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US20100216789A1 - Rho-kinase inhibitors - Google Patents

Rho-kinase inhibitors Download PDF

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US20100216789A1
US20100216789A1 US12/688,428 US68842810A US2010216789A1 US 20100216789 A1 US20100216789 A1 US 20100216789A1 US 68842810 A US68842810 A US 68842810A US 2010216789 A1 US2010216789 A1 US 2010216789A1
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alkyl
halogen
etoac
hplc
phenyl
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Dhanapalan Nagarathnam
Uday Khire
Davoud Asgari
Jianxing Shao
Xiao-Gao Liu
Chunguang Wang
Barry Hart
Olaf Weber
Mark Lynch
Lei Zhang
Lei Wang
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • 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.
  • 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.
  • Rho proteins controls the formation of actin stress fibers, thick bundles of actin filaments, and the clustering of integrins at focal adhesion complexes.
  • 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.
  • Rho proteins can control different biological responses.
  • Rho proteins are responsible for the calcium sensitization during smooth muscle contraction.
  • the Rho GTPases are responsible for the cellular responses to agonist such as lysophosphatidic acid (LPA), thrombin and thromboxane A 2 (Fukata, et al. Trends Pharcol Sci 2001, 22, 32).
  • LPA lysophosphatidic acid
  • thrombin thrombin
  • thromboxane A 2 thromboxane A 2
  • Agonist response is coupled through heterotrimeric G proteins G alpha12 or G alpha13 (Goetzl, et al. Cancer Res 1999, 59, 4732; Buhl, et al. J Biol Chem 1995, 270, 24631) though other receptors may be involved.
  • Rho GTPases Upon activation Rho GTPases activate a number of downstream effectors including PIPS-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).
  • 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.
  • Rho kinase mediates calcium sensitization and smooth muscle contraction and inhibition of Rho kinase blocks 5-HT and phenylephrine agonist induced muscle contraction.
  • Rho kinase 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).
  • 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.
  • cardiovascular diseases such as hypertension (Uehata, et al. Nature 1997, 389, 990), atherosclerosis (Retzer, et al.
  • Rho kinase activity has benefits for controlling cerebral vasospasms and ischemia following subarachnoid hemorrhage ( Pharma Japan 1995, 1470, 16).
  • Rho Kinase inhibitors 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.
  • 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.
  • the invention provides compounds of formula I
  • X is —(CH 2 ) x —, —O—(CH 2 ) n —, —S—(CH 2 ) n —, —NR 7 —CO—(CH 2 ) n —, —NR 7 —SO 2 —(CH 2 ) n —, —NR 7 —(CH 2 ) n —, or —(O)C—NR 7 —
  • each n is an integer which is independently 0, 1, 2 or 3
  • x is 0-3
  • p is 0-3
  • c are each independently —CR 5 ⁇ , —N ⁇ , or —NR 6 —, wherein one of a or c is —NR 6 —, and b is —CR 5 ⁇ or —N ⁇
  • A is H, halogen, —CO—OR 8 , —CO—R 8 , cyano, —OR 8 , —NR 8 R 9 , —CO—NR 8 R 9 ,
  • A may optionally be substituted up to 3 times by (i) C 1 -C 10 alkyl or C 2 -C 10 -alkenyl, each optionally substituted with halogen up to perhalo; (ii) C 3 -C 10 cycloalkyl; (iii) optionally substituted aryl; (iv) optionally substituted heteroaryl; (v) halogen; (vi) —CO—OR 8 ; (vii) —CO—R 8 ; (viii) cyano; (ix) —OR 8 , (x) —NR 8 R 13 ; (xi) nitro; (xii) —CO—NR 8 R 9 ; (xiii) —C 1-10 -alkyl-NR 8 R 9 ; (xiv) —NR 8 —CO—R 12 ; (xv) —NR 7 —CO—OR 9 ; (xvi) —NR 7 —SO 2 —R 9 ; (xvii) —
  • R 1 , and R 6 -R 11 are each independently H and C 1-6 alkyl
  • R 2 -R 5 are each independently (i) C 1-10 alkyl or C 2-10 -alkenyl each optionally substituted by amino, N-lower alkylamino, N,N-dilower alkylamino, N-lower alkanoylamino, hydroxy, cyano, —COOR 10 , —COR 14 , —OCOR 14 , —OR 10 , C 5-10 -heteroaryl, C 5-10 -heteroaryloxy, C 5-10 -heteroaryl-C 1-10 -alkoxy, or halogen up to perhalo; (ii) C 3 -C 10 cycloalkyl, in which 1-3 carbon atoms are optionally independently replaced by O, N or S; (iii) C 3-10 -cycloalkenyl; (iv) partially unsaturated C 5-10 -heterocycl
  • suitable aryl or heteroaryl groups include, but are not limited to, 5-12 carbon-atom aromatic rings or ring systems containing 1-3 rings, at least one of which is aromatic, in which one or more, e.g., 1-4 carbon atoms in one or more of the rings can be replaced by oxygen, nitrogen or sulfur atoms.
  • Each ring typically has 3-7 atoms.
  • aryl or heteroaryl can be 2- or 3-furyl, 2- or 3-thienyl, 2- or 4-triazinyl, 1-, 2- or 3-pyrrolyl, 1-, 2-, 4- or 5-imidazolyl, 1-, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isoxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 2-, 3- or 4-pyridyl, 2-, 4-, 5- or 6-pyrimidinyl; 1,2,3-triazol-1-, -4- or 5-yl, 1,2,4-triazol-1-, -3- or B5-yl, 1- or 5-tetrazolyl, 1,2,3-oxadiazol-4- or 5-yl, 1,2,4-oxadiazol-3- or 5-yl, 1,3,4-thiadiazol-2- or 5-yl, 1,2,4-oxadiazol-3- or 5-yl, 1,3,4-thiadiazol-2- or
  • Preferred moieties A include cyclohexyl; or C 5-12 -aryl or C 5-12 -heteroaryl each independently optionally substituted up to three times by (i) C 1 -C 10 -alkyl or C 2-10 -alkenyl each optionally substituted with halogen up to perhalo; (ii) C 3 -C 10 cycloalkyl; (iii) C 5-12 -aryl optionally substituted by 1-3 halogen atoms; (iv) C 5-12 -heteroaryl; (v) halogen; (vi) —CO—OR 8 ; (vii) —CO—R 8 ; (viii) cyano; (ix) —OR 8 ; (x) —NR 8 R 13 ; (xi) nitro; (xii) —CO—NR 8 R 9 ; (xiii) —C 1-10 -alkyl-NR 8 R 9 ; (xiv) —NR 8
  • moieties A include phenyl, pyridyl, pyrimidinyl, oxazolyl, furyl, thienyl, pyrrolyl, imidazolyl, isoxazolyl and pyrazinyl, each independently substituted up to three times by halogen, C 1-10 -alkyl, C 1-10 -alkoxyphenyl, naphthyl, —OR 10 ,
  • each Z independently is halogen, hydroxy, hydroxy-C 1-10 -alkyl, —CN, —NO 2 , C 1-10 -alkoxycarboxyl, —NR 10 —CO—R 11 , or —NR 10 —CO—OR 11 , as well as OR 10 , y is 0-3, more preferably 1-3, and R 4 is as described above.
  • Preferred moieties A additionally include
  • R 15 is H; phenyl optionally substituted by C 1-10 -alkyl, C 1-10 -alkoxy, C 1-10 -alkylcarboxyl, or halogen; benzyl; pyrimidyl or pyridyl; and R 16 is H, phenyl, —COOR 10 ,
  • Suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, sulphonic acid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicyclic acid, phenylacetic acid, and mandelic acid.
  • basic salts of inorganic and organic acids such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, sulphonic acid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicyclic
  • pharmaceutically acceptable salts include acid salts of inorganic bases, such as salts containing alkaline cations (e.g., Li + , Na + or K + ), alkaline earth cations (e.g., Mg + , Ca + or Ba + ), the ammonium cation, as well as acid salts of organic bases, including aliphatic and aromatic substituted ammonium, and quaternary ammonium cations, such as those arising from protonation or peralkylation of triethylamine, N,N-diethylamine, N,N-dicyclohexylamine, pyridine, N,N-dimethylaminopyridine (DMAP), 1,4-diazabiclo[2.2.2]octane (DABCO), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
  • a number of the compounds of Formula I possess asymmetric carbons and can therefore exist in racemic and optically active forms. Methods of separation of enantiomeric and diastereomeric mixtures are well known to one skilled in the art.
