Diarylhydantoin compounds

Abstract

To provide: diarylhydantoin compounds, including diarylthiohydantoins; methods for synthesizing them; and methods for using them in the treatment of hormone refractory prostate cancer.SOLUTION: A method for treating a hyperproliferative disorder comprises administering a pharmaceutical composition of the present invention to a subject in need of such treatment, thereby treating the hyperproliferative disorder. The invention provides a series of compounds having strong antagonistic activities with minimal agonistic activities against AR.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to diarylhydantoin compounds, including diarylthiohydantoins, and methods for their synthesis and use in the treatment of hormone-refractory prostate cancer. This application claims priority from U.S. provisional applications Nos. 60 / 756,552, 60 / 750,351, and 60 / 680,835, the specifications of which are incorporated herein by reference. [Background technology]

[0002] BACKGROUND OF THE INVENTION Prostate cancer is the most common cancer of incidence in Western men and the second leading cause of cancer death. When the cancer is locally confined, the disease is curable by surgery or radiation therapy. However, 30% of such cancers recur with distant metastatic disease, and others present with diagnostically advanced disease. Advanced disease is treated by so-called androgen deprivation therapy, castration, and / or administration of antiandrogens. Castration reduces circulating levels of androgens and reduces androgen receptor (AR) activity. Administration of antiandrogens blocks AR function by competing with androgen binding, thus reducing AR activity. Although initially effective, these treatments rapidly fail, and the cancer becomes hormone refractory.

[0003] Recently, AR overexpression has been identified and confirmed as the cause of hormone-resistant prostate cancer.See Non-Patent Document 1, which is incorporated herein by reference.AR overexpression is sufficient to cause the progression of hormone-sensitive prostate cancer to hormone-resistant prostate cancer, suggesting that AR inhibitors that are better than current drugs can delay the progression of prostate cancer.It has been demonstrated that AR and its ligand binding are necessary for the growth of hormone-resistant prostate cancer, indicating that AR is still the target for this disease.It has also been demonstrated that AR overexpression converts antiandrogens from antagonists to agonists in hormone-resistant prostate cancer (AR antagonists inhibit AR activity, and AR agonists stimulate AR activity).The data from this study explain why castration and antiandrogens fail to prevent the progression of prostate cancer and reveal the unrecognized characteristics of hormone-resistant prostate cancer.

[0004] Bicalutamide (brand name: Casodex) is the most commonly used antiandrogen. It has an inhibitory effect on AR in hormone-sensitive prostate cancer, but fails to suppress AR when the cancer becomes hormone-resistant. Two weaknesses of current antiandrogens account for their failure to prevent the progression of prostate cancer from the hormone-sensitive stage to hormone-resistant disease and their failure to effectively treat hormone-resistant prostate cancer. One is their weak antagonist activity, and the other is their strong agonist activity when AR is overexpressed in hormone-sensitive prostate cancer. Therefore, better AR inhibitors with stronger antagonist activity and minimal agonist activity are needed to slow disease progression and treat lethal hormone-sensitive prostate cancer.

[0005] Nonsteroidal antiandrogens, such as bicalutamide, have been preferred over steroidal compounds for prostate cancer because they are more selective and have fewer side effects. This class of compounds is described in many patents, such as U.S. Pat. Nos. 5,629,292, 5,629,293, 5,629,294, 5,629,295, 5,629,296, and 5,629,296. all of which are incorporated herein by reference.

[0006] Although U.S. Patent No. 5,929,633 contains broad claims covering a very large number of compounds, synthetic routes are presented for only a small portion of these compounds, and pharmacological data is presented for only two of them, so that one skilled in the art cannot readily envision other specific compounds.

[0007] Because the mechanism of hormone-resistant prostate cancer is unknown, there is no biological system to test the compounds described in these patents for their effect on hormone-resistant prostate cancer.In particular, the ability of AR overexpression in hormone-resistant prostate cancer to switch inhibitors from antagonist to agonist has not been recognized.Some novel properties of hormone-resistant prostate cancer have been reported in PCT application US04 / 42221 and US05 / 05529, which are incorporated herein by reference.PCT international application US05 / 05529 provides a methodology for identifying the androgen receptor antagonistic and agonistic properties of compounds.However, for each compound produced, the time-consuming process of determining the antagonistic and agonistic properties of the compound must be determined.In other words, there is no method for accurately predicting the properties relevant to treating prostate cancer from the chemical structure of the compound alone. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 4,097,578 [Patent Document 2] U.S. Patent No. 5,411,981 [Patent Document 3] U.S. Patent No. 5,705,654 [Patent Document 4] International Publication No. 97 / 00071 Pamphlet [Patent Document 5] International Publication No. 00 / 17163 Brochure [Patent Document 6] US Patent Application Publication No. 2004 / 0009969 [Patent Document 7] U.S. Patent No. 5,434,176 [Non-patent literature]

[0009] [Non-Patent Document 1] Chen, C, D., Welsbie, DS, Tran, C., Baek, SH, Chen, R., Vessella, R., Rosenfeld, MG, and Sawyers, CL, Molecular determinants of resistance to antiandrogen therapy, Nat. Med., 10:33-39, 2004. Summary of the Invention [Problem to be solved by the invention]

[0010] There is a need for novel thiohydantoin compounds with desirable pharmacological properties, and synthetic routes for their preparation.Because activity is sensitive to small structural changes, one compound may be effective in treating prostate cancer, while a second compound may not be effective, even if it differs only slightly from the first compound, i.e., by the replacement of a single substituent.

[0011] Identification of compounds with high potency to antagonize androgen activity and minimal agonist activity should overcome hormone-resistant prostate cancer (HRPC) and prevent or delay the progression of hormone-sensitive prostate cancer (HSPC).Therefore, there is a need in the art for the identification of selective modulators of the androgen receptor, such as modulators that are nonsteroidal, non-toxic, and tissue-selective. [Means for solving the problem]

[0012] (Summary of the Invention) The present invention provides a series of compounds that possess potent antagonist activity with minimal agonist activity against AR, which inhibit the growth of hormone-refractory prostate cancer.

[0013] The present invention provides, for example, the following. (Item 1) Chemical formula: [ka] A compound having the formula: wherein X is selected from the group consisting of trifluoromethyl and iodine; W is selected from the group consisting of O and NR5; R5 is H, methyl, and [ka] selected from the group consisting of wherein D is S or O, E is N or O, and G is alkyl, aryl, substituted alkyl, or substituted aryl; or D is S or O, and EG together are C1-C4 lower alkyl, R1 and R2 together contain 8 or fewer carbon atoms and are selected from the group consisting of alkyl, substituted alkyl including haloalkyl, and, together with the carbon to which they are attached, a cycloalkyl group or a substituted cycloalkyl group; R3 is selected from the group consisting of hydrogen, halogen, methyl, C1-C4 alkoxy, formyl, haloacetoxy, trifluoromethyl, cyano, nitro, hydroxyl, phenyl, amino, methylcarbamoyl, methoxycarbonyl, acetamido, methanesulfonamido, methanesulfonyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl, and C1-C6 alkyl or C1-C6 alkenyl, wherein the C1-C6 alkyl or C1-C6 alkenyl is optionally substituted with hydroxyl, methoxycarbonyl, cyano, amino, amido, nitro, carbamoyl, or substituted carbamoyl, including methylcarbamoyl, dimethylcarbamoyl, and hydroxyethylcarbamoyl; R4 is selected from the group consisting of hydrogen, halogen, alkyl, and haloalkyl; and R3 is not either methylaminomethyl or dimethylaminomethyl, (Item 2) R5 [ka] Item 1. The compound according to item 1, (Item 3) The compound according to item 1, having the chemical formula [ka] and wherein R3 is selected from the group consisting of hydroxy, methylcarbamoyl, methylcarbamoylpropyl, methylcarbamoylethyl, methylcarbamoylmethyl, methylsulfonecarbamoylpropyl, methylaminomethyl, dimethylaminomethyl, methylsulfonyloxymethyl, carbamoylmethyl, carbamoylethyl, carboxymethyl, methoxycarbonylmethyl, methanesulfonyl, 4-cyano-3-trifluoromethylphenylcarbamoylpropyl, carboxypropyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl, methoxycarbonyl, 3-cyano-4-trifluoromethylphenylcarbamoyl, hydroxyethylcarbamoylethyl, and hydroxyethoxycarbonylethyl; and Compounds wherein R10 and R11 are both H, or F and H, or H and F, respectively. (Item 4) The compound according to item 3, wherein R10 and R11 are both H. (Item 5) The compound according to item 3, wherein R10 and R11 are F and H, respectively. (Item 6) The compound according to item 3, wherein R3 is methylcarbamoyl. (Item 7) The compound according to item 3, wherein R3 is methylcarbamoyl, and R10 and R11 are F and H, respectively. (Item 8) The compound according to item 1, R1 and R2 are independently methyl or, together with the carbon to which they are attached, a cycloalkyl group of 4 to 5 carbon atoms; and R3 is selected from the group consisting of carbamoyl, alkylcarbamoyl, carbamoylalkyl, and alkylcarbamoylalkyl, and R4 is H or F. thing. (Item 9) Item 9. The compound according to item 8, wherein R4 is 3-fluoro. (Item 10) The compound according to item 1, R1 and R2 are independently methyl or, together with the carbon to which they are attached, a cycloalkyl group of 4 to 5 carbon atoms; and R3 is selected from the group consisting of cyano, hydroxy, methylcarbamoyl, methylcarbamoyl-substituted alkyl, methylsulfonecarbamoyl-substituted alkyl, methylaminomethyl, dimethylaminomethyl, methylsulfonyloxymethyl, methoxycarbonyl, acetamido, methanesulfonamido, carbamoyl-substituted alkyl, carboxymethyl, methoxycarbonylmethyl, methanesulfonyl, 4-cyano-3-trifluoromethylphenylcarbamoyl-substituted alkyl, carboxy-substituted alkyl, 4-(1,1-dimethylethoxy)carbonyl)-1-piperazinyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl, hydroxyethylcarbamoyl-substituted alkyl, hydroxyethoxycarbonyl-substituted alkyl, and 3-cyano-4-trifluoromethylphenylcarbamoyl, and R4 is F; compound. (Item 11) The compound according to item 1, having the chemical formula: [ka] and wherein R3 is selected from the group consisting of methylcarbonyl, methoxycarbonyl, acetamido, and methanesulfonamido, and R4 is selected from the group consisting of F and H. compound. (Item 12) The compound according to item 1, having the chemical formula: [ka] and wherein R4 is selected from the group consisting of F and H; compound. (Item 13) Item 1. The compound according to item 1, wherein R1 and R2 together with the carbon to which they are attached are And then, [ka] That is, compound. (Item 14) A compound selected from Tier 1 and Tier 2 compounds. (Item 15) Chemical formula: [ka] Item 1. The compound according to item 1, having the formula: (Item 16) Chemical formula: [ka] Item 1. The compound according to item 1, having the formula: (Item 17) Chemical formula: [ka] Item 1. The compound according to item 1, having the formula: (Item 18) Chemical formula: [ka] Item 1. The compound according to item 1, having the formula: (Item 19) Chemical formula: [ka] Item 1. The compound according to item 1, having the formula: (Item 20) 19. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of items 1 to 19 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. (Item 21) A pharmaceutical composition comprising a therapeutically effective amount of a compound according to item 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. (Item 22) A pharmaceutical composition comprising a therapeutically effective amount of a compound according to item 9 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. (Item 23) 21. A method for treating a hyperproliferative disorder, comprising administering the pharmaceutical composition of item 20 to a subject in need of such treatment, thereby treating the hyperproliferative disorder. (Item 24) 24. The method of claim 23, wherein the composition is administered at a dosage of the compound ranging from about 0.001 mg / kg body weight / day to about 100 mg / kg body weight / day. (Item 25) 24. The method of claim 23, wherein the composition is administered at a dosage of the compound ranging from about 0.01 mg / kg body weight / day to about 100 mg / kg body weight / day. (Item 26) 24. The method of claim 23, wherein the composition is administered at a dosage of the compound ranging from about 0.1 mg / kg body weight / day to about 10 mg / kg body weight / day. (Item 27) 24. The method of claim 23, wherein the composition is administered at a dosage of about 1 mg / kg body weight / day of the compound. (Item 28) 22. A method for treating a hyperproliferative disorder, comprising administering the composition of claim 21 to a subject in need of such treatment, thereby treating the hyperproliferative disorder. (Item 29) The composition is administered in a range of about 0.1 mg / kg body weight / day to about 10 mg / kg body weight / day. 29. The method of claim 28, wherein the compound is administered in a dosage amount. (Item 30) 29. The method of claim 28, wherein the composition is administered at a dosage of about 1 mg / kg body weight / day of the compound. (Item 31) 24. The method of claim 23, wherein the hyperproliferative disorder is hormone-refractory prostate cancer. (Item 32) 24. The method of claim 23, wherein the compound is administered by intravenous injection, by injection into a tissue, intraperitoneally, orally, or nasally. (Item 33) 29. The method of claim 28, wherein the composition is administered orally. (Item 34) 24. The method of claim 23, wherein the composition has a form selected from the group consisting of a solution, a dispersion, a suspension, a powder, a capsule, a tablet, a pill, a time-release capsule, a time-release tablet, and a time-release pill. (Item 35) 29. The method of claim 28, wherein the composition has a form selected from the group consisting of a capsule, a tablet, and a pill. (Item 36) Item 37. The method according to Item 28, wherein the compound is selected from the group consisting of RD162', RD162'', RD169, or RD170, or a pharmaceutically acceptable salt thereof. 29. The method of claim 28, wherein the compound is N-methyl-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluorobenzamide [RD162] or a pharmaceutically acceptable salt thereof. (Item 38) Chemical formula: [ka] A method for synthesizing a diaryl compound of the formula: In a first polar solvent, compound I [ka] Compound II [ka] to form a mixture; heating the mixture; adding a second solvent, which may be the same as or different from the first polar solvent, and an aqueous acid solution to the mixture; refluxing the mixture; cooling the mixture and combining with water; and separating the diaryl compound from the mixture. It encompasses wherein R51 comprises an alkyl chain of 1 to 4 carbon atoms, R52 is selected from the group consisting of cyano, hydroxy, methylcarbamoyl, methylcarbamoyl-substituted alkyl, methylsulfonecarbamoyl-substituted alkyl, methylaminomethyl, dimethylaminomethyl, methylsulfonyloxymethyl, methoxycarbonyl, 3-cyano-4-trifluoromethylphenylcarbamoyl, carbamoyl-substituted alkyl, carboxymethyl, methoxycarbonylmethyl, methanesulfonyl, 4-cyano-3-trifluoromethylphenylcarbamoyl-substituted alkyl, carboxy-substituted alkyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl, hydroxyethylcarbamoyl-substituted alkyl, and hydroxyethoxycarbonyl-substituted alkyl, and R53 is selected from the group consisting of F and H. (Item 39) 39. The method of claim 38, wherein R51 comprises an alkyl chain of 1 to 2 carbon atoms, R52 is selected from the group consisting of carbamoyl and methylcarbamoyl, and R53 is F. (Item 40) Chemical formula: [ka] A method for synthesizing a compound of the formula: combining 4-isothiocyanato-2-trifluoromethylbenzonitrile and N-methyl-4-(1-cyanocyclobutylamino)-2-fluorobenzamide in dimethylformamide to form a first mixture; heating the first mixture to form a second mixture; adding an alcohol and an acid to the second mixture to form a third mixture; refluxing the third mixture to form a fourth mixture; cooling the fourth mixture; combining the fourth mixture with water and extracting the organic layer; isolating the compound from the organic layer. The method includes: (Item 41) A method for synthesizing the compound [RD162'] according to item 16, comprising: combining N-methyl-2-fluoro-4-(1,1-dimethyl-cyanomethyl)-aminobenzamide and 4-isothiocyanato-2-trifluoromethylbenzonitrile in DMF and heating to form a first mixture; adding an alcohol and an acid to the first mixture to form a second mixture; refluxing the second mixture; cooling the second mixture; combining the second mixture with water and extracting the organic layer; isolating the compound from the organic layer. The method includes: (Item 42) A method for synthesizing the compound [RD162″] according to item 17, comprising: combining N-methyl-2-fluoro-4-(1-cyanocyclopentyl)aminobenzamide, 4-isothiocyanato-2-trifluoromethylbenzonitrile, and DMF and heating under reflux to form a first mixture; adding an alcohol and an acid to the first mixture to form a second mixture; refluxing the second mixture; cooling the second mixture; combining the second mixture with water and extracting the organic layer; isolating the compound from the organic layer. The method includes: (Item 43) A method for synthesizing the compound [RD169] according to item 18, comprising: combining N,N-dimethyl 4-[4-(1-cyanocyclobutylamino)phenyl]butanamide, 4-isothiocyanato-2-trifluoromethylbenzonitrile, and DMF and heating under reflux to form a first mixture; adding an alcohol and an acid to the first mixture to form a second mixture; refluxing the second mixture; cooling the second mixture; combining the second mixture with water and extracting the organic layer; isolating the compound from the organic layer. The method includes: (Item 44) A method for synthesizing the compound [RD170] according to item 19, comprising: combining DMSO, dichloromethane, and oxalyl chloride to form a first mixture; adding 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanamide to the first mixture to form a second mixture; adding triethylamine to the second mixture to form a third mixture; warming the third mixture and quenching with aqueous NH4Cl to form a fourth mixture; extracting an organic layer from the fourth mixture; isolating the compound from the organic layer. The method includes: (Item 45) Chemical formula: [ka] A compound having the formula: R5 is CN or NO2 or SO2R 11 and R6 is CF3, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogenated alkyl, halogenated alkenyl, halogenated alkynyl, or halogen; A is sulfur (S) or oxygen (O), B is O or S or NR8, R8 is H, methyl, aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic or heterocyclic non-aromatic, cycloalkyl, substituted cycloalkyl, SO2R 11 , N.R. 11 R 12 , (CO)OR 11 , (CO)NR 11 R 12 , (CO)R 11 , (CS)R 11 , (CS)NR 11 R 12 , (CS)OR 11 , [ka] selected from the group consisting of D is S or O, E is N or O, and G is alkyl, aryl, substituted alkyl, or substituted aryl; or D is S or O, and EG together are C1-C4 lower alkyl, R1 and R2 are independently alkyl, haloalkyl, hydrogen, aryl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogenated alkenyl, halogenated alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic or heterocyclic non-aromatic, cycloalkyl, substituted cycloalkyl, or R1 and R2 are joined to form a heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, [ka] and forming a ring which may be X is carbon or nitrogen and can be at any position within the ring; and R3, R4, and R7 are independently hydrogen, halogen, methyl, methoxy, formyl, haloacetoxy, trifluoromethyl, cyano, nitro, hydroxyl, phenyl, amino, methylcarbamoyl, methylcarbamoyl-substituted alkyl, dimethylcarbamoyl-substituted alkyl, methoxycarbonyl, acetamido, methanesulfonamino, carbamoyl-substituted alkyl, methanesulfonyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl, hydroxyethylcarbamoyl-substituted alkyl, hydroxyl-substituted alkyl, hydroxyl-substituted alkenyl, carbamoyl-substituted alkenyl, methoxycarbonyl-substituted alkyl, cyano-substituted alkyl, [ka] , aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogenated alkenyl, halogenated alkynyl, SO2R 11 , N.R. 11 R 12 , N.R. 12 (CO)OR 11 , NH(CO)NR 11 R 12 , N.R. 12 (CO)R 11 , O(CO)R 11 , O(CO)OR 11 , O(CS)R 11 , N.R. 12 (CS)R 11 , NH(CS)NR 11 R 12 , N.R. 12 (CS)OR 11, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic or substituted heterocyclic non-aromatic, cycloalkyl, substituted cycloalkyl, haloalkyl, methylsulfonecarbamoyl-substituted alkyl, methylaminomethyl, dimethylaminomethyl, methylsulfonyloxymethyl, methoxycarbonyl, acetamido, methanesulfonamido, carbamoyl-substituted alkyl, carboxymethyl, methoxycarbonylmethyl, methanesulfonyl, 4-cyano-3-trifluoromethylphenylcarbamoyl-substituted alkyl, carboxy-substituted alkyl, 4-(1,1-dimethylethoxy)carbonyl)-1-piperazinyl, hydroxyethylcarbamoyl-substituted alkyl, hydroxyethoxycarbonyl-substituted alkyl, 3-cyano-4-trifluoromethylphenylcarbamoyl; R 11 and R 12 are independently hydrogen, aryl, aralkyl, substituted aralkyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogenated alkyl, halogenated alkenyl, halogenated alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic or heterocyclic non-aromatic, cycloalkyl, or substituted cycloalkyl; or R 11 and R 12 can be joined to form a ring, which can be heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic, cycloalkyl, or substituted cycloalkyl. (Item 46) 46. ​​The method of claim 45, wherein the compound has substantial androgen receptor antagonist activity and no substantial agonist activity against hormone-refractory prostate cancer cells. (Item 47) 10. A method comprising: providing at least one compound according to item 45; measuring the inhibition of androgen receptor activity for said compound and determining whether said inhibition is above a first predetermined level; measuring the compound's stimulation of androgen receptor activity in hormone-refractory cancer cells and determining whether the stimulation is below a second predetermined level; selecting the compound if said inhibition is above said first predetermined level and said stimulation is below said second predetermined level. A method comprising: (Item 48) Item 48. The method of item 47, wherein the predetermined level is the level of bicalutamide. (Item 49) 48. The method of claim 47, wherein the step of measuring the inhibition comprises measuring the inhibitory concentration (IC50) in the AR-responsive receptor system or the prostate-specific antigen-secreting system. (Item 50) 48. The method of claim 47, wherein the step of measuring stimulation comprises measuring the fold induction by increasing the concentration in the AR-responsive receptor system or the prostate-specific antigen-secreting system. (Item 51) 48. The method of claim 47, wherein the step of measuring inhibition and / or stimulation comprises measuring the effect of the compound on tumor growth in an animal.

[0014] The present invention relates to a compound having the chemical formula:

[0015] [ka] and a compound having the formula: wherein X is selected from the group consisting of trifluoromethyl and iodo; W is selected from the group consisting of O and NR5; and R5 is H, methyl, and

[0016] [ka] selected from the group consisting of wherein D is S or O, E is N or O, and G is alkyl, aryl, substituted alkyl, or substituted aryl; or D is S or O and EG together are C1-C4 lower alkyl, R1 and R2 together contain 8 or fewer carbon atoms and are selected from the group consisting of alkyl, substituted alkyl including haloalkyl, and, together with the carbon to which they are attached, a cycloalkyl group or a substituted cycloalkyl group; R3 is hydrogen, halogen, methyl, C1-C4 alkoxy, formyl, haloacetoxy , trifluoromethyl, cyano, nitro, hydroxyl, phenyl, amino, methylcarbamoyl, methoxycarbonyl, acetamido, methanesulfonamido, methanesulfonyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl, and C1-C6 alkyl or alkenyl, wherein the C1-C6 alkyl or alkenyl is optionally substituted with hydroxyl, methoxycarbonyl, cyano, amino, amido, nitro, carbamoyl, or substituted carbamoyl, including methylcarbamoyl, dimethylcarbamoyl, and hydroxyethylcarbamoyl; R4 is selected from the group consisting of hydrogen, halogen, alkyl, and haloalkyl; and R3 is not either methylaminomethyl or dimethylaminomethyl.

