Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
  • C07D265/04—1,3-Oxazines; Hydrogenated 1,3-oxazines
  • C07D265/12—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems
  • C07D265/14—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems condensed with one six-membered ring
  • C07D265/18—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems condensed with one six-membered ring with hetero atoms directly attached in position 2
• • 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
  • C07D413/02—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
  • C07D413/10—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 linked by a carbon chain containing aromatic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/04—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

Definitions

• This invention relates to compounds that antagonists of the progesterone receptor, their preparation and utility.
• Intracellular receptors form a class of structurally related gene regulators known as "ligand dependent transcription factors" (R. M. Evans, Science, 240, 889, 1988).
• 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 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 ofthe hormone is an antagonist.
• PR antagonists may be used in contraception. In this context they may be administered alone (Ulmann, et al, Ann. N. Y. Acad. Sci., 261, 248, 1995), in combination with a PR agonist (Kekkonen, et al, Fertility and Sterility, 60, 610, 1993) or in combination with a partial ER antagonist such as tamoxifen (WO 96/19997 Al July 4, 1996). 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.
• 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 (Murphy, et al, J. Clin. Endo. Metab., 76, 513, 1993) and endometriosis (Kettel, et al, Fertility and Sterility, 56, 402, 1991).
• PR antagonists may also be useful in hormone replacement therapy for post menopausal patients in combination with a partial ER antagonist such as tamoxifen (US 5719136).
• PR antagonists such as mifepristone and onapristone, 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).
• the compounds of this invention have been shown to act as competitive inhibitors of progesterone binding to the PR and act as antagonists in functional models, either/or in-vitro and in-vivo. These compounds may be used for contraception, in the treatment of fibroids, endometriosis, breast, uterine, ovarian and prostate cancer, and post menopausal hormone replacement therapy.
• Narr et al. German Patent, DE 3633861, CA 109:22973 claimed that imidazobenzoxazinones, e.g. A, as cardotonics; Benzoxazin-2-ones, such as brofoxine (B), being active as an anxiolytic was reported by Hartmann et al. (Proc. West. Pharmacol. Soc. 21, 51-55 (1978)); More recently, a number of patents (e.g. Young et al. WO95/20389; Christ et al. W098/14436) claimed quinazolin-2-ones and benzoxazin-2-ones such as compound CI and C2 as inhibitors of HIV reverse transcriptase.
• the compounds in the present invention contain a pendent aromatic substituent.
• the aromatic substituents proved to be critical for the resultant compounds being active as progesterone receptor modulators and have broad structural diversity which may consist of aryl, substituted aryl, heteroaryl or substituted heteroaryl group.
• This invention provides compounds of Formula (I):
• R 1 and R 2 are independent substituents selected from the group of H, C, to C 6 alkyl, substituted C, to C 6 alkyl, C 2 to C 6 alkenyl, substituted C 2 to C 6 alkenyl, C 2 to
• R 1 and R 2 are fused to form: a) an optionally substituted 3 to 8 membered spirocyclic alkyl ring; b) an optionally substituted 3 to 8 membered spirocyclic alkenyl; or c) an optionally substituted 3 to 8 membered heterocyclic ring containing one to three heteroatoms from the group including O, S and N; the spirocyclic rings of a), b) and c) being optionally substituted by from 1 to 4 groups selected from fluorine, C, to C 6 alkyl, C, to C 6 alkoxy, C, to C 6 thioalkyl, -CF 3 ,
• R B is H, C, to C 3 alkyl, or substituted to C 3 alkyl
• R 3 is H, OH, NH 2 , C, to C 6 alkyl, substituted C, to C 6 alkyl, C 3 to C 6 alkenyl, substituted C, to C 6 alkenyl, alkynyl, or substituted alkynyl, COR c ;
• R c 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 4 is H, halogen, CN, N0 2 , C, to C 6 alkyl, substituted C, to C 6 alkyl, alkynyl, or substituted alkynyl, C, to C 6 alkoxy, substituted C, to C 6 alkoxy, amino, C, to C 6 aminoalkyl, or substituted C, to C 6 aminoalkyl;
• R 5 is selected from a) or b) a) R 5 is a trisubstituted benzene ring containing the substituents X, Y and
• X is taken from the group including halogen, CN, C, to C 3 alkyl, substituted C j to C 3 alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, C, to C 3 alkoxy, substituted C, to C 3 alkoxy, C, to C 3 thioalkoxy, substituted C, to C 3 thioalkoxy, amino, C, to C 3 aminoalkyl, substituted C, to C 3 aminoalkyl, N0 2 , C, to C 3 perfluoroalkyl, 5 or 6 membered heterocyclic ring containing 1 to 3 heteroatoms, COR D , OCOR D , or NR E COR D ; R D is H, C, to C 3 alkyl, substituted C x to C 3 alkyl, aryl, substituted aryl, C ⁇ to C 3 alkoxy, substituted C, to C 3 alkoxy, C, to C 3 aminoal
• R E is H, C, to C 3 alkyl, or substituted C, to C 3 alkyl; Y and Z are independent substituents taken from the group including H, halogen, CN, N0 2 , amino, aminoalkyl, C, to C 3 alkoxy, C, to C 3 alkyl, or C, to C 3 thioalkoxy; or b) R 5 is a five or six membered ring with 1 , 2, or 3 heteroatoms from the group including O, S, SO, S0 2 or NR 6 and containing one or two independent substituents from the group including H, halogen, CN, N0 2 amino, and C, to C 3 alkyl, C, to C 3 alkoxy, C, to C 3 aminoalkyl, COR F , or NR G COR F ;
• R F 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 G is H, C, to C 3 alkyl, or substituted C, to C 3 alkyl;
• R 6 is H or C, to C 3 alkyl; or pharmaceutically acceptable salt thereof.