  • the present invention encompasses any isolated racemic or optically active form of compounds described in Formula I which possess Rho-kinase inhibitory activity.
  • the invention also includes pharmaceutical compositions including a compound of Formula I, and a physiologically acceptable carrier.
  • the invention moreover encompasses treating indications mediated by Rho-kinase, by administering a compound of Formula I, or a pharmaceutical composition containing a compound of Formula I.
  • cardiovascular diseases such as hypertension, artherosclerosis, restenosis and cerebral ischemia, or vasospasm central nervous system disorders such as neuronal degeneration and spinal cord injury, erectile dysfunction, e.g., in patients who do not have satisfactory response to PDE-5 inhibitors, and cancer (e.g., tumor growth) mediated by Rho-kinase, by administering, e.g., to a host in need thereof, of an effective amount of a compound of Formula I.
  • Cancers and tumors mediated by Rho-kinase include cancers of the breast, colon, prostate, ovaries, brain and lung and their metastases.
  • a number of the compounds of Formula I possess asymmetric carbons and can therefore exist in racemic and optically active forms. Methods of separation of enantiomeric and diastereomeric mixtures are well known to one skilled in the art.
  • the present invention encompasses any isolated racemic or optically active form of compounds described in Formula I which possess Rho-kinase inhibitory activity.
  • the invention also includes pharmaceutical compositions including a compound of Formula I, and a physiologically acceptable carrier.
  • the invention moreover encompasses treating indications mediated by Rho-kinase, by administering a compound of Formula I, or a pharmaceutical composition containing a compound of Formula I.
  • cardiovascular diseases such as hypertension, artherosclerosis, restenosis and cerebral ischemia, or vasospasm central nervous system disorders such as neuronal degeneration and spinal cord injury, erectile dysfunction, e.g., in patients who do not have satisfactory response to PDE-5 inhibitors, and cancer (e.g., tumor growth) mediated by Rho-kinase, by administering, e.g., to a host in need thereof, of an effective amount of a compound of Formula I.
  • Cancers and tumors mediated by Rho-kinase include cancers of the breast, colon, prostate, ovaries, brain and lung and their metastases.
  • the compounds may be administered orally, topically, parenterally, by inhalation or spray, vaginally, rectally or sublingually in dosage unit formulations.
  • administration by injection includes intravenous, intraarticular, intramuscular, subcutaneous and parenteral injections, as well as use of infusion techniques.
  • Dermal administration may include topical application or transdermal administration.
  • One or more compounds may be present in association with one or more non-toxic pharmaceutically acceptable carriers and if desired other active ingredients.
  • compositions intended for oral use may be prepared according to any suitable method known to the art for the manufacture of pharmaceutical compositions.
  • Such compositions may contain one or more agents selected from the group consisting of diluents, sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; and binding agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • These compounds may also be prepared in solid, rapidly released form.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions containing the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions may also be used.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropyl-methyl cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol,
  • the compounds may also be in the form of non-aqueous liquid formulations, e.g., oily suspensions which may be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or peanut oil, or in a mineral oil such as liquid paraffin.
  • oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Compounds of the invention may also be administrated transdermally using methods known to those skilled in the art (see, for example: Chien; “Transdermal Controlled Systemic Medications”; Marcel Dekker, Inc.; 1987. Lipp et al. WO94/04157 3 Mar., 1994).
  • a solution or suspension of a compound of Formula I in a suitable volatile solvent optionally containing penetration enhancing agents can be combined with additional additives known to those skilled in the art, such as matrix materials and bacteriocides. After sterilization, the resulting mixture can be formulated following known procedures into dosage forms.
  • a solution or suspension of a compound of Formula I may be formulated into a lotion or salve.
  • Suitable solvents for processing transdermal delivery systems are known to those skilled in the art, and include lower alcohols such as ethanol or isopropyl alcohol, lower ketones such as acetone, lower carboxylic acid esters such as ethyl acetate, polar ethers such as tetrahydrofuran, lower hydrocarbons such as hexane, cyclohexane or benzene, or halogenated hydrocarbons such as dichloromethane, chloroform, trichlorotrifluoroethane, or trichlorofluoroethane.
  • Suitable solvents may also include mixtures of one or more materials selected from lower alcohols, lower ketones, lower carboxylic acid esters, polar ethers, lower hydrocarbons, halogenated hydrocarbons.
  • Suitable penetration enhancing materials for transdermal delivery system include, for example, monohydroxy or polyhydroxy alcohols such as ethanol, propylene glycol or benzyl alcohol, saturated or unsaturated C 8 -C 18 fatty alcohols such as lauryl alcohol or cetyl alcohol, saturated or unsaturated C 8 -C 18 fatty acids such as stearic acid, saturated or unsaturated fatty esters with up to 24 carbons such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tertbutyl or monoglycerin esters of acetic acid, capronic acid, lauric acid, myristinic acid, stearic acid, or palmitic acid, or diesters of saturated or unsaturated dicarboxylic acids with a total of up to 24 carbons such as diisopropyl adipate, diisobutyl adipate, diiso
  • Additional penetration enhancing materials include phosphatidyl derivatives such as lecithin or cephalin, terpenes, amides, ketones, ureas and their derivatives, and ethers such as dimethyl isosorbid and diethyleneglycol monoethyl ether.
  • Suitable penetration enhancing formulations may also include mixtures of one or more materials selected from monohydroxy or polyhydroxy alcohols, saturated or unsaturated C 8 -C 18 fatty alcohols, saturated or unsaturated C 8 -C 18 fatty acids, saturated or unsaturated fatty esters with up to 24 carbons, diesters of saturated or unsaturated discarboxylic acids with a total of up to 24 carbons, phosphatidyl derivatives, terpenes, amides, ketones, ureas and their derivatives, and ethers.
  • Suitable binding materials for transdermal delivery systems include polyacrylates, silicones, polyurethanes, block polymers, styrenebutadiene copolymers, and natural and synthetic rubbers.
  • Cellulose ethers, derivatized polyethylenes, and silicates may also be used as matrix components. Additional additives, such as viscous resins or oils may be added to increase the viscosity of the matrix.
  • compositions of the invention may also be in the form of oil-in-water emulsions.
  • the oil phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example, liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth, naturally-occurring phosphatides, for example, soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • sweetening agents for example glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • the compounds may also be administered in the form of suppositories for rectal or vaginal administration of the drug.
  • compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature or vaginal temperature and will therefore melt in the rectum or vagina to release the drug.
  • suitable non-irritating excipient include cocoa butter and polyethylene glycols.
  • the present pharmaceutical compositions may take any form which is suitable for administration to the penis either via injection into the corpora cavernosa or transurethral administration, or topically applied to the urethral meatus.
  • the pharmaceutical composition is suitably in the form of a saline solution.
  • the pharmaceutical composition is in a form suitable for transurethral administration, and in this case the composition is typically in the form of a solution, an ointment, or a suppository.
  • the pharmaceutical composition is administered 1 to 50 minutes, preferably 10 to 20 minutes, prior to the time of commencing sexual intercourse.
  • the daily oral dosage regimen will preferably be from 0.01 to 200 mg/Kg of total body weight.
  • the daily dosage for administration by injection including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg/Kg of total body weight.
  • the daily vaginal dosage regime will preferably be from 0.01 to 200 mg/Kg of total body weight.
  • the daily topical dosage regimen will preferably be from 0.01 to 200 mg administered between one to four times daily.
  • the transdermal concentration will preferably be that required to maintain a daily dose is of from 0.1 to 200 mg/Kg.
  • the daily inhalation dosage regimen will preferably be from 0.01 to 10 mg/Kg of total body weight.
