Rho-kinase inhibitors

FIELD OF THE INVENTION

[0001] The present invention relates to compounds and derivatives thereof, their synthesis, and their use as Rho-kinase inhibitors. These compounds of the present invention are useful for inhibiting tumor growth, treating erectile dysfunction, and treating other indications mediated by Rho-kinase, e.g., coronary heart disease.

BACKGROUND

[0002] The pathology of a number of human and animal diseases including hypertension, erectile dysfunction, coronary cerebral circulatory impairments, neurodegenerative disorders and cancer can be linked directly to changes in the actin cytoskeleton. These diseases pose a serious unmet medical need. The actin cytoskeleton is composed of a meshwork of actin filaments and actin-binding proteins found in all eukaryotic cells. In smooth muscle cells the assembly and disassembly of the actin cytoskeleton is the primary motor force responsible for smooth muscle contraction and relaxation. In non-muscle cells, dynamic rearrangements of the actin cytoskeleton are responsible for regulating cell morphology, cell motility, actin stress fiber formation, cell adhesion and specialized cellular functions such as neurite retraction, phagocytosis or cytokinesis (Van Aelst, et al. Genes Dev 1997, 11, 2295).

[0003] The actin cytoskeleton is controlled by a family of proteins that are a subset of the Ras superfamily of GTPases. This subset currently consists of RhoA through E and RhoG (refereed to collectively as Rho), Rac 1 and 2, Cdc42Hs and G25K and TC10 isoforms (Mackay, et al. J Biol Chem 1998, 273, 20685). These proteins are GTP (guanine nucleotide triphosphate) binding proteins with intrinsic GTPase activity. They act as molecular switches and cycles between inactive GDP (guanine nucleotide diphosphate) bound and active GTP bound states. Using biochemical and genetic manipulations, it has been possible to assign functions to each family member. Upon activation the Rho proteins controls the formation of actin stress fibers, thick bundles of actin filaments, and the clustering of integrins at focal adhesion complexes. When activated the Rac proteins control the formation of lamellopodia or membrane ruffles on the cell surface and Cdc42 controls filopodia formation. Together this family of proteins plays a critical part in the control of key cellular functions including cell movement, axonal guidance, cytokinesis, and changes in cell morphology, shape and polarity.

[0004] Depending on the cell type and the activating receptor, the Rho proteins can control different biological responses. In smooth muscle cells, Rho proteins are responsible for the calcium sensitization during smooth muscle contraction. In non-smooth muscle cells the Rho GTPases are responsible for the cellular responses to agonist such as lysophosphatidic acid (LPA), thrombin and thromboxane A2 (Fukata, et al. Trends Pharcol Sci 2001, 22, 32). Agonist response is coupled through heterotrimeric G proteins Galpha12 or Galpha13 (Goetzl, et al. Cancer Res 1999, 59, 4732; Buhl, et al. J Biol Chem 1995, 270, 24631) though other receptors may be involved. Upon activation Rho GTPases activate a number of downstream effectors including PIP5-kinase, Rhothekin, Rhophilin, PKN and Rho kinase isoforms ROCK-1/ROKbeta and ROCK-1/ROKalpha (Mackay and Hall J Biol Chem 1998, 273, 20685; Aspenstrom Curr Opin Cell Biol 1999, 11, 95; Amano, et al. Exp Cell Res 2000, 261, 44).

[0005] Rho kinase was identified as a RhoA interacting protein isolated from bovine brain (Matsui, et al. Embo J 1996, 15, 2208). It is a member of the myotonic dystrophy family of protein kinase and contains a serine/threonine kinase domain at the amino terminus, a coiled-coil domain in the central region and a Rho interaction domain at the carboxy terminus (Amano, et al. Exp Cell Res 2000, 261, 44). Its kinase activity is enhanced upon binding to GTP-bound RhoA and when introduced into cells, it can reproduce many of the activities of activated RhoA. In smooth muscle cells Rho kinase mediates calcium sensitization and smooth muscle contraction and inhibition of Rho kinase blocks 5-HT and phenylephrine agonist induced muscle contraction. When introduced into non-smooth muscle cells, Rho kinase induces stress fiber formation and is required for the cellular transformation mediated by RhoA (Sahai, et al. Curr Biol 1999, 9, 136). Rho kinase regulates a number of downstream proteins through phosphorylation, including myosin light chain (Somlyo, et al. J Physiol (Lond) 2000, 522 Pt 2, 177), the myosin light chain phosphatase binding subunit (Fukata, et al. J Cell Biol 1998, 141, 409) and LIM-kinase 2 (Sumi, et al. J Bio Chem 2001, 276, 670).

[0006] Inhibition of Rho kinase activity in animal models has demonstrated a number of benefits of Rho kinase inhibitors for the treatment of human diseases. Several patents have appeared claiming (+)-trans-4-(1-aminoethyl)-1-(pyridin-4-ylaminocarbonyl)cyclohexane dihydrochloride monohydrate (WO-00078351, WO-00057913) and substituted isoquinolinesulfonyl (EP-00187371) compounds as Rho kinase inhibitors with activity in animal models. These include models of cardiovascular diseases such as hypertension (Uehata, et al. Nature 1997, 389, 990), atherosclerosis (Retzer, et al. FEBS Lett 2000, 466, 70), restenosis (Eto, et al. Am J Physiol Heart Circ Physiol 2000, 278, H1744; Negoro, et al. Biochem Biophys Res Commun 1999, 262, 211), cerebral ischeia (Uehata, et al. Nature 1997, 389, 990; Seasholtz, et al. Circ Res 1999, 84, 1186; Hitomi, et al. Life Sci 2000, 67, 1929; Yamamoto, et al. J Cardiovasc Pharmacol 2000, 35, 203), cerebral vasospasm (Sato, et al. Circ Res 2000, 87, 195; Kim, et al. Neurosurgery 2000, 46, 440), penile erectile dysfunction (Chitaley, et al. Nat Med 2001, 7, 119), central nervous system disorders such as neuronal degeneration and spinal cord injury (Hara, et al. J Neurosurg 2000, 93, 94; Toshima, et al. Stroke 2000, 31, 2245) and in neoplasias where inhibition of Rho kinase has been shown to inhibit tumor cell growth and metastasis (Itoh, et al. Nat Med 1999, 5, 221; Somlyo, et al. Biochem Biophys Res Commun 2000, 269, 652), angiogenesis (Uchida, et al. Biochem Biophys Res Commun 2000, 269, 633; Gingras, et al. Biochem J 2000, 348 Pt 2, 273), arterial thrombotic disorders such as platelet aggregation (Klages, et al. J Cell Biol 1999, 144, 745; Retzer, et al. Cell Signal 2000, 12, 645) and leukocyte aggregation (Kawaguchi, et al. Eur J Pharmacol 2000, 403, 203; Sanchez-Madrid, et al. Embo J 1999, 18, 501), asthma (Setoguchi, et al. Br J Pharmacol 2001, 132, 111; Nakahara, et al. Eur J Pharmacol 2000, 389, 103), regulation of intraoccular pressure (Honjo, et al. Invest Ophthalmol Vis Sci 2001, 42, 137) and bone resorption (Chellaiah, et al. J Biol Chem 2000, 275, 11993; Zhang, et al. J Cell Sci 1995, 108, 2285).

[0007] The inhibition of Rho kinase activity in patients has benefits for controlling cerebral vasospasms and ischemia following subarachnoid hemorrhage (Pharma Japan 1995, 1470, 16).

SUMMARY OF THE INVENTION

[0008] The compounds and their derivatives presented in this invention are useful as Rho Kinase inhibitors and thus have utilities in the treatment of hypertension, atherosclerosis, restenosis, cerebral ischemia, cerebral vasospasm, neuronal degeneration, spinal cord injury, cancers of the breast, colon, prostate, ovaries, brain and lung and their metastases, thrombotic disorders, asthma, glaucoma and osteoporosis.

[0009] In addition, the compounds of the invention are useful to treat erectile dysfunction, i.e., erectile dysfunction mediated by Rho-kinase. Erectile dysfunction can be defined as an inability to obtain or sustain an erection adequate for intercourse, WO 94/28902, U.S. Pat. No. 6,103,765 and U.S. Pat. No. 6,124,461.