• Preferred compounds ofthis invention include those of Formula I
• R 1 is H, C, to C 6 alkyl, substituted C, to C 6 alkyl, C 3 to C 8 cycloalkyl, substituted C 3 to C 8 cycloalkyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, COR A , orNR B COR A ;
• R 2 is H, C, to C 6 alkyl, substituted C, to C 6 alkyl, C 2 to alkenyl, substituted C 2 to C 6 alkenyl, C 3 to C 8 cycloalkyl, substituted C 3 to C 8 cycloalkyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, COR A , or NR B COR A ; or R 1 and R 2 are fused to form spirocyclic alkyl as a 3 to 8 membered spirocyclic ring, substituted spirocyclic alkyl constructed by fusing R 1 and R 2 to form a
• R A 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 B is H, C, to C 3 alkyl, or substituted C, to C 3 alkyl;
• R 3 is H, OH, NH 2 , C, to C 6 alkyl, substituted C, to C 6 alkyl, C 3 to C 6 alkenyl, substituted C, to C 6 alkenyl, alkynyl, or substituted alkynyl, COR c ;
• R c is H, C, to C 4 alkyl, substituted C, to C 4 alkyl, aryl, substituted aryl, C, to C 4 alkoxy, substituted C, to C 4 alkoxy, C, to C 4 aminoalkyl, or substituted C, to C 4 aminoalkyl;
• R 4 is H, halogen, CN, N0 2 , C, to C 6 alkyl, substituted C, to C 6 alkyl, C, to C 6 alkoxy, substituted C, to C 6 alkoxy, amino, C, to C 6 aminoalkyl, or substituted C, to C 6 aminoalkyl;
• R 5 is a trisubstituted benzene ring containing the substituents X, Y and Z as shown below:
• X is taken from the group including halogen, 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 thioalkoxy, substituted C, to C 3 thioalkoxy, amino, C, to C 3 aminoalkyl, substituted C, to C 3 aminoalkyl, N0 2 , C, to C 3 perfluoroalkyl, 5 membered heterocyclic ring containing 1 to 3 heteroatoms, COR D , OCOR 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
• R E is H, C, to C 3 alkyl, or substituted C, to C 3 alkyl
• Y and Z are independent substituents taken from the group including H, halogen, CN, N0 2 , C, to C 3 alkoxy, C, to C 3 alkyl, or C, to C 3 thioalkoxy; or
• R 5 is a five or six membered ring with 1 , 2, or 3 heteroatoms from the group including O, S, SO, S0 2 or NR 6 and containing one or two independent substituents from the group including H, halogen, CN, N0 2 amino, and C ⁇ to C 3 alkyl, or C, to C 3 alkoxy;
• R 6 is H, or C, to C 3 alkyl; or pharmaceutically acceptable salt thereof.
• R 1 R 2 and are selected from the group which includes C, to C 3 alkyl, substituted C, to C 3 alkyl, spirocyclic alkyl constructed by fusing R 1 and R 2 to form a 3 to 6 membered spirocyclic ring;
• R 3 is H, OH, NH 2 , C, to C 6 alkyl, or substituted C, to C 6 alkyl, COR c ;
• R c is H, C, to C 4 alkyl, or C, to C 4 alkoxy;
• R 4 is H, halogen, CN, N0 2 , C, to C 3 alkyl, substituted C, to C 3 alkyl, C, to C 3 alkoxy, or substituted C, to C 3 alkoxy;
• R 5 is a disubstituted benzene ring containing the substituents X, and Y as shown below:
• X is taken from the group including halogen, CN, C, to C 3 alkoxy, C, to C 3 alkyl, N0 2 , C, to C 3 perfluoroalkyl, 5 membered heterocyclic ring containing 1 to 3 heteroatoms, C, to C 3 thioalkoxy;
• Y is a substituent from the group including H, halogen, CN, N0 2 , C, to C 3 alkoxy, C, to C 4 alkyl, C, to C 3 thioalkoxy; or
• R 5 is a five membered ring with the structure
• U is O, S, or NR 6 ,
• R 6 is H, or C, to C 3 alkyl, or C, to C 4 C0 2 alkyl;
• X' is from the group including halogen, CN, N0 2 , or C, to C 3 alkyl and C, to C 3 alkoxy, provided that when U is NR 6 , then X' is not CN;
• Y' is from the group including H and C, to C 4 alkyl; or
• R 5 is a six membered ring with the structure:
• X 1 is N or CX 2 ;
• X 2 is halogen, CN, alkoxy, or N0 2 ; or pharmaceutically acceptable salt thereof
• R 1 R 2 and are selected from the group which includes CH 3 and spirocyclic alkyl constructed by fusing R 1 and R 2 to form a 6 membered spirocyclic ring
• R 3 is H, OH, NH 2 , CH 3 , substituted methyl, or COR c ;
• R c is H, C, to C 3 alkyl, or C, to C 4 alkoxy;
• R 4 is H, halogen, N0 2 , CN, or C, to C 3 alkyl;
• R 5 is a disubstituted benzene ring containing the substituents X and Y as shown below:
• X is taken from the group including halogen, CN, methoxy, N0 2 , or 2- thiazole;
• Y is H or F
• R is a five membered ring with the structure:
• U is O, S, or NH
• X' is halogen, CN, or N0 2 , provided that when U is NR 6 , X' is not CN; Y' is H or C, to C 4 alkyl and pharmaceutically acceptable salts.