  • the particular method of administration will depend on a variety of factors, all of which are considered routinely when administering therapeutics. It will also be understood, however, that the specific dose level for any given patient will depend upon a variety of factors, including, the activity of the specific compound employed, the age of the patient, the body weight of the patient, the general health of the patient, the gender of the patient, the diet of the patient, time of administration, route of administration, rate of excretion, drug combinations, and the severity of the condition undergoing therapy.
  • the optimal course of treatment i.e., the mode of treatment and the daily number of doses of a compound of Formula I or a pharmaceutically acceptable salt thereof given for a defined number of days, can be ascertained by those skilled in the art using conventional treatment tests.
  • the present compounds and compositions exhibit Rho-kinase inhibitory activity, and are thus useful to treat the indications listed above, e.g., indications mediated by Rho-kinase.
  • indications mediated by Rho-kinase is meant diseases or conditions whose progression proceeds, at least in part, via the Rho pathway.
  • Rho-kinase inhibitory activity e.g., ROCK-1 inhibition
  • the kinase domain of human ROCK-1, amino acids 27-530, is isolated as a glutathione S-transferase fusion protein from Sf9 insect cells.
  • the protein is partially purified by glutathione Sepharose 4B (Pharmacia Biotech, Piscataway, N.J.) affinity purification.
  • Reactions is carried out in 96-well plates in a total volume of 100 uL containing 50 mM N-[2-Hydroryethyl]piperaxine-N′[2-ethanesulfonic acid] pH 7.5, 5 mM MgCl 2 , 1 mM dithiothreitol, 6 ⁇ M ATP, 0.2 ⁇ Ci [ 33 P]ATP (NEN, Boston, Mass.), 1 ⁇ g myelin basic protein and 0.1 ⁇ g ROCK-1. Test compounds are dissolved in 100% dimethylsulfoxide, diluted to the appropriated concentration and added to the reaction. The final concentration of dimethylsulfoxide did not exceed 0.5%. The reaction is run for one hour at room temperature.
  • the reaction is stopped with the addition of 7 mL of 1 N HCL, transferred to P30 membranes and the amount of [ 33 P]ATP, as counts per minute (c.p.m.) incorporated into the substrate, myelin basic protein, is read in a BetaPlate Reader (Packard. Instrument Co., Meriden, Conn.). (All reagents were purchased from Sigma Chemical Co., St. Louis, Mo. unless stated otherwise.) Percentage inhibition is measured by the amount of incorporation of radioactivity in the presence of the test compound when compared to the amount of incorporation in the absence of the test compound.
  • Inhibitory activity can also be evaluated by measurement of stress fiber formation, performed essentially as described by Ridley, A. J., and A. Hall, Cell 70:389-399 (1992).
  • Human fibrosarcoma HT1080 (CCL-121, American Type Culture Collection, Manassas, Va.) cells are plated on 22 ⁇ 22 mm #1 glass cover slips in six-well tissue culture plates (Costar) at 2.5 ⁇ 10 4 cells/well in Delbeco's modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal calf serum. Cells are maintained in a humidified, 5% CO 2 atmosphere at 37′C.
  • DMEM Delbeco's modified Eagle's Medium
  • test compounds are dissolved in 100% dimethylsulfoxide, diluted to the appropriated concentration and added to the culture medium 60 minutes prior to the induction of stress fiber formation. The final concentration of dimethylsulfoxide did not exceed 0.25%.
  • Stress fiber formation is induced by the addition of lysophosphatidic acid (1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate, Avanti Polar-Lipids, Alabaster, Al) to 10 ⁇ M final concentration in Delbeco's modified Eagle's Medium containing 0.1% fatty acid free bovine serum albumin for 15 minutes at 37° C.
  • Cells are fixed with 4% paraformaldeyhde (Poly Scientific, Bay Shore, N.J.) in phosphate buffered saline (PBS) for 15 minutes. Cells are then washed 3 times in PBS and them permeabilized using a solution containing 40 mM piperazine-N-N′ bis[2-ethanesulfonic acid], 50 mM N-[2-hydroryethyl]piperaxine-N′[2-ethanesulfonic acid], 0.1% Triton X-100, 75 mM NaCl, mM MgCl 2 , 0.5 mM EGTA, pH 7.2 for 2 minutes at room temperature.
  • PBS phosphate buffered saline
  • the cells are washed 3 times for 5 minutes each in PBS and then actin stress fibers are stained using 10 units/mL rhodamine phalloidin (Molecular Probes, Eugene, Oreg.) in PBS for 60 minutes at room temperature.
  • the cells are washed 3 times with PBS and the cover slips mounted on glass microscope slides.
  • the percentage of stress fiber positive cells on each slide was determined visually using a Nikon Labphoto-2 microscope. At least 100 cells were counted per slide and experiments were done in duplicate. Percentage inhibition is measured by counting the number of stress fiber positive cells in the presence of the test compound when compared to the number of stress fiber positive cells in the absence of the test compound.
  • the compounds of the invention can be made according to routine, conventional chemical methods, and/or as disclosed below, from starting materials which are either commercially available or produceable according to routine, conventional chemical methods.
  • General methods for the preparation of the compounds are given below, and the preparation of representative compounds is specifically illustrated in the Examples.
  • a mixture of compound 3 and a phenol, N-substituted amine, or N-substituted aniline (A-X—H. where X is O or NR 8 ) is heated to 140° C. for 2 hours.
  • the mixture is cooled to room temperature and is treated with ether to form precipitate or purified by silica gel column chromatography.
  • the precipitate is filtered, washed with ether several times, and is dried under high vacuum to provide product.
  • a heterocyclic aminoester of Formula 6 is acylated with an aromatic acid chloride or anhydride, in a base such as pyridine.
  • the amide ester product is converted to the amide acid of Formula 7 by hydrolysis, or if R′′ is methyl, by action of boron tribromide.
  • Conversion of the acid to the amide of Formula 8 is accomplished by reaction of 7 with ammonia in the presence of catalysts such as DMAP and EDC. Cyclization to 9 may be carried out by heating the diamide 8 in the presence of a base such as sodium hydroxide.
  • the chloro intermediate of Formula 10 is formed by treatment of 9 with a chlorinating agent such as phosphorous oxychloride.
  • a cyanoheterocyclic amine of Formula 10 may be directly converted to compounds of Formula 4 by heating with a Vilsmeier reagent, prepared in situ by treatment of an aromatic N,N-dimethyl amide with a phosphorous oxychloride or oxalyl chloride and the like.
  • Step 2 Preparation of N-(1H-indazol-5-yl)-N-(5- ⁇ 4-[(2-pyridinylamino)methyl]-phenyl ⁇ -1-benzothien-7-yl)amine
  • Step 1 Preparation of methyl 3-[(2-quinoxalinylcarbonyl)amino]-2-thiophenecarboxylate
  • Example 62 Utilizing the method described above for Example 62 and using the appropriate starting materials, the examples shown below in Table 2 were prepared.
  • the Vilsmeier Reagent was prepared by stirring of 3-methoxy-N,N-dimethylbenzamide (1.17 g, 7.9 mmol) and POCl 3 (3.0 g, 19.7 mmol) at 0° C. for 30 minutes. To this reagent was added 2-amino-4-methyl-3-furonitrile (1.0 g, 6.6 mmol) and dry dichloroethane (5.0 ml). The reaction mixture was heated to 40° C. and stirred at this temperature for 18 h. The mixture was then poured into ice water. After adjusting the pH of the solution to 9 via treatment with NaHCO 3 solution, the solution was extracted with dichloromethane. The organic layer was then dried and concentrated.
  • Step 4 Preparation of 2-(1,1′-biphenyl-3-yl)-N-(3-methyl-1H-indazol-5-yl)thieno[3,2-d]pyrimidin-4-amine
  • step 3 A procedure analogous for that of Example 1, step 3 was followed.
  • a mixture of the step 3 product (0.4 g, 1.3 mmol), 3-phenylphenylboronic acid, (0.3 g, 1.5 mmol) and sodium bicarbonate (0.33 g, 3.9 mmol) in DME/H 2 O (3/1, 56 mL) was flushed with Ar for 1 h, and Pd(dppf)Cl 2 was added.
