn) uterine homs are weighed separately. f. Analysis of Results:
• the increase in weight of the decidualized uterine horn is calculated by D-horn/C-horn and logarithmic transformation is used to maximize normality and homogeneity of variance.
• the Huber M-estimator is used to down weight the outlying transformed observations for both dose-response curve fitting and one-way analysis of variance.
• JMP software SAS Instimte, Inc.
• AS Instimte, Inc. is used for both oneway ANOVA and non-linear dose-response analyses, g.
• Body weight Mean total animal body weight (default-kg)
• D-hom Wet weight of decidualized uterine horn (default-mg)
• C-horn Wet weight of control uterine horn (default-mg)
• Antiprogestational activity Compounds that decrease EC 50 progesterone induced decidualization significantly (p ⁇ 0.05)
• EC 50 for uterine weight Concentration of compound that gives half-maximal increase in decidual response (default-mg/kg)
• IC 50 for uterine weight Concentration of compound that gives half-maximal decrease in EC 50 progesterone induced decidual response (default-mg/kg)
• reaction mixmre was poured into water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The organic layers were combined, washed with brine (50 mL) and dried over magnesium sulfate. The solution was filtered, concentrated in vacuo, and the residue was purified by HPLC (Zorbax PRO, C18, lOu, 15A, 50 X 250mm; 35% Water/65% AcCN; 254NM; AMB. temp.) to give the title compound (200 mg, 35%) as a white powder, mp 199.5-201 °C.
• reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The organic layers were combined, washed with brine (50 mL) and dried over magnesium sulfate. The solution was filtered, concentrated in vacuo, and the residue was purified by flash column chromatography to give the product (0.7 g, 86%) as a tan powder, mp 163- 165 °C.
• reaction mixmre was diluted with dichloromethane (100 mL) and filtered through celite. The filtrate was poured into water (100 mL) and the layers were separated. The organic layer was washed with brine (50 mL) and dried over magnesium sulfate. The solution was filtered, concentrated in vacuo and the residue was purified by flash column chromatography on silica gel (40% ethyl acetate/hexane) to give the title compound (415 mg, 37%) as a yellow powder, mp 265 °C (dec).
• reaction mixmre was diluted with dichloromethane (100 mL) and filtered through celite. The filtrate was poured into water (100 mL) and the layers were separated. The organic layer was washed with brine (50 mL) and dried over magnesium sulfate. The solution was filtered, concentrated in vacuo and the residue was purified by flash column chromatography on silica gel (40% ethyl acetate/hexane) to give the title compound (650 mg, 41%) as a yellow powder, mp 150 - 153 °C.
• 5-(3-chlorophenyl)-4-methylspiro[cyclohexane-l,3-r3H]indol1-2(lH)-one A solution of the above 5-bromo-4-methyl-oxindole (0.1 g, 0.34 mmol) and tetrakis(triphenylphosphine) palladium (0.05 g, 0.04 mmol) in dimethoxyethane (10 mL) was stined under N 2 for 20 min. To this mixture was then added 3- chlorophenylboronic acid (0.065 g, 0.41 mmol) and sodium carbonate (0.1 g, 1.0 mmol) in water (3 mL).
• Oxalyl chloride (4 eq, 1.0 mL, 1 1 mmol) in DCM (40 mL) at -78 °C was treated with DMSO (8 eq, 1.62 mL, 22 mmol). After 2 min a solution of 2-(l,2- dihydro-2-oxospiro[cyclohexane-l,3-[3H]indol]-5-yl)butan-l,4-diol (1 eq, 0.78 g, 2.9 mmol) in DMSO:DCM (1:3, 5 mL) was added followed 15 min later by addition of triethylamine (18 eq, 7.2 mL, 52 mmol).
• the solution was removed from the cooling bath and allowed to reach room temperamre.
• the solution was filtered through celite, concentrated in vacuo and redissolved in MeOH (10 mL). A large excess of ammonium acetate was added and the solution was heated to 60 °C for lh then stored in a refrigerator for 16h.
• the solution was partitioned between DCM and water.
• the title compound was prepared from CAT-817819 (1.9 g, 7.8 mmol ) and 2-bromochlorobenzene (1.0 g, 5.2 mmol) according to the method for Example 18 to afford the title compound (0.68 g, 42%) as an off white solid: mp.
• the title compound was prepared from CAT-830083 (0.9 g, 3.9 mmol ) and

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