• the compounds ofthis invention may contain an asymmetric carbon atom and some of the compounds ofthis 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 I, 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 one to eight carbon atoms, preferably one to six carbon atoms; "alkenyl” is intended to include both straight- and branched-chain alkyl group with at least one carbon-carbon double bond and two to eight carbon atoms, preferably two to six carbon atoms; “alkynyl” group is intended to cover both straight- and branched-chain alkyl group with at least one carbon-carbon triple bond and two to eight carbon atoms, preferably two to six carbon atoms.
• substituted alkyl refers to alkyl, alkenyl, and alkynyl as just described having from one to three substituents selected from the group including halogen, CN, OH, N0 2 , amino, aryl, heterocyclic, substituted aryl, substituted heterocyclic, alkoxy, aryloxy, substituted alkyloxy, alkylcarbonyl, alkylcarboxy, alkylamino, arylthio. These 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 refer 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 not limited to phenyl, naphthyl, biphenyl, anthryl, tetrohydronaphthyl, phenanthryl.
• substituted aryl refers to aryl as just defined having one to four substituents from the group including halogen, CN, OH, N0 2 , amino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryloxy, substituted alkyloxy, alkylcarbonyl, alkylcarboxy, alkylamino, or arylthio.
• heterocyclic is used herein to describe a stable 4- to 7-membered monocyclic or a stable multicyclic heterocyclic ring which is saturated, partially unsaturated, or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group including N, O, and S atoms.
• the N and S atoms may be oxidized.
• the heterocyclic ring also includes any multicyclic ring in which any of above defined heterocyclic rings is fused to an aryl ring.
• the heterocyclic ring may be attached at any heteroatom or carbon atom provided the resultant structure is chemically stable.
• heterocyclic groups include, for example, tetrahydrofuran, piperidinyl, piperazinyl, 2-oxopiperidinyl, azepinyl, pyrrolidinyl, imidazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, mo holinyl, indolyl, quinolinyl, thienyl, fiiryl, benzofuranyl, benzothienyl, thiamo ⁇ holinyl, thiamorpholinyl sulfoxide, and isoquinolinyl.
• substituted heterocyclic is used herein to describe the heterocyclic just defined having one to four substituents 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.
• alkoxy is used herein to refer to the OR group, where R is alkyl or substituted alkyl.
• aryloxy is used herein to refer to the OR group, where R is aryl or substituted aryl.
• alkylcarbonyl is used herein to refer to the RCO group, where R is alkyl or substituted alkyl.
• alkylcarboxy is used herein to refer to the COOR group, where R is alkyl or substituted alkyl.
• aminoalkyl refers to both secondary and tertiary amines wherein the alkyl or substituted alkyl groups, containing one to eight carbon atoms, which may be either same or different and the point of attachment is on the nitrogen atom.
• halogen refers to CI, Br, F, or I.
• the compounds of the present invention can be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases.
• These salts include, but are not limited to, the following salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and, as the case may be, such organic acids as acetic acid, oxalic acid, succinic acid, and maleic acid.
• Other salts include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium or magnesium in the form of esters, carbamates and other conventional "pro-drug" forms, which, when administered in such form, convert to the active moiety in vivo.
• This invention includes pharmaceutical compositions comprising one or more compounds of this invention and a pharmaceutically acceptable carrier or excipient.
• the invention also includes methods of treatment 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 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.
• Additional uses of the present progesterone receptor antagonists include the synchronization ofthe estrus in livestock.
• the compounds When the compounds are employed for the above utilities, they may be combined with one or more pharmaceutically acceptable carriers or excipients, for example, solvents, diluents and the like, and may be administered orally in such forms as tablets, capsules, dispersible powders, granules, or suspensions containing, for example, from about 0.05 to 5% of suspending agent, syrups containing, for example, from about 10 to 50% of sugar, and elixirs containing, for example, from about 20 to 50% ethanol, and the like, or parenterally in the form of sterile injectable solutions or suspensions containing from about 0.05 to 5% suspending agent in an isotonic medium.
• Such pharmaceutical preparations may contain, for example, from about 25 to about 90% of the active ingredient in combination with the carrier, more usually between about 5% and 60% by weight.
• the effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration and the severity of the condition being treated. However, in general, satisfactory results are obtained when the compounds of the invention are administered at a daily dosage of from about 0.5 to about 500 mg/kg of animal body weight, preferably given in divided doses two to four times a day, or in a sustained release form. For most large mammals, the total daily dosage is from about 1 to 100 mg, preferably from about 2 to 80 mg.
• Dosage forms suitable for internal use comprise from about 0.5 to 500 mg of the active compound in intimate admixture with a solid or liquid pharmaceutically acceptable carrier. This dosage regimen may be adjusted to provide the optimal therapeutic response.