  • the solution was heated to reflux for 48 h at 100° C.
  • the crude product was purified by silica gel chromatography to afford a white solid (0.35 g, 65%).
  • Rf 0.2 (EtOAc/hexane, 1/1).
  • Step 5 Preparation of N-[2-(1-benzothien-2-yl)pyrido[2,3-d]pyrimidin-4-yl]-N-(1H-indazol-5-yl)amine

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Abstract

Disclosed are 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.

Description

    FIELD OF THE INVENTION
  • 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
  • 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).
  • 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.
  • 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 PIPS-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).
  • 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).
  • 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 ischemia (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 Opthalmol 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).
  • 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
  • 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.
  • 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.
  • The invention provides compounds of formula I
  • Figure US20100216789A1-20100826-C00001
  • wherein X is —(CH2)x—, —O—(CH2)n—, —S—(CH2)n—, —NR7—CO—(CH2)n—, —NR7—SO2—(CH2)n—, —NR7—(CH2)n—, or —(O)C—NR7—,
    each n is an integer which is independently 0, 1, 2 or 3,
    x is 0-3
    p is 0-3
    a and c are each independently —CR5═, —N═, or —NR6—, wherein one of a or c is —NR6—, and b is —CR5═ or —N═;
    A is H, halogen, —CO—OR8, —CO—R8, cyano, —OR8, —NR8R9, —CO—NR8R9, —NR8—CO—R9, —NR8—CO—OR9, —NR8—SO2—R9, —SR8, —SO2—R8, —SO2—NR8R9, NR8—CO—NHR9,
    or
    A is a 3-20 atom, preferably 5-15 atom, cyclic or polycyclic moiety, e.g., containing 1-4 rings, which optionally contain 1-3 N, O or S atoms per ring, and may optionally be aryl or heteroaryl. A may optionally be substituted up to 3 times by (i) C1-C10 alkyl or C2-C10-alkenyl, each optionally substituted with halogen up to perhalo; (ii) C3-C10 cycloalkyl; (iii) optionally substituted aryl; (iv) optionally substituted heteroaryl; (v) halogen; (vi) —CO—OR8; (vii) —CO—R8; (viii) cyano; (ix) —OR8, (x) —NR8R13; (xi) nitro; (xii) —CO—NR8R9; (xiii) —C1-10-alkyl-NR8R9; (xiv) —NR8—CO—R12; (xv) —NR7—CO—OR9; (xvi) —NR7—SO2—R9;
    (xvii) —SR8; —SO2—R8; (xix) —SO2—NR8R9; or (xx) NR8—CO—NHR9;
    Ring B represents a fused 5- or 6-membered heterocyclic ring containing 1-2 O,N, and/or S atoms and 1-5 C atoms.
    R1, and R6-R11 are each independently H and C1-6 alkyl,
    R2-R5 are each independently (i) C1-10 alkyl or C2-10-alkenyl each optionally substituted by amino, N-lower alkylamino, N,N-dilower alkylamino, N-lower alkanoylamino, hydroxy, cyano, —COOR10, —COR14, —OCOR14, —OR10, C5-10-heteroaryl, C5-10-heteroaryloxy, C5-10-heteroaryl-C1-10-alkoxy, or halogen up to perhalo; (ii) C3-C10 cycloalkyl, in which 1-3 carbon atoms are optionally independently replaced by O, N or S; (iii) C3-10-cycloalkenyl; (iv) partially unsaturated C5-10-heterocyclyl; (v) aryl; (vi) heteroaryl; (vii) halogen; (viii) —CO—OR10; (ix) —OCOR10; (x) —OCO2R10; (xi) —CHO; (xii) cyano; (xiii) —OR16;
    (xiv) —NR10R15; (xv) nitro; (xvi) —CO—NR10R11; (xvii) —NR10—CO—R12; (xviii) —NR10—CO—OR11; (xix) —NR10—SO2—R12; (xx) —SR16; (xxi) —SOR16; (xxii) —SO2—R16; (xxiii) —SO2—NR10R11; (xxiv) NR10—CO—NHR11; (xxv) amidino; (xxvi) guanidino; (xxvii) sulfo; (xxviii) —B(OH)2; (xxix) —OCON(R10)2; or (xxx) —NR10CON(R10)2;
    R12 is H, C1-6-alkyl or C5-10-aryl,
    R13 is H, C1-6-alkyl or C1-6-alkoxy,
    R14 is lower alkyl or phenyl;
    R15 is lower alkyl, halogen, amino, N-lower alkyl amino, N,N-dilower alkylamino, N-lower alkanoylamino, OH, CN, COOR10, —COR14 or —OCOR14;
    R16 is hydrogen, C1-6-alkyl optionally substituted by halogen, up to perhalo, or C5-10-heteroaryl; and
    with the provisos that A is not hydrogen when x is 0;
    —X-A is not CH3 when B represents a thieno[3,2b]fused ring, and b and c are —CR5═, and a is NH;
    and A is not phenyl when X is NH, B forms an imidazo fused ring, and -a-b-c- is —CR5═N—NR6— or —NR6═N—CR5—.
    In formula I, suitable aryl or heteroaryl groups, e.g., for A, include, but are not limited to, 5-12 carbon-atom aromatic rings or ring systems containing 1-3 rings, at least one of which is aromatic, in which one or more, e.g., 1-4 carbon atoms in one or more of the rings can be replaced by oxygen, nitrogen or sulfur atoms. Each ring typically has 3-7 atoms. For example, aryl or heteroaryl can be 2- or 3-furyl, 2- or 3-thienyl, 2- or 4-triazinyl, 1-, 2- or 3-pyrrolyl, 1-, 2-, 4- or 5-imidazolyl, 1-, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isoxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 2-, 3- or 4-pyridyl, 2-, 4-, 5- or 6-pyrimidinyl; 1,2,3-triazol-1-, -4- or 5-yl, 1,2,4-triazol-1-, -3- or B5-yl, 1- or 5-tetrazolyl, 1,2,3-oxadiazol-4- or 5-yl, 1,2,4-oxadiazol-3- or 5-yl, 1,3,4-thiadiazol-2- or 5-yl, 1,2,4-oxadiazol-3- or 5-yl, 1,3,4-thiadiazol-2- or 5-yl, 1,3,4-thiadiazol-3- or 5-yl, 1,2,3-thiadiazol-4- or 5-yl, 2-, 3-, 4-, 5- or 6-2H-thiopyranyl, 2-, 3- or 4-4H-thiopyranyl, 3- or 4-pyridazinyl, pyrazinyl, 2-, 3-, 4-, 5-, 6- or 7-benzofuryl, 2-, 3-, 4-, 5-, 6- or 7-benzothienyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-indolyl, 1-, 2-, 4- or 5-benzimidazolyl, 1-, 3-, 4-, 5-, 6- or 7-benzopyrazolyl, 2-, 4-, 5-, 6- or 7-benzoxazolyl, 3-, 4-, 5-, 6- or 7-benzisoxazolyl, 1-, 3-, 4-, 5-, 6- or 7-benzothiazolyl, 2-, 4-, 5-, 6- or 7-benzisothiazolyl, 2-, 4-, 5-, 6- or 7-benz-1,3-oxadiazolyl, 2-, 3-, 4-, 5-, 6-, 7- or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-, 8-isoquinolinyl, 1-, 2-, 3-, 4- or 9-carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-acridinyl, or 2-, 4-, 5-, 6-, 7- or 8-quinazolinyl, or additionally optionally substituted phenyl, 2- or 3-thienyl, 1,3,4-thiadiazolyl, 3-pyrryl, 3-pyrazolyl, 2-thiazolyl or 5-thiazolyl, etc.