[0017] R5 is

[0018] [ka] It could be.

[0019] This compound has the chemical formula

[0020] [ka] and wherein R3 is selected from the group consisting of hydroxy, methylcarbamoyl, methylcarbamoylpropyl, methylcarbamoylethyl, methylcarbamoylmethyl, methylsulfonecarbamoylpropyl, methylaminomethyl, dimethylaminomethyl, methylsulfonyloxymethyl, carbamoylmethyl, carbamoylethyl, carboxymethyl, methoxycarbonylmethyl, methanesulfonyl, 4-cyano-3-trifluoromethylphenylcarbamoylpropyl, carboxypropyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl, methoxycarbonyl, 3-cyano-4-trifluoromethylphenylcarbamoyl, hydroxyethylcarbamoylethyl, and hydroxyethoxycarbonylethyl; and R10 and R11 are both H, or respectively F and H, or H and F. In certain embodiments, R10 and R11 can both be H, or respectively F and H. R3 can be methylcarbamoyl.

[0021] In some embodiments, R1 and R2 are independently methyl or, together with the carbon to which they are attached, a cycloalkyl group of 4 to 5 carbon atoms, and R3 is selected from the group consisting of carbamoyl, alkylcarbamoyl, carbamoylalkyl, and alkylcarbamoylalkyl, and R4 is H or F, or R4 is 3-fluoro.

[0022] In other embodiments, R1 and R2 are independently methyl or together with the carbon to which they are attached are a cycloalkyl group of 4 to 5 carbon atoms; R3 is selected from the group consisting of cyano, hydroxy, methylcarbamoyl, methylcarbamoyl-substituted alkyl, methylsulfonecarbamoyl-substituted alkyl, methylaminomethyl, dimethylaminomethyl, methylsulfonyloxymethyl, methoxycarbonyl, acetamido, methanesulfonamido, carbamoyl-substituted alkyl, carboxymethyl, methoxycarbonylmethyl, methanesulfonyl, 4-cyano-3-trifluoromethylphenylcarbamoyl-substituted alkyl, carboxy-substituted alkyl, 4-(1,1-dimethylethoxy)carbonyl)-1-piperazinyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl, hydroxyethylcarbamoyl-substituted alkyl, hydroxyethoxycarbonyl-substituted alkyl, and 3-cyano-4-trifluoromethylphenylcarbamoyl; and R4 is F.

[0023] The compounds of the present invention have the chemical formula:

[0024] [ka] and wherein R3 is selected from the group consisting of methylcarbonyl, methoxycarbonyl, acetamido, and methanesulfonamido, and R4 is selected from the group consisting of F and H.

[0025] The compounds of the present invention have the chemical formula:

[0026] [ka] and wherein R4 is selected from the group consisting of F and H.

[0027] In an embodiment of the invention, R1 and R2, together with the carbon to which they are attached, are:

[0028] [ka] .

[0029] Compounds of the invention may be those listed below in Tier 1, Tier 2, Tier 3, and / or Tier 4. Specific compounds of the invention include:

[0030] [ka] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound according to any of the preceding compounds, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

[0031] The present invention encompasses methods for treating hyperproliferative disorders, comprising administering such pharmaceutical compositions to a subject in need of such treatment, thereby treating the hyperproliferative disorder. The hyperproliferative disorder may be hormone-refractory prostate cancer. Dosages may range from about 0.001 mg / kg body weight / day to about 100 mg / kg body weight / day, about 0.01 mg / kg body weight / day to about 100 mg / kg body weight / day, about 0.1 mg / kg body weight / day to about 10 mg / kg body weight / day, or about 1 mg / kg body weight / day.

[0032] The compound may be administered intravenously, by tissue injection, intraperitoneally, orally, or nasally. The composition may have a form selected from the group consisting of a solution, dispersion, suspension, powder, capsule, tablet, pill, time-release capsule, time-release tablet, and time-release pill.

[0033] The compound administered can be selected from the group consisting of RD162', RD162'', RD169, or RD170, or a pharmaceutically acceptable salt thereof. The compound administered can be RD162 or a pharmaceutically acceptable salt thereof.

[0034] The present invention relates to a compound having the chemical formula:

[0035] [ka] The present invention provides a method for synthesizing a diaryl compound of the formula: In a first polar solvent, compound I

[0036] [ka] Compound II

[0037] [ka] to form a mixture, heating the mixture, adding a second solvent, the same as or different from the first polar solvent, and an aqueous acid to the mixture, refluxing the mixture, cooling the mixture and combining with water, and isolating the diaryl compound from the mixture, wherein R51 comprises an alkyl chain of 1 to 4 carbon atoms, and R52 is selected from the group consisting of cyano, hydroxy, methylcarbamoyl, methylcarbamoyl-substituted alkyl, methylsulfonecarbamoyl-substituted alkyl, methylaminomethyl, dimethylaminomethyl, and the like. , methylsulfonyloxymethyl, methoxycarbonyl, 3-cyano-4-trifluoromethylphenylcarbamoyl, carbamoyl-substituted alkyl, carboxymethyl, methoxycarbonylmethyl, methanesulfonyl, 4-cyano-3-trifluoromethylphenylcarbamoyl-substituted alkyl, carboxy-substituted alkyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl, hydroxyethylcarbamoyl-substituted alkyl, and hydroxyethoxycarbonyl-substituted alkyl; and R53 is selected from the group consisting of F and H.

[0038] R51 can include an alkyl chain of 1 to 2 carbon atoms, R52 can be selected from the group consisting of carbamoyl and methylcarbamoyl, and R53 can be F.

[0039] The present invention relates to a compound having the chemical formula:

[0040] [ka] the method includes the steps of: mixing 4-isothiocyanato-2-trifluoromethylbenzonitrile and N-methyl-4-(1-cyanocyclobutylamino)-2-fluorobenzamide in dimethylformamide to form a first mixture; heating the first mixture to form a second mixture; adding an alcohol and an acid to the second mixture to form a third mixture; refluxing the third mixture to form a fourth mixture; cooling the fourth mixture; combining the fourth mixture with water and extracting the organic layer; and isolating the compound from the organic layer.

[0041] Similarly, the present invention provides a method for synthesizing RD162', comprising the steps of mixing N-methyl-2-fluoro-4-(1,1-dimethyl-cyanomethyl)-aminobenzamide and 4-isothiocyanato-2-trifluoromethylbenzonitrile in DMF and heating to form a first mixture; and treating as described above.

[0042] The present invention also provides a method for synthesizing RD162″, the method comprising: 4-fluoro-4-(1-cyanocyclopentyl)aminobenzamide, 4-isothiocyanato-2-trifluoromethylbenzonitrile, and DMF and heating under reflux to form a first mixture, and treating as above.

[0043] The present invention further provides a method of synthesizing RD169, comprising combining and heating under reflux N,N-dimethyl 4-[4-(1-cyanocyclobutylamino)phenyl]butanamide, 4-isothiocyanato-2-trifluoromethylbenzonitrile, and DMF to form a first mixture; and treating as described above.

[0044] The present invention provides a method for synthesizing RD170, the method comprising: mixing DMSO, dichloromethane, and oxalyl chloride to form a first mixture; adding 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanamide to the first mixture to form a second mixture; adding triethylamine to the second mixture to form a third mixture; warming the third mixture and quenching with aqueous NH4Cl to form a fourth mixture; extracting an organic layer from the fourth mixture; and isolating the compound from the organic layer.

[0045] Further compounds according to the invention have the formula:

[0046] [ka] wherein R5 is CN or NO2 or SO2R11, R6 is CF3, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkyl halide, alkenyl halide, alkynyl halide, halogen, A is sulfur (S) or oxygen (O), B is O or S or NR8, R8 is H, methyl, aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic or heterocyclic non-aromatic, cycloalkyl, substituted cycloalkyl, SO2R11, NR11R12, (CO)OR11, (CO)NR11R12, (CO)R11, (CS)R11, (CS)NR11R12, (CS)OR11,

[0047] [ka] selected from the group consisting of D is S or O, E is N or O, and G is alkyl, aryl, substituted alkyl, or substituted aryl; or D is S or O, and EG together are C1-C4 lower alkyl, R1 and R2 are independently alkyl, haloalkyl, hydrogen, aryl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogenated alkenyl, halogenated alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic or substituted heterocyclic non-aromatic, cycloalkyl, substituted cycloalkyl, or R1 and R2 are joined to form a heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl,

[0048] [ka] and forming a ring which may be X is carbon or nitrogen and can be in any position within the ring; and R3, R4, and R7 are independently hydrogen, halogen, methyl, methoxy, formyl, haloacetoxy, trifluoromethyl, cyano, nitro, hydroxyl, phenyl, amino, methylcarbamoyl, methylcarbamoyl-substituted alkyl, dimethylcarbamoyl-substituted alkyl, methoxycarbonyl, acetamido, methanesulfonamino, carbamoyl-substituted alkyl, methanesulfonyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl, hydroxyethylcarbamoyl-substituted alkyl, hydroxyl-substituted alkyl, hydroxyl-substituted alkenyl, carbamoyl-substituted alkenyl, methoxycarbonyl-substituted alkyl, cyano-substituted alkyl,

[0049] [ka] , aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogenated alkenyl, halogenated alkynyl, SO2R11, NR11R12, NR12(CO)OR11, NH(CO)NR11R12, NR12(CO)R11, O(CO)R11, O(CO)OR11, O(CS)R11, NR12(CS)R11, NH(CS)NR11R12, NR12(CS)OR11, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic or heterocyclic non-aromatic, cycloalkyl, substituted cycloalkyl, haloal alkyl, methylsulfonecarbamoyl-substituted alkyl, methylaminomethyl, dimethylaminomethyl, methylsulfonyloxymethyl, methoxycarbonyl, acetamido, methanesulfonamido, carbamoyl-substituted alkyl, carboxymethyl, methoxycarbonylmethyl, methanesulfonyl, 4-cyano-3-trifluoromethylphenylcarbamoyl-substituted alkyl, carboxy-substituted alkyl, 4-(1,1-dimethylethoxy)carbonyl)-1-piperazinyl, hydroxyethylcarbamoyl-substituted alkyl, hydroxyethoxycarbonyl-substituted alkyl, 3-cyano-4-trifluoromethylphenylcarbamoyl; R and R are independently hydrogen, aryl, aralkyl, substituted aralkyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogenated alkyl, halogenated alkenyl, halogenated alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or non-aromatic, substituted heteroaromatic or non-aromatic, cycloalkyl, or substituted cycloalkyl, or R and R can be joined to form a ring which can be heteroaromatic or non-aromatic, substituted heteroaromatic, cycloalkyl, or substituted cycloalkyl.

[0050] Such compounds have substantial androgen receptor antagonist activity and no substantial agonist activity against hormone-refractory prostate cancer cells.

[0051] The present invention includes a method, which comprises the steps of: providing at least one such compound; measuring the inhibition of androgen receptor activity of the compound and determining whether the inhibition is above a first predetermined level; measuring the stimulation of androgen receptor activity in hormone-resistant cancer cells of the compound and determining whether the stimulation is below a second predetermined level; and selecting the compound if the inhibition is above the first predetermined level and the stimulation is below the second predetermined level. The predetermined level can be the level of bicalutamide. Measuring the inhibition can include measuring the inhibitory concentration (IC50) in the AR-responsive receptor system or the prostate-specific antigen secretion system. Measuring the stimulation can include measuring the induction fold of the AR-responsive receptor system or the prostate-specific antigen secretion system by increasing the concentration. The method for measuring inhibition and / or stimulation can include measuring the effect of the compound on tumor growth in animals.

[0052] The following figures present the results of pharmacological testing of certain compounds. [Brief explanation of the drawings]

[0053] [Figure 1] Graph showing that bicalutamide exhibits agonistic effects on LNCaP-AR. The agonistic activity of bicalutamide in hormone-resistant prostate cancer cells overexpressing AR. LNCaP-AR cells overexpressing AR were treated with increasing concentrations of bicalutamide in the absence of DMSO or R1881 as a vehicle. The activity of the AR-responsive reporter was measured. [Figure 2] Figure 1 shows the antagonist assay of bicalutamide on LNCaP-AR. The agonist activity of bicalutamide in hormone-sensitive prostate cancer. LNCaP cells were treated with increasing concentrations of bicalutamide in the absence of DMSO or R1881 as vehicle. The activity of the AR-responsive reporter was measured. [Figure 3]1 is a graph showing the effect of compounds on LNCaP-AR. [Figure 4] 1 is a graph showing the effect of compounds on LNCaP-AR. [Figure 5] 1 is a graph showing the inhibitory effect on LNCaP-AR. [Figure 6] In Figure 6, Example 5-3b is RD7, and Example 7-3b is RD37. Figure 6. Inhibition of proliferation of AR-overexpressing LNCaP cells. AR-overexpressing and androgen-deprived LNCaP cells were treated with increasing concentrations of test substances in the presence of DMSO as vehicle or 100 pM R1881. After 4 days of incubation, cell proliferation was measured by MTS assay. [Figure 7] In Figure 7, Example 5-3b is RD7, and Example 7-3b is RD37. Inhibitory effect on the growth of AR-overexpressing LNCaP xenograft model. Mice bearing established LN-AR xenograft tumors were randomly divided and orally treated with the indicated compounds once daily. Tumor size was measured by internal diameter. (A) Mice were treated with 1 mg / kg bicalutamide, Example 7-3b, or vehicle for 44 days. (B) Mice were treated with vehicle, 0.1, 1, or 10 mg / kg Example 7-3b for 44 days. [Figure 8] In Figure 8, Example 5-3b is RD7, and Example 7-3b is RD37. Inhibitory effect on PSA expression in AR-overexpressing LNCaP xenograft model. Mice were orally treated with vehicle, 0.1, 1, or 10 mg / kg of Example 7-3b once a day for 44 days. Tumors were removed from mice after 44 days of treatment, tumor lysates were extracted, and PSA levels in tissue lysates were determined by ELISA. [Figure 9]In Figure 9, Example 5-3b is RD7, and Example 7-3b is RD37. Inhibitory effects on growth and PSA in hormone-resistant LAPC4 xenograft models. Mice bearing established tumors were randomly divided and orally treated with 1 mg / kg bicalutamide, Example 7-3b, or vehicle once daily for 17 days. (A) Tumor size was measured by internal diameter. (B) Tumors were removed from the mice after 17 days of treatment, tumor lysates were extracted, and PSA levels in the tissue lysates were determined by ELISA. [Figure 10] In Figure 10, Example 5-3b is RD7, and Example 7-3b is RD37. Inhibitory effect on the proliferation of hormone-sensitive prostate cancer cells. Androgen-deprived LNCaP cells were treated with increasing concentrations of test substance in the presence of DMSO as vehicle or 1 pM R1881. After 4 days of incubation, cell proliferation was measured by MTS assay. [Figure 11] Graph of tumor size. AR-overexpressing LNCaP cells were subcutaneously injected into the flanks of castrated SCID mice. When tumors reached approximately 100 mm 3 , they were randomly divided into five groups. Each group had nine animals. After they reached this tumor volume, they were orally administered either vehicle, bicalutamide, or RD162 at 10 or 50 mg / kg daily. Tumors were measured three-dimensionally for width, length, and depth using calipers. [Figure 12] Figure 1 shows the experimental results of tumor size. On day 18, animals were imaged via an optical CCD camera 3 hours after the last dose of treatment. ROIs were drawn around the tumor for luciferase activity measurements in photons / second. The right panel shows the ROI measurements. [Figure 13] 1 is a graph showing the pharmacokinetic curves of RD162 from intravenous administration (top curve) and oral administration (bottom curve). [Figure 14] 1 is a graph showing PSA uptake measured for LN-AR cells after treatment with various doses of several compounds. [Figure 15]A table is presented providing some characteristics of the compounds. Figure 15 also presents a graph providing the pharmacokinetic characteristics of some compounds according to the serum concentration of the compound as a function of time. [Figure 16] This is a chart showing prostate weights after treatment with various compounds. 10, 25, or 50 mg of compound per kilogram of body weight was administered per day, as indicated by the bar labels. Compounds were administered to healthy FVB mice. After 14 days of compound treatment, urogenital tract weights were determined by removing and weighing the seminal vesicles, prostate, and bladder. Three mice were administered a given compound to obtain the data presented by the bars in the chart. One set of mice was not treated with compound; data are presented in the bar labeled "Untreated." Another set of mice was treated with vehicle solution only; data are presented in the bar labeled "Vehicle." [Figure 17] 7 is a graph presenting a PSA assay performed according to the experimental protocol presented in FIG. 6. [Figure 18] 1 is a graph presenting the effect of different dose regimens of RD162 on tumor volume. [Figure 19] 1 is a graph presenting the rate of photon emission associated with luciferase activity on day 17 compared to the rate on day 0 after treatment with RD162 at doses of 0.1, 1, and 10 mg / kg body weight / day, and without RD162. [Figure 20] Results are presented for an experiment in which SCID mice were injected with the LN-AR(HR) cell line to induce tumor growth. One set of mice was treated with the compound RD162 at a dose of 10 mg / kilogram body weight / day; the other set of mice was treated with vehicle solution only. (A) Relative tumor volume as a function of time for each set of mice. (B) Images of each set of mice with luciferase activity and associated photon emission at day 31 shown as color contours. (C) Rates of luciferase activity and associated photon emission at several time points for each set of mice. [Figure 21]1 is a graph presenting PSA uptake associated with LN-AR cells treated with various concentrations of RD162, RD162′, RD162″, and RD170, and vehicle solution. [Figure 22] 1 is a graph presenting PSA uptake associated with LN-CaP cells treated with various concentrations of RD37, RD131, RD162, bicalutamide, and DMSO. [Figure 23] Results are presented for experiments conducted using wild-type non-transgenic mice (WT), castrated luciferase transgenic mice (Cast), and non-castrated luciferase transgenic mice (Intact). Data are shown for castrated luciferase transgenic mice treated with implanted testosterone pellets, resulting in a 90-day release period of 12.5 mg / kilogram body weight (T / Cast). Data are shown for non-castrated luciferase transgenic mice treated with implanted testosterone pellets, resulting in a 90-day release period of 12.5 mg / kilogram body weight (Intact+T). Data are shown for castrated luciferase transgenic mice treated with implanted testosterone pellets and bicalutamide (BIC+T / Cast) or RD162 (RD162+T / Cast) at 10 mg / kilogram body weight / day. (A) Genitourinary tract weight on day 14. (B) Photon emission rate on day 14. In all cases, a hormone-resistant disease state was not induced. [Figure 24] 1 is a graph of luciferase activity in L1AR cell lines dosed with various compounds administered at concentrations ranging from 125 nmol to 1000 nmol. [Figure 25] 1 is a graph of luciferase activity in the LN / AR cell line for various compounds administered at concentrations ranging from 1.25 to 10 μmol. [Figure 26] 1 is a graph of luciferase activity in the 4AR cell line for various compounds administered at concentrations ranging from 1.25 to 10 μmol. [Figure 27]1 is a graph of PSA levels in the 1AR cell line for various compounds administered at concentrations ranging from 1.25 to 10 μmol. [Figure 28] 1 is a graph of PSA levels in the LN / AR cell line for various compounds administered at concentrations ranging from 125 nmol to 1000 nmol. [Figure 29] 1 is a graph of luciferase activity for various compounds administered at concentrations ranging from 125 nmol to 1000 nmol. DETAILED DESCRIPTION OF THE INVENTION

[0054] (Detailed explanation) Embodiments of the present invention are discussed in more detail below. In describing the embodiments, specific terminology is used for the sake of clarity. However, the present invention is not intended to be limited to the specific terminology so selected. Those skilled in the relevant art will recognize that other equivalents are available and other methods can be developed without departing from the spirit and scope of the present invention. All references cited herein are incorporated by reference as if each were individually incorporated.

[0055] (Synthesis of diarylhydantoin compounds) The present invention relates to a compound of the formula

[0056] [ka] where R71 comprises an alkyl chain of 1 to 4 carbon atoms. For example, R72 can be carbamoyl, e.g., -(CO)NH2, or methylcarbamoyl, e.g., -(CO)NHCH3. An amide group bonded to another structure at the carbonyl carbon atom is called a carbamoyl substituent. For example, R73 can be a fluorine or hydrogen atom. That is, the fluorine atom can be bonded to any one of the carbons on the right aryl ring that are not bonded to the R72 substituent or the nitrogen atom. Alternatively, the fluorine atom cannot be bonded to the carbon on the right aryl ring that is not bonded to the R72 substituent or the nitrogen atom. For example, a hydrogen atom can be bonded to each of the carbons on the right aryl ring that are not bonded to the R72 substituent or the nitrogen atom.

[0057] For example, as further presented below (see, e.g., Figures 3, 5, 11-13), the chemical formula

[0058] [ka] Compounds having the formula: surprisingly exhibited potent antagonist activity while possessing minimal agonist activity for AR overexpressed in hormone-refractory prostate cancer.

[0059] A list of several compounds according to the present invention is presented in Tables 5-11. These compounds are grouped into tiers, with Tier 1 to Tier 3 compounds predicted to be superior to bicalutamide for the treatment of prostate cancer, Tier 4 compounds comparable in efficacy to bicalutamide, and Tier 5 and Tier 6 compounds inferior to bicalutamide for the treatment of prostate cancer. A more detailed description of the protocol used to rank these compounds into tiers is presented below.

[0060] (definition) As used herein, the term "alkyl" refers to a branched or unbranched hydrocarbon chain, preferably having from about 1 to about 8 carbons, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, 2-methylpentyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, etc. Substituted alkyl includes alkyl groups that may be attached to such a chain and are optionally substituted with one or more functional groups, such as hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio, cyano, alkylthio, heterocyclyl, aryl, heteroaryl, carboxyl, carvalcoyl, alkyl, alkenyl, nitro, amino, alkoxyl, amido, etc., to form trifluoromethyl, 3-hydroxyhexyl, 2-carboxypropyl, 2-fluoroethyl, carboxymethyl, cyanobutyl, etc.