• 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 prefe ⁇ ed pharmaceutical compositions from the standpoint of ease of preparation and administration are solid compositions, particularly tablets and hard- filled or liquid-filled capsules. Oral administration ofthe compounds is prefe ⁇ ed.
• active compounds may also be administered parenterally or intraperitoneally.
• Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
• Dispersions can also be prepared in glycerol, liquid, polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• 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. In all cases, the form must be sterile and must be fluid to the extent that easy syringe ability exits.
• 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.
• ethanol e.g., glycerol, propylene glycol and liquid polyethylene glycol
• suitable mixtures thereof e.g., glycerol, propylene glycol and liquid polyethylene glycol
• the compounds of this invention are generally prepared by employing the suitable coupling reaction as a final step.
• a latent coupling precursor such as alkoxy group which can be converted into OTf group suitable in the coupling reaction.
• a suitable organo metallic reagent e.g. Grignard reagent
• suitable nonprotic solvents which include but not limited to THF or ether to give ortho-amino carbinol 2 under an inert atmosphere such as argon or nitrogen at -78 °C to room temperature.
• Ring closure of carbinol 2 to yield benzoxazin-2-ones 3 is commonly effected by a condensing agent such as carbonyldiimidazole, phosgene, dimethylcarbonate, or diethylcarbonate in a suitable nonprotic solvent such as THF at temperatures ranging from room temperature to 65 °C.
• a condensing agent such as carbonyldiimidazole, phosgene, dimethylcarbonate, or diethylcarbonate
• a suitable nonprotic solvent such as THF
• transition metallic catalyst e.g., palladium or nickel complex often with phosphino ligands, e.g., Ph 3 P, 1,1 '- bis(diphenylphosphino)fe ⁇ ocene, 1 ,2-bis(diphenylphosphino)ethane or palladium salt such as palladium acetate.
• phosphino ligands e.g., Ph 3 P
• 1,1 '- bis(diphenylphosphino)fe ⁇ ocene 1,1 '- bis(diphenylphosphino)fe ⁇ ocene
• 1 ,2-bis(diphenylphosphino)ethane or palladium salt such as palladium acetate.
• an appropriately substituted nucleophilic reagent e.g., aryl boronic acid, arylstannane, or aryl zinc compound, is coupled with benzoxazinones 3 to give 4.
• the commonly used bases include but not limited to sodium bicarbonate, sodium carbonate, potassium phosphate, barium carbonate, potassium acetate, or cesium fluoride.
• the most commonly used solvents in these reactions include benzene, DMF, isopropanol, toluene, ethanol, DME, ether, acetone or a mixture of any one of these solvent and water.
• the coupling reaction is generally executed under an inert atmosphere such as nitrogen or argon at temperatures ranging from room temperature to the boiling point ofthe solvent or solvent system or mixture.
• Benzoxazinones 3 can be converted into a nucleophile such as boronic acid which can be coupled with an appropriate electrophile, e.g., aryl bromide or aryl iodide, to yield 4 employing the coupling reaction condition as described above.
• a nucleophile such as boronic acid
• an appropriate electrophile e.g., aryl bromide or aryl iodide
• the transformation of 3 into 5 can be effected by treating 3 with an organo metallic reagent, e.g., n-BuLi, in a nonprotic solvent such as THF or ether followed by quenching the reaction solution with a suitable electrophile such as trimethyl borate, triisopropyl borate, bishexalkyl tin reagent, or zinc chloride at temperatures ranging from-78 °C to reflux temperature under an inert atmosphere such as argon or nitrogen.
• organo metallic reagent e.g., n-BuLi
• a suitable electrophile such as trimethyl borate, triisopropyl borate, bishexalkyl tin reagent, or zinc chloride
• Scheme la illustrates an alternative approach leading to the benzoxazinones 3.
• an appropriate aniline 1 is protected with a suitable alkoxy carbonyl protective group including but not limited to allyloxy carbonyl, t-butoxy carbonyl, benzoxy carbonyl, ethoxy carbonyl, or methoxy carbonyl in a suitable solvent such as THF, acetonitrile, with or without presence of a base either as a catalyst or as an acid scavenger.
• the protected aniline is then treated with a suitable organo metallic reagent such as organo lithium agent or Grignard reagent in the same fashion as to prepare compound 2 to give the carbinol 6.
• Coupling of amide 8 with an aryl electrophile such as aryl boronic acid or arylstannane to give 9 can be effected by employing a typical coupling reaction such as Suzuki, Stille coupling procedure in a similar fashion as described for the preparation of benzoxazinones 4.
• a typical coupling reaction such as Suzuki, Stille coupling procedure in a similar fashion as described for the preparation of benzoxazinones 4.
• organo metallic compounds e.g., alkyllithium, alkynyllithium, aryllithium, or their Grignard counterpart in a nonprotic solvent such as THF or ether under an inert atmosphere such as argon or nitrogen at -78 ° to room temperature affords amino ketone 10.
• Conversion of ketone 10 to carbinol 11 can be effected by treatment of 10 with an organo metallic reagent such as alkyl, alkynyl, or aryl Grignard compound in a nonprotic solvent such as THF or ether under an inert atmosphere such as argon or nitrogen at -78 °C to room temperature.
• Conversion of ketone 10 to carbinol 11 can also be effected by reduction of ketone group of 10 to the carbinol moiety of 11 using an appropriate reducing reagent such as lithium aluminum hydride, sodium borohydride in a suitable solvent such as THF, ether, or anhydrous alcohol under an inert atmosphere in the temperature ranging from 0 °C to the boiling point of the solvent.