    Preferred moieties A include cyclohexyl; or C5-12-aryl or C5-12-heteroaryl each independently optionally substituted up to three times by (i) C1-C10-alkyl or C2-10-alkenyl each optionally substituted with halogen up to perhalo; (ii) C3-C10 cycloalkyl; (iii) C5-12-aryl optionally substituted by 1-3 halogen atoms; (iv) C5-12-heteroaryl; (v) halogen; (vi) —CO—OR8; (vii) —CO—R8; (viii) cyano; (ix) —OR8; (x) —NR8R13; (xi) nitro; (xii) —CO—NR8R9; (xiii) —C1-10-alkyl-NR8R9; (xiv) —NR8—CO—R12; (xv) —NR8—CO—OR9; (xvi) —NR8—SO2—R9; (xvii) —SR8; (xviii) —SO2—R8; (xix) —SO2—NR8R9, or (xx) NR8—CO—NHR9.
    Further preferred moieties A include phenyl, pyridyl, pyrimidinyl, oxazolyl, furyl, thienyl, pyrrolyl, imidazolyl, isoxazolyl and pyrazinyl, each independently substituted up to three times by halogen, C1-10-alkyl, C1-10-alkoxyphenyl, naphthyl, —OR10,
  • Figure US20100216789A1-20100826-C00002
  • as well as
  • Figure US20100216789A1-20100826-C00003
  • wherein each Z independently is halogen, hydroxy, hydroxy-C1-10-alkyl, —CN, —NO2, C1-10-alkoxycarboxyl, —NR10—CO—R11, or —NR10—CO—OR11, as well as OR10,
    y is 0-3, more preferably 1-3,
    and R4 is as described above.
    Preferred moieties A additionally include
  • Figure US20100216789A1-20100826-C00004
  • as well as
  • Figure US20100216789A1-20100826-C00005
  • wherein R15 is H; phenyl optionally substituted by C1-10-alkyl, C1-10-alkoxy, C1-10-alkylcarboxyl, or halogen; benzyl; pyrimidyl or pyridyl; and R16 is H, phenyl, —COOR10,
  • Figure US20100216789A1-20100826-C00006
  • The present invention is also directed to pharmaceutically acceptable salts of formula I. Suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, sulphonic acid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicyclic acid, phenylacetic acid, and mandelic acid. In addition, pharmaceutically acceptable salts include acid salts of inorganic bases, such as salts containing alkaline cations (e.g., Li+, Na+ or K+), alkaline earth cations (e.g., Mg+, Ca+ or Ba+), the ammonium cation, as well as acid salts of organic bases, including aliphatic and aromatic substituted ammonium, and quaternary ammonium cations, such as those arising from protonation or peralkylation of triethylamine, N,N-diethylamine, N,N-dicyclohexylamine, pyridine, N,N-dimethylaminopyridine (DMAP), 1,4-diazabiclo[2.2.2]octane (DABCO), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
  • A number of the compounds of Formula I possess asymmetric carbons and can therefore exist in racemic and optically active forms. Methods of separation of enantiomeric and diastereomeric mixtures are well known to one skilled in the art. The present invention encompasses any isolated racemic or optically active form of compounds described in Formula I which possess Rho-kinase inhibitory activity.
  • The invention also includes pharmaceutical compositions including a compound of Formula I, and a physiologically acceptable carrier.
  • The invention moreover encompasses treating indications mediated by Rho-kinase, by administering a compound of Formula I, or a pharmaceutical composition containing a compound of Formula I. Thus, the invention encompasses treating cardiovascular diseases such as hypertension, artherosclerosis, restenosis and cerebral ischemia, or vasospasm central nervous system disorders such as neuronal degeneration and spinal cord injury, erectile dysfunction, e.g., in patients who do not have satisfactory response to PDE-5 inhibitors, and cancer (e.g., tumor growth) mediated by Rho-kinase, by administering, e.g., to a host in need thereof, of an effective amount of a compound of Formula I. Cancers and tumors mediated by Rho-kinase include cancers of the breast, colon, prostate, ovaries, brain and lung and their metastases.
  • A number of the compounds of Formula I possess asymmetric carbons and can therefore exist in racemic and optically active forms. Methods of separation of enantiomeric and diastereomeric mixtures are well known to one skilled in the art. The present invention encompasses any isolated racemic or optically active form of compounds described in Formula I which possess Rho-kinase inhibitory activity.
  • The invention also includes pharmaceutical compositions including a compound of Formula I, and a physiologically acceptable carrier.
  • The invention moreover encompasses treating indications mediated by Rho-kinase, by administering a compound of Formula I, or a pharmaceutical composition containing a compound of Formula I. Thus, the invention encompasses treating cardiovascular diseases such as hypertension, artherosclerosis, restenosis and cerebral ischemia, or vasospasm central nervous system disorders such as neuronal degeneration and spinal cord injury, erectile dysfunction, e.g., in patients who do not have satisfactory response to PDE-5 inhibitors, and cancer (e.g., tumor growth) mediated by Rho-kinase, by administering, e.g., to a host in need thereof, of an effective amount of a compound of Formula I. Cancers and tumors mediated by Rho-kinase include cancers of the breast, colon, prostate, ovaries, brain and lung and their metastases.
  • The compounds may be administered orally, topically, parenterally, by inhalation or spray, vaginally, rectally or sublingually in dosage unit formulations. The term ‘administration by injection’ includes intravenous, intraarticular, intramuscular, subcutaneous and parenteral injections, as well as use of infusion techniques. Dermal administration may include topical application or transdermal administration. One or more compounds may be present in association with one or more non-toxic pharmaceutically acceptable carriers and if desired other active ingredients.
  • Compositions intended for oral use may be prepared according to any suitable method known to the art for the manufacture of pharmaceutical compositions. Such compositions may contain one or more agents selected from the group consisting of diluents, sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; and binding agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. These compounds may also be prepared in solid, rapidly released form.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions containing the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions may also be used. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropyl-methyl cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring and coloring agents, may also be present.
  • The compounds may also be in the form of non-aqueous liquid formulations, e.g., oily suspensions which may be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or peanut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Compounds of the invention may also be administrated transdermally using methods known to those skilled in the art (see, for example: Chien; “Transdermal Controlled Systemic Medications”; Marcel Dekker, Inc.; 1987. Lipp et al. WO94/04157 3 Mar., 1994). For example, a solution or suspension of a compound of Formula I in a suitable volatile solvent optionally containing penetration enhancing agents can be combined with additional additives known to those skilled in the art, such as matrix materials and bacteriocides. After sterilization, the resulting mixture can be formulated following known procedures into dosage forms. In addition, on treatment with emulsifying agents and water, a solution or suspension of a compound of Formula I may be formulated into a lotion or salve.
  • Suitable solvents for processing transdermal delivery systems are known to those skilled in the art, and include lower alcohols such as ethanol or isopropyl alcohol, lower ketones such as acetone, lower carboxylic acid esters such as ethyl acetate, polar ethers such as tetrahydrofuran, lower hydrocarbons such as hexane, cyclohexane or benzene, or halogenated hydrocarbons such as dichloromethane, chloroform, trichlorotrifluoroethane, or trichlorofluoroethane. Suitable solvents may also include mixtures of one or more materials selected from lower alcohols, lower ketones, lower carboxylic acid esters, polar ethers, lower hydrocarbons, halogenated hydrocarbons.
  • Suitable penetration enhancing materials for transdermal delivery system are known to those skilled in the art, and include, for example, monohydroxy or polyhydroxy alcohols such as ethanol, propylene glycol or benzyl alcohol, saturated or unsaturated C8-C18 fatty alcohols such as lauryl alcohol or cetyl alcohol, saturated or unsaturated C8-C18 fatty acids such as stearic acid, saturated or unsaturated fatty esters with up to 24 carbons such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tertbutyl or monoglycerin esters of acetic acid, capronic acid, lauric acid, myristinic acid, stearic acid, or palmitic acid, or diesters of saturated or unsaturated dicarboxylic acids with a total of up to 24 carbons such as diisopropyl adipate, diisobutyl adipate, diisopropyl sebacate, diisopropyl maleate, or diisopropyl fumarate. Additional penetration enhancing materials include phosphatidyl derivatives such as lecithin or cephalin, terpenes, amides, ketones, ureas and their derivatives, and ethers such as dimethyl isosorbid and diethyleneglycol monoethyl ether. Suitable penetration enhancing formulations may also include mixtures of one or more materials selected from monohydroxy or polyhydroxy alcohols, saturated or unsaturated C8-C18 fatty alcohols, saturated or unsaturated C8-C18 fatty acids, saturated or unsaturated fatty esters with up to 24 carbons, diesters of saturated or unsaturated discarboxylic acids with a total of up to 24 carbons, phosphatidyl derivatives, terpenes, amides, ketones, ureas and their derivatives, and ethers.