[0061] Unless otherwise indicated, the term "cycloalkyl," as used herein, alone or as part of another group, includes saturated or partially unsaturated (containing one or more double bonds) cyclic hydrocarbon groups containing 1 to 3 rings, including monocyclic alkyls, bicyclic alkyls, and tricyclic alkyls containing a total of 3 to 20 carbons forming the ring, preferably 3 to 10 carbons forming the ring, which may be fused to one or two aromatic rings as described for aryl, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, and cyclododecyl, cyclohexenyl. "Substituted cycloalkyl" includes cycloalkyl groups optionally substituted with one or more substituents, such as halogen, alkyl, alkoxy, hydroxy, aryl, aryloxy, arylalkyl, cycloalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, thiol, and / or alkylthio, and / or any of the substituents included within the definition of "substituted alkyl." For example,

[0062] [ka] And so on.

[0063] Unless otherwise indicated, the term "alkenyl," as used herein, by itself or as part of another group, refers to a straight or branched chain group of 2 to 20, or 2 to 12, and more preferably 2 to 8 carbons in the linear chain, containing one or more double bonds in the linear chain, such as, for example, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl, 4-dodecenyl, 4,8,12-tetradecatrienyl, and the like. "Substituted alkenyl" includes alkenyl groups optionally substituted with one or more substituents, such as those included above in the definitions of "substituted alkyl" and "substituted cycloalkyl."

[0064] Unless otherwise indicated, the term "alkynyl," as used herein, by itself or as part of another group, refers to a straight or branched chain group of 2 to 20, or 2 to 12, and more preferably 2 to 8 carbons in the linear chain, containing one or more triple bonds in the linear chain, such as, for example, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, 4-decynyl, 3-undecynyl, 4-dodecynyl, etc. "Substituted alkynyl" includes alkynyl groups optionally substituted with one or more substituents, such as those included above in the definitions of "substituted alkyl" and "substituted cycloalkyl."

[0065] The terms "arylalkyl," "arylalkenyl," and "arylalkynyl," alone or as part of another group, refer to alkyl, alkenyl, and alkynyl groups, as described above, having an aryl substituent. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, phenethyl, benzhydryl, naphthylmethyl, and the like. "Substituted arylalkyl" includes arylalkyl groups in which the aryl portion is optionally substituted with one or more substituents, such as those included within the definition of "substituted alkyl" and "substituted cycloalkyl."

[0066] The terms "arylalkyl," "arylalkenyl," and "arylalkynyl," alone or as part of another group, refer to alkyl, alkenyl, and alkynyl groups, as described above, having an aryl substituent. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, phenethyl, benzhydryl, naphthylmethyl, and the like. "Substituted arylalkyl" includes arylalkyl groups in which the aryl portion is optionally substituted with one or more substituents, such as those included within the definition of "substituted alkyl" and "substituted cycloalkyl."

[0067] The terms "halogen" or "halo," as used herein, alone or as part of another group, refer to chlorine, bromine, fluorine, and iodine.

[0068] The terms "halogenalkyl," "halogenated alkenyl," and "halogenated alkynyl," as used herein, alone or as part of another group, refer to "alkyl," "alkenyl," and "alkenyl" that is substituted with one or more atoms selected from chlorine, bromine, fluorine, and iodine.

[0069] Unless otherwise indicated, the terms "aryl" or "Ar," as used herein, alone or as part of another group, refer to monocyclic and polycyclic aromatic groups containing 6 to 10 carbons in the ring portion (e.g., phenyl or naphthyl, including 1-naphthyl and 2-naphthyl) and optionally containing 1 to 3 additional rings fused to a carbocyclic or heterocyclic ring (e.g., an aryl ring, a cycloalkyl ring, a heteroaryl ring, or a cycloheteroalkyl ring).

[0070] "Substituted aryl" includes aryl groups that are optionally substituted with one or more functional groups, such as halo, haloalkyl, alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, trifluoromethyl, trifluoromethoxy, alkynyl, cycloalkyl-alkyl, cycloheteroalkyl, cycloheteroalkylalkyl, aryl, heteroaryl, arylalkyl, aryloxy, aryloxyalkyl, arylalkoxy, alkoxycarbonyl, arylcarbonyl, arylalkenyl, aminocarbonylaryl, arylthio, arylsulfinyl, arylazo, heteroarylalkyl, heteroarylalkenyl, heteroarylheteroaryl, heteroaryloxy, hydro oxy, nitro, cyano, amino, substituted amino containing one or two substituents (which may be alkyl, aryl, or any of the other aryl compounds referred to in this definition), thiol, alkylthio, arylthio, heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylcarbonyl, arylcarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, alkylcarbonylamino, arylcarbonylamino, arylsulfinyl, arylsulfinylalkyl, arylsulfonylamino, or arylsulfoneaminocarbonyl and / or any alkyl substituent as set forth herein.

[0071] Unless otherwise indicated, the terms "heterocyclic" or "heterocycle," as used herein, refer to a stable, unsubstituted or substituted, 5- to 10-membered monocyclic ring system, which may be saturated or unsaturated, and which consists of carbon atoms and from 1 to 4 heteroatoms selected from N, O, or S, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heterocycle may be attached at any heteroatom or carbon atom that results in the creation of a stable structure. Examples of such heterocyclic groups include, but are not limited to, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolyl, pyrrolidinyl, furanyl, thienyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, thiadiazolyl, tetrahydropyranyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl. The term "heteroaromatic," as used herein, alone or as part of another group, refers to a 5- or 7-membered aromatic ring containing 1, 2, 3, or 4 heteroatoms, such as nitrogen, oxygen, or sulfur, and such ring is fused to an aryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring (e.g., "Substituted heteroaryl" includes heteroaryl groups optionally substituted with 1 to 4 substituents, such as those included within the definition of "substituted alkyl" and "substituted cycloalkyl." Examples of heteroaryl groups include:

[0072] [ka] etc. [Example]

[0073] Example 1 4-Isothiocyanato-2-trifluoromethylbenzonitrile, (1a) 4-Amino-2-trifluoromethylbenzonitrile (2.23 g, 12 mmol) was added portionwise over 15 minutes to a well-stirred heterogeneous mixture of thiophosgene (1 ml, 13 mmol) in water (22 ml) at room temperature. Stirring was continued for another hour. The reaction medium was extracted with chloroform (3 × 15 ml). The combined organic layers were dried over MgSO and evaporated to dryness under reduced pressure to give the desired product, 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a), as a brownish solid, which was used as such for the next step (2.72 g, 11.9 mmol, 99%).

[0074] Example 2 2-1). (4-Aminophenyl)carbamic acid tert-butyl ester, (2a) An aqueous solution of potassium carbonate (1.52 g, 11 mmol) in 5 ml of water was added to a solution of 1,4-diaminobenzene (3.24 g, 30 mmol) in THF (30 ml) and DMF (10 ml). To this mixture, di-tert-butyl pyrocarbonate, BocO (2.18 g, 10 mmol) was added dropwise over 0.5 h. The reaction mixture was stirred at room temperature for an additional 4 h. The mixture was then poured into cold water (40 ml) and extracted with chloroform (3 × 50 ml). The combined organic layers were dried over MgSO and concentrated to give a brown residue, which was subjected to flash chromatography (dichloromethane / acetone, 4:1) to give (4-aminophenyl)carbamic acid tert-butyl ester, 2a, as a yellow solid (1.98 g, 9.5 mmol, 95%) (yield based on BocO).

[0075] 2-2).{4-[(1-cyano-1-methylethyl)amino]phenyl}carbamic acid tert-butyl ester, 2b A mixture of 2a (0.83 g, 4 mmol), acetone cyanohydrin (4 mL), and MgSO (2 g) was heated to 80 °C and stirred for 2.5 h. After cooling to room temperature, compound 2b was crystallized in water (30 mL). The solid was filtered, dried, and purified to give tert-butyl {4-[(1-cyano-1-methylethyl)amino]phenyl}carbamate. The ester, 2b, was obtained (1.08 g, 3.9 mmol, 98%).

[0076] 2-3).{4-[3-(4-cyano-3-trifluoromethylphenyl)-4-imino-5,5-dimethyl-2-thioxo-imidazolidin-1-yl]phenyl}carbamic acid tert-butyl ester, (2c) Triethylamine (0.202 g, 2 mmol) was added to a solution of 1a (0.456 g, 2 mmol) and 2b (0.57 g, 2 mmol) in dry THF (5 ml). The reaction mixture was stirred at room temperature for 15 hours and then concentrated to give a dark residue, which was subjected to flash chromatography (ethyl ether / acetone, 97:3) to give {4-[3-(4-cyano-3-trifluoromethylphenyl)-4-imino-5,5-dimethyl-2-thioxo-imidazolidin-1-yl]phenyl}carbamic acid tert-butyl ester, (2c) (0.15 g, 0.3 mmol, 15%).

[0077] 2-4). 4-[3-(4-aminophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 2d, [RD9]. A mixture of 2c (0.15 g, 0.3 mmol) in 3N aqueous HCl (1 mL) and methanol (4 mL) was heated to reflux for 2 h. After cooling to room temperature, the reaction mixture was poured into cold water (5 mL) and extracted with dichloromethane (8 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane / acetone, 9:1) to give 4-[3-(4-aminophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 2d, [RD9] (0.118 g, 0.29 mmol, 97%) as a yellow solid.

[0078] [ka] 2-5).4-[3-(4-azidophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 2e, [RD10] An aqueous solution of sulfuric acid (25% by weight, 1 ml) was added to a solution of 2d (0.10 g, 0.25 mmol) in acetone (1 ml) at −5° C. An aqueous solution of NaNO (0.024 g, 0.35 mmol in 0.5 ml of water) was slowly added to the above mixture over 0.1 h. The reaction mixture was allowed to stir for an additional 1 h at −5° C., and then an aqueous solution of NaN (0.02 g, 0.3 mmol in 0.3 ml of water) was added dropwise. Upon completion of the addition, the reaction medium was allowed to warm to room temperature and stirred for an additional 3 h. The product was extracted with dichloromethane (3×5 ml). The combined organic layers were dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 4-[3-(4-azidophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 2e, [RD10] as a yellowish solid (0.08 g, 0.18 mmol, 72%).

[0079] [ka] Example 3 3-1). 2-(4-hydroxyphenylamino)-2-methylpropanenitrile, 3a A mixture of 4-aminophenol (1.09 g, 10 mmol), acetone cyanohydrin (10 mL), and MgSO (2 g) was heated to 80 °C and stirred for 4 h. After concentration of the medium under vacuum, compound 3a was crystallized from water (20 mL). The solid was filtered and dried to give 2-(4-hydroxyphenylamino)-2-methylpropanenitrile, 3a (1.69 g, 9.6 mmol, 96%).

[0080] 3-2). 4-[3-(4-Hydroxyphenyl)-5-imino-4,4-dimethyl-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 3b. Triethylamine (0.101 g, 1 mmol) was added to a solution of 1a (0.456 g, 2 mmol) and 3a (0.352 g, 2 mmol) in dry THF (5 mL). The mixture was stirred at 0° C. for 48 h and then concentrated to give a dark residue, which was subjected to flash chromatography (dichloromethane / acetone, 85:15) to give 4-[3-(4-hydroxyphenyl)-5-imino-4,4-dimethyl-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 3b (0.274 g, 0.68 mmol, 34%).

[0081] 3-3).4-[3-(4-hydroxyphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 3c, [RD8] A mixture of 3b (0.202 g, 0.5 mmol) in 2 N aqueous HCl (2 mL) and methanol (5 mL) was heated to reflux for 2 h. After cooling to room temperature, the reaction mixture was poured into cold water (10 mL) and extracted with ethyl acetate (10 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane / acetone, 9:1) to give 4-[3-(4-hydroxyphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 3c, [RD8] (0.198 g, 0.49 mmol, 98%) as a white powder.

[0082] [ka] Example 4 Chloroacetic acid 4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]phenyl ester, 4a, [RD13] Chloroacetyl chloride (0.045 g, 0.4 mmol) was added to a mixture of 3c (0.101 g, 0.25 mmol) and triethylamine (0.041 g, 0.41 mmol) in dry THF (1.5 mL). The mixture was stirred at room temperature for 4 h. Triethylamine hydrochloride was removed by filtration. The filtrate was concentrated and chromatographed (dichloromethane / acetone, 95:5) to give 84% chloroacetic acid 4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]phenyl ester, 4a, [RD13], as a white powder (0.101 g, 0.21 mmol).

[0083] [ka] Example 5 5-1a). 2-Methyl-2-(4-methylphenyl)aminopropanenitrile, 5a A mixture of p-toluidine (1.07 g, 10 mmol) and acetone cyanohydrin (10 ml) was heated to 80° C. and stirred for 4 h. The medium was concentrated and dried under vacuum to give 2-methyl-2-(4-methylphenyl)aminopropanenitrile, 5a, as a brown solid (1.72 g, 9.9 mmol, 99%).

[0084] 5-1b). 2-Methyl-2-(4-methylphenyl)aminopropanenitrile, 5a Sodium cyanide (0.735 g, 15 mmol) was added to a mixture of p-toluidine (1.07 g, 10 mmol) and acetone (1.16 g, 20 mmol) in 90% acetic acid (10 mL). The reaction mixture was stirred at room temperature for 12 hours, and then ethyl acetate (50 mL) was added. The organic layer was washed with water (4 × 30 mL), dried over magnesium sulfate, and concentrated to dryness in vacuo to give 2-methyl-2-(4-methylphenyl)aminopropanenitrile, 5a, as a brown solid (1.65 g, 9.5 mmol, 95%).

[0085] 5-2). 4-[3-(4-methylphenyl)-5-imino-4,4-dimethyl-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 5b Triethylamine (0.101 g, 1 mmol) was added to a solution of 1a (0.456 g, 2 mmol) and 5a (0.348 g, 2 mmol) in dry THF (3 mL). The reaction mixture was stirred at 0° C. for 2 days and then concentrated to give a dark residue, which was subjected to flash chromatography (dichloromethane / acetone, 95:5) to give 4-[3-(4-methylphenyl)-5-imino-4,4-dimethyl-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 5b (0.136 g, 0.34 mmol, 17%).

[0086] 5-3a). 4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 5c A mixture of 5b (0.121 g, 0.3 mmol) in 2 N aqueous HCl (2 mL) and methanol (5 mL) was heated to reflux for 2 h. After cooling to room temperature, the reaction mixture was poured into cold water (10 mL) and extracted with ethyl acetate (10 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 5c, as a white powder (0.118 g, 0.294 mmol, 98%).

[0087] 5-3b). 4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethyl-benzonitrile, 5c, [RD7] A mixture of 1a (0.547 g, 2.4 mmol) and 5a (0.348 g, 2 mmol) in dry DMF (0.6 mL) was stirred for 36 hours. To this mixture, methanol (20 mL) and 2N HCl (5 mL) were added. This second mixture was refluxed for 6 hours. After cooling to room temperature, the mixture was poured into cold water (30 mL) and extracted with ethyl acetate (40 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethyl-benzonitrile, 5c, [RD7] (0.596 g, 1.48 mmol, 74%) as a white powder.

[0088] [ka] Example 6 6-1). 2-Methyl-2-phenylaminopropanenitrile, 6a. A mixture of aminobenzene (0.931 g, 10 mmol) and acetone cyanohydrin (2 mL) was heated to reflux and stirred for 20 hours. After cooling to room temperature, the reaction mixture was poured into ethyl acetate (40 mL) and washed with cold water (2 × 30 mL). The organic layer was dried over MgSO4 and concentrated to dryness under vacuum to give 2-methyl-2-phenylaminopropanenitrile, 6a (1.51 g, 9.4 mmol, 94%) as a brown slurry.

[0089] 6-2). 4-[3-phenyl-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 6b, [RD10] A mixture of 1a (0.274 g, 1.2 mmol) and 6a (0.160 g, 1 mmol) in dry DMF (0.2 mL) was stirred for 48 h. To this mixture, methanol (10 mL) and 2 N HCl (3 mL) were added. This second mixture was refluxed for 6 h. After cooling to room temperature, the reaction mixture was poured into cold water (20 mL) and extracted with ethyl acetate (20 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 4-[3-phenyl-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 6b, [RD10] (0.276 g, 0.71 mmol, 71%) as a white powder.

[0090] [ka] Example 7 7-1a). 1-(4-methylphenyl)aminocyclobutanenitrile, 7a Sodium cyanide (0.147 g, 3 mmol) was added to p-toluidine (0.214 g, 2 mmol) and cyclobutanone (0.21 g, 3 mmol) in 90% acetic acid (3 mL). The reaction mixture was stirred at room temperature for 12 hours, and then 20 mL of ethyl acetate was added. The organic layer was washed with water (3 × 10 mL), dried over magnesium sulfate, and concentrated to dryness in vacuo to give 1-(4-methylphenyl)aminocyclobutanenitrile, 7a, as a brown solid (0.343 g, 1.84 mmol, 92%).

[0091] 7-1b). 1-(4-methylphenyl)aminocyclobutanenitrile, 7a Trimethylsilyl cyanide (0.93 ml, 7 mmol) was added dropwise to a mixture of p-toluidine (0.535 g, 5 mmol) and cyclobutanone (0.42 g, 6 mmol). The reaction mixture was stirred at room temperature for 6 h and then concentrated in vacuo to give a brown liquid, which was chromatographed (dichloromethane) to give 1-(4-methylphenyl)aminocyclobutanenitrile, 7a, as a yellowish solid (0.912 g, 4.9 mmol, 98%).

[0092] 7-2). 4-(8-Imino-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 7b To a solution of 1a (2.28 g, 10 mmol) in dry DMF (3 ml) was added a solution of 7a (1.764 g, 9 mmol) in dry DMF (3 ml) slowly over 20 h at room temperature. The medium was stirred for another 4 h. After evaporation of DMF, the residue was chromatographed (dichloromethane / acetone, 95:5) to give 4-(8-imino-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 7b (1.937 g, 4.68 mmol, 52%).

[0093] 7-3a). 4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 7c [RD37] A mixture of 7b (0.041 g, 0.1 mmol) in 2N aqueous HCl (3 mL) and methanol (1 mL) was heated to reflux for 2 h. After cooling to room temperature, the reaction mixture was poured into cold water (5 mL) and extracted with ethyl acetate (6 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 7c (0.04 g, 0.096 mmol, 96%) as a white powder.

[0094] 7-3b). 4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 7c, [RD37] A mixture of 1a (0.912 g, 4 mmol) and 7a (0.558 g, 3 mmol) in dry DMF (0.5 mL) was stirred at room temperature for 24 hours. To this mixture, methanol (30 mL) and 2N aqueous HCl (6 mL) were added. This second mixture was refluxed for 6 hours. After cooling to room temperature, the reaction mixture was poured into cold water (50 mL) and extracted with ethyl acetate (60 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 7c, as a white powder (0.959 g, 2.31 mmol, 77%).

[0095] [ka] Example 8 8-1). 1-(4-methylphenyl)aminocyclopentanenitrile, 8a Trimethylsilyl cyanide (0.865 ml, 7 mmol) was added dropwise to a mixture of p-toluidine (0.535 g, 5 mmol) and cyclopentanone (0.589 g, 7 mmol). The reaction mixture was stirred at room temperature for 6 h and then concentrated in vacuo to give a brown liquid, which was chromatographed (dichloromethane) to give 1-(4-methylphenyl)aminocyclopentanenitrile, 8a, as a yellowish solid (0.981 g, 4.9 mmol, 98%).

[0096] 8-2).4-(4-oxo-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile, 8b, [RD35] A mixture of 1a (0.296 g, 1.3 mmol) and 8a (0.2 g, 1 mmol) in dry DMF (0.2 mL) was stirred for 48 h. To this mixture, methanol (10 mL) and 2N aqueous HCl (3 mL) were added. This second mixture was refluxed for 6 h. After cooling to room temperature, the reaction mixture was poured into cold water (20 mL) and extracted with ethyl acetate (30 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 4-(4-oxo-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile, 8b, [RD35] as a white powder (0.3 g, 0.7 mmol, 70%).

[0097] [ka] Example 9 9-1). 1-(4-methylphenyl)aminocyclohexanenitrile, 9a Sodium cyanide (0.147 g, 3 mmol) was added to a mixture of p-toluidine (0.214 g, 2 mmol) and cyclohexanone (0.294 g, 3 mmol) in 90% acetic acid (3 mL). The reaction mixture was stirred at room temperature for 12 hours, and then 20 mL of ethyl acetate was added. The organic layer was washed with water (3 × 10 mL), dried over magnesium sulfate, and concentrated to dryness in vacuo to give 1-(4-methylphenyl)aminocyclohexanenitrile, 9a (0.398 g, 1.86 mmol, 93%) as a brown solid.

[0098] 9-2). 4-(4-Imino-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile, 9b Triethylamine (0.05 g, 0.5 mmol) was added to a solution of 1a (0.228 g, 1 mmol) and 9a (0.214 g, 1 mmol) in dry THF (2 mL). The reaction mixture was stirred at room temperature for 2 days and then concentrated to give a dark residue, which was subjected to flash chromatography (dichloromethane / acetone, 95:5) to give 4-(4-imino-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile, 9b (0.035 g, 0.08 mmol, 8%).

[0099] 9-3). 4-(4-oxo-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile, 9c, [RD48] A mixture of 9b (0.035 g, 0.08 mmol) in 2N aqueous HCl (1 mL) and methanol (3 mL) was heated to reflux for 2 h. After cooling to room temperature, the reaction mixture was poured into cold water (5 mL) and extracted with ethyl acetate (6 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 4-(4-methyl-2-pyrrolidone). To obtain 1-(4-methylphenyl)-2-oxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile, 9c, [RD48], as a white powder (0.034 g, 0.076 mmol, 95%).

[0100] [ka] Example 10 10-1). 1-(4-methylphenyl)aminocyclohexanenitrile, 10a Sodium cyanide (0.147 g, 3 mmol) was added to p-toluidine (0.214 g, 2 mmol) and cycloheptanone (0.337 g, 3 mmol) in 90% acetic acid (3 mL). The reaction mixture was stirred at room temperature for 12 hours, and then 20 mL of ethyl acetate was added. The organic layer was washed with water (3 × 10 mL), dried over magnesium sulfate, and concentrated to dryness in vacuo to give 1-(4-methylphenyl)aminocyclohexanenitrile, 10a, as a brown solid (0.438 g, 1.92 mmol, 96%).

[0101] 10-2): 4-(4-Imino-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]undec-3-yl)-2-trifluoromethylbenzonitrile, 10b Triethylamine (0.05 g, 0.5 mmol) was added to a solution of 1a (0.228 g, 1 mmol) and 9a (0.228 g, 1 mmol) in dry THF (2 mL). The reaction mixture was stirred at room temperature for 2 days and then concentrated to give a dark residue, which was subjected to flash chromatography (dichloromethane / acetone, 95:5) to give 4-(4-imino-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]undec-3-yl)-2-trifluoromethylbenzonitrile, 10b (0.036 g, 0.08 mmol, 8%).