• Ring closure of carbinol 11 to produce the compounds of this invention can be accomplished with condensing agents such as carbonyldiimidazole, phosgene, dimethylcarbonate, or diethylcarbonate in a suitable nonprotic solvent such as THF at temperatures ranging from room temperature to 65 °C.
• CDI or triphosgene, THF 0 degrees C to 65 degrees C
• ortho-amino ketone 10 can be prepared by treatment of ortho- amino benzonitrile 14 with an organo metallic compound such as organo lithium reagent or Gringard reagent in a suitable solvent such as THF or ether under an inert atmosphere such as argon or nitrogen at temperatures ranging from -78 °C to room temperature as illustrated in Scheme III.
• organo metallic compound such as organo lithium reagent or Gringard reagent
• a suitable solvent such as THF or ether
• an inert atmosphere such as argon or nitrogen
• Benzonitrile 14 can be readily prepared from an appropriately substituted benzonitrile such as bromobenzonitrile 13 using a suitable coupling reaction such as Stille or Suzuki protocol carried out in a similar fashion as described for the preparation ofthe Weinreb amide 9.
• Scheme IV depicts an approach to prepare benzoxazinones with a low perfluoroalkyl substituent at position-4, e.g. R ⁇ is trifluorormethyl group.
• An appropriately substituted chloroaniline 15 was protected with a suitable protective reagent such as pivaloyl chloride or di-tert-butyl pyrocarbonate to give protected aniline 16 in a suitable solvent such as acetonitrile, acetone, THF, methylene chloride, or a mixture of solvent such as methylene chloride and water under an inert atmosphere such as argon or nitrogen at temperatures ranging from 0 °C to 70 °C.
• a suitable base such as sodium carbonate, sodium bicarbonate, or potassium carbonate may be needed when the reaction produces an acid as a side-product such as hydrochloride.
• an appropriate alkyllithium such as n- butyllithium or -y-butyllithium followed by reaction with a low perfluorocarboxy derivatives, e.g., trifluoroacetyl chloride, 1 -(trifluoroacetyl)-imidazole, or ethyl trifluoroacetate in a nonprotic solvent such as ether or THF under an inert atmosphere such as argon or nitrogen at -78 °C to ambient temperature gives the protective ortho- amino ketones.
• an appropriate alkyllithium such as n- butyllithium or -y-butyllithium
• a low perfluorocarboxy derivatives e.g., trifluoroacetyl chloride, 1 -(trifluoroacetyl)-
• Subsequent removal of the protecting group can be effected by reaction of protected amino ketones with a suitable acid such as TFA, 3N aqueous hydrochloride solution in a suitable solvent such as methylene chloride or water at 0 °C to boiling point ofthe solvent to afford ortho-amino ketone 17.
• a suitable acid such as TFA, 3N aqueous hydrochloride solution in a suitable solvent such as methylene chloride or water at 0 °C to boiling point ofthe solvent to afford ortho-amino ketone 17.
• a suitable acid such as TFA, 3N aqueous hydrochloride solution in a suitable solvent such as methylene chloride or water at 0 °C to boiling point ofthe solvent to afford ortho-amino ketone 17.
• the preparation of 6-chlorobenzoxazinones 19 from 17 can be accomplished in the same fashion as described for the synthesis of benzoxazinone 12 from ketone 10.
• the coupling reaction of 19 with an appropriate aryl boronic acid can be accomplished in the presence of a suitable base such as potassium phosphate and a catalyst of nickel (0 or II) complex, e.g. a nickel complex of dppe, dppf, or triphenylphosphine.
• a suitable base such as potassium phosphate
• a catalyst of nickel (0 or II) complex e.g. a nickel complex of dppe, dppf, or triphenylphosphine.
• the most commonly used solvents in the reaction include dioxane or THF.
• the coupling reaction is generally executed under an inert atmosphere such as nitrogen or argon at temperatures ranging from ambient temperature to 95 °C.
• alkyl or substituted alkyl derivatives 20 can be formed by treatment of carbamate 12 or 6 with a suitable base such as sodium hydride in suitable solvent such as DMF under an inert atmosphere such as argon or nitrogen followed by addition of an appropriate electrophile such as alkyl or substituted alkyl bromide, iodide, or triflate.
• Such a transformation of 12 or 6 at position- 1 can also be effected using biphasic conditions as indicated in scheme V in which alkylation is executed using a biphasic catalyst such as tributylammonium bromide in a suitable solvent such as acetonitrile.
• a biphasic catalyst such as tributylammonium bromide in a suitable solvent such as acetonitrile.
• Another example of this type of modification includes, but is not limited to, the one depicted in scheme V where heating 12 or 6 with triethyl orthoformate affords 1 -substituted derivatives of compound 12 or 6.
• the acylation or carboxylation of the compound 12 or 6 at position- 1 to give compound 21 can be readily effected by treatment of 12 or 6 with a suitable acylating or carboxylating reagent such as di-t-butyl dicarbonate in the presence of a suitable basic catalyst such as DMAP in a suitable solvent such as acetonitrile under an inert atmosphere such as argon or nitrogen.
• a suitable acylating or carboxylating reagent such as di-t-butyl dicarbonate in the presence of a suitable basic catalyst such as DMAP in a suitable solvent such as acetonitrile under an inert atmosphere such as argon or nitrogen.