  • Suitable binding materials for transdermal delivery systems are known to those skilled in the art and include polyacrylates, silicones, polyurethanes, block polymers, styrenebutadiene copolymers, and natural and synthetic rubbers. Cellulose ethers, derivatized polyethylenes, and silicates may also be used as matrix components. Additional additives, such as viscous resins or oils may be added to increase the viscosity of the matrix.
  • Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example, liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth, naturally-occurring phosphatides, for example, soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • The compounds may also be administered in the form of suppositories for rectal or vaginal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature or vaginal temperature and will therefore melt in the rectum or vagina to release the drug. Such materials include cocoa butter and polyethylene glycols.
  • Moreover, for treatment of erectile dysfunction, the present pharmaceutical compositions may take any form which is suitable for administration to the penis either via injection into the corpora cavernosa or transurethral administration, or topically applied to the urethral meatus. In the case of injection into the corpora cavernosa, the pharmaceutical composition is suitably in the form of a saline solution. Preferably, the pharmaceutical composition is in a form suitable for transurethral administration, and in this case the composition is typically in the form of a solution, an ointment, or a suppository. Typically, the pharmaceutical composition is administered 1 to 50 minutes, preferably 10 to 20 minutes, prior to the time of commencing sexual intercourse.
  • For all regimens of use disclosed herein for compounds of Formula I, the daily oral dosage regimen will preferably be from 0.01 to 200 mg/Kg of total body weight. The daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg/Kg of total body weight. The daily vaginal dosage regime will preferably be from 0.01 to 200 mg/Kg of total body weight. The daily topical dosage regimen will preferably be from 0.01 to 200 mg administered between one to four times daily. The transdermal concentration will preferably be that required to maintain a daily dose is of from 0.1 to 200 mg/Kg. The daily inhalation dosage regimen will preferably be from 0.01 to 10 mg/Kg of total body weight.
  • It will be appreciated by those skilled in the art that the particular method of administration will depend on a variety of factors, all of which are considered routinely when administering therapeutics. It will also be understood, however, that the specific dose level for any given patient will depend upon a variety of factors, including, the activity of the specific compound employed, the age of the patient, the body weight of the patient, the general health of the patient, the gender of the patient, the diet of the patient, time of administration, route of administration, rate of excretion, drug combinations, and the severity of the condition undergoing therapy. It will be further appreciated by one skilled in the art that the optimal course of treatment, i.e., the mode of treatment and the daily number of doses of a compound of Formula I or a pharmaceutically acceptable salt thereof given for a defined number of days, can be ascertained by those skilled in the art using conventional treatment tests.
  • The present compounds and compositions exhibit Rho-kinase inhibitory activity, and are thus useful to treat the indications listed above, e.g., indications mediated by Rho-kinase. By indications mediated by Rho-kinase is meant diseases or conditions whose progression proceeds, at least in part, via the Rho pathway.
  • Rho-kinase inhibitory activity, e.g., ROCK-1 inhibition, can be evaluated as follows:
  • The kinase domain of human ROCK-1, amino acids 27-530, is isolated as a glutathione S-transferase fusion protein from Sf9 insect cells. The protein is partially purified by glutathione Sepharose 4B (Pharmacia Biotech, Piscataway, N.J.) affinity purification. Reactions is carried out in 96-well plates in a total volume of 100 uL containing 50 mM N-[2-Hydroryethyl]piperaxine-N′[2-ethanesulfonic acid] pH 7.5, 5 mM MgCl2, 1 mM dithiothreitol, 6 μM ATP, 0.2 μCi [33P]ATP (NEN, Boston, Mass.), 1 μg myelin basic protein and 0.1 μg ROCK-1. Test compounds are dissolved in 100% dimethylsulfoxide, diluted to the appropriated concentration and added to the reaction. The final concentration of dimethylsulfoxide did not exceed 0.5%. The reaction is run for one hour at room temperature. The reaction is stopped with the addition of 7 mL of 1 N HCL, transferred to P30 membranes and the amount of [33P]ATP, as counts per minute (c.p.m.) incorporated into the substrate, myelin basic protein, is read in a BetaPlate Reader (Packard. Instrument Co., Meriden, Conn.). (All reagents were purchased from Sigma Chemical Co., St. Louis, Mo. unless stated otherwise.) Percentage inhibition is measured by the amount of incorporation of radioactivity in the presence of the test compound when compared to the amount of incorporation in the absence of the test compound.
  • Inhibitory activity can also be evaluated by measurement of stress fiber formation, performed essentially as described by Ridley, A. J., and A. Hall, Cell 70:389-399 (1992). Human fibrosarcoma HT1080 (CCL-121, American Type Culture Collection, Manassas, Va.) cells are plated on 22×22 mm #1 glass cover slips in six-well tissue culture plates (Costar) at 2.5×104 cells/well in Delbeco's modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal calf serum. Cells are maintained in a humidified, 5% CO2 atmosphere at 37′C. After 24 hours the culture medium is removed and replaced with medium without 10% fetal calf serum and the cells cultured for an additional 48 hours. Test compounds are dissolved in 100% dimethylsulfoxide, diluted to the appropriated concentration and added to the culture medium 60 minutes prior to the induction of stress fiber formation. The final concentration of dimethylsulfoxide did not exceed 0.25%. Stress fiber formation is induced by the addition of lysophosphatidic acid (1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate, Avanti Polar-Lipids, Alabaster, Al) to 10 μM final concentration in Delbeco's modified Eagle's Medium containing 0.1% fatty acid free bovine serum albumin for 15 minutes at 37° C. Cells are fixed with 4% paraformaldeyhde (Poly Scientific, Bay Shore, N.J.) in phosphate buffered saline (PBS) for 15 minutes. Cells are then washed 3 times in PBS and them permeabilized using a solution containing 40 mM piperazine-N-N′ bis[2-ethanesulfonic acid], 50 mM N-[2-hydroryethyl]piperaxine-N′[2-ethanesulfonic acid], 0.1% Triton X-100, 75 mM NaCl, mM MgCl2, 0.5 mM EGTA, pH 7.2 for 2 minutes at room temperature. The cells are washed 3 times for 5 minutes each in PBS and then actin stress fibers are stained using 10 units/mL rhodamine phalloidin (Molecular Probes, Eugene, Oreg.) in PBS for 60 minutes at room temperature. The cells are washed 3 times with PBS and the cover slips mounted on glass microscope slides. The percentage of stress fiber positive cells on each slide was determined visually using a Nikon Labphoto-2 microscope. At least 100 cells were counted per slide and experiments were done in duplicate. Percentage inhibition is measured by counting the number of stress fiber positive cells in the presence of the test compound when compared to the number of stress fiber positive cells in the absence of the test compound.
  • Using the above protocols, all of the compounds as disclosed herein are determined to have Rho-kinase inhibitory activity.
  • The compounds of the invention can be made according to routine, conventional chemical methods, and/or as disclosed below, from starting materials which are either commercially available or produceable according to routine, conventional chemical methods. General methods for the preparation of the compounds are given below, and the preparation of representative compounds is specifically illustrated in the Examples.
    • ABBREVIATIONS AND ACRONYMS
  • When the following abbreviations are used herein, they have the following meaning:
    • Ac2O acetic anhydride
    • anhy anhydrous
    • n-BuOH n-butanol
    • t-BuOH t-butanol
    • CD3OD methanol-d4
    • Celite® diatomaceous earth filter agent, ®Celite Corp.