[0102] 10-3).4-(4-oxo-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]undec-3-yl)-2-trifluoromethylbenzonitrile, 10c, [RD49] A mixture of 9b (0.036 g, 0.08 mmol) in 2 N aqueous HCl (1 mL) and methanol (3 mL) was heated to reflux for 2 h. After cooling to room temperature, the reaction mixture was poured into cold water (5 mL) and extracted with ethyl acetate (6 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 10c (0.034 g, 0.075 mmol, 94%) as a white powder.

[0103] [ka] Example 11 11-1). 1-(4-hydroxyphenyl)aminocyclobutanenitrile, 11a Trimethylsilyl cyanide (0.93 ml, 7 mmol) was added dropwise to a mixture of 4-hydroxyaniline (0.545 g, 5 mmol) and cyclobutanone (0.42 g, 6 mmol). The reaction mixture was stirred at room temperature for 6 h and then concentrated in vacuo to give a brown liquid, which was chromatographed (dichloromethane:acetone, 98:2) to give 11a (0.903 g, 4.8 mmol, 96%) as a yellowish solid.

[0104] 11-2).4-(8-oxo-6-thioxo-5-(4-hydroxyphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 11b, [RD58] A mixture of 1a (0.57 g, 2.5 mmol) and 7a (0.376 g, 2 mmol) in dry DMF (0.5 mL) was stirred at room temperature for 40 h. To this mixture was added methanol (30 mL) and aqueous HCl (5 mL). This second mixture was refluxed for 6 h. After cooling to room temperature, the reaction mixture was poured into cold water (40 mL) and extracted with ethyl acetate (50 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane:acetone, 98:2) to give 11b (0.659 g, 1.58 mmol, 79%) as a white powder.

[0105] [ka] Example 12 12-1). 1-(4-Biphenylamino)cyclobutanecarbonitrile, 12a. Trimethylsilyl cyanide (0.2 ml, 1.5 mmol) was added dropwise to a mixture of 4-biphenylamine (0.169 g, 1 mmol) and cyclobutanone (0.098 g, 1.4 mmol). The reaction mixture was stirred at room temperature for 6 hours and then concentrated in vacuo to give a brown liquid, which was chromatographed (dichloromethane) to give 12a (0.24 g, 0.97 mmol, 97%) as a white solid.

[0106] 12-2).4-(8-oxo-6-thioxo-5-(4-biphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 12b [RD57] A mixture of 1a (0.137 g, 0.6 mmol) and 12a (0.124 g, 0.5 mmol) in dry DMF (0.2 mL) was stirred at room temperature for 3 days. To this mixture, methanol (5 mL) and 2N aqueous HCl (1 mL) were added. This second mixture was refluxed for 6 hours. After cooling to room temperature, the reaction mixture was poured into cold water (10 mL) and extracted with ethyl acetate (15 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 12b as a white powder (0.162 g, 0.34 mmol, 68%).

[0107] [ka] Example 13 13-1). 1-(2-naphthylamino)cyclobutanecarbonitrile, 13a Trimethylsilyl cyanide (0.27 ml, 2 mmol) was added dropwise to a mixture of 2-aminonaphthalene (0.143 g, 1 mmol) and cyclobutanone (0.098 g, 1.4 mmol). The reaction mixture was stirred at room temperature for 12 h and then concentrated in vacuo to give a brown liquid, which was chromatographed (dichloromethane) to give 13a (0.209 g, 0.94 mmol, 94%) as a yellow solid.

[0108] 13-2).4-(8-oxo-6-thioxo-5-(4-biphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 12b, [RD85] 1a (0.137 g, 0.6 mmol) and 13a (0.111 g, 0.5 mmol) in dry DMF (0.2 mL) were stirred at room temperature for 3 days. To this mixture, methanol (5 mL) and aqueous HCl (1 mL) were added. This second mixture was refluxed for 6 hours. After cooling to room temperature, the reaction mixture was poured into cold water (10 mL) and extracted with ethyl acetate (15 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 12b (0.146 g, 0.325 mmol, 65%) as a white powder.

[0109] [ka] Example 14 14-1). 2-(4-methyl-2-pyridineamino)-2-methylpropanenitrile, 14a Trimethylsilyl cyanide (0.27 ml, 2 mmol) was added dropwise to a mixture of 2-amino-4-methylpyridine (0.108 g, 1 mmol) and acetone (0.58 g, 10 mmol). The reaction mixture was stirred at room temperature for 6 days and then concentrated in vacuo to give a brown liquid, which was chromatographed (dichloromethane:acetone, 60:40) to give 14a (0.133 g, 0.76 mmol, 76%) as a white solid.

[0110] 14-2).4-[4,4-dimethyl-3-(4-methylpyridin-2-yl)-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 14b, [RD83] A mixture of 1a (0.91 g, 0.4 mmol) and 14a (0.053 g, 0.3 mmol) in dry DMF (0.2 mL) was stirred at room temperature for 6 days. To this mixture was added methanol (5 mL) and aqueous HCl (1 mL). This second mixture was refluxed for 5 hours. After cooling to room temperature, the reaction mixture was poured into cold water (10 mL) and extracted with ethyl acetate (15 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 14b as a white powder (0.07 g, 0.174 mmol, 58%).

[0111] [ka] Example 15 15-1). 2-(2-pyridineamino)-2-methylpropanenitrile, 15a Trimethylsilyl cyanide (0.27 ml, 2 mmol) was added dropwise to a mixture of 2-aminopyridine (0.094 g, 1 mmol) and acetone (0.58 g, 10 mmol). The reaction mixture was stirred at room temperature for 6 days and then concentrated in vacuo to give a brown liquid which was chromatographed (dichloromethane:acetone, 60:40) to give 15a as a white solid (0.131 g, 0.81 mmol, 81%).

[0112] 15-2).4-[4,4-dimethyl-3-(4-pyridin-2-yl)-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 15b, [RD82] A mixture of 1a (0.91 g, 0.4 mmol) and 15a (0.048 g, 0.3 mmol) in dry DMF (0.3 mL) was stirred at room temperature for 10 days. To this mixture was added methanol (5 mL) and aqueous HCl (1 mL). This second mixture was refluxed for 5 h. After cooling to room temperature, the reaction mixture was poured into cold water (10 mL) and extracted with ethyl acetate (15 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 15b as a white powder (0.059 g, 0.153 mmol, 51%).

[0113] [ka] Example 16 16-1). 1-(5-methyl-2H-pyrazol-3-ylamino)-cyclobutanecarbonitrile, 16a Trimethylsilyl cyanide (0.532 ml, 4.0 mmol) was added dropwise to a mixture of 3-amino-5-methylpyrazole (0.194 g, 2.0 mmol) and cyclobutanone (0.154 g, 2.2 mmol). The reaction mixture was stirred at room temperature for 40 h and then concentrated in vacuo to give a dark liquid, which was chromatographed (dichloromethane) to give 16a (0.267 g, 1.52 mmol, 76%) as an off-white powder.

[0114] 16-2).4-[5-(5-methyl-2H-pyrazol-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl]-2-trifluoromethyl-benzonitrile, 16b, [RD84] A mixture of 1a (0.0684 g, 0.3 mmol) and 16a (0.053 g, 0.3 mmol) in dry DMF (0.2 mL) was stirred at room temperature for 4 days. To this mixture, methanol (10 mL) and 2 N aqueous HCl (2 mL) were added. This second mixture was refluxed for 5 hours. After cooling to room temperature, the reaction mixture was poured into cold water (30 mL) and extracted with ethyl acetate (30 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane:acetone, 97:3) to give 16b (0.0826 g, 0.2 mmol, 67%) as a white powder.

[0115] [ka] Example 17 4-[3-(4-hydroxyphenyl)-4,4-dimethyl-2,5-dithioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 17a, [RD59] A mixture of 3c (0.081 g, 0.2 mmol) and Lawesson's reagent (0.097 g, 0.24 mmol) in toluene (3 mL) was heated to reflux for 15 h. After cooling to room temperature, the reaction mixture was poured into cold water (10 mL) and extracted with ethyl acetate (10 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:pentane, 9:1) to give 17a (0.0185 g, 0.044 mmol, 22%) as a white powder.

[0116] [ka] Example 18 4-[3-(4-hydroxyphenyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, 18a, [RD60] Hydrogen peroxide 30% (3 mL, 26 mmol) was added dropwise to a solution of 3c (0.121 g, 0.4 mmol) in glacial acetic acid (3 mL). The mixture was stirred at room temperature for 12 h, and then 20 mL of ethyl acetate was added. The organic layer was washed with water (3 × 15 mL), dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 18a (0.102 g, 0.261 mmol, 87%) as a white powder.

[0117] [ka] Example 19 19-1). 3-Fluoro-2-methyl-2-(4-methylphenyl)aminopropionitrile, 19a Trimethylsilyl cyanide (0.146 ml, 1.1 mmol) was added dropwise to a mixture of p-toluidine (0.107 g, 1 mmol) and fluoroacetone (0.082 g, 1.1 mmol). The reaction mixture was stirred at room temperature for 12 h and then concentrated in vacuo to give a brown liquid, which was chromatographed (dichloromethane) to give 19a (0.179 g, 0.93 mmol, 93%) as a yellowish solid.

[0118] 19-2). 4-(4-Fluoromethyl-4-methyl-5-oxo-2-thioxo-3-(4-methylphenyl)imidazolidin-1-yl)-2-trifluoromethylbenzonitrile, 19b, [RD68] A mixture of 1a (0.16 g, 0.7 mmol) and 19a (0.096 g, 0.5 mmol) in dry DMF (0.3 mL) was stirred at room temperature for 48 hours. To this mixture, methanol (10 mL) and 2 N aqueous HCl (2 mL) were added. This second mixture was refluxed for 6 hours. After cooling to room temperature, the reaction mixture was poured into cold water (30 mL) and extracted with ethyl acetate (30 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 19b (0.168 g, 0.4 mmol, 80%) as a white powder.

[0119] [ka] Example 20 20-1). 2-Methyl-2-(4-trifluoromethylphenyl)aminopropanenitrile, 20a A mixture of 4-trifluoromethylaniline (1.61 g, 10 mmol), acetone cyanohydrin (5 mL), and magnesium sulfate (2 g) was heated to 80° C. and stirred for 12 hours. Ethyl acetate (50 mL) was added to the mixture, which was then washed with water (3×30 mL). The organic layer was dried over MgSO4 and concentrated to dryness under vacuum to give 20a as a brown solid (2.166 g, 9.5 mmol, 95%).

[0120] 20-2). 4-(4,4-dimethyl-5-oxo-2-thioxo-3-(4-trifluoromethylphenyl)imidazolidin-1-yl)-2-trifluoromethylbenzonitrile, 20b, [RD66] A mixture of 1a (0.114 g, 0.5 mmol) and 20a (0.092 g, 0.4 mmol) in dry DMF (0.3 mL) was stirred at room temperature for 48 hours. To this mixture, methanol (10 mL) and aqueous HCl (3 mL) were added. This second mixture was refluxed for 6 hours. After cooling to room temperature, the reaction mixture was poured into cold water (20 mL) and extracted with ethyl acetate (20 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 20b (0.117 g, 0.256 mmol, 64%) as a white powder.

[0121] [ka] Example 21 21-1). 3-Chloro-2-chloromethyl-2-(4-methylphenyl)aminopropanenitrile, 21a Trimethylsilyl cyanide (0.27 mL, 2 mmol) was added dropwise to a mixture of p-toluidine (0.107 g, 1 mmol) and 1,3-dichloroacetone (0.254 g, 2 mmol). The reaction mixture was heated to 80° C. and stirred for 6 h. 20 mL of ethyl acetate was added to the mixture, which was then washed with water (2×20 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 21a as a brown powder (0.192 g, 0.79 mmol, 79%).

[0122] 21-2). 4-(4,4-bischloromethyl-5-oxo-2-thioxo-3-(4-methylphenyl)imidazolidin-1-yl)-2-trifluoromethylbenzonitrile, 21b, [RD67] A mixture of 1a (0.16 g, 0.7 mmol) and 21a (0.122 g, 0.5 mmol) in dry DMF (0.5 mL) was stirred at room temperature for 10 days. To this mixture, methanol (10 mL) and 2 N aqueous HCl (2 mL) were added. This second mixture was refluxed for 6 hours. After cooling to room temperature, the reaction mixture was poured into cold water (20 mL) and extracted with ethyl acetate (30 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 21b as a white powder (0.09 g, 0.19 mmol, 38%).

[0123] [ka] Example 22 22-1). 1-(4-methylphenyl)aminocyclohexanenitrile, 22a Sodium cyanide (0.245 g, 5 mmol) was added to a mixture of anthranilic acid (0.411 g, 3 mmol) and acetone (1 mL, 13.6 mmol) in 90% acetic acid (3 mL). The reaction mixture was stirred at room temperature for 12 hours, and then 50 mL of ethyl acetate was added. The organic layer was washed with brine (3 × 30 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone, 90:10) to give 22a (0.551 g, 2.7 mmol, 90%) as a brown solid.

[0124] 22-2). 2-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]benzoic acid, 22b, [RD65] A mixture of 1a (0.114 g, 0.2 mmol) and 22a (0.103 g, 0.5 mmol) in dry DMF (0.5 mL) was stirred at room temperature for 3 days. To this mixture, methanol (10 mL) and 2N aqueous HCl (3 mL) were added. This second mixture was refluxed for 6 hours. After cooling to room temperature, the reaction mixture was poured into cold water (20 mL) and extracted with ethyl acetate (30 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (ethyl acetate:pentane, 2:1) to give 22b (0.143 g, 0.33 mmol, 66%) as a white powder.

[0125] [ka] Example 23 23-1). 1-(2-methylphenyl)aminocyclobutanenitrile, 23a Trimethylsilyl cyanide (0.66 ml, 5 mmol) was added dropwise to a mixture of p-toluidine (0.321 g, 3 mmol) and cyclobutanone (0.28 g, 4 mmol). The reaction mixture was stirred at room temperature for 6 h and then concentrated in vacuo to give a brown liquid, which was chromatographed (dichloromethane) to give 23a (0.541 g, 2.91 mmol, 97%) as a yellowish solid.

[0126] 23-2).4-(8-oxo-6-thioxo-5-(2-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 23b, [RD71] A mixture of 1a (0.114 g, 0.5 mmol) and 23a (0.093 g, 0.5 mmol) in dry DMF (0.3 mL) was stirred at room temperature for 3 days. To this mixture, methanol (10 mL) and 2N aqueous HCl (3 mL) were added. This second mixture was refluxed for 6 hours. After cooling to room temperature, the reaction mixture was poured into cold water (20 mL) and extracted with ethyl acetate (30 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 23b (0.116 g, 0.28 mmol, 56%) as a white powder.

[0127] [ka] Example 24 24-1). 1-Aminocyclopentanecarbonitrile, 24a Anhydrous ammonia was bubbled into a mixture of cyclopentanone (0.452 g) and trimethylsilyl cyanide (0.66 mL, 5 mmol). Excess ammonia was refluxed using a dry ice-acetone condenser. After 1 h of reflux, ammonia was degassed from the medium, and the remaining mixture was then concentrated under vacuum to give 24a (0.522 g, 4.75 mmol, 95%) as a colorless liquid.

[0128] 24-2). 4-(4-Imino-2-thioxo-1,3-diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile, 24b Triethylamine (0.101 g, 0.1 mmol) was added to a solution of 1a (0.684 g, 3 mmol) and 24a (0.33 g, 3 mmol) in dry THF (5 mL). The reaction mixture was stirred at room temperature for 5 h and then concentrated to give a brown residue, which was subjected to flash chromatography (dichloromethane / acetone, 93:7) to give 24b (0.741 g, 2.19 mmol, 73%).

[0129] 24-3).4-(4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile, 24c, [RD77] A mixture of 24b (0.741 g, 2.19 mmol) in 2N aqueous HCl (4 mL) and methanol (20 mL) was heated to reflux for 1 h. After cooling to room temperature, the reaction mixture was poured into cold water (20 mL) and extracted with ethyl acetate (40 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 24c (0.72 g, 2.12 mmol, 97%) as a white powder.

[0130] [ka] Example 25 25).4-[1-(4-nitrophenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl]-2-trifluoromethylbenzonitrile, 25a, [RD55] A mixture of 25c (0.0678 g, 0.2 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (0.05 g, 0.33 mmol), and 4-fluoronitrobenzene (0.056 g, 0.4 mmol) in dimethylformamide (0.5 mL) was placed under argon in a sealed tube and heated at 130 °C for 40 h. The reaction mixture was poured into ethyl acetate (5 mL) and washed with water (2 × 10 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 25a as a white powder (0.038 g, 0.084 mmol, 42%).

[0131] [ka] Example 26 26).4-[1-(4-cyanophenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl]-2-trifluoromethylbenzonitrile, 26a, [RD54] A mixture of 24c (0.0678 g, 0.2 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (0.061 g, 0.4 mmol), and 4-fluorocyanobenzene (0.048 g, 0.4 mmol) in dimethylformamide (0.5 mL) was placed under argon in a sealed tube and heated at 140 °C for 5 days. The reaction mixture was poured into ethyl acetate (5 mL) and washed with water (2 × 10 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 26a as a white powder (0.023 g, 0.052 mmol, 26%).

[0132] [ka] Example 27 27-1). 1-Methyl-4-(4-methylphenylamino)piperidine-4-carbonitrile, 27a Sodium cyanide (0.318 g, 6.5 mmol) was added to a mixture of p-toluidine (0.535 g, 5 mmol) and 1-methyl-4-piperidinone (0.678 g, 6 mmol) in 90% acetic acid (5 mL). The mixture was stirred at room temperature for 6 hours, and then 100 mL of dichloromethane was added. The organic layer was washed with 2 N NaOH solution (2 × 50 mL), dried over magnesium sulfate, concentrated, and chromatographed (DCM and then acetone) to give 27a (0.722 g, 3.15 mmol, 63%).

[0133] 27.2). 4-(4-Imino-8-methyl-2-thioxo-1-(4-methylphenyl)-1,3,8-triazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile, 27b Triethylamine (0.02, 0.2 mmol) was added to a solution of 1a (0.228 g, 1 mmol) and 27a (0.114 g, 0.5 mmol) in dry THF (2 mL). The mixture was stirred at room temperature for 20 h and then concentrated to give a dark residue, which was subjected to flash chromatography (dichloromethane / acetone, 90:10, followed by acetone) to give 27b (0.059 g, 0.13 mmol, 26%).

[0134] 27-3): 4-(8-methyl-4-oxo-2-thioxo-1-(4-methylphenyl)-1,3,8-triazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile, 27c, [RD53] A mixture of 27b (0.059 g, 0.13 mmol) in 2N aqueous HCl (1 mL) and methanol (3 mL) was heated to reflux for 2 h. After cooling to room temperature, the reaction mixture was poured into cold water (5 mL) and extracted with ethyl acetate (10 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone, 60:40) to give 27c (0.055 g, 0.012 mmol, 92%) as a white powder.

[0135] [ka] Example 28 4-(8-methyl-4-oxo-2-thioxo-1,3,8-triazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile, 28a, [RD52] Compound 28a was synthesized according to the procedure described in US Pat. No. 5,958,936.

[0136] [ka] Example 29 4-[3-(4-hydroxybutyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile, RU59063 Compound RU59063 was synthesized according to the procedure described by Teutsch et al. [J. Steroid. Biochem. Molec. Biol. 1994, 48(1), 111-119].

[0137] [ka] Example 30 30-1). 1-Methylaminocyclobutanecarbonitrile, 30a Methylamine was bubbled into a refrigerated mixture of cyclobutanone (0.21 g, 3 mmol) and trimethylsilyl cyanide (0.396 g, 4 mmol) until the volume doubled. The mixture was stirred for 3 h and then concentrated to dryness to give 30a (0.33 g, quantitative).

[0138] 30-2).4-(5-methyl-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 30b, [RD73] A mixture of 1a (0.114 g, 0.5 mmol) and 30a (0.055 g, 0.5 mmol) in dry DMF (0.2 mL) was stirred at room temperature for 0.5 h. To this mixture, 10 mL of methanol and 2 mL of 2 N HCl were added. This second mixture was refluxed for 2 h. After cooling to room temperature, the reaction mixture was poured into cold water (20 mL) and extracted with ethyl acetate (30 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 30b (0.148 g, 0.435 mmol, 87%) as a white powder.

[0139] [ka] 30-3).4-(5-methyl-6,8-dioxo-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile, 30c, [RD74] Hydrogen peroxide (2 mL, 30%) was added to a mixture of 30b (0.068 g, 0.2 mmol) in glacial acetic acid (3 mL). After stirring at room temperature for 10 h, the reaction mixture was poured into ethyl acetate (20 mL) and then washed with water (2 × 20 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone) to give 30c as a white powder (0.057 g, 0.176 mmol, 88%).

[0140] [ka] Example 31 31-1). 1-Methylaminocyclopentanecarbonitrile, 31a. Methylamine was bubbled into a refrigerated mixture of cyclopentanone (0.252 g, 3 mmol) and trimethylsilyl cyanide (0.396 g, 4 mmol) until the volume doubled. The mixture was stirred for 3 h and then concentrated to dryness to give 31a (0.372 g, quantitative).

[0141] 31-2). 4-(1-methyl-4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile, 31b, [RD75] A mixture of 1a (0.114 g, 0.5 mmol) and 31a (0.062 g, 0.5 mmol) in dry DMF (0.2 mL) was stirred at room temperature for 0.5 h. To this mixture, 10 mL of methanol and 2 mL of 2 N HCl were added. This second mixture was refluxed for 2 h. After cooling to room temperature, the reaction mixture was poured into cold water (20 mL) and extracted with ethyl acetate (30 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 31b (0.159 g, 0.45 mmol, 90%) as a white powder.

[0142] [ka] 31-3).4-(1-methyl-2,4-dioxo-1,3-diaza-spiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile, 31c, [RD76] Hydrogen peroxide (2 mL, 30%) was added to a mixture of 31b (0.07 g, 0.2 mmol) in glacial acetic acid (3 mL). After stirring at room temperature for 10 h, the reaction mixture was poured into ethyl acetate (20 mL) and then washed with water (2 × 20 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone) to give 31c as a white powder (0.057 g, 0.168 mmol, 84%).

[0143] [ka] Example 32 4-(8-methylimino-6-thioxo-5-p-tolyl-5,7-diaza-spiro[3.4]oct-7-yl)-2-trifluoromethyl-benzonitrile, 32a, [RD90] A mixture of 7b (0.042 g, 0.1 mmol), DBU (0.023 g, 0.15 mmol), and iodomethane (0.073 g, 0.5 mmol) in DMF (0.3 mL) was stirred at room temperature for 15 h. After evaporation of the DMF, the medium was chromatographed (dichloromethane) to give 32a (0.011 g, 0.026 mmol, 26%) as a white powder.