• the amination of position- 1 of compound 12 or 6 to give 22 can be accomplished using a suitable aminating reagent such as chloroamine in the presence of a suitable base, such as sodium hydride, in a suitable solvent, such as THF or diethyl ether, following literature procedures (Metlesics et al.
• reaction mixture was cooled to ambient temperature and quenched with a saturated aqueous ammonium chloride solution (20 mL). Ethyl acetate (50 mL) was added and organic layer was separated. The aqueous layer was extracted with ethyl acetate (3x15 mL). The combined organic layers were washed with brine and dried with MgS0 4 .
• EXAMPLE 8 6-(4-Chloro-phenyl)-4,4-dimethyI-l,4-dihydro-benzo[d][l,3]- oxazin-2-one Prepared according to Procedure A from 6-bromo-4,4-dimethyl-l,4-dihydro- benzo[d][l,3]oxazin-2-one and 4-chlorophenyl boronic acid.
• reaction mixture was cooled to ambient temperature and quenched with a saturated aqueous ammonium chloride solution (20 mL). Ethyl acetate (100 mL) was added and organic layer was separated. The aqueous layer was extracted with ethyl acetate (3x30 mL). The combined organic layers were washed with brine and dried with MgS0 4 .
• the white solid was washed with the distilled water (3x20 mL) and dissolved in a mixture of ethyl acetate (10 mL) and methanol (10 mL). The solution was applied on a pad of silica gel and eluted with a mixture of ethyl acetate and hexane (1 : 1).
• reaction solution was sti ⁇ ed at -78 °C for 30 minutes and treated with anhydrous DMF (100 mL). The mixture was allowed to warm to ambient temperature and was quenched with IN aqueous hydrochloride solution (1L). The product was extracted with ethyl acetate (3x450 mL). The extracts were washed with water, brine and dried (MgS0 4 ). After removal of solvent in vacuo, the title 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 33 2-Amino-5-bromo--/V-methoxy-7V-methyIbenzamide To a mixture of N, O-dimethylhydroxylamine hydrochloride (9.42g, 96 mmol) and triethyl amine (13.5 mL, 96 mmol) in ethanol and water (100 mL/10 mL) was added a solution of 5-bromoisatoic anhydride (20g, 74 mmol) in ethanol and water (100 mL/10 mL) at ambient temperature under nitrogen. The reaction mixture was heated at reflux for 3 hours.
• EXAMPLE 37 6-(3-ChlorophenyI)-4-methyI-4-propyn-l-yl-l,4-dihydro-benzofdl[l,3]oxazin-2- one Prepared from l-(4-amino-3'-chloro-biphenyl-3-yl)-ethanone and propynyl- magnesium bromide followed by treatment with CDI according to Procedure C.
• EXAMPLE 38 6-(3-Chlorophenyl)-4-ethynyl-4-methyl-l,4-dihydro-benzo[d][l,31oxazin-2-one Prepared from l-(4-amino-3'-chloro-biphenyl-3-yl)-ethanone (0.2g, 0.82 mmol) and ethynylmagnesium bromide followed by treatment with CDI according to procedure C.
• EXAMPLE 39 6-(3-ChIorophenyl)-4-methyl-4-phenyl-l,4-dihydro-benzo[d][l,3]oxazin-2-one Prepared from l-(4-amino-3'-chloro-biphenyl-3-yl)-ethanone (0.2g, 0.82 mmol) and phenylmagnesium bromide followed by treatment with CDI according to Procedure C.
• EXAMPLE 46 l-(2-Amino-5-chIoro-phenyl)-2,2,2-trifluoro-ethanone To a solution of N-(4-chlorophenyl)-2,2-dimethylpropanamide (6Jg, 30 mmol) in anhydrous THF (100 mL) under nitrogen at 0 °C was added a solution of n- BuLi (2.5M, 30 mL, 70 mmol) in hexane in a dropwise fashion. After addition, the solution was kept stirring at 0 °C for 40 minutes and treated with a solution of 1- (trifluoroacetyl)imidazole (9 mL, 78 mmol) in anhydrous THF (10 mL).
• 1- (trifluoroacetyl)imidazole 9 mL, 78 mmol
• reaction mixture was warmed to ambient temperature and kept for 18 hours.
• a saturated aqueous ammonium chloride solution 50 mL
• ethyl acetate 100 mL
• the organic layer was separated and the solvent was removed in vacuo.
• the residue obtained was suspended in 3N aqueous hydrochloride solution (50 mL) and heated at reflux overnight.
• the reaction solution was cooled to room temperature and treated with a cold ammonium hydroxide solution to a pH above 8.
• the aqueous mixture was extracted with ethyl acetate (3x50 mL) and the organic layers were washed with brine and dried (MgS0 4 ).
• 2-carbonitriIe 4-Dicyclopropyl-l,4-dihydro-2-oxo-2H-3,l-benzoxazin-6-yl)boronic acid was prepared from 2-amino-5-bromobenzoic acid according to Example 1, 2, and 4.
• the title compound was prepared according to Procedure B from (4,4- dicyclopropyl-l,4-dihydro-2-oxo-2H-3,l-benzoxazin-6-yl)boronic acid and 4-bromo- 2-thiophene carbonitrile.