[0010] The invention involves compounds of the following structures:

[0011] The compounds of the Formula I can be made according to routine, conventional chemical methods, and/or as disclosed below, from starting materials which are either commercially available or producible according to routine, conventional chemical methods. Methods for the preparation of the compounds are given below in the Examples.

[0012] In the following examples, all temperatures are set forth uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

[0013] The entire disclosure of all applications, patents and publications, cited above or below, and Provisional Application No. 60/349,986, filed Jan. 23, 2002, are hereby incorporated by reference.

Abbreviations and Acronyms

[0014] When the following abbreviations are used herein, they have the following meaning:

1Ac2Oacetic anhydrideanhyanhydrousn-BuOHn-butanolt-BuOHt-butanolCD3ODmethanol-d4Celite ®diatomaceous earth filter agent, ® Celite Corp.CH2Cl2methylene chlorideCI-MSchemical ionization mass spectroscopyconcconcentrateddecdecompositionDMEdimethoxyethaneDMFN,N-dimethylformamideDMSOdimethylsulfoxideELSDevaporative light scattering detectorEtOAcethyl acetateEtOHethanol (100%)Et2Odiethyl etherEt3NtriethylamineHPLChigh performance liquid chromatography-electrospray massES-MSspectroscopyNMM4-methylmorpholinePh3PtriphenylphosphinePd(dppf)Cl2[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)Pd(PPh3)4tetrakis(triphenylphosphine)palladium(0)Pd(OAc)2palladium acetateP(O)Cl3phosphorous oxychlorideRTretention time (HPLC0)rtroom temperatureTHFtetrahydrofuranTFAtrifluoroacetic acidTLCthin layer chromatography

EXPERIMENTAL EXAMPLES

[0015] All reactions were performed in flame-dried or oven-dried glassware under a positive pressure of dry argon, and were stirred magnetically unless otherwise indicated. Sensitive liquids and solutions were transferred via syringe or cannula, and introduced into reaction vessels through rubber septa. Commercial grade reagents and solvents were used without further purification. Thin layer chromatography (TLC) was performed on Analtech UNIPLATE™ pre-coated glass-backed silica gel 60 A F-254 250 μm plates. Column chromatography (flash chromatography) was performed on a Biotage system using 32-63 micron, 60 A, silica gel pre-packed cartridges. Proton (1H) nuclear magnetic resonance (NMR) spectra were measured with a Varian (300 MHz) spectrometer with residual protonated solvent (CHCl3 δ 7.26; MeOH δ 3.30; DMSO δ 2.49) as standard. Low-resolution mass spectra (MS) were either obtained as electron impact (El) mass spectra or as fast atom bombardment (FAB) mass spectra.

[0016] The IUPAC name was obtained using the ACD/I Lab Web service.

[0017] A. Preparation of Chloropyrimidine Intermediates

Intermediate A1

Preparation of 2-amino-4-chloro-5,6-dimethyl-pyrimidine

[0018]

[0019] Step 1. Preparation of 2-amino-5,6-dimethyl-4-pyrimidinone

[0020] To a solution of ethyl 2-acetoacetate (6.0 g, 41.6 mmol) and guanidine carbonate (5.6 g, 31.2 mmol) in EtOH (32 mL) was added 12 N HCl (350 μL). The mixture was refluxed for 16 h. After the reaction was cooled to room temperature, the solid was collected by filtration and washed with EtOH. A solution of the solid in 1 N NaOH was refluxed for 3 h. After the reaction was cooled to room temperature, the aqueous mixture was adjusted to pH=5 with concentrated acetic acid. The resulting precipitate was collected by filtration, washed with water and then with hexanes, and dried under vacuum. Desired compound (6.34 g, 45.6 mmol; 100% yield); 1H NMR (D2O; NaOD) δ 1.47 (s, 3H), 1.29-1.30 (m, 2H), 1.22 (s, 3H); ES MS [M+H]+=140.

[0021] Step 2. Preparation of 2-amino-4-chloro-5,6-dimethyl-pyrimidine

[0022] The product of the previous step (2.0 g, 14.4 mmol) and phosphorus oxychloride (6 mL, 57.5 mmol), was refluxed for 4 h. The reaction was cooled to rt and poured over ice. The mixture was separated and the aqueous layer was extracted with chloroform (3×75 mL). The aqueous mixture was adjusted to pH=9 with concentrated ammonium hydroxide. The resulting solid product was collected by filtration, washed with water, and dried under vacuum. Desired compound (963 mg, 6.1 mmol; 43% yield); mp=212-220° C.; ES MS [M+H]+=158; TLC (CH2Cl2—MeOH, 90:10); Rf=0.72.

Intermediate A2

Preparation of 2-amino-4-chloro-6-(4-pyridyl)pyrimidine

[0023]

[0024] Step 1. Preparation of 2-amino-4-hydroxy-6-(4-pyridyl)pyrimidine

[0025] A solution of guanidine carbonate (7.1 g, 39 mmol, 1.5 eq), ethyl isonicotinoyl acetate (10 g, 51.76 mmol), and hydrochloric acid (0.75 mL, 9.0 mmol) in absolute ethanol (80 mL) was refluxed under argon overnight. The precipitate formed was filtered, washed with ethanol and dried. The solid was then dissolved in 1 N NaOH (100 mL) and refluxed for 2 h. The reaction mixture was then cooled to room temperature, acidified with glacial acetic acid, and the solid formed was filtered and dried to afford the desired product as a white solid (5.45 g, 56%). 1H-NMR (DMSO-d6) δ 6.24 (s, 1H), 6.79 (bs, 2H), 7.85 (d, J=5.1 Hz, 2H), 8.62 (d, J=5.3 Hz, 2H), 11.22 (bs, 1H).

[0026] Step 2. Preparation of 2-amino-4-chloro-6-(4-pyridyl)pyrimidine:

[0027] A solution of 2-amino-4-hydroxy-6-(4-pyridyl)pyrimidine (5.45 g, 29 mmol) in POCl3 (12 mL) was refluxed under argon for 5 h. The reaction mixture was cooled to room temperature, poured over ice, and allowed to stir at room temperature for 2 h to ensure the quenching of POCl3. At this time, the mixture was made basic upon addition of 1 N NaOH and the brown solid was filtered to afford 4.52 g of crude product, which was used without further purification (NMR analysis showed 1:1 product/starting material). The filtrate formed more solid upon standing at room temperature (1 g, NMR analysis showed 2:1 product/starting material). 1H-NMR (DMSO-d6) δ 7.34 (bs, 2H), 7.38 (s, 1H), 7.99 (d, J=4.2 Hz, 2H), 8.72 (d, J=4.6 Hz, 2H).

Intermediate A3

Preparation of 2-amino-4-chloro-6-(2-thienyl)pyrimidine

[0028]

[0029] Step 1. Preparation of ethyl-2-(thiophene-2-oyl)acetate.

[0030] A solution of thiophene-2-carboxylic acid (8.9 g, 68.5 mmol), 2,2-dimethyl-1,3-dioxane-4,6-dione (12.0 g, 81.6 mmol), and 4-dimethylaminopyridine (17.0 g, 138 mmol) in dry CH2Cl2 (100 mL) was cooled to 0° C. and treated with a solution of 1,3-dicyclohexylcarbodiimide (75 mL, 1.0 M in CH2Cl2, 75 mmol). The reaction was allowed to stir at room temperature for 2 h and the dicyclohexylurea was then filtered and washed with CH2Cl2. The filtrate was concentrated at reduced pressure and the residue was dissolved in absolute ethanol (400 mL). The solution was then treated with a solution of p-toluenesulfonic acid monohydate (32 g, 168 mmol) in absolute ethanol (100 mL) and refluxed under argon for 1 h. At this time, the ethanol was removed at reduced pressure and the residue was dissolved in EtOAc and washed sequentially with H2O (300 mL), saturated NaHCO3 (200 mL), 1 N HCl (200 mL), saturated NaCl, and dried (MgSO4). The solvent was removed at reduced pressure and the residue was filtered through a pad of silica with 10% EtOAc/90% hexanes to afford the desired product as an oil (13 g, 96%). TLC (20% EtOAc/80% hexane) Rf 0.21; 1H-NMR (DMSO-d6) δ 1.17 (t, J=7.01, 3H), 4.06-4.14 (m, 4H), 7.25 (t, J=5.1 Hz, 1H), 7.98 (d, J=3.8 Hz, 1H), 8.06 (d, J=4.9 Hz, 1H).