[0144] [ka] Example 33 1-[3-(4-cyano-3-trifluoromethyl-phenyl)-5,5-dimethyl-2-thioxo-1-p-tolyl-imidazolidin-4-ylidene]-3-ethyl-thiourea, 33a, [RD91] A mixture of 5b (0.06 g, 0.149 mmol), ethyl thioisocyanate (0.087 g, 1 mmol), and CuI (0.01 g, 0.05 mmol) in DMF (0.1 mL) was heated in a microwave oven for 45 min. The medium was then washed with brine and extracted with ethyl acetate. The organic layer was dried over MgSO, concentrated, and chromatographed (HPLC, alumina column) to give 33a (0.054 g, 0.108 mmol, 72%) as a white powder.

[0145] [ka] Example 34 1-[7-(4-cyano-3-trifluoromethyl-phenyl)-6-thioxo-5-p-tolyl-5,7-diaza-spiro[3.4]oct-8-ylidene]-3-phenyl-thiourea, 34a, [RD92] 7b (0.021 g, 0.05 mmol) and phenylthiocyante (0.027 g, 0.2 mmol) in DMF (0.3 ml) were stirred for 2 days at 60° C. After evaporation of the DMF, the medium was chromatographed (dichloromethane) to give 34a as a white powder (0.015 g, 0.028 mmol, 57%).

[0146] [ka] Example 35 1-(4-cyano-3-trifluoromethyl-phenyl)-3-[7-(4-cyano-3-trifluoromethyl-phenyl)-6-thioxo-5-p-tolyl-5,7-diaza-spiro[3.4]oct-8-ylidene]-thiourea, 35a, [RD93] 1a (0.5.02 g, 2.2 mmol) and 7a (0.186 g, 1 mmol) in DMF (1 mL) were stirred at room temperature. After stirring for 20 h, the mixture was concentrated under reduced pressure to give an orange viscous liquid, which was chromatographed (dichloromethane:acetone, 99:1) to give 35a (0.269 g, 0.42 mmol, 42%) as a yellow powder.

[0147] [ka] Example 36 36-1). 1-(4-hydroxymethylphenylamino)-cyclobutanecarbonitrile, 36a Trimethylsilyl cyanide (0.66 ml, 5 mmol) was added dropwise to a mixture of 4-aminobenzoic acid (0.492 g, 4 mmol) and cyclobutanone (0.35 g, 5 mmol) in dichloromethane (10 ml). The reaction mixture was stirred at room temperature for 6 h and then concentrated under reduced pressure to give a brown liquid, which was chromatographed (dichloromethane) to give 36a (0.677 g, 3.36 mmol, 84%) as a brown solid.

[0148] 36-2).4-[8-(4-hydroxymethylphenyl)-5-oxo-7-thioxo-6-azaspiro[3.4]oct-6-yl]-2-trifluoromethyl-benzonitrile, 36b, [RD110] A mixture of 1a (0.342 g, 1.5 mmol) and 36a (0.21 g, 1 mmol) in dry DMF (0.5 mL) was stirred at room temperature for 24 hours. To this mixture, methanol (20 mL) and 2N aqueous HCl (5 mL) were added. This second mixture was refluxed for 6 hours. After cooling to room temperature, the reaction mixture was poured into cold water (40 mL) and extracted with ethyl acetate (60 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane:acetone, 90:10) to give 36b (0.296 g, 0.69 mmol, 69%) as a white powder.

[0149] [ka] Example 37 4-[5-(4-formylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethyl-benzonitrile, 37a, [RD114] To a mixture of 36b (0.303 g, 0.7 mmol) and Dess-Martin periodinane (0.417 g, 1 mmol) in dichloromethane (5 mL) was added pyridine (1.01 g, 1 mmol). The mixture was stirred at room temperature for 2 h, and then ethyl ether (10 mL) was added to precipitate the reaction by-product. After filtration and concentration under reduced pressure, the mixture was chromatographed (dichloromethane:acetone, 95:5) to give 37a (0.24 g, 0.56 mmol, 80%) as a white powder.

[0150] [ka] Example 38 4-{5-[4-(1-hydroxyethyl)-phenyl]-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl}-2-trifluoromethyl-benzonitrile, 38a [RD116] A mixture of 37a (0.043 g, 0.1 mmol) and dry THF (1 mL) in a flame-dried flask was placed under argon and cooled to −78°C. Methylmagnesium iodide (1.1 mL, 0.1 M) was then added. The mixture was stirred at −78°C for 30 min and allowed to warm slowly to room temperature. The medium was washed with water (3 mL) and extracted with ethyl acetate (10 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane:acetone, 95:5) to give 38a (0.037 g, 0.082 mmol, 82%) as a white powder.

[0151] [ka] Example 39 3-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-acrylic acid ethyl ester, 39a [RD117] A mixture of 37a (0.043 g, 0.1 mmol) and (carbethoxyethylidene)triphenylphosphorane (0.039 g, 0.12 mmol) in dichloromethane (2 mL) was stirred at room temperature for 10 h. The medium was concentrated and chromatographed (dichloromethane) to give 39a (0.048 g, 0.096 mmol, 96%) as a white powder.

[0152] [ka] Example 40 4-{5-[4-(3-hydroxypropenyl)-phenyl]-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl}-2-trifluoromethylbenzonitrile, 40a [RD120] To a mixture of 39a (0.05 g, 0.1 mmol) in dichloromethane (2 mL) was added a solution of diisobutylaluminum hydride in THF (0.11 mL, 1 M, 0.11 mmol) at −78° C. The mixture was stirred at −78° C. for 3 h. After warming to room temperature, the mixture was washed with aqueous sodium thiosulfate and extracted with ethyl acetate. The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane:acetone, 95:5) to give 40a (0.040 g, 0.089 mmol, 89%) as a white powder.

[0153] [ka] Example 41 41-1) 3-[4-(1-cyanocyclobutylamino)-phenyl]-propionic acid, 41a(41-1) Trimethylsilyl cyanide (0.4 g, 4 mmol) was added dropwise to a mixture of 3-(4-aminophenyl)-propionic acid (0.33 g, 2 mmol), cyclobutanone (0.35 g, 5 mmol), and sodium sulfate (1 g) in 1,4-dioxane (5 mL). The mixture was stirred for 15 h. After filtration to remove the sodium sulfate, the medium was concentrated under vacuum to give a brown liquid, which was chromatographed (dichloromethane:acetone, 50:50) to give 41a (0.472 g, 1.93 mmol, 97%) as a yellowish solid.

[0154] 41-2) 3-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-propionic acid methyl ester, 41b(41-2) [RD128] A mixture of 1a (0.661 g, 2.9 mmol) and 41a (0.472 g, 1.93 mmol) in dry DMF (2 mL) was stirred at room temperature for 15 h. To this mixture was added methanol (10 mL) and aqueous HCl (5 mL, 2 M). This second mixture was refluxed for 3 h. After cooling to room temperature, the reaction mixture was poured into cold water (10 mL) and extracted with ethyl acetate (3 × 30 mL). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane) to give 41b (0.582 g, 1.19 mmol, 62%) as a white powder.

[0155] [ka] 41-3) 3-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-propionic acid, 41c(41-3) [RD132] A mixture of 41b (0.487 g, 1 mmol) and sodium hydroxide solution (10 ml, 2 M) in methanol (10 ml) was stirred at room temperature for 5 h. Methanol was evaporated. The residue was adjusted to pH = 5 with aqueous HCl (2 M) and then extracted with ethyl acetate (3 × 50 ml). The organic layer was dried over MgSO and concentrated to dryness to give 41c (0.472 g, 0.99 mmol, 99%).

[0156] 41-4) 3-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-propionamide, 41d(41-4) [RD133] To a suspension of 41c (0.094 g, 0.2 mmol) in THF (10 ml) was added thionyl chloride (0.019 ml, 0.26 mmol) at −5° C. The medium was stirred at −5° C. for 1 h. Ammonia was then bubbled into the mixture. Excess ammonia was condensed under a reflux condenser at −78° C. for 30 min and then evaporated. The medium was filtered. The filtrate was concentrated and chromatographed (dichloromethane:acetone, 70:30) to give 41d (0.09 g, 0.19 mmol, 95%) as an off-white powder.

[0157] [ka] 41-5) 3-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-N-methyl-propionamide, 41e(41-5) [RD134] To a suspension of 41c (0.094 g, 0.2 mmol) in THF (10 ml) was added thionyl chloride (0.019 ml, 0.26 mmol) at −5° C. The mixture was stirred at −5° C. for 1 h. Methylamine was bubbled through the mixture at −5° C. for 30 min. The mixture was filtered. The filtrate was concentrated and chromatographed (dichloromethane:acetone, 75:25) to give 41e (0.092 g, 0.19 mmol, 95%) as an off-white powder.

[0158] [ka] 41-6) 3-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-N-(2-hydroxyethyl)-propionamide, 41f(41-6) [RD135] To a suspension of 41c (0.094 g, 0.2 mmol) in THF (10 ml) was added thionyl chloride (0.019 ml, 0.26 mmol) at −5° C. The medium was stirred at −5° C. for 1 h. 2-aminoethanol (0.0183 g, 0.03 mmol) was then added to the mixture at −5° C. After stirring for another 30 min, the medium was filtered. The filtrate was concentrated and chromatographed (dichloromethane:acetone, 50:50) to give 41f (0.093 g, 0.18 mmol, 90%) as an off-white powder.

[0159] [ka] 42-1) 4-[4-(1-cyanocyclobutylamino)-phenyl]-butyric acid, 42a Trimethylsilyl cyanide (0.50 g, 5 mmol) was added dropwise to a mixture of 4-(4-aminophenyl)-butyric acid (0.537 g, 3 mmol), cyclobutanone (0.35 g, 5 mmol), and sodium sulfate (1 g) in 1,4-dioxane (10 ml). The mixture was stirred for 15 h. After filtration to remove the sodium sulfate, the medium was concentrated under vacuum to give a brown liquid, which was chromatographed (dichloromethane:acetone, 50:50) to give 42a (0.665 g, 2.58 mmol, 86%) as a yellowish solid.

[0160] 42-2) 4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-butyric acid methyl ester, 42b [RD129] A mixture of 1a (0.547 g, 2.4 mmol) and 42a (0.342 g, 1.5 mmol) in dry DMF (2 mL) was stirred at room temperature for 15 h. To this mixture was added methanol (10 mL) and aqueous HCl (5 mL, 2 M). This second mixture was refluxed for 3 h. After cooling to room temperature, the reaction mixture was poured into cold water (10 mL) and extracted with ethyl acetate (3 × 30 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane) to give 42b (0.594 g, 1.18 mmol, 79%) as a white powder.

[0161] [ka] 42-3) 4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl {methyl}-butyric acid, 42c [RD141] A mixture of 42b (0.501 g, 1 mmol) and sodium hydroxide solution (10 ml, 2 M) in methanol (10 ml) was stirred at room temperature for 5 h. Methanol was evaporated. The residue was adjusted to pH = 5 with aqueous HCl (2 M) and then extracted with ethyl acetate (3 × 50 ml). The organic layer was dried over MgSO and concentrated to dryness to give 42c (0.482 g, 0.99 mmol, 99%), the structure of which is illustrated in Formula 5.

[0162] [ka] 42-4) 4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-butyramide, 42d [RD130] To a suspension of 42c (0.097 g, 0.2 mmol) in THF (10 ml) was added thionyl chloride (0.019 ml, 0.26 mmol) at −5° C. The medium was stirred at −5° C. for 1 h. Ammonia was then bubbled into the mixture. Excess ammonia was condensed under a reflux condenser at −78° C. for 30 min and then evaporated. The medium was filtered. The filtrate was concentrated and chromatographed (dichloromethane:acetone, 70:30) to give 42d (0.093 g, 0.19 mmol, 95%) as an off-white powder.

[0163] [ka] 42-5) 4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-N-methyl-butyramide, 42e [RD131] To a suspension of 42c (0.097 g, 0.2 mmol) in THF (10 ml) was added thionyl chloride (0.019 ml, 0.26 mmol) at −5° C. The medium was stirred at −5° C. for 1 h. The mixture was stirred. Methylamine was bubbled through the mixture for 30 minutes at -5°C. The medium was filtered. The filtrate was concentrated and chromatographed (dichloromethane:acetone, 75:25) to give 42e (0.095 g, 0.19 mmol, 95%) as an off-white powder.

[0164] [ka] 42-6) N-(4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}-butanoyl)-methanesulfonamide, 42f [RD157] A mixture of 4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}butanoic acid (42c) (0.049 g, 0.1 mmol), 2,4,6-trichlorobenzoyl chloride (0.244 g, 1 mmol), 4-dimethylaminopyridine (0.122 g, 1 mmol), and methanesulfonamide (0.019 g, 0.2 mmol) in dichloromethane was stirred at room temperature for 20 hours. The mixture was concentrated and chromatographed (dichloromethane:acetone, 80:20) to give N-(4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}-butanoyl)-methanesulfonamide (42f) [RD157] (0.053 g, 0.094 mmol, 94%) as a white powder, the structure of which is depicted in Formula 8.

[0165] [ka] 42-7) N-methyl-4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-6,8-dioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}butyramide, 42g [RD158] Hydrogen peroxide (30%, 0.4%) was added dropwise to a solution of N-methyl-4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}butanamide (42e) (0.032 g, 0.064 mmol) in glacial acetic acid (0.5 ml). The mixture was stirred at room temperature for 5 hours, then washed with water and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, concentrated, and chromatographed (dichloromethane:acetone, 80:20) to give N-methyl-4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-6,8-dioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}butyramide (42g) [RD158] (0.029 g, 0.06 mmol, 94%) as a white powder, the structure of which is shown in Formula 9.

[0166] [ka] Example 43 43-1) 4-(4-aminophenyl)-piperazine-1-carboxylic acid tert-butyl ester, 43a A mixture of 4-iodoaniline (0.654 g, 3 mmol), piperazine-1-carboxylic acid tert-butyl ester (0.67 g, 3.6 mmol), potassium phosphate (1.272 g, 6 mmol), ethylene glycol (0.33 mL), and copper iodide (0.03 g, 0.15 mmol) in 2-propanol (3 mL) was placed under argon in a sealed tube and heated at 80 °C for 30 h. After cooling to room temperature, the medium was washed with water (50 mL) and extracted with ethyl acetate (100 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone, 70:30) to give 43a (0.36 g, 1.3 mmol, 43%) as a yellow powder.

[0167] 43-2) 4-[4-(1-cyanocyclobutylamino)phenyl]-piperazine-1-carboxylic acid tert-butyl ester, 43b Trimethylsilyl cyanide (0.3 g, 3 mmol) was added dropwise to a mixture of 43a (0.415 g, 1.5 mmol), cyclobutanone (0.21 g, 3 mmol), and sodium sulfate (1 g) in dichloromethane (5 mL). The mixture was stirred for 15 h. After filtration to remove the sodium sulfate, the medium was concentrated in vacuo to give a brown liquid, which was chromatographed (dichloromethane:acetone, 75:25) to give 43b (0.448 g, 1.26 mmol, 84%) as a yellow solid.

[0168] 43-3) 4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-imino-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-piperazine-1-carboxylic acid tert-butyl ester, 43c [RD139] and 4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-(4-cyano-3-trifluoromethyl-phenylthiocarbamoylimino)-6-thioxo-5 ,7-Diazaspiro[3.4]oct-5-yl]-phenyl}-piperazine-1-carboxylic acid tert-butyl ester, 43d [RD140] 1a (0.228 g, 1 mmol) and 43b (0.472 g, 0.63 mmol) in dry DMF (1 mL) were stirred at room temperature for 20 h. The mixture was concentrated and chromatographed (dichloromethane:acetone, 90:10) to give 43c (0.173 g, 0.296 mmol, 47%) as an off-white powder, the structure of which is illustrated in Formula 10, and 43d (0.169 g, 0.21 mmol, 33%) as a yellow powder, the structure of which is illustrated in Formula 11.

[0169] [ka] 43-4) 4-[8-oxo-5-(4-piperazin-1-yl-phenyl)-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethylbenzonitrile, 43e [RD137] A mixture of 43c (0.117 g, 0.2 mmol), methanol (5 mL), and aqueous HCl (2 mL, 2 M) was refluxed for 2 h. After cooling to room temperature, the mixture was poured into cold water (10 mL) and extracted with ethyl acetate (3 × 30 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone, 50:50, then methanol:acetone, 50:50) to give 43e (0.089 g, 0.184 mmol, 92%) as a white powder.

[0170] [ka] 43-5) 4-{5-[4-(4-methanesulfonylpiperazin-1-yl)-phenyl]-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl}-2-trifluoromethylbenzonitrile, 43f [RD138] A mixture of 43e (0.049 g, 0.1 mmol), methanesulfonyl chloride (0.012 mL, 0.15 mmol), and triethylamine (0.15 mL) in dichloromethane was stirred at room temperature for 5 hours. The medium was filtered. The filtrate was concentrated and chromatographed (dichloromethane:acetone, 95:5) to give 43f (0.042 g, 0.074 mmol, 74%) as a white powder.

[0171] [ka] Example 44 44-1) 3-{4-[7-(4-cyano-3-trifluoromethyl-phenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-acrylic acid, 44a A solution of 39a (0.025 g, 0.05 mmol) and sodium hydroxide (2 ml, 2 M) in methanol (2 ml) was stirred at room temperature for 5 h. Methanol was evaporated. The residue was adjusted to pH = 5 with aqueous HCl (2 M) and then extracted with ethyl acetate (3 × 50 ml). The organic layer was dried over MgSO and concentrated to dryness to give 44a (0.02 g, 0.042 mmol, 85%).

[0172] 44-2) 3-{4-[7-(4-cyano-3-trifluoromethyl-phenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-acrylamide, 44b [RD119] To a suspension of 44b (0.02 g, 0.042 mmol) in THF (1 ml) was added thionyl chloride (0.007 ml, 0.1 mmol) at −5° C. The medium was stirred at −5° C. for 1 hour. Ammonia was then bubbled into the mixture. Excess ammonia was removed by The mixture was refluxed at 78° C. for 30 minutes and then evaporated. The medium was filtered. The filtrate was concentrated and chromatographed (dichloromethane:acetone, 70:30) to give 44b (0.014 g, 0.03 mmol, 71%) as an off-white powder.

[0173] [ka] (Example 45 [RD145]) Trimethylsilyl cyanide (0.4 g, 4 mmol) was added dropwise to a mixture of 4-methanesulfonylphenylamine hydrochloride (0.415 g, 2 mmol), cyclobutanone (0.28 g, 4 mmol), and sodium sulfate (1 g) in DMF (3 mL). The mixture was stirred at 120 °C for 15 h. After filtration to remove the sodium sulfate, the filtrate was washed with brine and extracted with ethyl acetate. The organic layer was concentrated and chromatographed (dichloromethane:acetone, 90:10) to give 1-(4-methanesulfonylphenylamino)cyclobutanecarbonitrile (45a) (0.116 g, 0.44 mmol, 22%) as a yellowish solid. 4-Methanesulfonylphenylamine was also recovered (0.201 g, 1.17 mmol, 59%).

[0174] 4-Isothiocyanato-2-trifluoromethylbenzonitrile (1a) (0.0.141 g, 0.62 mmol) and 1-(4-methanesulfonylphenylamino)cyclobutanecarbonitrile (45a) (0.11 g, 0.42 mmol) in dry DMF (2 ml) were stirred at room temperature for 3 days. To this mixture, methanol (10 ml) and 2N aqueous HCl (5 ml) were added. This second mixture was refluxed for 3 hours. After cooling to room temperature, the mixture was poured into cold water (10 ml) and extracted with ethyl acetate (3 x 30 ml). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone, 97:3) to give 4-[5-(4-methanesulfonylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethylbenzonitrile (45b) [RD145] as a white powder (0.031 g, 0.065 mmol, 15%), the structure of which is illustrated in Formula 14.

[0175] [ka] Example 46 Trimethylsilyl cyanide (0.69 g, 7 mmol) was added dropwise to a mixture of 4-aminophenylacetic acid (0.755 g, 5 mmol) and cyclobutanone (0.49 g, 7 mmol) in dioxane (20 mL). The mixture was stirred at 80 °C for 8 h. The mixture was concentrated and chromatographed (dichloromethane:acetone, 60:40) to give [4-(1-cyanocyclobutylamino)phenyl]acetic acid (46a) as a white powder (1.138 g, 4.95 mmol, 99%).

[0176] 46-1)RD146 A mixture of 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a) (0.638 g, 2.8 mmol) and [4-(1-cyanocyclobutylamino)phenyl]acetic acid (46a) (0.46 g, 2.0 mmol) in DMF (5 mL) was stirred at room temperature for 15 hours. To this mixture was added methanol (20 mL) and 2N aqueous HCl (10 mL). This second mixture was refluxed for 1 hour. After cooling to room temperature, the mixture was poured into cold water (10 mL) and extracted with ethyl acetate (3 × 50 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (pure dichloromethane, then dichloromethane:acetone, 95:5) to give {4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}acetic acid methyl ester (46b) [RD146] as a white powder (0.532 g, 1.124 mmol, 56%), the structure of which is illustrated in Formula 15.

[0177] [ka] 46-2)RD147 A mixture of {4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}acetic acid methyl ester (46b) (0.095 g, 0.2 mmol) and sodium hydroxide (1 ml, 2 M) in methanol (2 ml) was stirred at room temperature for 2 hours. The methanol was evaporated. The residue was adjusted to pH 5 with 2 M aqueous HCl, and then the mixture was extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over MgSO4 and concentrated to dryness to give {4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}acetic acid (46c) [RD147] (0.087 g, 0.19 mmol, 95%), the structure of which is illustrated in Formula 16.

[0178] [ka] 46-3)RD148 Thionyl chloride (0.238 g, 2 mmol) was added dropwise to a mixture of {4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}acetic acid (46c) (0.357 g, 0.777 mmol) in THF (5 mL) cooled to 0 °C. The mixture was stirred at room temperature for 1 hour, and then ammonia was bubbled through the mixture. Excess ammonia was condensed using a reflux condenser at −78 °C for 30 minutes and then evaporated. The medium was filtered, and the filtrate was concentrated and chromatographed (dichloromethane:acetone, 70:30) to give 2-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}acetamide (46d) [RD148] as an off-white powder (0.345 g, 0.75 mmol, 97%), the structure of which is depicted in Formula 17.