    • CH2Cl2 methylene chloride
    • CI-MS chemical ionization mass spectroscopy
    • conc concentrated
    • dec decomposition
    • DME dimethoxyethane
    • DMF N,N-dimethylformamide
    • DMSO dimethylsulfoxide
    • ELSD evaporative light scattering detector
    • EtOAc ethyl acetate
    • EtOH ethanol (100%)
    • Et2O diethyl ether
    • Et3N triethylamine
    • HPLC ES-MS high performance liquid chromatography-electrospray mass spectroscopy
    • NMM 4-methylmorpholine
    • Ph3P triphenylphosphine
    • Pd(dppf)Cl2 [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)
    • Pd(PPh3)4 tetrakis(triphenylphosphine)palladium(0)
    • Pd(OAc)2 palladium acetate
    • P(O)Cl3 phosphorous oxychloride
    • RT retention time (HPLC0
    • rt room temperature
    • THF tetrahydrofuran
    • TFA trifluoroacetic acid
    • TLC thin layer chromatography
  • General Methods of Preparation
  • Figure US20100216789A1-20100826-C00007
  • A mixture of compounds 1 and 2, and potassium acetate in THF/water is stirred at room temperature overnight. Water is added to the mixture resulting in the formation of a precipitate. The precipitate is washed with water, filtered, and dried under high vacuum to afford 3.
  • Figure US20100216789A1-20100826-C00008
  • A mixture of compound 3, ethylene glycol dimethyl ether/water, aryl boronic acid and sodium bicarbonate is degassed with argon for 15 minutes and Pd(dppf)2Cl2 is added. The mixture is heated to reflux overnight. After cooling to room temperature, CH2Cl2 and H2O are added to the mixture. The organic and aqueous layers are separated and the aqueous layer is extracted with CH2Cl2, and the combined organic layers are dried over anhydrous sodium sulfate. The organic solvent is removed under reduced pressure and the crude product is purified by silica gel chromatography of HPLC to afford compound 4.
  • Figure US20100216789A1-20100826-C00009
  • A mixture of compound 3 and a phenol, N-substituted amine, or N-substituted aniline (A-X—H. where X is O or NR8) is heated to 140° C. for 2 hours. The mixture is cooled to room temperature and is treated with ether to form precipitate or purified by silica gel column chromatography. The precipitate is filtered, washed with ether several times, and is dried under high vacuum to provide product.
  • Figure US20100216789A1-20100826-C00010
  • A heterocyclic aminoester of Formula 6 is acylated with an aromatic acid chloride or anhydride, in a base such as pyridine. The amide ester product is converted to the amide acid of Formula 7 by hydrolysis, or if R″ is methyl, by action of boron tribromide. Conversion of the acid to the amide of Formula 8 is accomplished by reaction of 7 with ammonia in the presence of catalysts such as DMAP and EDC. Cyclization to 9 may be carried out by heating the diamide 8 in the presence of a base such as sodium hydroxide. The chloro intermediate of Formula 10 is formed by treatment of 9 with a chlorinating agent such as phosphorous oxychloride.
  • Figure US20100216789A1-20100826-C00011
  • A cyanoheterocyclic amine of Formula 10 may be directly converted to compounds of Formula 4 by heating with a Vilsmeier reagent, prepared in situ by treatment of an aromatic N,N-dimethyl amide with a phosphorous oxychloride or oxalyl chloride and the like.
  • Without further-elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
  • In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.
  • The entire disclosure of all applications, patents and publications, cited above or below, and of corresponding U.S. Provisional Application Ser. No. 60/277,974, filed Mar. 23, 2001, U.S. Provisional Application 60/315,341 filed Aug. 29, 2001, U.S. Provisional Application Ser. No. 60/315,338 filed Aug. 29, 2001, and U.S. Provisional Application No. 60/346,628, filed Jan. 10, 2002 are hereby incorporated by reference.
    • EXAMPLES Preparation of 2-(5-chloro-2-thienyl)-N-(1H-indazol-5-yl)thieno[3,2-d]pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00012
    • Step 1: Preparation of 2,3-dichlorothieno[3,2-d]pyrimidine
  • Figure US20100216789A1-20100826-C00013
  • A solution of thienoyleneurea (6.0 g) and N,N-dimethylaniline (2.9 mL) in phosphorus oxychloride (35 mL) was heated at 125° C. (oil bath) for 22 h under argon. The solution cooled to 50° C. and was poured into cold water (0° C., 8.0 mL) while vigorously stirring. The precipitate was filtered, washed with water, and dissolved in EtOAc. The organic solution was filtered, washed with water, and the organic phase was dried over MgSO4, filtered and concentrated to afford a yellow precipitate (4.5 g, 61% yield).
    • Step 2: Preparation of 4-(N-5-aminoindazole)-2-chloro-thieno[3,2-d]pyrimidine
  • Figure US20100216789A1-20100826-C00014
  • A mixture of the compound for step 1 (4.0 g, 19.5 mmol), 5-aminoindazole (3.2 g, 23.4 mmol), and potassium acetate (2.5 g, 25.4 mmol) in THF/water (100 mL/50 mL) was stirred rt overnight. The THF was removed under reduced pressure and the residue was partitioned between EtOAc and water. The organic phase was washed with aqueous NH4Cl, dried over MgSO4, filtered. The filtrate was poured onto a silica gel column and eluted with EtOAc. The solvent was concentrated by rotary evaporation and a gray precipitate was obtained (3.9 g, 65% yield). Rf=0.28 (EtOAc/hexanes, 1/1).
  • Step 3: Preparation of 2-(5-chloro-2-thienyl)-N-(1H-indazol-5-yl)thieno[3,2-d]-pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00015
  • A mixture of the compound from step 2 (6.9 g, 23 mmol), 5-chlorothienyl boronie acid (3.7 g, 23 mmol) and sodium bicarbonate (5.8 g, 69 mmol) in DME/H2O (3/1, 300 mL) was flushed with Ar for 1 h. Pd(dppf)Cl2 (1.8 g, 2.3 mmol) was added and the mixture was heated to reflux for 48 h. The solvent was removed by rotary evaporation and the crude product was purified by silica gel chromatography to afford of yellow solid (3.5 g, 40% yield). Rf=0.20 (EtOAc/hexane, 1/1). 1H NMR (methanol-d4) δ 8.05 (s, 1H), 8.00 (s, 1H), 7.89 (d, 1H, J=3 Hz), 7.68-7.59 (m, 2H), 7.50 (d, 1H, J=3 Hz), 7.29 (1H, d, J=3 Hz), 6.95 (d, 1H, J=3 Hz).
    • Examples 2-55
  • Using an procedure analogous to that described for example 1 and reacting 4-(N-5-aminoindazole)-2-chlorothieno[3,2-d]pyrimidine and the appropriate boronic acid or ester, the compounds described in Table 1 below were prepared.