• EXAMPLE 60 6-(3,5-difluoro-phenyl)-4,4-dimethyI-l,4-dihydrobenzo-[d][l,3]oxazin-2-one Prepared according to procedure B from (4,4-dimethyl-l,4-dihydro-2-oxo-2H- 3,l-benzoxazin-6-yl)boronic acid and l-bromo-3,5-difluorobenzene.
• the reaction was cooled to room temperature and ethyl acetate (100 mL) was added. The organic layers were washed twice with aqueous ammonium chloride (100 mL) and once with brine (100 mL), dried over magnesium sulfate and concentrated.
• N-(tert-Butoxycarbonylamino)-3-fluorobenzoic acid (Takagishi et al. Synlett 4, 360-2 (1992); mp 159-161 °C) was deprotected using trifluoroacetic acid to give o- amino benzoic acid which was treated with methylmagnesium bromide to afford o- amino dimethyl carbinol.
• the o-amino dimethyl carbinol (2.23 g, 13.2 mmol) was treated with U ' -carbonyldiimidizole (2.8 g, 17.2 mmol) in THF (20 mL) at 50 °C for 12 hours.
• reaction mixture was cooled to room temperature and ethyl acetate (100 mL) was added.
• the organic layer was washed twice with aqueous ammonium chloride (100 mL) and once with brine (100 mL), dried over magnesium sulfate and concentrated.
• EXAMPLE 75 3-[3-(4,4-Dimethyl-2-oxo-l,4-dihydro-2H-benzo[d][l,3]oxazin-6-yl)-phenyl]- propynenitrile
• acetonitrile and water 9 mL/3 mL/0.5 mL
• sodium iodide 11 mg, 0.072 mmol
• Chlorotrimethylsilane was then added to the above mixture in a dropwise manner. After addition, the mixture was heated at 50 °C for 72 hours. The reaction mixture was then cooled to rt and treated with 0.5 N aqueous HCl cold solution (50 mL). The precipitate obtained was collected on a filter and washed with water.
• 8-Fluoro-(l,4-dihydro-4,4-dimethyl-2-oxo-2H-3,l-benzoxin-6-yl)boronic acid was prepared from 6-bromo-8-Fluoro-4,4-dimethyl-dihydro-benzo[d][l,3]oxazin-2- one using the procedure of example 4.
• the title compound was prepared from 8-fluoro-(l,4-dihydro-4,4-dimethyl-2- oxo-2H-3,l-benzoxin-6-yl)boronic acid and 4-bromo-2-cyanothiophene according to procedure B.
• the reaction mixture was poured into aqueous saturated sodium bicarbonate solution (100 mL) and extracted with ethyl acetate (3 x 100 mL). The organic layers were combined, washed with water (100 mL) and 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 (30% ethyl acetate/hexane) to give the title compound as an off-white powder (0.7 g, 62%): mp 176 °C.
• reaction mixture was poured into water (50 mL) and extracted with ethyl ether (2 x 50 mL). The organic layers were combined, washed with brine (30 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 (30% ethyl acetate/hexane) to give a yellow oil which crystallized from 5% ethyl acetate/hexane to give the title compound as a bright yellow powder (350 mg, 45%): mp 125 °C.
• EXAMPLE 108 4-(l, 2-Dihydro-2-oxospiro[4H-3,l-benzoxazine-4,l-cyclohexan]-6-yl)-2- thiophenecarbonitrile Prepared according to Procedure B from spiro-(4, l '-cyclohexane-l,4-dihydro- 2-oxo-2H-3,l-benzoxazin-6-yl) boronic acid and 3-bromo-5-cyanothiophene. White crystals: mp 230-232 °C. IR (KBr) 2200 cm '.
• the compounds of this invention were tested in the relevant assay as described below and their potency are in the range of 0.01 nM to 5 ⁇ M in the in vitro assays and 0.001 to 300 mg kg in the in vivo assays.
• the in-vitro biology is determined by (1) competitive Radioligand Binding: using the A-form ofthe 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.
• hPR Binding assay This assay is carried out in accordance with: Pathirana, C; Stein, R.B.; Berger, T.S.; Fenical, W.; Ianiro, T.; Mais, D.E.; Tones, A.; Glodman, M.E., Nonsteroidal human progesterone receptor modulators from the marine alga cymoplia barbata, J. Steroid Biochem. Mol. Biol., 1992, 41, 733-738.
• the object ofthis 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 ofthe 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.
• Cells are then treated with reference or test compounds in experimental medium. Compounds are tested for antiprogestational activity in the presence of 3 nM progesterone. Twenty-four hr. after treatment, the medium is discarded, cells are washed three times with D-PBS (GIBCO, BRL). Fifty ⁇ l of cell lysis buffer (Promega, Madison, WI) is added to each well and the plates are shaken for 15 min in a Titer Plate Shaker (Lab Line Instrument, Inc.). Luciferase activity is measured using luciferase reagents from Promega. c. Analysis of Results:
• Each treatment consists of at least 4 replicates.
• Log transformed data are used for analysis of variance and nonlinear dose response curve fitting for both agonist and antagonist modes. Huber weighting is used to downweight the effects of outliers.
• EC 50 or IC 50 values are calculated from the retransformed values.
• JMP software SAS Institute, Inc. is used for both one-way analysis of variance and non-linear response analyses.
• Progesterone and trimegestone 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.
• Progestational activity Compounds that increase PRE-luciferase activity significantly (p ⁇ 0.05) compared to vehicle control are considered active.