[0179] [ka] 46-4)RD149 Thionyl chloride (0.238 g, 2 mmol) was added dropwise to a mixture of {4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}acetic acid (46c) (0.357 g, 0.777 mmol) in THF (5 mL) cooled to 0° C. The mixture was stirred at room temperature for 1 hour, and then methylamine (0.5 mL) was added to the mixture. The mixture was stirred for an additional 2 hours. The medium was filtered, and the filtrate was concentrated and chromatographed (dichloromethane:acetone, 80:20) to give N-methyl-2-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}acetamide (46e) [RD149] as an off-white powder (0.348 g, 0.738 mmol, 95%), the structure of which is depicted in Formula 18.

[0180] [ka] Example 47 N-{4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]phenyl}methanesulfonamide (47a) [RD150] 4-[3-(4-aminophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzene in dichloromethane (1 ml) A mixture of 2d (0.02 g, 0.05 mmol), methanesulfonyl chloride (0.009 g, 0.075 mmol), and pyridine (0.006 g, 0.075 mmol) was stirred at room temperature for 15 hours. The mixture was washed with water (2 mL) and extracted with ethyl acetate (5 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (HPLC, alumina column) to give N-{4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]phenyl}methanesulfonamide (47a) [RD150] as a white powder (0.009 g, 0.018 mmol, 36%), the structure of which is shown in Formula 2.

[0181] [ka] Example 48 N-{4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]phenyl}acetamide, 48a, [RD151] A mixture of 4-[3-(4-aminophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile (2d) [RD9] (0.008 g, 0.02 mmol), acetyl chloride (0.004 g, 0.03 mmol) and triethylamine (0.003 g, 0.03 mmol) in dichloromethane (1 ml) was stirred at 0° C. for 2 h. The mixture was concentrated and chromatographed (dichloromethane:acetone, 90:10) to give N-{4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]phenyl}acetamide, 48a, [RD151] as a white powder (0.007 g, 0.016 mmol, 80%), the structure of which is illustrated in Formula 3.

[0182] [ka] Example 49 Concentrated sulfuric acid was slowly added to a mixture of 4-aminobenzoic acid (4 g, 29.2 mmol) in methanol cooled to 0 °C. After the addition, the mixture was stirred at room temperature for 5 h. The mixture was washed with a saturated solution of sodium bicarbonate and extracted with ethyl acetate. The organic layer was dried over MgSO and concentrated in vacuo to give 4-aminobenzoic acid methyl ester (49a) as an off-white solid (4.22 g, 27.9 mmol, 96%).

[0183] A mixture of 4-aminobenzoic acid methyl ester (0.32 g, 2.12 mmol), acetone cyanohydrin (3 mL), and sodium sulfate (1 g) was refluxed for 15 h. After filtration to remove the sodium sulfate, the filtrate was washed with brine and extracted with ethyl acetate. The organic layer was concentrated and chromatographed (dichloromethane:acetone, 60:40) to give 4-[(cyanodimethylmethyl)-amino]-benzoic acid methyl ester (49b) as a white solid (0.398 g, 1.95 mmol, 92%).

[0184] 49-1)RD152 4-Isothiocyanato-2-trifluoromethylbenzonitrile (1a) (0.228 g, 1 mmol) and 4-[(cyanodimethylmethyl)-amino]-benzoic acid methyl ester (49b) (0.14 g, 0.64 mmol) in DMF (2 ml) were heated at 60° C. for 12 hours under microwave irradiation. To this mixture was added methanol (6 ml) and 2 N aqueous HCl (2 ml). This second mixture was refluxed for 4 hours. After cooling to room temperature, the reaction mixture was poured into cold water (10 ml) and extracted with ethyl acetate (3×30 ml). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane; dichloromethane:acetone, 75:25) to give 4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]benzoic acid methyl ester (49c) [RD152] as a white powder (0.18 g, 0.4 mmol, 63%), the structure of which is illustrated in Formula 19.

[0185] [ka] 49-2)RD153 A mixture of 4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]benzoic acid methyl ester (49c) (0.02 g, 0.0435 mmol) and methylamine (2 mL, distilled from its 40% aqueous solution) was kept at −20° C. for 15 h. After evaporation of the methylamine, the mixture was chromatographed (dichloromethane:acetone, 80:20) to give 4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]-N-methylbenzamide (49d) [RD153] (0.01 g, 0.0224, 51%), the structure of which is depicted in Formula 20. Ester 4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]benzoic acid methyl The ethyl ester (49c) was also recovered (0.08 g, 0.0179 mmol, 41%).

[0186] [ka] Example 50 50-1)RD154 A mixture of 4-[8-(4-hydroxymethylphenyl)-5-oxo-7-thioxo-6-azaspiro[3.4]oct-6-yl]-2-trifluoromethyl-benzonitrile (36b) (0.086 g, 0.2 mmol) and methanesulfonyl anhydride (0.07 g, 0.4 mmol) in dichloromethane (1 mL) was stirred at room temperature for 15 h. The mixture was concentrated and chromatographed (dichloromethane:acetone, 98:2) to give methanesulfonic acid 4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]phenylmethyl ester (50a) [RD154] as a white powder (0.089 g, 0.175 mmol, 88%), the structure of which is shown in Formula 22.

[0187] [ka] 50-2) RD155 Methylamine (0.5 ml) was bubbled into methanesulfonic acid 4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]phenylmethyl ester (50a) (0.059 g, 0.115 mmol) in THF (3 ml) cooled to −78° C. After 1 h of reaction at −78° C., the mixture was concentrated and chromatographed (dichloromethane:acetone, 95:5; methanol) to give 4-[5-(4-methylaminomethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethylbenzonitrile (50b) [RD155] as a white powder (0.042 g, 0.095 mmol, 82%), the structure of which is depicted in Formula 23.

[0188] [ka] 50-3)RD156 A mixture of methanesulfonic acid 4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]phenylmethyl ester (50a) (0.02 g, 0.039 mmol) and dimethylamine (0.5 mL; distilled from its 40% aqueous solution) in THF (1 mL) was stirred at −78° C. for 2 h. The mixture was concentrated and chromatographed (dichloromethane:acetone, 95:5; acetone) to give 4-[5-(4-dimethylaminomethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethylbenzonitrile (50c) [RD156] as a white powder (0.017 g, 0.037 mmol, 95%), the structure of which is depicted in Formula 24.

[0189] [ka] Example 51 Sodium cyanide (0.245 g, 5 mmol) was added to 4-aminobenzoic acid (0.274 g, 2 mmol) and cyclobutanone (0.21 g, 3 mmol) in 90% acetic acid (4.5 mL). The reaction mixture was stirred at room temperature for 15 hours. The mixture was washed with aqueous HCl (pH 2) and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated to dryness in vacuo to give 4-(1-cyanocyclobutylamino)benzoic acid (51a) as a white solid (0.426 g, 1.97 mmol, 99%).

[0190] 51-1) RD159 and RD160 4-Isothiocyanato-2-trifluoromethylbenzonitrile in DMF (2 ml) A mixture of 1a (0.51 g, 2.22 mmol) and 4-(1-cyanocyclobutylamino)benzoic acid 51a (0.343 g, 1.59 mmol) was heated to 60° C. under microwave irradiation and stirred for 16 hours. To this mixture was added methanol (10 ml) and 2 M aqueous HCl (5 ml). This second mixture was refluxed for 12 hours. After cooling to room temperature, the reaction mixture was poured into cold water (20 ml) and extracted with ethyl acetate (3×30 ml). The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane:acetone, 95:5) to give 4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-benzoic acid methyl ester (51b) [RD159] as a white powder (0.09 g, 0.196 mmol, 12%). Its structure is depicted in Formula 25, and N-(3-cyano-4-trifluoromethylphenyl)-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]benzamide (51b') [RD160] was obtained as a white powder (0.28 g, 0.45 mmol, 29%), the structure of which is depicted in Formula 26.

[0191] [ka] 51-2)RD161 A mixture of 4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-benzoic acid methyl ester (51b) (0.046 g, 0.1 mmol) and methylamine (1 mL distilled from its 40% aqueous solution) was kept at −20° C. for 15 h. After evaporation of the methylamine, the mixture was chromatographed (dichloromethane:acetone, 80:20) to give N-methyl-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]benzamide (51c) [RD161] (0.041 g, 0.085, 84%), the structure of which was confirmed. This is illustrated in Chemical Formula 27.

[0192] [ka] (Example 52 [RD162]) Thionyl chloride (2.38 g, 20 mmol) was slowly added to a solution of 2-fluoro-4-nitrobenzoic acid (2.97 g, 16 mmol) in DMF (50 ml) cooled to -5 °C. The mixture was stirred for another hour at -5 °C. Methylamine (0.62 g, 20 mmol; freshly distilled from its 40% aqueous solution) was added to the reaction medium. This second mixture was stirred for another hour. Ethyl acetate (300 ml) was added to the mixture, which was washed with brine (3 × 150 ml). The organic layer was dried over MgSO4 and concentrated to give N-methyl-2-fluoro-4-nitrobenzamide (52a) as a yellow solid (2.89 g, 14.6 mmol, 91%).

[0193] [ka] A mixture of N-methyl-2-fluoro-4-nitrobenzamide (52a) (2.89 g, 14.6 mmol) and iron (5.04 g, 90 mmol) in ethyl acetate (40 mL) was refluxed for 1 h. The solid particles were filtered off. The filtrate was washed with water and extracted with ethyl acetate. The organic layer was dried over MgSO4, concentrated, and chromatographed (dichloromethane:acetone, 95:5) to give N-methyl-2-fluoro-4-aminobenzamide (52b) as an off-white solid (2.3 g, 13.7 mmol, 94%).

[0194] [ka] Sodium cyanide (1.47 g, 30 mmol) was added to a mixture of N-methyl-2-fluoro-4-aminobenzamide (52b) (1.68 g, 10 mmol) and cyclobutanone (1.4 g, 20 mmol) in 90% acetic acid (20 mL). The reaction mixture was stirred at 80 °C for 24 hours. The mixture was washed with water and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated to dryness under vacuum. The solid was washed with a 50:50 mixture of ethyl ether and hexane (10 mL) to remove cyclobutanone cyanohydrin and, after filtration, N-methyl-4-(1-cyanocyclobutylamino)-2-fluorobenzamide (52b) was obtained. This gave fluorobenzamide (52c) (2.19 g, 8.87 mmol, 89%).

[0195] [ka] A mixture of 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a) (2.16 g, 9.47 mmol) and N-methyl-4-(1-cyanocyclobutylamino)-2-fluorobenzamide (52c) (1.303 g, 5.27 mmol) in DMF (20 ml) was heated at 80° C. for 16 hours under microwave irradiation. To this mixture was added methanol (50 ml) and 2N aqueous HCl (20 ml). This second mixture was refluxed for 3 hours. After cooling to room temperature, the reaction mixture was poured into cold water (100 ml) and extracted with ethyl acetate (150 ml). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone, 95:5) to give N-methyl-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluorobenzamide (52d) [RD162] as a yellow powder (1.43 g, 3.0 mmol, 57%), the structure of which is illustrated in Formula 28.

[0196] [ka] (Example 53 [RD163]) 4-Nitro-3-fluorophenol (0.314 g, 2 mmol) and iron (0.56 g, 10 mmol) in ethyl acetate (4 mL) and acetic acid (2 mL) were refluxed for 3 hours. The solid particles were filtered off. The filtrate was washed with water and extracted with ethyl acetate. The organic layer was dried over MgSO4 and concentrated to give 4-amino-3-fluorophenol (53a) as a brown solid (0.25 g, 19.6 mmol, 98%).

[0197] [ka] Sodium cyanide (0.194 g, 4 mmol) was dissolved in 4-amino-3-fluorophenol (0.29 g, 2.28 mmol) and cyclobutanoic acid in 90% acetic acid (3 ml). The resulting mixture was added to a mixture of 1-(2-fluoro-4-hydroxyphenylamino)-cyclobutanecarbonitrile (53b) (0.175 g, 2.5 mmol). The reaction mixture was stirred at room temperature for 15 hours. The medium was washed with water and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, concentrated, and chromatographed (dichloromethane:acetone, 90:10) to give 1-(2-fluoro-4-hydroxyphenylamino)-cyclobutanecarbonitrile (53b) (0.271 g, 1.31 mmol, 58%) as an off-white solid.

[0198] [ka] A mixture of 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a) (0.228 g, 1.0 mmol) and 1-(2-fluoro-4-hydroxyphenylamino)-cyclobutanecarbonitrile (53b) (0.145 g, 0.7 mmol) in dry DMF (2 ml) was stirred at room temperature for 24 hours. To this mixture was added methanol (10 ml) and 2 M aqueous HCl (2 ml). This second mixture was refluxed for 1 hour. After cooling to room temperature, the reaction mixture was poured into cold water (10 ml) and extracted with ethyl acetate (50 ml). The organic layer was dried over MgSO, concentrated, and chromatographed (pure dichloromethane, and then dichloromethane:acetone, 90:10) to give 4-[5-(2-fluoro-4-hydroxyphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethylbenzonitrile (53c) [RD163] as an off-white powder (0.17 g, 0.39 mmol, 56%), the structure of which is illustrated in Formula 29.

[0199] [ka] (Example 54 [RD168]) A mixture of 4-nitro-2-fluorobenzonitrile (1.83 g, 5 mmol) and iron (1.68 g, 6 mmol) in a mixture of acetic acid (40 mL) and ethyl acetate (40 mL) was refluxed for 2 hours. The solids were filtered off, and the filtrate was washed with water and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, concentrated, and chromatographed (dichloromethane:acetone, 95:5) to give 4-amino-2-fluorobenzonitrile (54a) (0.653 g, 4.8 mmol, 96%).

[0200] Sodium cyanide (0.74 g, 15 mmol) was added to a mixture of 4-amino-2-fluorobenzonitrile (1.36 g, 10 mmol) and cyclopentanone (1.26 g, 15 mmol) in 90% acetic acid (10 ml). The reaction mixture was stirred at room temperature for 3 hours, then heated to 80 °C and stirred for an additional 5 hours. The mixture was washed with water and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, concentrated, and chromatographed (dichloromethane:acetone, 97:3) to give 4-(1-cyanocyclopentylamino)-2-fluorobenzonitrile (54b) as a yellow solid (2.07 g, 9.03 mmol, 90%).

[0201] [ka] 4-Isothiocyanato-2-trifluoromethylbenzonitrile (1a) (0.171 g, 0.75 mmol) and 4-(1-cyanocyclopentylamino)-2-fluorobenzonitrile (54b) (0.115 g, 0.5 mmol) in dry DMF (1 ml) were heated to 60° C. for 48 hours under microwave irradiation. To this mixture was added methanol (3 ml) and 2 M aqueous HCl (2 ml). This second mixture was refluxed for 1 hour. After cooling to room temperature, the reaction mixture was poured into cold water (10 ml) and extracted with ethyl acetate (15 ml). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone, 98:2) to give 4-[1-(4-cyano-3-fluorophenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl]-2-trifluoromethylbenzonitrile (54c) [RD168] as an off-white powder (0.017 g, 0.037 mmol, 7%), the structure of which is illustrated in Formula 30.

[0202] [ka] Example 55 [RD136 and RD142] Additional diarylhydantoin compounds can be synthesized, including the following compounds illustrated in formulas 35 and 36:

[0203] [ka] (Example 56 [RD162']) In the following, air- or moisture-sensitive reactions were performed under an argon atmosphere using oven-dried glassware and standard syringe / septum techniques. Reactions were monitored using SiO2 TLC plates under UV light (254 nm), followed by visualization with p-anisaldehyde or ninhydrin staining solutions. Column chromatography was performed on silica gel 60. 1 H NMR was measured at 400 MHz in CDCl3 unless otherwise stated, and data are reported in ppm (δ): chemical shift (multiplicity, integral, coupling constant, Hz) from an internal standard (TMS, 0.0 ppm).

[0204] [ka] Periodic acid (1.69 g, 7.41 mmol) was dissolved in acetonitrile (25 mL) by vigorous stirring, and then chromium trioxide (0.16 g, 1.60 mmol) was dissolved in the solution. 2-Fluoro-4-nitrotoluene (0.33 g, 2.13 mmol) was added to the above solution with stirring. A white precipitate immediately formed accompanied by an exothermic reaction. After stirring for 1 hour, the supernatant liquid of the reaction mixture was decanted into a flask, and the solvent was removed by evaporation. The residue was dissolved in methylene chloride (2 × 30 mL) and water ( The organic layer was dried over MgSO4 and concentrated to give 2-fluoro-4-nitrobenzoic acid (Formula 37) (0.32 mg, 81%) as a white solid.

[0205] [ka] Thionyl chloride (0.15 g, 1.30 mmol) was slowly added to a solution of 2-fluoro-4-nitrobenzoic acid (Formula 37) (0.20 g, 1.10 mmol) in DMF (5 mL) cooled to −5° C. The mixture was stirred for an additional 1 h at −5° C. Excess methylamine (freshly distilled from its 40% aqueous solution) was added to the reaction medium. This second mixture was stirred for an additional 1 h. Ethyl acetate (50 mL) was added to the mixture, which was washed with brine (2 × 50 ml). The organic layer was dried over MgSO4 and concentrated to give N-methyl-2-fluoro-4-nitrobenzamide (Formula 38) (0.18 g, 85%) as a yellowish solid.

[0206] [ka] N-methyl-2-fluoro-4-nitrobenzamide (Formula 38) (0.18 g, 0.91 mmol) and iron (0.31 g, 5.60 mmol) in ethyl acetate (5 mL) and acetic acid (5 mL) were refluxed for 1 hour. The solid particles were filtered off. The filtrate was washed with water and extracted with ethyl acetate. The organic layer was dried over MgSO4 and concentrated, and the residue was purified by SiO2 column chromatography (dichloromethane:acetone, 95:5) to give N-methyl-2-fluoro-4-aminobenzamide (Formula 39) (0.14 g, 92%) as an off-white solid.

[0207] [ka] A mixture of N-methyl-2-fluoro-4-aminobenzamide (Formula 39) (96 mg, 0.57 mmol), acetone cyanohydrin (0.3 mL, 3.14 mmol), and magnesium sulfate (50 mg) was heated to 80 °C and stirred for 12 hours. To this mixture was added ethyl acetate (25 mL), which was then washed with water (2 × 25 mL). The organic layer was dried over MgSO and concentrated, and the residue was purified using SiO column chromatography (dichloromethane:acetone, 95:5) to give N-methyl-2-fluoro-4-(1,1-dimethyl-cyanomethyl)-aminobenzamide (Formula 40) (101 mg, 75%) as a white solid.

[0208] [ka] 4-Amino-2-trifluoromethylbenzonitrile (2.23 g, 12 mmol) was added portionwise over 15 minutes to a well-stirred heterogeneous mixture of thiophosgene (1 mL, 13 mmol) in water (22 mL) at room temperature. Stirring was continued for another hour. The reaction medium was extracted with chloroform (3 × 15 mL). The combined organic layers were dried over MgSO4 and evaporated to dryness under reduced pressure to give the desired product 4-isothiocyanato-2-trifluoromethylbenzonitrile (Formula 41) (2.72 g, 11.9 mmol, 99%) as a brownish solid, which was used as such for the next step.

[0209] [ka]

[0210] [ka] 56-1)RD162' A mixture of N-methyl-2-fluoro-4-(1,1-dimethylcyanomethyl)-aminobenzamide (Formula 40) (30 mg, 0.13 mmol) and 4-isothiocyanato-2-trifluoromethylbenzonitrile (Formula 41) (58 mg, 0.26 mmol) in DMF (1 mL) was heated at 100 °C for 11 h under microwave irradiation. To this mixture was added methanol (20 mL) and 1N aqueous HCl (5 mL). This second mixture was refluxed for 1.5 h. After cooling to room temperature, the reaction mixture was poured into cold water (50 mL) and extracted with ethyl acetate (50 mL). The organic layer was dried over MgSO and concentrated, and the residue was purified using SiO column chromatography (dichloromethane:acetone, 95:5) to give RD162' (Formula 42) (15 mg, 25%) as colorless crystals.

[0211] [ka] Example 57

[0212] [ka] A mixture of N-methyl-2-fluoro-4-aminobenzamide (Formula 39) (62 mg, 0.37 mmol), cyclopentanone (0.07 mL, 0.74 mmol), and TMSCN (0.1 mL, 0.74 mmol) was heated to 80 °C and stirred for 13 hours. Ethyl acetate (2 × 20 mL) was added to the mixture, which was then washed with water (2 × 20 mL). The organic layer was dried over MgSO and concentrated, and the residue was purified using silica gel column chromatography (dichloromethane:acetone, 95:5) to give N-methyl 2-fluoro-4-(1-cyanocyclopentyl)aminobenzamide (Formula 43) (61 mg, 63%) as a white solid.

[0213] [ka]

[0214] [ka] 57-1)RD162'' A mixture of N-methyl 2-fluoro-4-(1-cyanocyclopentyl)aminobenzamide (Formula 43) (57 mg, 0.22 mmol) and 4-isothiocyanato-2-trifluoromethyl benzonitrile (0.15 g, 0.65 mmol) in DMF (3 mL) was heated at 130 °C under microwave irradiation (open vessel) for 12 hours. To this mixture was added methanol (20 mL) and 1N aqueous HCl (5 mL). This second mixture was refluxed for 1.5 hours. After cooling to room temperature, the reaction mixture was poured into cold water (50 mL) and extracted with ethyl acetate (50 mL). The organic layer was dried over MgSO4, concentrated, and the residue was purified using silica gel column chromatography (dichloromethane:acetone, 95:5) to give 4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.4]nonan-1-yl)-2-fluoro-N-methylbenzamide, RD162'' (chemical formula) as a pale yellowish solid. The compound of formula 44 was obtained (8 mg, 7%).

[0215] [ka] Example 58

[0216] [ka] Trifluoroacetic anhydride (0.85 mL, 6.14 mmol) was added to a solution of 4-(4-aminophenyl)butyric acid (0.5 g, 2.79 mmol) in chloroform (10 mL) at 0 °C. The mixture was warmed to room temperature and stirred for 3 h. The mixture was partitioned with chloroform (20 mL) and water (20 mL). The organic layer was dried over MgSO and concentrated, and the residue was purified using silica gel column chromatography (dichloromethane:acetone, 9:1) to give 4-[4-(2,2,2-trifluoroacetylamino)phenyl]butanoic acid (Formula 45) (0.53 g, 69%).

[0217] [ka] Thionyl chloride (71 mg, 0.60 mmol) was slowly added to a solution of 4-[4-(2,2,2-trifluoroacetylamino)phenyl]butanoic acid (Formula 45) (0.15 g, 0.55 mmol) in DMF (5 mL) cooled to −5° C. The mixture was stirred for an additional 1 h at −5° C. Excess dimethylamine (freshly distilled from its 40% aqueous solution) was added to the reaction medium. This second mixture was stirred for an additional 1 h. Ethyl acetate (50 mL) was added to the mixture, which was washed with brine (2 × 50 mL). The organic layer was dried over MgSO4 and concentrated to give N,N-dimethyl 4-[4-(2,2,2-trifluoroacetylamino)phenyl]butanamide (Formula 46) as a yellowish solid (0.17 g, quantitative).