[0031] Step 2. Preparation of 2-amino-4-hydroxy-6-(2-thienyl)pyrimidine.

[0032] The procedure was similar to that used for Intermediate A2, step 1, using ethyl-2-(thienyl-2-oyl)acetate as starting material. (43% yield). TLC (6% MeOH/94% CH2Cl2) Rf 0.23; MS ES 194 [M+H]+; 1H-NMR (DMSO-d6) δ 6.06 (s, 1H), 6.70 (bs, 2H), 7.11 (t, J=4.9 Hz, 1H), 7.64 (d, J=4.9 Hz, 1H), 7.70 (d, J=3.6 Hz, 1H), 10.95 (bs, 1H).

[0033] Step 3. Preparation of 2-amino-4-chloro-6-(2-thienyl)pyrimidine.

[0034] The procedure was similar to that of Intermediate A2, step 2, using 2-amino-4-hydroxy-6-(2-thiophene)pyrimidine as starting material. It afforded 33% yield after purification on silica with 15% EtOAc/85% hexanes. TLC (20% EtOAc/80% hexanes) Rf 0.29; 1H-NMR (DMSO-d6) δ 7.16-7.23 (m, 4H), 7.77 (dd, J=0.8, 5.0 Hz, 1H), 7.98 (dd, J=1.0, 3.8 Hz, 1H).

Intermediate A4

Preparation of 2-amino-4-chloro-6-(2-furyl)pyrimidine

[0035]

[0036] Step 1. Preparation of 2-amino-4-hydroxy-6-(2-furyl)pyrimidine.

[0037] The general procedure for the preparation of Intermediate A2, (step 1) was used; (37% yield). MS (ES) 178 [M+H]+.

[0038] Step 2. Preparation of 2-amino-4-chloro-6-(2-furyl)pyrimidine.

[0039] A solution of 2-amino-4-hydroxy-6-(2-furyl)pyrimidine (1.40 g, 7.9 mmol) in POCl3 (4 mL) was refluxed under argon for 2 h. The POCl3 was distilled; the residue was diluted with EtOAc and poured over iced saturated NaHCO3. The layers were separated and the aqueous was extracted with EtOAc (100 mL). The combined extracts was washed with saturated NaCl, dried (MgSO4), and the solvent removed at reduced pressure to afford 0.5 g of crude product, which was used without further purification. TLC (20% EtOAc/80% hexane) Rf 0.26; 1H-NMR (DMSO-d6) δ 6.68 (dd, J=1.7, 3.4 Hz, 1H), 6.94 (s, 1H), 7.25 (dd, J=1, 3.7 Hz, 1H), 7.91 (dd, J=0.8, 1.9 Hz, 1H).

Intermediate A5

Preparation of 6-benzyl-4-chloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-amine

[0040]

[0041] Step 1. Preparation of 2-amino-7-benzyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4(3H)-one.

[0042] To EtOH (16 mL) cooled to 0° C. (ice/H2O bath) was added Na spheres (204 mg, 8.9 mmol). The mixture was stirred until all Na dissolved. Methyl 1-benzyl-4-oxo-3-piperidine-carboxylate hydrochloride (3.0 g, 10.1 mmol) and guanidine carbonate (1.4 g, 7.6 mmol) were added. The mixture was refluxed for 16 h. After the reaction was cooled to room temperature, the solid was collected by filtration, washed with EtOH, and dried under vacuum. Desired compound (2.58 g, 10.0 mmol; 99+% yield); mp=202-212° (dec.); ES MS [M+H]+=257; TLC (CH2Cl2—MeOH, 90:10); Rf=0.20.

[0043] Step 2. Preparation of 6-benzyl-4-chloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-amine.

[0044] A solution of the product from step 1 (3.5 g, 13.7 mmol) in POCl3 (52 mL) was refluxed under argon for 5 h. The reaction mixture was cooled to room temperature, poured over ice, and allowed to stir at room temperature for 2 h to ensure the quenching of POCl3. At this time, the mixture was made basic upon addition of ammonium hydroxide and was extracted with CH2Cl2 (3×200 mL). The combined organics were washed with 1N NaOH followed by brine, dried (MgSO4), and concentrated under reduced pressure. The residue was taken up in benzene and was made acidic upon the addition of 1N HCl in diethyl ether. The brown solid was filtered to afford 0.35 g of crude product, which was used without further purification. ES MS [M+H]+=275.

Intermediate A6

Preparation of 2-amino-6-(trifluoromethyl)-4-pyrimidinyl 4-methylbenzenesulfonate

[0045]

[0046] To a solution of 2-amino-6-(trifluoromethyl)-4(3H)-pyrimidinone (250 mg, 1.4 mmol), triethylamine (196 μL, 1.4 mmol), N,N-dimethylaminopyridine (17 mg, 0.14 mmol), in CH2Cl2 (13 mL) cooled to 0° C. was added p-toluenesulfonyl chloride (534 mg, 2.8 mmol). The mixture was stirred at room temperature for 16 h. The mixture was diluted with CH2Cl2, washed with H2O (2×20 mL) followed by brine, dried (Na2SO4), evaporated, and dried under vacuum. Desired compound (466 mg, 1.4 mmol; 99+% yield; ES MS [M+H]+=140.

[0047] Using the above methods for the preparation of A1-A6 and substituting the appropriate starting materials, the following pyrimidine intermediates were also prepared.

2TABLE 1Chloropyrimidine Intermediates A

Intermediate No.R1R2R3Physical Data A7MeHNH2Aldrich A8EtHNH2Aldrich or Lancaster A9HHNH2AldrichA10t-BuHNH2mp = 109-113° C.; ES MS [M + H]+ = 186; TLC(90:10 CH2Cl2/MeOH); Rf = 0.37.A11MeClNH2Aldrich or Lancaster?A12—(CH)4—NH21H NMR(DMSO-d6)δ6.60(s, 2H), 2.55−2.29(m, 4H), 1.68-1.56(m, 4H)A13—(CH)5—NH21H NMR(DMSO-d6)δ6.65(s, 2H), 2.72−2.58(m, 4H), 1.74-1.60(m, 2H), 1.55-1.34(m, 4H)A14—(CH)3—NH21H NMR(DMSO-d6)δ6.73(s, 2H), 2.72−2.57(m, 4H), 1.89(sept, J=7.0, 2H)A15i-PrHNH2mp = 104-112° C.; 1H NMR(D2O)δ6.11(s, 1H),2.23−2.11(m, 1H), 0.46(d, J=6.2Hz, 6H); ESMS [M + H]+ = 172A16CH3HPh-NH—A17PhHNH2A183-pyridylHNH2A192-pyridylHNH2A203-NO2-PhHNH2A21ClHNH2Aldrich

[0048] B. Preparation of Arylamine Intermediates

Intermediate B1

Preparation of 1-(4-pyridinyl)-1H-indol-5-amine

[0049]

[0050] Step 1. Preparation of 5-nitro-1-(4-pyridinyl)-1H-indole

[0051] To a solution of 5-nitroindole (7.0 g, 43.2 mmol) and 4-chloropyridine hydrochloride (7.8 g, 51.8 mmol) in DMF (43 mL) was added potassium tert-butoxide (12.1 g, 108.0 mmol), portionwise. The reaction was heated at 100° C. for 48 h. The mixture was allowed to cool to room temperature and poured into water (400 mL). The resulting solid was removed by filtration and dried under vacuum. Desired compound (6.04 g, 25.3 mmol; 58% yield); 1H NMR (DMSO-d6) δ 8.76 (dd, J=1.7, 4.5, 2H), 8.68 (d, J=2.2, 1H), 8.06-8.13 (m, 2H), 7.92 (d, J=9.2, 1H), 7.75 (dd, J=1.5, 4.6, 2H), 7.07 (dd, J=0.9, 3.5, 1H); ES MS [M+H]+=240.