• Antiprogestational activity Compounds that decrease 3 nM progesterone induced PRE-luciferase activity significantly (p ⁇ 0.05)
• EC 50 Concenfration of a compound that gives half-maximal increase PRE- luciferase activity (default-nM) with SE.
• IC 50 Concentration of a compound that gives half-maximal decrease in 3 nM progesterone induced PRE-luciferase activity (default-nM) with SE.
• T47D cell proliferation assay The objective ofthis 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). b.
• Treatment medium Minimum Essential Medium (MEM) (#51200-038GIBCO, BRL) phenol red-free supplemented with 0.5% charcoal stripped fetal bovine serum, lOOU/ml penicillin, 200 mg/ml streptomycin, and 2 mM GlutaMax (GIBCO, BRL). c. Cell culture
• Antiprogestins are tested in the presence of 0.03 nM trimegestone, the reference progestin agonist. Twenty-four hours after treatment, the medium is discarded and cells are labeled with 10 mM BrdU (Amersham Life Science, Arlington Heights, IL) in treatment medium for 4 hr. d. Cell Proliferation Assay
• BrdU incorporation is measured using a cell proliferation ELISA kit (#RPN 250, Amersham Life Science) according to manufacturer's instructions. Briefly, cells are fixed in an ethanol containing fixative for 30 min, followed by incubation in a blocking buffer for 30 min to reduce background. Peroxidase-labeled anti-BrdU antibody is added to the wells and incubated for 60 min. The cells are rinsed three times with PBS and incubated with 3,3'5,5'-tetramethylbenzidine (TMB) substrate for 10-20 min depending upon the potency of tested compounds. Then 25 ⁇ l of 1 M sulfuric acid is added to each well to stop color reaction and optical density is read in a plate reader at 450 nm within 5 min. e. Analysis of Results:
• 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
• T47D cell alkaline phosphatase assay The purpose ofthis 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. Culture medium: DMEM:F12 (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). b. Alkaline phosphatase assay buffer:
• Frozen T47D cells were thawed in a 37°C water bath and diluted to 280,000 cells/ml in culture 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 in a 5%> C0 2 /humidified atmosphere for 24 hr. d.
• Alkaline Phosphatase Enzyme Assay At the end of treatment, the medium was removed from the plate and fifty ⁇ l of assay buffer I was added to each well. The plates were shaken in a titer plate shaker for 15 min. Then 150 ⁇ l of assay buffer II was added to each well. Optical density measurements were taken at 5 min intervals for 30 min at a test wavelength of 405 nM. e. Analysis of Results: Analysis of dose-response data For reference and test compounds, a dose response curve is generated for dose (X-axis) vs. the rate of enzyme reaction (slope) (Y-axis).
• Progesterone and trimegestone 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.
• 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 ofthis 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 ofthe test compounds in oil
• 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 ofthe 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 ofthe uterine horns by scratching the antimesometrial luminal epithelium with a blunt 21 G needle. The confralateral horn 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-horn) and control (C-hom) uterine horns 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 Institute, Inc.
• JMP software is used for both oneway ANOVA and non-linear dose-response analyses, g. Reference Compounds:
• Body weight Mean total animal body weight (default-kg)
• D-horn 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 Concenfration of compound that gives half-maximal decrease in EC 50 progesterone induced decidual response (default-mg/kg)
• THF was treated at -78 °C under a blanket of nitrogen with t-butyllithium (7.4 mL, 12.5 mmol).
• the reaction solution was then slowly warmed to -20 °C, kept stirring for 1.5 hours, and treated with cyclobutanone (lmL, 13.4 mmol).
• the mixture was warmed to rt and quenched with brine (30 mL) and IN aqueous hydrogen chloride solution (10 mL) was added Ethyl acetate was added and the organic layer was separated and dried (MgS0 4 ). After removal ofthe solvent, the residue was purified by flash column chromatography (hexane: ethyl acetate/3 : 1 ) to give the alcohol which was used in next step without further purification.
• the reaction mixture was allowed to cool to ambient temperature, treated with 3-bromo-5-fluoro-benzonitrile (1.2 g, 6 mmol), [1,1 '- bis(diphenylphosphino)- fe ⁇ ocene]palladium (II) chloride (1 :1 complex with methylene chloride, 0.5g, 0.6 mmol), and sodium carbonate (2g, 19 mmol) in water (10 mL).
• the resulted solution was heated at 85 °C for 3 hours under a blanket of nitrogen, cooled to rt, and treated with brine (50 mL). Ethyl acetate (100 mL) was added, organic layer was separated, dried (MgS0 4 ), and evaporated.
• the title compound was prepared according to the procedure B from 3- bromo-5-fluorophenylacetonitrile and (l,4-dihydro-4,4-dimethyl-2-oxo-2H-3,l- benzoxazin-6-yl)boronic acid. A white solid was obtained and recrystallized from ethanol ether: mp 218-220.
• the title compound was prepared from 5-Bromo-2-fluorophenylacetonitrile and ( l,4-dihydro-4,4-dimethyl-2-oxo-2H-3,l-benzoxaxin-6-yl)boronic acid.
• EXAMPLE 145 4-(l ,4-Dihydro-4,4-dimethyl-2-oxo-2H-3, 1 -benzoxazin-6-yl)-2- furancarboxaldehyde oxime

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