[0218] [ka] A 1N NaOH solution (3 mL) was added to a solution of N,N-dimethyl 4-[4-(2,2,2-trifluoroacetylamino)phenyl]butanamide (Formula 46) (0.17 g, 0.55 mmol) in methanol (2 mL) at room temperature. The mixture was stirred for 14 hours. The mixture was partitioned with chloroform (25 mL) and water (25 mL). The organic layer was dried over MgSO4 and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 9:1) to give N,N-dimethyl 4-[4-(2,2,2-trifluoroacetylamino)phenyl]butanamide (Formula 46) (0.17 g, 0.55 mmol) as a white solid. 4-(4-aminophenyl)butanamide (formula 47) was obtained (74 mg, 66%).

[0219] [ka] A mixture of N,N-dimethyl 4-(4-aminophenyl)butanamide (Formula 47) (74 mg, 0.36 mmol), cyclobutanone (54 mg, 0.78 mmol), and TMSCN (77 mg, 0.78 mmol) was heated to 80 °C and stirred for 15 h. Ethyl acetate (2 × 20 mL) was added to the mixture, which was then washed with water (2 × 20 mL). The organic layer was dried over MgSO and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 9:1) to give N,N-dimethyl 4-(4-aminophenyl)butanamide (Formula 47) as a white solid. 4-[4-(1-cyanocyclobutylamino)phenyl]butanamide (chemical formula 48) was obtained (58 mg, 57%).

[0220] [ka] A mixture of N,N-dimethyl 4-[4-(1-cyanocyclobutylamino)phenyl]butanamide (Formula 48) (58 mg, 0.20 mmol) and 4-isothiocyanato-2-trifluoromethyl benzonitrile (74 mg, 0.32 mmol) in DMF (3 mL) was heated under reflux for 2 hours. To this mixture was added methanol (20 mL) and 1N aqueous HCl (5 mL). This second mixture was refluxed for 1.5 hours. After cooling to room temperature, the reaction mixture was poured into cold water (50 mL) and extracted with ethyl acetate (50 mL). The organic layer was dried over MgSO4, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 95:5) to give 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)-N,N-dimethylbutanamide, RD169 (Formula 49) as a pale yellowish solid (44 mg, 42%).

[0221] [ka] Example 59

[0222] [ka] A mixture of 4-(4-aminophenyl)butyric acid (0.20 g, 1.12 mmol), cyclobutanone (0.17 mL, 2.23 mmol), and TMSCN (0.30 mL, 2.23 mmol) was heated to 80 °C and stirred for 13 h. Ethyl acetate (2 × 30 mL) was added to the mixture, which was then washed with water (2 × 30 mL). The organic layer was dried over MgSO and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 9:1) to give 4-[4-(1-cyanocyclobutylamino)phenyl]butanoic acid (Formula 50) as a yellowish solid (0.21 g, 74%).

[0223] [ka] A mixture of 4-[4-(1-cyanocyclobutylamino)phenyl]butanoic acid (Formula 50) (0.21 g, 0.83 mmol) and 4-isothiocyanato-2-trifluorobenzonitrile (0.25 g, 1.08 mmol) in toluene (10 mL) was heated under reflux for 1 hour. 1N aqueous HCl solution (5 mL) was added to the medium. This second mixture was refluxed for 1.5 hours. After cooling to room temperature, the reaction mixture was poured into cold water (50 mL) and extracted with ethyl acetate (50 mL). The organic layer was dried over MgSO4, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:acetone, 95:5) to give 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanoic acid, RD141 (Formula 51) (60 mg, 15%).

[0224] [ka] Example 60

[0225] [ka] To a solution of 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanoic acid, RD141 (Formula 51) (60 mg, 0.12 mmol) in DMF (3 mL) was added thionyl chloride (0.01 mL, 0.15 mmol) at 0° C. The mixture was stirred at 0° C. for 1 h. Ammonia was then bubbled through the mixture. The mixture was partitioned with ethyl acetate (25 mL) and water (25 mL). The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone, 70:30) to give 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanamide, RD130 (Formula 52) as a white powder (37 mg, 61%).

[0226] [ka] Example 61

[0227] [ka] A solution of DMSO (0.01 mL, 0.12 mmol) in dry dichloromethane (1 mL) was added to a stirred solution of oxalyl chloride (0.01 mL, 0.09 mmol) in dry dichloromethane (2 mL) at −78° C. After 15 minutes, a dichloromethane solution of 4-(4-(7-(4-cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanamide, RD130 (Formula 52) (35 mg, 0.07 mmol) was added to the reaction mixture. Stirring was continued at −78° C. for 20 minutes, and then triethylamine (0.03 mL, 0.22 mmol) was added. After 30 minutes at −78° C., the reaction mixture was warmed to room temperature, and then the reaction was quenched with saturated aqueous NH4Cl. The reaction mixture was diluted with dichloromethane and extracted with dichloromethane. The organic layer was dried over MgSO, concentrated, and chromatographed (dichloromethane:acetone, 95:5) to give 4-(5-(4-(3-cyanopropyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-2-(trifluoromethyl)benzonitrile, RD170 (Formula 53) (29 mg, 87%) as a viscous oil.

[0228] [ka] One of skill in the art can modify and / or combine the syntheses described herein to make other diarylhydantoin compounds.

[0229] Compounds of the invention also include those having the following formula:

[0230] [ka] where R is hydrogen, aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkyl halide, alkenyl halide, alkynyl halide, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or non-aromatic, substituted heteroaromatic or non-aromatic, cycloalkyl, substituted cycloalkyl, halogen, SO2R 11 , N.R. 11 R 12 , N.R. 12 (CO)OR 11 , NH(CO)NR 11 R 12 , N.R. 12 (CO)R 11 , O(CO)R 11 , O(CO)OR 11 , O(CS)R 11 , N.R. 12 (CS)R 11 , NH(CS)NR 11 R 12 , N.R. 12 (CS)OR 11 is selected from.

[0231] R1 and R2 are independently selected from hydrogen, aryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogenated alkyl, halogenated alkenyl, halogenated alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic or heterocyclic non-aromatic, cycloalkyl, substituted cycloalkyl.

[0232] R1 and R2 can be joined to form a ring which can be a heterocycle, a substituted heterocycle, a cycloalkyl, or a substituted cycloalkyl.

[0233] R3 is aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic or heterocyclic non-aromatic, cycloalkyl, substituted cycloalkyl, SO2R 11 , N.R. 11 R 12 , (CO)OR 11 , (CO)NR 11 R 12 , (CO)R 11 , (CS)R 11 , (CS)R 11 , (CS)NR 11 R 12 , (CS)OR 11 is selected from.

[0234] R5 is CN or NO2 or SO2R 11 is.

[0235] R6 is CF3, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogenated alkyl, halogenated alkenyl, halogenated alkynyl, or halogen.

[0236] A is a sulfur atom (S) or an oxygen atom (O).

[0237] B is O or S or NR3.

[0238] X is carbon or nitrogen and can be in any position on the ring.

[0239] R 11 and R 12 are independently hydrogen, aryl, aralkyl, substituted aralkyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkyl halide, alkenyl halide, alkynyl halide, arylalkyl, arylalkenyl, arylalkynyl, heteroaromatic or heterocyclic non-aromatic, substituted The heterocyclic ring is selected from substituted heteroaromatic or substituted heterocyclic non-aromatic, cycloalkyl, and substituted cycloalkyl.

[0240] R 11 and R 12 can be joined to form a ring which can be heteroaromatic or heterocyclic non-aromatic, substituted heteroaromatic, cycloalkyl, substituted cycloalkyl.

[0241] (Pharmacological testing of compounds) The compound whose synthetic route is described above is identified through screening for AR antagonist activity and agonist activity against hormone-resistant prostate cancer, using screening procedures similar to those in PCT application US04 / 42221 and US05 / 05529, which are incorporated herein by reference.Many compounds show strong antagonist activity, with minimal agonist activity, against the AR that is overexpressed in hormone-resistant prostate cancer.

[0242] In vitro biological assays (Effect of compounds on AR by reporter assay) Compounds were tested in hormone-resistant prostate cancer cell lines using an artificial AR-responsive reporter system. In this system, prostate cancer LNCaP cells were engineered to stably express AR at levels approximately five-fold higher than endogenous levels. This exogenous AR has similar properties to the endogenous AR in that both are stabilized by the synthetic androgen R1881. AR-overexpressing cells were also engineered to stably incorporate an AR-responsive reporter, and the reporter activity of these cells exhibited characteristics of hormone-resistant prostate cancer. It responded to low concentrations of the synthetic androgen R1881 and was only inhibited by high concentrations of bicalutamide, demonstrating its agonist activity (Figure 1 and Table 2). Consistent with published data, bicalutamide inhibited the AR-responsive reporter and had no agonist activity in hormone-sensitive prostate cancer cells (Figure 2).

[0243] We investigated the antagonist activity of the compound synthesized as described above in the presence of 100 pM R1881. Engineered LNCaP cells (LNCaP-AR, also abbreviated as LN-AR) were maintained in Iscove's medium containing 10% fetal bovine serum (FBS). Two days before drug treatment, the cells were grown in Iscove's medium containing 10% charcoal-stripped FBS (CS-FBS) to deplete androgens. The cells were split and grown in Iscove's medium containing 10% CS-FBS with 100 pM R1881 and increasing concentrations of test compounds. After two days of incubation, reporter activity was assayed.

[0244] Table 1 lists the IC50 of these compounds for inhibiting AR in hormone-resistant prostate cancer.The control substance bicalutamide has an IC50 of 889nM.Most of the identified compounds (diarylthiohydantoins) have an IC50 between 100 and 200nM when inhibiting AR in hormone-resistant prostate cancer.In contrast, for example, the antiandrogenic compounds (RD73-RD77) listed in U.S. Patent No. 5,705,654, for example, in Examples 30-2, 30-3, 31-2, 31-3 and 24-3, do not have inhibitory activity against AR in this system.

[0245] [Table 1-1]

[0246] [Table 1-2]

[0247] [Table 1-3]

[0248] [Table 1-4]

[0249] [Table 1-5] One previously unrecognized characteristic of AR overexpression in hormone-refractory prostate cancer is its ability to switch from antagonist to agonist. Therefore, only compounds with minimal or no agonist activity have been recognized as antiandrogens for this disease. To determine the agonist activity of different compounds, we examined their stimulatory activity on the AR using an AR response reporter as a measure in the LN-AR system in the absence of R1881. Table 2 lists the agonist activity of various compounds. Consistent with previous results, bicalutamide activated the AR in hormone-refractory prostate cancer. Diarylthiohydantoin derivatives such as Examples 7-3b (RD37), 33 (RD91), 34 (RD92), and 35 (RD93) did not have agonist activity. In contrast, RU59063, as well as other antiandrogen compounds listed as examples in U.S. Pat. No. 5,705,654, such as Examples 30-2, 30-3, 31-2, 31-3, and 24-3 (RD73-RD77), potently activated AR in hormone-refractory prostate cancer.

[0250] [Table 2-1]

[0251] [Table 2-2] To test the specificity of AR inhibitors, selective compounds were tested in LNCaP cells with overexpression of glucocorticoid receptor (GR), which is the closest member of each receptor family to AR. These cells also contain a GR-responsive reporter, and reporter activity was induced by the GR agonist dexamethasone, and this induction was blocked by the GR inhibitor RU486. Example 7-3b (RD37) (4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile) had no effect on GR in this system.

[0252] (Effect of compounds on AR by measuring secreted levels of prostate-specific antigen (PSA)) It is well established that PSA level is an indicator of AR activity in prostate cancer. To investigate whether compounds affect AR function in physiological environments, we determined the secretion level of endogenous PSA induced by R1881 in AR-overexpressing LNCaP cells (LNCaP-AR, also abbreviated as LN-AR). LNCaP-AR cells are a lymph node carcinoma line of prostate cells transformed with a plasmid that expresses androgen receptor. LNCaP-AR cells were maintained in Iscove's medium containing 10% FBS. Two days before drug treatment, cells were grown in Iscove's medium containing 10% CS-FBS to deplete androgen. Cells were split and grown in Iscove's medium containing 10% CS-FBS with appropriate concentrations of R1881 and test compounds. After 4 days of incubation, secreted PSA levels were assayed using a PSA ELISA kit (American Qualex, San Clemente, CA).

[0253] Secreted PSA levels in LNCaP-AR cells were potently induced by 25 pM R1881. In contrast, PSA was not induced in parental LNCaP cells until the R1881 concentration reached 100 pM. This is consistent with our previous report that AR in hormone-refractory prostate cancer is hypersensitive to androgens. Dose-dependent inhibition of AR activity was performed to determine the IC50 of various compounds in inhibiting PSA expression. The results are listed in Table 1. The IC50s for PSA expression of selected compounds were closely similar to those measured by reporter assay, confirming that diarylhydantoin derivatives are potent inhibitors of AR in hormone-refractory prostate cancer.

[0254] We also investigated the agonist activity of selected compounds against AR in hormone-refractory prostate cancer using secreted PSA as a surrogate marker. To do this, androgen-deprived, AR-overexpressing LNCaP cells were cultured as described above in the absence of R1881. The cells were incubated with increasing concentrations of compounds, the synthesis of which is described in, and PSA secreted into the culture medium was measured after 4 days.

[0255] Table 3 lists the agonist activity of selected compounds.Consistent with the results obtained from reporter assay, diarylthiohydantoin derivatives, such as Example 7-3b (RD37), 33 (RD91), 34 (RD92) and 35 (RD93), do not have agonist activity.In contrast, RU59063 and other antiandrogen compounds listed as examples in U.S. Patent No. 5,705,654, such as Example 30-2 (RD73), 30-3 (RD74) and 31-2 (RD75), stimulate PSA expression in hormone-resistant prostate cancer.

[0256] [Table 3-1]

[0257] [Table 3-2] (Effect of compounds on AR mitochondrial activity by MTS assay) LNCaP-AR cells were maintained in Iscove's medium containing 10% FBS. Compounds were examined for their effects on hormone-refractory prostate cancer growth. Overexpressed LNCaP cells were used because these cells behave like hormone-refractory prostate cancer cells in vitro and in vivo (1). We measured mitochondrial activity, a surrogate for proliferation, by MTS assay. LNCaP cells with overexpressed AR (LN-AR) were maintained in Iscove's medium containing 10% FBS. Two days before drug treatment, cells were grown in Iscove's medium containing 10% CS-FBS to deprive them of androgen. Cells were split and grown in Iscove's medium containing 10% CS-FBS with the appropriate concentration of R1881 and increasing concentrations of test compound. After 4 days of incubation, cell growth was monitored by MTS (Promega, Madison, WI).

[0258] Consistent with the reporter and PSA assays, the growth of AR-overexpressing LNCaP cells was stimulated by 25 μM R1881, whereas parental cells were not stimulated until the R1881 concentration reached 100 μM. Figure 2 shows the inhibitory effects of selected compounds on hormone-refractory prostate cancer growth in the presence of 100 μM R1881. The current clinical drug, bicalutamide, did not inhibit hormone-refractory prostate cancer. In contrast, Example 5-3b (RD7) (4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethyl-benzonitrile) and Example 7-3b (RD37) (4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile) inhibited hormone-refractory prostate cancer with high potency.

[0259] We demonstrated that growth inhibition in the MTS assay was caused by targeting AR. To examine whether or not AR-expressing prostate cancer cells can be inhibited, Example 5-3b (RD7) (4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile) and Example 7-3b (RD37) (4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile) were tested in DU-145 cells, a prostate cancer cell line lacking AR expression. These compounds did not have a growth inhibitory effect on DU-145 cells. These compounds did not have a growth inhibitory effect on two commonly used breast cancer cells, MCF7 and SkBr3, or on the normal mouse fibroblast cell line 3T3, and therefore did not inhibit cells other than AR-expressing prostate cancer cells.

[0260] Examples of in vitro biological activities of diarylthiohydantoin derivatives are shown in Figures 3, 4, and 5. For example, based on relative luciferase activity, Figure 3 shows that the compounds were ranked from most active to least active at a concentration of 500 nM as follows: RD152 > RD153 > RD145 > RD163 > RD161 = RD162 > bicalutamide. For example, based on relative PSA levels, Figure 4 shows that the compounds were ranked from most active to least active at a concentration of 500 nM as follows: RD138 > RD131 > RD37 > RD133 > RD134 > RD137 > RD138 > RD135 > bicalutamide. For example, based on relative MTS units, Figure 5 shows that the compounds were ranked in order from most to least active at a concentration of 500 nM as follows: RD168 > RD37 > RD141 > RD162 > bicalutamide.

[0261] (Inhibitory effect on hormone-refractory prostate cancer xenograft tumors) To examine whether diarylhydantoin derivatives have in vivo efficacy against hormone-refractory prostate cancer, Example 7-3b (RD37) (4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile) was used. First, we examined this compound against xenograft tumors established from AR-overexpressing LNCaP cells. Engineered cells in Matrigel (Collaborative Biomedical) were injected subcutaneously into the flanks of castrated male SCID mice. Tumor size was measured weekly in three dimensions using calipers. After xenograft tumors were established (tumor size was at least 40 mm), 3 Tumor-bearing mice were randomly assigned and orally treated once daily with various doses of the compound. Consistent with clinical observations, the current clinical drug bicalutamide did not inhibit the growth of hormone-refractory prostate cancer (similar to vehicle) (Fig. 7a). In contrast, Example 7-3b (RD37) (4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile) potently inhibited the growth of these tumors (Fig. 7a), and this inhibition was dose-dependent (Fig. 7b). Furthermore, Example 7-3b (RD37) inhibited PSA expression, a clinical marker for hormone-refractory prostate cancer (Fig. 8).

[0262] Example 7-3b (RD37) (4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile) was also tested in another xenograft model of hormone-resistant prostate cancer, hormone-resistant LAPC4. This model was established from the passage of hormone-sensitive prostate cancer in castrated mice, and it mimics the clinical progression of prostate cancer (2). Similar to the findings using the AR-overexpressing LNCaP xenograft model, the current clinical drug bicalutamide did not inhibit growth and PSA expression (similar to vehicle) in the hormone-resistant LAPC4 xenograft model (Figures 9a and 9b). In contrast, Example 7- 3b(RD37) potently inhibited the growth and PSA expression of these tumors (FIGS. 9a and 9b).

[0263] (Inhibitory effect on the growth of hormone-sensitive prostate cancer cell lines) To determine whether diarylthiohydantoin derivatives also inhibit hormone-sensitive prostate cancer cells, we tested several selected compounds against the proliferation of LNCaP cells by measuring mitochondrial activity (MTS). In contrast to having no effect on the growth of hormone-resistant prostate cancer cells, the current clinical drug bicalutamide moderately inhibited hormone-sensitive LNCaP cells in a dose-dependent manner. Example 5-3b (RD7) (4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethyl-benzonitrile) and Example 7-3b (RD37) (4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile) inhibited hormone-sensitive prostate cancer with 10-fold greater potency than bicalutamide ( FIG. 10 ).

[0264] In vivo biological assays All animal experiments were conducted by the Animal Research Committee The study was conducted in accordance with the guidelines of the University of California at Los Angeles. Animals were purchased from Taconic and maintained in a laminar flow tower with defined flora colonies. LNCaP-AR and LNCaP-vector cells were maintained in RPMI medium supplemented with 10% FBS. 100 μL of 1:1 Matrigel:RPMI medium was used. 6 Cells were injected subcutaneously into the flanks of intact or castrated SCID mice. Tumor size was measured weekly in three dimensions (length x width x depth) using calipers. When tumor size reached approximately 100 mm 3 When the tumor reached 18 days of age, mice were randomly assigned to treatment groups. The drug was given orally at 10 mg / kg and 50 mg / kg daily. To obtain pharmacodynamic readouts, animals were imaged via an optical CCD camera 3 hours after the last dose of treatment. ROIs were drawn around the tumor for luciferase activity measurements in photons / second. The right panel was a representation of the ROI measurements. The data are shown in Figures 11 and 12. RD162 over 18 days was effective in disrupting tumor growth and even causing tumor shrinkage, clearly more effective than bicalutamide.

[0265] The pharmacokinetics of bicalutamide, 4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-toluene [RD37], N-methyl-4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}butanamide [RD131], and N-methyl-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluorobenzamide (52d) [RD162] were evaluated in vivo using 8-week-old FVB mice purchased from Charles River Laboratories. Mice were divided into three groups for each time point: two mice were not treated with the drug, and the other two mice were treated with the vehicle solution. Each group was treated with 10 mg of the drug per kg of body weight.

[0266] Drugs were dissolved in a 1:5:14 mixture of DMSO:PEG400:HO (vehicle solution) and administered to mice through the tail vein. To dilate the tail vein, animals were warmed under a heat lamp for approximately 20 minutes before treatment. Each mouse was placed in a mouse restrainer (Fisher Sci. Cat. No. 01-288-32A), and 200 μL of drug in vehicle solution was injected into the dilated tail vein. After drug administration, the mice were treated at different time points: 5 minutes, 30 minutes, 2 At 1 h, 6 h, and 16 h, animals were euthanized via CO inhalation. Animals were bled via cardiac puncture (1 ml BD syringe + 27G 5 / 8 needle) immediately after CO exposure. For oral administration, drugs were dissolved in a 50:10:1:989 mixture of DMSO:carboxymethylcellulose:Tween 80:HO and then administered orally via a feeding syringe.

[0267] Serum samples were analyzed by HPLC (Waters 600 pump, Waters 600 controller, and Waters 2487 detector) equipped with an Alltima C18 column (3μ, 150 mm × 4.6 mm) to determine drug concentrations. RD37, RD131, and RD162 compounds were detected at 254 nm wavelength, and bicalutamide was detected at 270 nm wavelength.

[0268] Samples for HPLC analysis were prepared according to the following procedure: -Blood cells were separated from serum by centrifugation. To 400 μL of serum, 80 μL of a 10 μM solution of the internal standard and 520 μL of acetonitrile were added. Precipitation occurred. - The mixture was vortexed for 3 minutes and then placed under ultrasound for 30 minutes. -Solid particles were filtered off or separated by centrifugation. The filtrate was dried under a stream of argon. Samples were reconstituted to 80 μL with acetonitrile before analysis by HPLC to determine drug concentration. -Drug standard curves were used to improve accuracy.

[0269] The plasma concentrations of RD162 as a function of time from intravenous and oral administration are shown in Figure 13. The steady-state concentrations (Css) of bicalutamide, RD131, and RD162 are shown in Table 4. The steady-state concentrations of RD162 were essentially as good as those of bicalutamide and substantially better than those of RD131.