[0052] Step 2. Preparation of 1-(4-pyridinyl)-1H-indol-5-amine

[0053] A mixture of the product from step 1 (8.27 g, 34.6 mmol) and 10% palladium-on-charcoal catalyst (827 mg) in ethyl acetate (166 mL) and EtOH (9 mL) was stirred under hydrogen at atmospheric pressure for 48 h. Further catalyst (414 mg) was added and the reaction was stirred for 24 h. Again, further catalyst (414 mg) was added and the reaction was stirred an additional 24 h. The catalyst was removed by filtration through diatomaceous earth and the solvent removed from the filtrate by evaporation. The residue was triturated with ether, collected by filtration, and dried under vacuum. Desired compound (4.67 g, 22.3 mmol; 65% yield); mp=149-154° C.; ES MS [M+H]+=210; TLC (CH2Cl2-MeOH, 95:5); Rf=0.29.

Intermediate B2

Preparation of 4-[(4-aminophenyl)sulfanyl]phenol

[0054]

[0055] Step 1. Preparation of 4-[(4-nitrophenyl)sulfanyl]phenol.

[0056] To a solution of nitrobenzenesulfonyl chloride (4 g, 21 mmol) in ether (25 mL) was added phenol (1.97 g, 20 mmol) as a solution in ether (25 mL). After being stirred for 15 h at rt, the mixture was concentrated to afford a crude solid which was recrystallized from acetic acid. Desired compound (4.0 g, 16.2 mmol, 76% yield). TLC (Hexanes/EtOAc, 70:30); Rf=0.54.

[0057] Step 2. Preparation of 4-[(4-aminophenyl)sulfanyl]phenol.

[0058] To a solution of the product of step 1 (4 g, 16.2 mmol) in EtOH (500 mL) was added SnCl2.2H2O (18.3 g, 81 mmol) The solution was warmed to reflux. After being stirred for 3 h, the mixture was allowed to cool to rt, and the volatiles were removed by rotary evaporation. The resultant slurry was suspended in EtOAc, and solid NaHCO3 was added. Subsequently, the mixture was filtered, and the filtered solid was washed thoroughly with EtOAc. The organic filtrate was washed with water, and the aqueous washes were extracted with EtOAc. The combined organic extracts were washed with brine, dried (MgSO4), filtered, and concentrated to afford an orange solid, which was used without additional purification. Desired compound (3.0 g, 13.8 mmol, 86% yield).

[0059] TLC (Hexanes/EtOAc, 70:30); Rf=0.34.

Intermediate B3

Preparation of (3-aminophenyl)[4-(methylsulfanyl)phenyl]methanone

[0060]

[0061] Step 1. Preparation of [4-(methylsulfanyl)phenyl](3-nitrophenyl)methanone

[0062] 3-nitrobenzoylchloride (5.0 g, 26.94 mmol) was added to a solution of thioanisole (3.16 ml, 26.94 mmol) and 1,2-dichlorethane (95 mL). The resulting reaction mixture was cooled to 0° C. (ice/H2O bath) and 0.5 equivalents of aluminum trichloride (1.8 g, 13.47 mmol) was added. The reaction was allowed to stir for 15 min at this temperature and the cold bath was removed followed by addition of the remaining equivalents of AlCl3 (2.51 g, 18.87). The reaction solution turned a dark greenish/yellow and was allowed to stir at room temp. for 18 h, after which time the reaction was quenched slowly with H2O (50 mL). The mixture was diluted with CH2Cl2 (50 mL) and washed with H2O (3×50 mL), and the combined organic phases were washed with satd NaHCO3 (50 mL), dried (MgSO4) and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography (EtOAc/hexane, ¼) to afford 3.3 g (44%) of 4-(methylsulfanyl)phenyl](3-nitrophenyl)methanone as a solid. EI-LRMS m/z 274 (M+); TLC Rf 0.68 (EtOAc/Hex, ⅔).

[0063] Step 2. Preparation of (3-aminophenyl)[4-(methylsulfanyl)phenyl]methanone

[0064] Prepared analogously to Intermediate B2, step 2. The crude product was purified by flash column chromatography, eluting with 70:30 Hexanes/EtOAc. TLC: (Hexanes/EtOAc, 70:30); Rf=0.15.

Intermediate B4

Preparation of 4-(4-aminophenoxy)phenol

[0065]

[0066] Step 1. Preparation of 4-(4-nitrophenoxy)phenol

[0067] A mixture of p-nitrofluorobenzene (25 g, 0.177 mol), dihydroquinone (19.5 g, 0.177 mol), and sodium hydroxide (7.08 g, 0.177 mol) in EtOH/H2O (1:1 v/v, 176 mL) was heated at reflux for 20 h, and subsequently allowed to cool to room temperature. The mixture was filtered, the filtrate was made acidic with dilute aqueous HCl, and the resultant precipitate filtered to afford the crude product as a yellow solid. The desired product was recrystallized from EtOH. (15 g, 0.064 mol, 37% yield). TLC (Hexanes/EtOAc, 70:30); Rf=0.44.

[0068] Step 2. Preparation of 4-(4-aminophenoxy)phenol

[0069] To a solution of the product of step 1 in EtOH (100 mL) was added 10% palladium on carbon (200 mg). After being stirred under an atmosphere of hydrogen overnight, the mixture was filtered through Celite®. The volatiles were removed from the filtrate to provide the crude product which was purified by flash column chromatography eluting with Hexanes/EtOAc (85:15, followed by 75:25). Desired product (1.5 g, 7.45 mmol, 86%). TLC (Hexanes/EtOAc, 70:30); Rf=0.41.

Intermediate B5

Preparation of 4-(4-pyridinylthio)aniline

[0070]

[0071] To a solution of 4-aminothiophenol (20.2 g, 156.5 mmol) in anhydrous DMF (200 mL) was added 4-chloropyridine hydrochloride (24.4 g, 161.0 mmol) followed by potassium carbonate (44 g, 318.4 mmol). The reaction mixture was heated at 80° C. overnight, then diluted with ethyl acetate (400 mL) and water (400 mL). The aqueous layer was back-extracted with ethyl acetate (2×200 mL). The combined organic layers were washed with a saturated aqueous NaCl solution (200 mL), dried over anhy MgSO4, and concentrated under reduced pressure. The residue was filtered through a pad of silica with ethyl acetate and the resulting material was triturated with an ethyl ether/hexane solution to afford the desired product (24.7 g, 78%). TLC (50% ethyl acetate/50% hexane) Rf 0.25; 1H-NMR (DMSO-d6) δ 5.67 (bs, 2H), 6.65 (d, J=8.4 Hz, 2H), 6.88 (d, J=6.2 Hz, 2H), 7.19 (d, J=8.4 Hz, 2H), 8.27 (d, J=6.2 Hz, 2H), MS[M+H]+=203.

Intermediate B6

Preparation of 4-[2-(4-pyridinyl)ethyl]aniline

[0072]

[0073] Step 1. Preparation of 4-[(E)-2-(4-nitrophenyl)ethenyl]pyridine

[0074] To an oven dried 500 mL 3-necked flask was added (4-nitrobenzyl)triphenylphosphonium bromide (15 g, 30.42 mmol) followed by the addition of THF (100 mL). The solution was cooled to 0° C. in an ice bath. Potassium t-butoxide (3.9 g, 33.02 mmol) was then added in one portion resulting in an orange suspension. The suspension was maintained at 0° C. while a solution of 4-pyridine-2-carboxaldehyde (2.7 g, 24.70 mmol) in THF (20 mL) was added in 10 minutes. The ice bath was removed and the reaction was stirred at room temperature for 2 h. At this time, the reaction was quenched with saturated ammonium chloride solution (50 mL) and stirred for 15 minutes. The mixture was then extracted with ethyl acetate (2×100 mL), the combined extracts was washed with saturated aqueous NaCl solution (100 mL) and dried (MgSO4). The solvent was removed at reduced pressure and the residue was chromatographed on silica with 0-50% ethyl acetate in hexanes to afford the desired product (1.8 g, 32%). TLC (50% ethyl acetate/50% hexane) Rf 0.28; 1H-NMR (DMSO-d6) δ 6.84 (d, J=12.4 Hz, 1H), 6.96 (d, J=12.4 Hz, 1H), 7.14 (d, J=6.2 Hz, 2H), 7.45 (d, J=8.7 Hz, 2H), 8.15 (d, J=8.7 Hz, 2H), 8.47 (d, J=6.2 Hz, 2H).

[0075] Step 2. Preparation of 4-[2-(4-pyridinyl)ethyl]aniline

[0076] To a dry 50 mL flask flushed with argon was added 10% Pd on carbon (285 mg) followed by the addition of ethanol (12 mL) and the product from step 1 (1.8 g, 8.0 mmol). At this time, the argon line was replaced with a hydrogen balloon and the reaction was stirred overnight. The mixture was filtered through a pad of Celite® and the filtrate was concentrated at reduced pressure. The solid residue was triturated with ethyl ether/hexanes to afford the desired product (1.2 g, 67%). TLC (4% acetone/96% methylene chloride) Rf 0.09; 1H-NMR (DMSO-d6) δ 2.67-2.83 (m, 4H), 4.83 (bs, 2H), 6.45 (d, J=8.2 Hz, 2H), 6.84 (d, J=8.2 Hz, 2H), 7.20 (d, J=6 Hz, 2H), 8.41 (d, J=6 Hz, 2H).

Intermediate B7

Preparation of 3-fluoro-4-(4-pyridinylsulfanyl)aniline

[0077]

[0078] Step 1. Preparation of 4-[(2-fluoro-4-nitrophenyl)sulfanyl]pyridine.

[0079] A solution of 4-mercaptopyridine (4.2 g, 35.6 mmol), 3,4-difluoronitrobenzene (5.7 g, 35.7 mmol), and potassium carbonate (12.4 g, 89.7 mmol) in anhydrous DMF (40 mL) was stirred at 40° C. and under argon for 3 h. TLC showed complete reaction. The mixture was diluted with ethyl acetate (100 mL) and water (100 mL) and the aqueous layer was back-extracted with ethylacetate (2×100 mL). The organic layers were washed with a saturated NaCl solution (100 mL), dried (MgSO4), and concentrated under reduced pressure. The crude product was purified by column chromatography with 50% ethyl acetate/50% hexanes. It afforded the desired product as a yellow solid (6.3 g, 71%). TLC (50% EtOAc/50% hexane) Rf 0.53; 1H-NMR (DMSO-d6) δ 7.27 (dd, J=0.76, 4.2 Hz, 2H), 7.78 (dt, J=0.76, 7.2 Hz, 1H), 8.11-8.15 (m, 1H), 8.28-8.33 (m, 1H), 8.5 (dd, J=1.4, 4.6 Hz, 2H), MS [M+H]+=251.

[0080] Step 2. Preparation of 3-fluoro-4-(4-pyridinylsulfanyl)aniline.

[0081] A slurry of 3-fluoro-4-pyridinylthio)nitrobenzene (6.3 g, 25.2 mmol), iron powder (6.0 g, 107.4 mmol), acetic acid (100 mL), and water (1 mL) were stirred at room temperature overnight. The mixture was diluted with Et2O (100 mL) and water (100 mL). The aqueous phase was adjusted to pH 5 with a 4 N NaOH solution. The combined organic layers were washed with an aqueous saturated NaCl solution (100 mL), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by column chromatography with 50% ethyl acetate/50% hexanes. It afforded the desired product as a white solid (4.8 g, 86%). TLC (50% EtOAc/50% hexane) Rf 0.28; 1H-NMR (DMSO-d6) δ 6.04 (bs, 2H), 6.47-6.51 (m, 2H), 6.91 (d, J=6.1 Hz, 2H), 7.22 (t, J=8.4 Hz, 1H), 8.30 (d, J=6.4 Hz, 2H).

[0082] Using similar methods to those described for the preparation of Intermediates B1-B7, the following additional compounds were also prepared:

3TABLE 2Arylamine Intermediates B

IntermediateNo.Z(R5)nXAPhysical PropertiesB8CHH(4)-S—CH2—pyrid-4-ylTLC Rf = 0.12(50% EtOAc/50% hexanes).1H NMR(DMSO-d6)δ3.91(s, 2H), 5.26(bs,2H), 6.44(d, J=8.7Hz, 2H), 6.96(d, J=8.7Hz, 2H), 7.12(d, J=6.3Hz, 2H), 8.40 (d, J=6.0Hz, 2H).B9CH3-CF3(4)-S—pyrid-4-ylTLC Rf = 0.10(50% EtOAc/50% hexanes).1H NMR(DMSO-d6)δ6.21(bs, 2H), 6.84-6.87(m, 3H), 7.10(d, J=2.4Hz, 1H), 7.39(d, J=8.4Hz, 1H), 8.29(d, J=6.3Hz, 2H).B10CHH(4)-O—isoquinolin-5-ylB11CH3-F(4)-O—isoquinolin-5-yl1H NMR(DMSO-d6)δ5.42(bs, 2H), 6.41-6.55(m, 2H), 6.81-7.05(m, 2H), 7.48-7.54(m, 1H), 7.73-7.76(m, 1H), 8.06-8.08(m,1H), 8.54-8.56(m, 1H), 9.32(s, 1H).B12CH3,5-(Cl)2(4)-O—isoquinolin-5-ylTLC Rf = 0.29(45% EtOAc/55% hexanes).1H NMR(DMSO-d6)δ5.73(bs, 2H), 6.69(dd, J=1.1 and 8.0Hz, 1H), 6.75(s, 2H),7.51(t, J=7.7Hz, 1H), 7.78(d, J=8.2Hz,1H), 8.12(d, J=5.9Hz, 1H), 8.58(d, J=5.6Hz, 1H), 9.34(bs, 1H).B13CHH(4)-S—pyrid-4-ylTLC Rf = 0.07(100% EtOAc). 1H NMR(DMSO-d6)δ5.84(bs, 2H), 6.95-6.99(m,3H), 7.32(d, J=8.6Hz, 1H), 8.00(d, J=2.8Hz, 1H), 8.31(d, J=4.7Hz, 2H).B14CH3,5-(Cl)2(4)-S—pyrid-4-yl1H NMR(DMSO-d6)δ6.30(bs, 2H), 6.82(s,2H), 6.89, (d, J=6.0Hz, 2H), 8.33(d, J=6.1Hz, 2H).B15CH2,5-(F)2(4)-S—pyrid-4-ylB16CH3-Cl(4)-S—pyrid-4-ylB17CHH(4)-S—isoquinolin-5-ylB18CHH(4)-CH2—S—pyrid-4-ylB19CHH(4)-S—pyrid-3-ylB20CHH(3)-S—pyrid-4-ylB21CHH(4)-O—quinolin-5-yl

[0083] C. Preparation of Examples of the Invention

Example 1

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0084]

[0085] A suspension of 2-amino-4-chloro-6-methylpyrimidine (Intermediate A7, 0.2 g, 1.3 mmol), 3-fluoro-4-(4-pyridinylthio)aniline (Intermediate B7, 0.3 g, 1.3 mmol), and K2CO3 (0.2 g, 1.3 mmol) in o-xylene (1.3 mL) was heated to 100° C. in a 5 mL reaction-vial overnight. The reaction mixture was diluted with MeOH and coated on silica and purified by MPLC (Biotage) with 5-7% MeOH in CH2Cl2. It afforded 74 mg of product (18% yield). TLC (6% MeOH/94% CH2Cl2) Rf 0.29; MS ES 328 [M+H]+; 1H-NMR (DMSO-d6) δ 2.12 (s, 3H), 5.92 (s, 1H), 6.38 (bs, 2H), 6.96 (d, J=5.1 Hz, 2H), 7.39-7.52 (m, 2H), 8.26 (d, J=11.9 Hz, 1H), 8.33 (d, J=4.8 Hz, 2H), 9.55 (bs, 1H).