[0270] [Table 4] (Ranking of compounds in tiers) Tables 5-10 present diarylhydantoin compounds classified into Tiers 1-6. Table 11 presents diarylhydantoin compounds that were not placed in any tier. Compound placement into tiers was based on available data coupled with analytical judgment. Data considered included in vitro assays (AR-responsive reporter system in LNCaP cell line, PSA level measurement, MTS mitochondrial assay) and in vivo experiments (tumor size measured directly or by luminescence induced by a luciferase reporter gene, pharmacokinetic assay based on blood plasma levels). Not all compounds were subjected to each assay. Not all data generated are shown. Judgment was applied when ranking compounds relative to each other for their utility in treating prostate cancer, especially when ranking two compounds for which the same experiment had not been performed. Characteristics considered in establishing the ranking include AR antagonist activity, lack of AR agonism in hormone-refractory cells, interference with tumor growth, tumor shrinkage, and pharmacokinetic behavior, with longer residence time in the blood being advantageous.

[0271] (Tier1) Generally, Tier 1 compounds are diarylthiohydantoins with a disubstituted left-hand aryl ring that is disubstituted on the right-hand hydantoin carbon and has either an oxygen or N-substituent on the left-hand hydantoin carbon. The amino substituent is predicted to hydrolyze to oxygen in aqueous solution, as encountered in biological systems in vitro and in vivo. RD100 has good activity with an iodine in place of the CF3 substituent on the left-hand aryl ring.

[0272] Tier 1 compounds (see Table 5) were determined to be much better than bicalutamide for treating prostate cancer. However, RD37 and RD131 were found to be rapidly metabolized, i.e., they have a short residence time in the blood. RD162 had desirable pharmacokinetics.

[0273] Figure 17 shows that PSA levels for LNCaP cells remained the same or increased under treatment with bicalutamide compared to treatment with vehicle solution, while PSA levels decreased under treatment with RD162. Figure 18 illustrates that tumors continued to increase in size under treatment with vehicle solution. In contrast, treatment with RD162 at a dose of 1 mg / kg body weight / day reduced the rate of tumor growth, and tumor size appeared to stabilize after approximately 17 days. Treatment with RD162 at a dose of 10 mg / kg body weight / day reduced tumor size over time. Figure 19 illustrates that treatment with RD162 at a dose of 10 mg / kg body weight / day reduced photon emission associated with luciferase activity. Figure 20 shows that treatment with RD162 at this dose reduced or stabilized tumor size and reduced photon emission associated with luciferase activity.

[0274] Figure 21 shows that the PSA level of LN-AR cells was reduced under treatment with RD162, RD162', RD162'', RD169, and RD170 at doses of 100, 200, 500, and 1000 nM. Furthermore, the higher the dose, the lower the PSA level. Figure 23 shows the urogenital tract weight and the rate of photon emission associated with luciferase activity for intact and castrated mice at the beginning or 14 days after treatment with bicalutamide or RD162. Weight and photon emission increased for both intact and castrated mice. Treatment of castrated mice with RD162 resulted in a decrease in weight and photon emission compared to untreated castrated mice, similar to treatment with bicalutamide.

[0275] Therefore, Tier 1 compounds are particularly advantageous for use as AR antagonists and as therapeutic agents for hormone-resistant prostate cancer.They can be useful for treating other AR-related diseases or conditions, such as benign prostatic hyperplasia, hair loss and acne.These and related compounds can also be useful as modulators of other nuclear receptors, such as glucocorticoid receptors, estrogen receptors and peroxisome proliferator-activated receptors, and as therapeutic agents for diseases in which nuclear receptors play a role, such as breast cancer, ovarian cancer, diabetes, heart disease and metabolic-related diseases.They can be useful in assays (for example, as standards), or as intermediates or as prodrugs.

[0276] [Table 5-1]

[0277] [Table 5-2]

[0278] [Table 5-3] (Tier 2) Although Tier 2 compounds (see Table 6) were significantly better than bicalutamide for treating prostate cancer, there was evidence that RD54 may act as an agonist. Figure 3 illustrates that compounds RD145, RD152, RD153, RD162, and RD163 in Tier 1 and RD161 in Tier 2, administered at concentrations ranging from 125 nM to 1000 nM, acted to reduce luciferase activity in LNCaP-AR cells, whereas DMSO control solutions and bicalutamide solutions had little or no effect. Figure 4 illustrates, for example, that at a concentration of 1000 nM, compounds RD37 and RD131 in Tier 1 caused a greater reduction in PSA levels in LNCaP-AR cells than compounds RD133, RD134, and RD138 in Tier 2. Figure 11 shows tumor volume versus time, demonstrating that tumors continued to grow under treatment with bicalutamide or vehicle solution, whereas tumors decreased in size under treatment with RD162 in Tier 1. Figure 12 illustrates that photon emission associated with luciferase activity remained roughly the same or increased under treatment with bicalutamide, whereas it decreased under treatment with RD162, compared to treatment with vehicle solution. Figure 14 illustrates that PSA levels decreased under treatment with RD131 and RD162, whereas there was little or no decrease in PSA levels under treatment with bicalutamide. Figure 15 shows the IC for RD37, RD131, and RD162 in Tier 1. 50 However, IC for bicalutamide 50 The figure shows that it was much lower than

[0279] Generally, Tier 2 compounds are structurally similar to Tier 1 compounds, but have different substituents on the right aryl ring.Tier 2 compounds are advantageous for use as AR antagonists and as therapeutic agents for hormone-resistant prostate cancer.They can be useful for treating other AR-related diseases or conditions, such as benign prostatic hyperplasia, hair loss, and acne.These and related compounds can also be useful as modulators of other nuclear receptors, such as estrogen receptors and peroxisome proliferator-activated receptors, and as therapeutic agents for diseases in which nuclear receptors play a role, such as breast cancer, ovarian cancer, diabetes, heart disease, and metabolic-related diseases.They can be useful in assays (for example, as standards), or as intermediates, or as prodrugs.

[0280] [Table 6-1]

[0281] [Table 6-2] (Tier3) The Tier 3 compounds (see Table 7) were judged to be slightly better than bicalutamide for treating prostate cancer. RD133, RD134, and RD138 (in Tier 2) caused a greater reduction in PSA levels in LNCaP-AR cells than RD135 and RD137 in Tier 3. All of these compounds caused a greater reduction in PSA levels than bicalutamide.

[0282] Other Tier 3 compounds (not shown) were not diarylthiohydantoins and were comparable in activity to the prior art monoarylhydantoin compounds RD2, RD4, and RD5.

[0283] Therefore, Tier 3 compounds are useful as AR antagonists and as therapeutic agents for hormone-resistant prostate cancer.They can be useful for treating other AR-related diseases or conditions, such as benign prostatic hyperplasia, hair loss and acne.These and related compounds can also be useful as modulators of other nuclear receptors, such as estrogen receptors and peroxisome proliferator-activated receptors, and as therapeutic agents for diseases in which nuclear receptors play a role, such as breast cancer, ovarian cancer, diabetes, heart disease and metabolic-related diseases.They can be useful in assays (for example, as standards), or as intermediates or as prodrugs.

[0284] [Table 7] (Tier 4) Tier 4 compounds (see Table 8) were determined to be only similar to bicalutamide for treating prostate cancer. RD39 and RD40 of Tier 4 and RD37 of Tier 1 differ, for example, only in the substituents on the right lower carbon of the hydantoin ring. Substituents on the right aryl ring may also affect activity.

[0285] Several Tier 4 compounds (including those shown and others not shown) were not diaryl compounds (lacking a right-side aryl ring), were not thiohydantoins, and were not disubstituted on the carbon on the right lower portion of the hydantoin ring and / or had a substituent other than oxygen or amide on the lower carbon on the left side of the hydantoin ring. This provides evidence of the surprising advantages of diarylthiohydantoins that are disubstituted on the right lower carbon of the hydantoin ring and have oxygen or amide on the lower carbon on the left side of the hydantoin ring.

[0286] Therefore, Tier 4 compounds can be useful as AR antagonists and as therapeutic agents for hormone-resistant prostate cancer, at least to a degree comparable to bicalutamide.They can be useful for treating other AR-related diseases or conditions, such as benign prostatic hyperplasia, hair loss, and acne.These and related compounds can also be useful as modulators of other nuclear receptors, such as estrogen receptors and peroxisome proliferator-activated receptors, and as therapeutic agents for diseases in which nuclear receptors play a role, such as breast cancer, ovarian cancer, diabetes, heart disease, and metabolic-related diseases.They can be used in assays (for example, as standards), or as intermediates, or as therapeutic agents for other diseases. These compounds may be useful as prodrugs or as anti-cancer drugs.

[0287] [Table 8-1]

[0288] [Table 8-2]

[0289] [Table 8-3] (Tier 5) Tier 5 compounds (see Table 9) were inactive or nearly inactive and therefore worse than bicalutamide for treating prostate cancer. Substituents on the right aryl ring are important for determining activity.

[0290] Several Tier 5 compounds (some of which are shown and some of which are not) were not diaryl compounds (lacking a right-side aryl ring), were not thiohydantoins, and were not disubstituted on the carbon on the right lower portion of the hydantoin ring and / or had a substituent other than oxygen or amide on the lower carbon on the left side of the hydantoin ring. This provides evidence of the surprising advantages of diarylthiohydantoins that are disubstituted on the right lower carbon of the hydantoin ring and have oxygen or amide on the lower carbon on the left side of the hydantoin ring. In particular, the terminal substituent (CHNR) in RD155, RD156, and RD158 x R y , where R x,y =H or methyl) does not appear to contribute to activity in these compounds.

[0291] Although Tier 5 compounds are not desirable for treating prostate cancer or as AR antagonists, these and related compounds can also be useful as modulators of other nuclear receptors, such as estrogen receptors and peroxisome proliferator-activated receptors, and as therapeutic agents for diseases in which nuclear receptors play a role, such as breast cancer, ovarian cancer, diabetes, heart disease, and metabolic-related diseases.They can be useful in assays (e.g., as standards), or as intermediates or prodrugs.

[0292] [Table 9] (Tier 6) Tier 6 compounds (see Table 10) are inactive or nearly inactive and are more potent agonists and therefore are more effective than bicalutamide for treating prostate cancer. The results were much worse. The comparative compounds ranked very poorly compared to the compounds of the present invention. Notably, RD72, which has a chlorine substituent on the left aryl ring, had very poor activity, while RD7, which has trifluoromethane, and RD100, which has iodine, were ranked in Tier 1. The results for Tier 6 compounds provide evidence of the surprising advantages of diarylthiohydantoins, which are disubstituted on the lower carbon on the right side of the hydantoin ring, have oxygen or amide on the lower carbon on the left side of the hydantoin ring, and have specific substituents on the left aryl ring.

[0293] Tier 6 compounds are not desirable for the treatment of prostate cancer or as AR antagonists.

[0294] [Table 10] (non-tier compounds) For some compounds, there was insufficient experimental data to rank them. These non-tiered compounds are presented in Table 11.

[0295] Based on the data and methods of the present invention, and applying a survey of many compounds, including some not shown herein, several observations can be made regarding off-tier compounds. Comparative example RD1 is predicted to be in Tier 3, which includes comparative examples RD3-RD5. RD89 is predicted to be hydrolyzed to RD37 (Tier 1) and therefore should have comparable activity. RD104 is predicted to be hydrolyzed to RD58 (Tier 1) and therefore should have comparable activity. RD105 is predicted to be hydrolyzed to RD8 (Tier 1), and RD139 and RD140 are predicted to be hydrolyzed to RD138 (Tier 2) and therefore should have comparable activity.

[0296] [Table 11-1]

[0297] [Table 11-2] In summary, a novel compound has been identified and prepared that shows evidence of being significantly superior to bicalutamide in treating prostate cancer.

[0298] (Sensitivity of anticancer activity of compounds to structural differences) The present inventors have determined that what may appear to be small changes in the structure of hydantoin compounds can result in large changes in the compound's performance in treating prostate cancer. For example, RD161 and RD162 differ only by a single fluorine substituent on the aryl ring; RD162 is Tier 1, while RD161 is Tier 2. Both are better than bicalutamide for treating prostate cancer, but RD162 is superior. However, RD149, which differs from RD161 only by having an additional carbon atom between the methylcarbamoyl group and the aryl ring, is comparable to bicalutamide for treating prostate cancer and is ranked in Tier 4. The effects of RD161, RD162, and RD149 on luciferase activity can be seen in Figure 24. At a given compound concentration, luciferase activity upon exposure to RD161 and RD162 is less than that upon exposure to RD149.

[0299] RD9 differs from RD8 only in that a hydroxyl group is replaced with an amino group. However, RD8 is a Tier 1 and is much better than bicalutamide for treating prostate cancer, while RD9 is a Tier 4 and is comparable to bicalutamide. The effects of RD8 and RD9 on luciferase activity in the 1AR cell line can be seen in Figure 27. For a given dose, luciferase activity upon exposure to RD8 is lower than luciferase upon exposure to RD9. The effects of RD8 and RD9 on luciferase activity in the 4AR cell line can be seen in Figure 26. For a given dose, luciferase activity upon exposure to RD8 is lower than luciferase upon exposure to RD9. The effects of RD8 and RD9 on PSA levels in the LN / AN cell line can be seen in Figure 25. For a given dose, PSA levels upon exposure to RD8 are lower than PSA levels upon exposure to RD9.

[0300] RD130 and RD131 are substituted only by the methyl substituent on the end of the carbamoyl group. Although both compounds differ from each other and are ranked in Tier 1, RD131 has been found to be particularly advantageous. RD129 is identical to RD130, except that the amino group is replaced with a methoxy group. However, RD129 is ranked in Tier 3. RD128 is similar to RD129, but has one less carbon in the chain connecting the ester group to the aryl ring; RD128 is ranked in Tier 3. The effects of RD130, RD131, RD128, and RD129 on PSA levels in LN / AR cell lines can be seen in Figure 28. For a given concentration, PSA levels upon exposure to RD130 and RD131 are lower than those upon exposure to RD128 and RD129.

[0301] RD153 and RD155 differ from each other in that the former has a methylcarbamoyl group attached to the aryl ring and a dimethyl substituent attached to the thiohydantoin group, while the latter has a methylamino group attached to the right aryl ring and a cyclobutyl substituent attached to the thiohydantoin group. RD153 is a Tier 1 compound and is much better than bicalutamide for treating prostate cancer, while RD155 is a Tier 5 compound and is inactive or nearly inactive for treating prostate cancer. The effects of RD153 and RD155 on luciferase activity in LN / AR cell lines can be seen in Figure 29. For a given concentration, luciferase activity upon exposure to RD153 is lower than that upon exposure to RD155.

[0302] RD58 and RD60 differ from each other in the substitution of a thio for an oxo group and a dimethyl for a cyclobutyl substituent. RD58 is Tier 1, while RD60 is Tier 4.

[0303] Pharmaceutical Compositions and Administration The compounds of the present invention are useful as pharmaceutical compositions prepared using a therapeutically effective amount of a compound of the present invention, as defined herein, and a pharmaceutically acceptable carrier or diluent.

[0304] The diarylhydantoin compounds of the present invention can be formulated as pharmaceutical compositions and administered to a subject in need of treatment, e.g., a mammal such as a human patient, in a variety of forms adapted to the selected route of administration, e.g., oral, nasal, intraperitoneal, or parenteral, intravenous, intramuscular, topical, or subcutaneous routes, or by injection into a tissue.

[0305] Thus, the diarylhydantoin compounds of the present invention may be administered systemically, for example, orally, in combination with a pharmaceutically acceptable vehicle, such as an inert diluent, or an assimilable edible carrier, or by inhalation or injection. They may be enclosed in hard or soft-shell gelatin capsules, compressed into tablets, or incorporated directly into the food of the patient's diet. For oral therapeutic administration, the diarylhydantoin compounds may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, elixirs, capsules, suspensions, syrups, wafers, and the like. The diarylhydantoin compounds may be combined with a finely divided inert powder carrier and inhaled or injected by the subject. Such compositions and preparations should contain at least 0.1% of the diarylhydantoin compound. The percentage of the compositions and preparations may, of course, vary and may conveniently be between about 2% and about 60% of the weight of a given unit dosage form. The amount of diarylhydantoin compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0306] The tablets, troches, pills, capsules and the like may also contain: a binder such as tragacanth, acacia, cornstarch or gelatin; an excipient such as dicalcium phosphate. Disintegrating agents such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to the above-mentioned materials, a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or to modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetener, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or orange flavoring. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the diarylhydantoin compounds may be incorporated into sustained-release preparations and devices. For example, the diarylhydantoin compounds may be incorporated into time-release capsules, time-release tablets, and time-release pills.

[0307] Diarylhydantoin compounds can also be administered intravenously or intraperitoneally by infusion or injection.Diarylhydantoin compound solutions can be prepared in water, optionally mixed with a non-toxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, and in oil.Under normal conditions of storage and use, these preparations can contain preservatives to prevent the growth of microorganisms.

[0308] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing diarylhydantoin compounds, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, and may be encapsulated in liposomes as needed. In all cases, the final dosage form should be sterile, liquid, and stable under the conditions of manufacture and storage. Liquid carriers or vehicles may be solvents or liquid dispersion media, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The appropriate fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0309] Sterile injectable solutions are prepared by incorporating the diarylhydantoin compound in the required amount in a suitable solvent with various other ingredients listed above, if required, and then sterilizing by filtration.In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces a powder of the active ingredient and any additional desired ingredients that are present in the previously sterile-filtered solution.

[0310] For topical administration, the diarylhydantoin compounds can be applied in pure form, but it is generally desirable to administer them to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which can be solid or liquid.

[0311] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, and the like. Examples of suitable solid carriers include cellulose, silica, alumina, etc. Other solid carriers include non-toxic polymer nanoparticles or microparticles. Useful liquid carriers include water, alcohol, or glycol, or a blend of water / alcohol / glycol, in which the diarylhydantoin compound can be dissolved or dispersed at an effective level, optionally with the aid of a non-toxic surfactant. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given application. The resulting liquid composition can be applied from absorbent pads, used in impregnated bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprays.

[0312] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified mineral substances may also be utilized with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for direct application to the user's skin.

[0313] Examples of useful dermatological compositions that can be used to deliver diarylhydantoin compounds to the skin are known in the art; see, for example, Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157), and Wortzman (U.S. Pat. No. 4,820,508), all of which are incorporated herein by reference.

[0314] The useful dosage of the compound of formula I can be determined by comparing their in vitro activity and in vivo activity in animal models.Methods for extrapolating effective dosages in mice and other animals to humans are known in the art; for example, see U.S. Patent No. 4,938,949, which is incorporated herein by reference.

[0315] For example, the concentration of the diarylhydantoin compound in a liquid composition such as a lotion may be about 0.1 to 25% by weight, or about 0.5 to 10% by weight, and in a semi-solid or solid composition such as a gel or powder may be about 0.1 to 5% by weight, or about 0.5 to 2.5% by weight.

[0316] The amount of diarylhydantoin compound required for therapeutic use will vary not only with the particular salt selected, but also with the route of administration, the condition being treated, and the age and condition of the patient, and is ultimately at the discretion of the physician or clinician.

[0317] The effective dosage and route of administration of the agents of the present invention are conventional. The exact amount of the agent (effective dose) varies from subject to subject, depending on, for example, the species, age, weight, and general or clinical condition of the subject, the severity or mechanism of any disorder being treated, the specific agent or vehicle used, the method and schedule of administration, etc. The therapeutically effective dose can be determined empirically by conventional procedures known to those skilled in the art. For example, see The Pharmacological Basis of Therapeutics, edited by Goodman and Gilman, Macmillan Publishing. Co., New York. For example, an effective dose can be estimated initially either in cell culture assays or in an appropriate animal model. The animal model may also be used to determine an appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutic dose can also be selected by analogy with the dosage for a comparable therapeutic agent.

[0318] The particular mode of administration and dosing regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic.) Treatment may involve daily or multiple daily doses of the compound for a period ranging from several days to several months or even years.

[0319] Generally, however, a suitable dose ranges from about 0.001 to about 100 mg / kg body weight per day, e.g., from about 0.01 to about 100 mg / kg body weight, e.g., greater than about 0.1 mg / kg body weight, or from about 1 to about 10 mg per kilogram body weight of the recipient per day. For example, a suitable dose may be about 1 mg / kg, 10 mg / kg, or 50 mg / kg body weight per day.

[0320] The diarylhydantoin compounds are conveniently administered in unit dosage form, for example, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, or about 100 mg of active ingredient per unit dosage form.

[0321] The diarylhydantoin compound can be administered to achieve a peak plasma concentration of, for example, about 0.5 to about 75 μM, about 1 to about 50 μM, about 2 to about 30 μM, or about 5 to about 25 μM. Exemplary desired plasma concentrations include at least 0.25, 0.5, 1, 5, 10, 25, 50, 75, 100, or 200 μM or less. For example, plasma levels can be about 1 to 100 micromolar or about 10 to about 25 micromolar. This can be achieved, for example, by intravenous injection of a 0.05 to 5% solution of the diarylhydantoin compound, optionally in saline, or orally administered as a bolus containing about 1 to 100 mg of the diarylhydantoin compound. Desired blood levels may be maintained by continuous infusion to provide about 0.00005-5 mg / kg body weight / hour, e.g., at least 0.00005, 0.0005, 0.005, 0.05, 0.5, or 5 mg / kg / hour, or less. Alternatively, such levels can be obtained by intermittent infusion containing about 0.0002-20 mg / kg body weight, e.g., at least 0.0002, 0.002, 0.02, 0.2, 2, 20, or 50 mg / kg body weight, or less, of the diarylhydantoin compound / kg body weight.

[0322] The diarylhydantoin compounds may conveniently be presented in a single dose or in divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day, which sub-dose itself may be further divided, into a number of loosely spaced administrations, for example, multiple inhalations from an inhaler.

[0323] Many of the compounds identified above show little or no activity for hormone-resistant prostate cancer cells.Because these compounds are strong AR inhibitors, they can not only be used to treat prostate cancer, but also can be used to treat other AR-related diseases or conditions, such as benign prostatic hyperplasia, hair loss and acne.Because AR belongs to the family of nuclear receptors, these compounds can serve as the backbone for the synthesis of drugs that target other nuclear receptors, such as estrogen receptor and peroxisome proliferator-activated receptor.Therefore, they can be further developed for other diseases that nuclear receptors play a role in, such as breast cancer, ovarian cancer, diabetes, heart disease and metabolic-related diseases.

[0324] The embodiments illustrated and discussed herein are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be construed as limiting the scope of the invention. All examples presented are representative and non-limiting. The above-described embodiments of the invention may be modified or adapted without departing from the invention, as will be appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

Claims

[Claim 1] The invention described in the specification.

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