Example 2

Preparation of 6-ethyl-N4-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]-2,4-pyrimidinediamine

[0086]

[0087] 2-Amino-4-chloro-6-ethylpyrimidine (Intermediate A8, 55.1 mg, 0.25 mmol) and Intermediate B7(39.4 mg, 0.25 mmol) were suspended in 0.01 M aqueous HCl (500 μL). The mixture was refluxed for 6 h. The reaction was cooled to room temperature and the solvent was evaporated by vacuum. The residue was purified by reversed phase chromatography on a YMC Pack-pro C18 column (trademark) eluting with acetonitrile/H2O (10:90-90:10 gradient). The compound was further purified by preparative TLC eluting with CH2Cl2-MeOH (90:10). Desired compound (2.9 mg, 0.0085 mmol; 34% yield); 1H NMR (Methanol-d4) 8.16 (dd, J=1.7, 4.7, 2H), 8.00-8.04 (m, 1H), 7.37 (m, 2H), 6.93 (dd, J=1.8, 4.9, 2H), 5.91 (s, 1H), 2.39 (q, J=7.7, 2H), 1.13 (t, J=7.5, 3H); ES MS [M+H]+342; TLC (CH2Cl2-MeOH, 90:10); Rf=0.48.

Examples 3-26

[0088] Using the above procedures, the following examples of pyridines were synthesized and are summarized in Table 3.

4TABLE 3

IntermediateIntermediatePyrimidoneAmineEx. No.(A)(B)R1R2R4Analytical Data 3A9B1HHamp = 245-247° C.; 1H NMR(Methanol-d4)8.79(d, J=5.7, 2H), 8.32(s, 1H), 8.13 (d, J=5.9, 2H), 7.97(d, J=9.2, 1H), 7.88(d, J =3.6, 1H), 7.71(d, J=7.3, 1H), 7.56(d, J=9.1, 1H), 6.94(d, J=4.0, 1H), 6.33(d, J=7.6, 1H); ES MS [M + H]+= 303. 4A1B1CH3CH2—Hamp = 230-233° C.; 1H NMR(DMSO-d6)12.43(s, 1H), 10.51(s, 1H), 8.79(d, J=6.3,2H), 8.22(s, 1H), 7.87-8.23(m, 5H), 7.46(d, J=8.4, 1H), 6.85(d, J=3.3, 1H), 6.14(s,1H), 2.51-2.61(m, 2H), 1.19(t, J=7.5,3H); ES MS [M + H]+ = 331. 5A1B1CH3—Clamp = 238-241° C.; ES MS [M + H]+ = 351;TLC: Rf = 0.71(CH2Cl2—MeOH, 95:5). 6A1B1—(CH2)4—a1H NMR(DMSO-d6)11.75(s, 1H), 10.59(s,1H), 8.78(d, J=5.4, 2H), 8.21(s, 1H), 7.87-7.97(m, 5H), 7.47(d, J=8.1, 1H), 6.85 (d,J=3.4, 1H), 6.21(s, 1H), 1.29(s, 9H); ESMS [M + H]+ = 359. 7A1B1—(CH2)3)3—a1H NMR(Methanol-d4)8.80 (d, J 6.4,1H), 8.21(d, J=6.6, 1H), 8.16(s, 1H), 7.99(d, J=8.9, 1H), 7.90(d, J=3.3, 1H), 7.60(d, J=8.9, IH), 6.96(d, J=4.0, 1H), 2.93-2.99(m, 2H), 2.86(s, 2H), 2.23-2.29(m,2H); ES MS [M + H]+ = 343; TLC: Rf = 0.46(CH2Cl2—MeOH, 90:10. 8AldrichB1HHb1H NMR(Methanol-d4)8.13(dd, J=1.4,4.6, 2H), 7.80(dd, J=2.0, 6.7, 2H), 7.71(d,J=6.4, 1H),7.38-7.42(m, 2H),6.92(dd, J=1.6, 4.7, 2H), 6.01(d, J=6.1, 1H); ES MS[M + H]+ = 296; TLC: Rf = 0.28(CH2Cl2—MeOH; 90:10); 9A1B1(CH3)3C—Hbmp = 126-129° C.; ES MS [M + H]+ = 352;TLC: Rf = 0.62(CH2Cl2—MeOH, 90:10.10A1B1CH3—Clb1H NMR(Methanol-d4)8.43(d, J=5.9,2H), 7.93-7.96(m, 2H), 7.68-7.71(m,2H), 7.46(d, J=6.6, 2H), 2.51(s, 3H); ESMS [M + H]+ = 344.11A1B1—(CH2)4—bmp = 321-324° C.; 1H NMR(DMSO-d6)9.35(s, 1H), 8.37(dd, J=1.4, 4.7, 2H), 7.91(d, J=8.9, 2H), 7.58(d, J=8.4, 2H), 7.02(dd, J=1.5, 4.6, 2H), 3.33(brs, 4H), 2.60(brs, 2H), 1.77(brs, 4H); ES MS [M + H]+ = 350.12AldrichB1HHc1H NMR(Methanol-d4)8.26(dd, J=1.4, 4.7,2H), 8.11-8.16m, 1H), 7.84(d, J=6.2, 1H),7.45-7.50(m, 2H), 7.04(dd, J=1.6, 4.9,2H), 6.11(d, J=6.1, 1H); ES MS [M + H]+=314; TLC: Rf = 0.40(CH2Cl2—MeOH, 90:10).13A1B1(CH3)3C—Hc1H NMR(Methanol-d4)8.25-8.27(m, 2H),8.11-8.16(m, 1H), 7.46-7.48(m, 2H), 7.03(d, J=4.9, 2H), 6.15(s, 1H), 1.28(s, 9H);ESMS [M + H]+ = 370; TLC: Rf = 0.55(CH2Cl2—MeOH, 90:10).14A1B1—(CH2)4—cmp = 248-250° C., ES MS [M +H]+ = 368;TLC: Rf = 0.56(CH2Cl2—MeOH, 90:10).15A1B1—(CH2)5—c1H NMR(Methanol-d4)8.78(d, J=6.6, 2H),8.52(d, J=5.9, 1H), 8.34(brs, 1H), 7.76(d,J=6.9, 2H), 7.42(d, J=6.1, 1H), 7.23-7.33(m, 1H), 7.11(brs, 1H), 6.51-6.58(m, 3H),2.85-2.87(m, 2H), 2.60-2.63(m, 1H), 2.31-2.34(m, 2H), 1.42-1.75(m, 6H); ES MS[M + H]+ = 382.16A1B1CH3—Hamp = 254-256° C., TLC: Rf = 0.03(95:5CH2Cl2/MeOH).17A7B16CH3—HeTLC: Rf = 0.23(6% MeOH/94% CH2Cl2);LC MS [M + H]+ = 344; (3.37 min)18A7B10CH3—HhTLC: Rf = 0.39(6% MeOH/94% CH2Cl2)LC MS [M + H]+ = 345; (3.37 min)19A7B17CH3HgTLC: Rf = 0.44(6% MeOH/94% CH2Cl)2LC′MS[M + H]+360; (2.64 min)20A17B7PhHcTLC: Rf = 0.26(4% MeOH/96% CH2Cl2);LCMS: ES[M + H]+ 390; (2.76 min)21A18B143-pyridylHfTLC: Rf = 0.37(6% MeOH/94% CH2Cl2);LCMS: ES m/z 441(1.65 min)22A18B53-pyridylHbTLC: Rf = 0.35(4% MeOH/96% CH2Cl2);LCMS: ES[M + H]+ 373; (2.61 min)23A2B54-pyridylHbTLC: Rf = 0.13(4% MeOH/96% CH2Cl2);LCMS: ES[M + H]+ 373; (2.56 min)24A17B11PhHiTLC: Rf = 0.21(2% MeOH/98% CH2Cl2);LCMS: ES[M + H]+ 424; (2.75 min)25A2B114-pyridylHiTLC: Rf = 0.35(6% MeOH/94% CH2Cl2);LCMS: ES[M + H]+ 425; (2.60 min)26A7B5CH3HbLCMS: ES[M + H]+ 310; (3.53 min)

[0089] By selecting combinations of the appropriate Intermediates A1-A21 with Intermediates B1-B17, a variety of products were prepared in like manner and are described in Example 27-31.

Example 27

[0090] Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-{4-[(4-pyridinylmethyl)sulfanyl]phenyl}amine

[0091] Prepared in 34% yield from Intermediate A7 and B8: TLC (7% MeOH in CH2Cl2) Rf 0.36; MS (ES) 324 [M+H]+; 1H-NMR (DMSO-d6) δ 2.09 (s, 3H), 4.12 (s, 2H), 5.87 (s, 1H), 6.33 (bs, 2H), 7.19 (d, J=8.5 Hz, 2H), 7.23 (d, J=5.8 Hz, 2H), 7.64 (d, J=8.5 Hz, 2H), 8.43 (d, J=5.3 Hz, 2H), 9.20 (bs, 1H).

Example 28

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-{4-[(4-pyridinylsulfanyl)methyl]phenyl}amine

[0092]

[0093] Prepared in 6% yield from Intermediate A7 and B18:TLC (7% MeOH in CH2Cl2) Rf 0.38; MS (ES) 342 [M+H]+; 1H-NMR (DMSO-d6) δ 2.06 (s, 3H), 4.30 (s, 2H), 5.84 (s, 1H), 6.13 (bs, 2H), 7.27-7.31 (m, 4H), 7.63 (d, J=7.9 Hz, 2H), 8.33 (d, J=6.1 Hz, 2H), 8.99 (bs, 1H).

Example 29

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-{4-[2-(4-pyridinyl)ethyl]phenyl}amine

[0094]

[0095] Prepared in 30% yield from A7 and B6: TLC (8% MeOH in CH2Cl2) Rf 0.34; MS (ES) 306 [M+H]+; 1H-NMR (DMSO-d6) δ 2.06 (s, 3H), 2.83-2.87 (m, 4H), 5.82 (s, 1H), 6.09 (bs, 2H), 7.07 (d, J=8.5 Hz, 2H), 7.21 (d, J=5.8 Hz, 2H), 7.54 (d, J=8.5 Hz, 2H), 8.41 (d, J=6.2 Hz, 2H), 8.87 (s, 1H).

Example 30

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-[4-(4-pyridinylsulfanyl)-3-(trifluoromethyl)phenyl]amine

[0096]

[0097] Prepared in 1.2% yield from A7 and B9: TLC (7% MeOH in CH2Cl2) Rf 0.39; MS (ES) 378 [M+H]+; 1H-NMR (DMSO-d6) δ 2.03 (s, 3H), 5.94 (s, 1H), 6.33 (bs, 2H), 6.91 (d, J=6.5 Hz, 2H), 7.64 (d, J=8.9 Hz, 1H), 8.19 (d, J=2.2 Hz, 1H), 8.33 (d, J=5.9 Hz, 2H), 8.37 (dd, J=2.1, 8.6 Hz, 1H), 9.66 (s, 1H).

Example 31

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-[4-(5-isoquinolinyloxy)phenyl]amine

[0098]

[0099] Prepared in 30% yield from A7 and B10: TLC (6% MeOH in CH2Cl2) Rf 0.39; MS (ES) 344 [M+H]+; 1H-NMR (DMSO-d6) δ 2.08 (s, 3H), 5.85 (s, 1H), 6.11 (bs, 2H), 7.02-7.08 (m, 3H), 7.58 (t, J=8.1 Hz, 1H), 7.74 (d, J=8.6 Hz, 2H), 7.84 (d, J=8.2 Hz, 1H), 7.98 (d, J=5.8 Hz, 1H), 8.54 (d, J=5.9 Hz, 1H), 9.03 (bs, 1H), 9.35 (bs, 1H).

Example 32

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-[3-fluoro-4-(5-isoquinolinyloxy)phenyl]amine

[0100]

[0101] A suspension of 2-amino4-chloro-6-methylpyrimidine (Intermediate A7, 0.14 g, 1.0 mmol), 3-fluoro-4-(5-isoquinolin-oxy)aniline (Intermediate B10, 0.25 g, 1.0 mmol), and HCl (0.1 mL) in H2O (1.0 mL) was heated to 70° C. in a 5 mL reaction vial overnight. The reaction mixture was diluted with MeOH, treated with saturated NaHCO3, and coated on silica and purified by MPLC (Biotage) with 5% MeOH in CH2Cl2. It afforded 52 mg of product (14% yield). TLC (6% MeOH/94% CH2Cl2) Rf 0.45; MS (ES) 362 [M+H]+; 1H-NMR (DMSO-d6) δ 2.10 (s, 3H), 5.88 (s, 1H), 6.29 (bs, 2H), 6.93 (d, J=7.9 Hz, 1H), 7.22 (t, J=8.9 Hz, 1H), 7.34 (dd, J=1.7, 8.9 Hz, 1H), 7.55 (t, J=8.1 Hz, 1H), 7.82 (d, J=8.3 Hz, 1H), 8.08 (d, J=5.6 Hz, 1H), 8.21 (dd, J=2.6, 14.2 Hz, 1H), 8.58 (d, J=5.9 Hz, 1H), 9.30 (bs, 1H), 9.36 (s, 1H).

[0102] Using the above-described method for Example 32, Examples 33-41 were similarly prepared.

Example 33

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-[3,5-dichloro-4-(4-pyridinylsulfanyl)phenyl]amine

[0103]

[0104] Prepared in 10% yield from A7 and B14: TLC (5% MeOH in CH2Cl2) Rf 0.14; MS (ES) 378 [M+H]+; 1H-NMR (DMSO-d6) δ 2.14 (s, 3H), 5.92 (s, 1H), 6.49 (bs, 2H), 6.93 (d, J=6.1 Hz, 2H), 8.13 (s, 2H), 8.35 (d, J=6.2 Hz, 2H), 9.63 (bs, 1H).

Example 34

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-[3,5-dichloro-4-(5-isoquinolinyloxy)phenyl]amine

[0105]

[0106] Prepared in 58% yield from A7 and B12: TLC (70% EtOAc/30% hexanes) Rf 0.18; MS (ES) 412 [M+H]+; 1H-NMR (DMSO-d6) δ 2.13 (s, 3H), 5.89 (s, 1H), 6.38 (bs, 2H), 6.73 (d, J=7.6 Hz, 1H), 7.52 (t, J=7.9 Hz, 1H), 7.82 (d, J=8.2 Hz, 1H), 8.05 (s, 2H), 8.16 (d, J=5.8 Hz, 1H), 8.61 (d, J=5.9 Hz, 1H), 9.36 (s, 1H), 9.42 (s, 1H).

Example 35

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-[6-(4-pyridinylsulfanyl)-3-pyridinyl]amine

[0107]

[0108] Prepared in 16% yield from A7 and B13: TLC (6% MeOH in CH2Cl2) Rf 0.16; MS (ES) 311 [M+H]+; 1H-NMR (DMSO-d6) δ 2.11 (s, 3H), 5.91 (s, 1H), 6.34 (bs, 2H), 7.15 (d, J=6.2 Hz, 2H), 7.48 (d, J=8.8 Hz, 1H), 8.30 (dd, J=2.5, 8.4 Hz, 1H), 8.38 (d, J=6.1 Hz, 2H), 9.00 (d, J=2.4 Hz, 1H), 9.45 (bs, 1H).

Example 36

Preparation of N-(2-amino-6-phenyl-4-pyrimidinyl)-N-[4-(4-pyridinylsulfanyl)phenyl]amine

[0109]

[0110] Prepared in 65% yield from A17. TLC (4% MeOH in CH2Cl2) Rf 0.22; MS (ES) 372 [M+H]+; 1H-NMR (DMSO-d6) δ 6.46 (bs, 2H), 6.55 (s, 1H), 6.96 (d, J=4.8 Hz, 2H), 7.46-7.49 (m, 5H), 7.91-8.00 (m, 4H), 8.32 (d, J=4.9 Hz, 2H), 9.57 (bs, 1H).

Example 37

Preparation of N-[2-amino-6-(3-pyridinyl)-4-pyrimidinyl]-N-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0111]

[0112] Prepared in 45% yield. TLC (4% MeOH in CH2Cl2) Rf 0.27; MS (ES) 391 [M+H]+; 1H-NMR (DMSO-d6) δ 6.58 (s, 1H), 6.70 (bs, 2H), 6.99 (d, J=6.4 Hz, 2H), 7.46-7.57 (m, 3H), 8.23-8.36 (m, 4H), 8.65 (d, J=4.4 Hz, 1H), 9.09 (s, 1H), 9.86 (bs, 1H).

Example 38

Preparation of N-[2-amino-6-(4-pyridinyl)-4-pyrimidinyl]-N-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0113]

[0114] Prepared in 22% yield from A2 and B7:. TLC (6% MeOH in CH2Cl2) Rf 0.32; MS (ES) 391 [M+H]+; 1H-NMR (DMSO-d6) δ 6.63 (s, 1H), 6.74 (bs, 2H), 6.99 (d, J=5.8 Hz, 2H), 7.46-7.58 (m, 2H), 7.85 (d, J=5.8 Hz, 2H), 8.31-8.36 (m, 3H), 6.70 (d, J=4.3 Hz, 2H), 9.92 (bs, 1H).

Example 39

Preparation of N-[2-amino-6-(4-pyridinyl)-4-pyrimidinyl]-N-[3,5-dichloro-4-(4-pyridinylsulfanyl)phenyl]amine

[0115]

[0116] Prepared in 0.4% yield from A2 and B14: TLC (4% MeOH in CH2Cl2) Rf 0.15; 1H-NMR (DMSO-d6) δ 6.60 (s, 1H), 6.82 (bs, 2H), 6.95 (d, J=5.9 Hz, 2H), 7.85 (d, J=5.9 Hz, 2H), 8.18 (s, 2H), 8.36 (d, J=3.8 Hz, 2H), 8.71 (d, J=4.7 Hz, 2H), 9.99 (bs, 1H).

Example 40

Preparation of N-[2-amino-6-(3-pyridinyl)-4-pyrimidinyl]-N-[3-fluoro-4-(5-isoquinolinyloxy)phenyl]amine

[0117]

[0118] Prepared in 54% yield from A18 and B11: TLC (5% MeOH in CH2Cl2) Rf 0.34; MS (ES) 425 [M+H]+; 1H-NMR (DMSO-d6) δ 6.53 (s, 1H), 6.59 (bs, 2H), 6.96 (d, J=7.4 Hz, 11H), 7.26 (t, J=9.6 Hz, 1H), 7.38-7.59 (m, 3H), 7.83 (d, J=8.2 Hz, 1H), 8.09 (d, J=5.7 Hz, 1H), 8.23-8.31 (m, 2H), 8.58-8.65 (m, 2H), 9.09 (bs, 1H), 9.37 (bs, 1H), 9.61 (bs, 1H).

Example 41

Preparation of N-[2-amino-6-(2-pyridinyl)-4-pyrimidinyl]-N-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0119]

[0120] Prepared in 60% yield from A19 and B7: TLC (50% EtOAc/50% hexanes) Rf 0.14; MS (ES) 391 [M+H]+; 1H-NMR (DMSO-d6) δ 6.63 (bs, 2H), 6.98 (d, J=6.6 Hz, 2H), 7.17 (s, 1H), 7.45-7.57 (m, 3H), 7.93 (dt, J=1.9, 7.7 Hz, 1H), 8.23-8.37 (m, 4H), 8.65-8.67 (m, 1H), 9.90 (bs, 1H).

Example 42

Preparation of N-(2-amino-6-ethyl-4-pyrimidinyl)-N-[4-(4-pyridinylsulfanyl)phenyl]amine

[0121]

[0122] A mixture of Intermediate B5(50.6 mg, 0.250 mmol) and 2-amino-4-ethyl-6-chloropyrimidine (Intermediate A8, 39.4 mg, 0.25 mmol) in 0.01 M aqueous HCl (500 μL) was refluxed for 6 h. The reaction was cooled to room temperature and the solvent was evaporated by vacuum. The residue was purified by reverse phase chromatography on a YMC Pack-pro® C18 column eluting with acetonitrile/H2O (10:90-90:10 gradient) to give the desired compound (13.0 mg, 0.040 mmol; 16% yield); mp=181-186° C.; ES MS [M+H]+=324; TLC (CH2Cl2—MeOH, 95:5); Rf=0.41.

Example 43

Preparation of of N-(2-amino-6-chloro-4-pyrimidinyl)-N-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0123]

[0124] 2-Amino-4,6 dichloropyrimidine (A21, 12 mmol) and 3-fluoro-4-(4-pyridinylthio)-aniline (B7,12 mmol) were suspended in water (150 mL) and treated with 10 drops of concentrated hydrochloric acid. The mixture was stirred at 100° C. overnight. The clear solution was then neutralized with ammonium hydroxide. The precipitated yellow product was filtered, washed with water, and purified by column chromatography with 1-3% MeOH in CH2Cl2 to give the desired product as a white solid (1.98 g, 47%).

Example 44

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-[2,5-difluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0125]

[0126] The compound was prepared using a similar method used for the preparation of Example 1 (described above) from A7 and B15: HPLC/MS: [M+H]+ 346.1 m/z. Retention time (HPLC/MS)=1.39 min.

Example 45

Preparation of N-(2-amino-6-chloro-4-pyrimidinyl)-N-[3,5-dichloro-4-(4-pyridinylsulfanyl)phenyl]amine

[0127]

[0128] The compound was prepared using a similar method used for the preparation of Example 43 (described above) from A21 and B14: HPLC/MS: [M+H]+ 399.0 m/z. Retention time (HPLC/MS)=3.02 min.

Example 46

Preparation of N-(2-amino-6-chloro-4-pyrimidinyl)-N-[3-fluoro-4-(5-isoquinolinyloxy)phenyl]amine

[0129]

[0130] The product was prepared using a similar method used for the preparation of Example 43 (described above) from A21 and B11: HPLC/MS: [M+H]+ 382.1 m/z. Retention time (HPLC/MS)=2.91 min.

Examples 47-49

Using procedures similar to the above examples and using the appropriate Intermediates A and B, the following examples were prepared in like manner:

[0131] Example 47

Preparation of N-(2-amino-6-methyl-4-pyrimidinyl)-N-[1-(4-pyridinyl)-1H-indol-5-yl]amine

[0132]

[0133] Prepared by reaction of Intermediate A7 and B1.

Example 48

Preparation of N-(2-anilino-6-methyl-4-pyrimidinyl)-N-[1-(4-pyridinyl)-1H-indol-5-yl]amine

[0134]

[0135] Prepared by reaction of Intermediate A16 and B1.

Example 49

Preparation of N-(2-anilino-6-methyl-4-pyrimidinyl)-N-[4-fluoro-3-(4-pyridinylsulfanyl)phenyl]amine

[0136]

[0137] Prepared by reaction of Intermediate A16 and B7.

Rho Kinase Biochemical Assay

[0138] ROCK-1 activity criteria: 0 no effect (<40% inhibition), 1 effect (>40% inhibition). The assay tests for inhibition of ROCK-1 phosphorylation of MBP (Myelin Basic Protein). The reaction (100 μl final volume) is carried out in polypropylene 96-well plates in 50 mM HEPES buffer pH 7.5 containing 5 mM MgCl2 and 1 mM DTT. For each well, gstROCK1 (0.25 μgs of BAYER DRT gstROCK1) is combined with MBP (1 μg) in reaction buffer (70 μL combined volume). Inhibitors (5 μL of 20×conc. in 40% DMSO) are added to each well to give an 8 point dose response range from 1.0 μM to 0.5 nM. The reaction is begun by adding 25 μL of ATP (4x=12 μM) in reaction buffer containing 0.8 μCi of 33P gamma-ATP (4×) for a final concentration of 3 μM cold and 0.2 μCi hot ATP. Plates were incubated for 1 hour at room temperature with the reaction being stopped by addition of 7 μL of 1 N HCl. The radioactively labeled MBP was transferred to P30 filtermats (EG&G Wallac), washed in 1% phosphoric acid followed by brief washes in water. The filtermats were then dried and the incorporation of 33P detected by liquid scintillation counting. Background 33P incorporation is determined by ROCK1 autophosphorylation without MBP. The data are expressed as percent inhibition: % inhibition=1−((cpm with inhibitor−background)/(cpm without inhibitor−background))*100.

[0139] The entire disclosure of all applications, patents and publications, cited herein and of U.S. Provisional Application Serial No. 60/349,986, filed Jan. 23, 2002 is incorporated by reference herein.

Rho-kinase inhibitors

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Provisional Applications (1)