  • TABLE 1
    Figure US20100216789A1-20100826-C00016
    Ex. No. Ar1 Note
    2
    Figure US20100216789A1-20100826-C00017
         		1
    3
    Figure US20100216789A1-20100826-C00018
         		2
    4
    Figure US20100216789A1-20100826-C00019
         		3
    5
    Figure US20100216789A1-20100826-C00020
         		4
    6
    Figure US20100216789A1-20100826-C00021
         		5
    7
    Figure US20100216789A1-20100826-C00022
         		6
    8
    Figure US20100216789A1-20100826-C00023
         		7
    9
    Figure US20100216789A1-20100826-C00024
         		8
    10
    Figure US20100216789A1-20100826-C00025
         		9
    11
    Figure US20100216789A1-20100826-C00026
         		10
    12
    Figure US20100216789A1-20100826-C00027
         		11
    13
    Figure US20100216789A1-20100826-C00028
         		12
    14
    Figure US20100216789A1-20100826-C00029
         		13
    15
    Figure US20100216789A1-20100826-C00030
         		14
    16
    Figure US20100216789A1-20100826-C00031
         		15
    17
    Figure US20100216789A1-20100826-C00032
         		16
    18
    Figure US20100216789A1-20100826-C00033
         		17
    19
    Figure US20100216789A1-20100826-C00034
         		18
    20
    Figure US20100216789A1-20100826-C00035
         		19
    21
    Figure US20100216789A1-20100826-C00036
         		20
    22
    Figure US20100216789A1-20100826-C00037
         		21
    23
    Figure US20100216789A1-20100826-C00038
         		22
    24
    Figure US20100216789A1-20100826-C00039
         		23
    25
    Figure US20100216789A1-20100826-C00040
         		24
    26
    Figure US20100216789A1-20100826-C00041
         		25
    27
    Figure US20100216789A1-20100826-C00042
         		26
    28
    Figure US20100216789A1-20100826-C00043
         		27
    29
    Figure US20100216789A1-20100826-C00044
         		28
    30
    Figure US20100216789A1-20100826-C00045
         		29
    31
    Figure US20100216789A1-20100826-C00046
         		30
    32
    Figure US20100216789A1-20100826-C00047
         		31
    33
    Figure US20100216789A1-20100826-C00048
         		32
    34
    Figure US20100216789A1-20100826-C00049
         		33
    35
    Figure US20100216789A1-20100826-C00050
         		34
    36
    Figure US20100216789A1-20100826-C00051
         		35
    37
    Figure US20100216789A1-20100826-C00052
         		36
    38
    Figure US20100216789A1-20100826-C00053
         		37
    39
    Figure US20100216789A1-20100826-C00054
         		38
    40
    Figure US20100216789A1-20100826-C00055
         		39
    41
    Figure US20100216789A1-20100826-C00056
         		40
    42
    Figure US20100216789A1-20100826-C00057
         		41
    43
    Figure US20100216789A1-20100826-C00058
         		42
    44
    Figure US20100216789A1-20100826-C00059
         		43
    45
    Figure US20100216789A1-20100826-C00060
         		44
    46
    Figure US20100216789A1-20100826-C00061
         		45
    47
    Figure US20100216789A1-20100826-C00062
         		46
    48
    Figure US20100216789A1-20100826-C00063
         		47
    49
    Figure US20100216789A1-20100826-C00064
         		48
    50
    Figure US20100216789A1-20100826-C00065
         		49
    51
    Figure US20100216789A1-20100826-C00066
         		50
    52
    Figure US20100216789A1-20100826-C00067
         		51
    53
    Figure US20100216789A1-20100826-C00068
         		52
    54
    Figure US20100216789A1-20100826-C00069
         		53
    55
    Figure US20100216789A1-20100826-C00070
         		54
    56
    Figure US20100216789A1-20100826-C00071
         		55
    57
    Figure US20100216789A1-20100826-C00072
         		56
    58
    Figure US20100216789A1-20100826-C00073
         		57
    1 HPLC/MS: (M + H)+ m/z 378.4. Rf = 0.39 (50% EtOAc/Hex). 1H NMR (DMSO): δ 13.1 (s, 1H), 9.8 (s, 1H), 8.3-8.4 (m, 2H), 8.2 (d, J = 5.7 Hz, 1H), 8.1 (s, 2H), 7.5-7.7 (m, 5H)
    2 HPLC/MS: (M + H)+ m/z 421.4. Rf = 0.25 (50% EtOAc/Hex). 1H NMR (CD3OD): δ 8.7 (m, 1H), 8.4 (d, J = 8.4 Hz, 2H), 8.2-8.3 (m, 7H), 7,6-7.7 (m, 4H)
    3 HPLC/MS: (M + H)+ m/z 450.4. Rf = 0.50 (1/1, EtOAc/Hex).
    4 HPLC/MS: (M + H)+ m/z 438.4. Rf = 0.40 (1/1, EtOAc/Hex).
    5 HPLC/MS; (M + H)+ 420.4 m/z. Rf = 0.33 (1/1, EtOAc/Hex). 1H NMR (DMSO): δ 13.0 (s, 1H), 9.6 (s, 1H), 8.7 (s, 1H), 8.4 (d, J = 8.7, 1H), 8.3 (s, 1H), 8.2 (d, J = 5.7, 1H), 8.0 (s, 1H), 7.8-7.4 (m, 10H)
    6 HPLC/MS: (M + H)+ m/z 378.4. Rf = 0.62 (1/1, EtOAc/Hex). 1NNMR (DMSO and CH2Cl2): δ 12.9 (s, 1H), 9.7 (s, 1H), 8.3-8.4 (m, 3H), 8.0-8.1 (m, 4H), 7.3-7.7 (m, 3H)
    7 HPLC/MS: (M + H)+ m/z 421.4. Rf = 0.24 (1/1, EtOAc/Hex). 1H NMR (CD3OD): δ 9.1 (s, 1H), 8.7 (m, 1H), 8.6 (dd, 1H), 8.4-8.5 (d, J = 8.1 Hz, 2H), 8.3 (d, J = 6.0 Hz, 1H), 8.2 (s, 1H), 8.1 (m, 1H), 8.0 (m, 2H), 7.9 (m, 1H), 7.6-7.7 (m, 2H), 7.6 (d, J = 5.1, 1H)
    8 HPLC/MS: (M + H)+ m/z 438.4. Rf = 0.41 (1/1, EtOAc/Hex). 1H NMR (CD3OD): δ 8.3-8.4 ( . . . J = 8.4 Hz, 2H), 8.3 (d, J = 5.4 Hz, 1H), 8.2 (s, 1H), 8.1 (s, 1H), 7.8-7.9 (d, J = 8.4 Hz, 2H), 7.6-7.8 (m, 4H), 7.5-7.6 (d, J = 5.4, 1H), 7.2-7.3 (m, 2H)
    9 HPLC/MS: (M + H)+ m/z 454.4. Rf = 0.33 (1/1, EtOAc/Hex).
    10 HPLC/MS: (M + H)+ m/z 454.5. Rf = 0.40 (1/1, EtOAc/Hex).
    11 HPLC/MS: (M + H)+ m/z 454.4. Rf = 0.42 (1/1, EtOAc/Hex).
    12 HPLC/MS: (M + H)+ m/z 488.4. Rf = 0.43 (1/1, EtOAc/Hex).
    13 HPLC/MS: (M + H)+ m/z 465.4. Rf = 0.36 (1/1, EtOAc/Hex). 1H NMR (CD3OD): δ 8.6 (t, J = 1.8 Hz, 1H), 8.4 (d, J = 8.7, 2H), 8.3 (m, 2H), 8.1-8.2 (m, 3H), 8.0 (d, J = 10.5, 2H), 7.7-7.8 (m, 3H), 7.5-7.6 (d, J = 5.4 Hz, 1H)
    14 HPLC/MS: (M + H)+ m/z 468.4. Rf = 0.38 (1/1, EtOAc/Hex).
    15 HPLC/MS: (M + H)+ m/z 480.4. Rf = 0.37 (1/1, EtOAc/Hex).
    16 HPLC/MS: (M + H)+ m/z 480.4. Rf = 0.30 (1/1, EtOAc/Hex).
    17 HPLC/MS: (M + H)+ m/z 470.5. Rf = 0.36 (1/1, EtOAc/Hex). 1H NMR (CD3OD): δ 8.3-8.4 (m, 2H, 1H), 8.2 (s, 1H), 8.1 (s, 1H), 7.9-8.0 (m, 2H), 7.8-7.9 (m, 1H), 7.7-7.8 (m, 4H), 7.4-7.6 (m, 6H)
    18 HPLC/MS: (M + H)+ m/z 477.5. Rf = 0.13 (1/1, EtOAc/Hex).
    19 HPLC/MS: (M + H)+ m/z 462.5. Rf = 0.33 (1/1, EtOAc/Hex).
    20 HPLC/MS: (M + H)+ m/z 479.3. Rf = 0.39 (1/1, EtOAc/Hex).
    21 HPLC/MS: (M + H)+ m/z 434.4. Retention time (HPLC): Rt = 4.73. 1H NMR (CD3OD): δ 8.1-8.4 (m, 6H), 7.4-7.7 (m, 7H)
    22 HPLC/MS: (M + H)+ m/z 465.3. Retention time (HPLC, CH3CN/H2O/0.1% TFA): Rt = 2.79
    23 HPLC/MS: (M + H)+ m/z 426.4. Retention time (HPLC): Rt = 2.55
    24 HPLC/MS: (M + H)+ m/z 480.4. Retention time (HPLC): Rt = 2.31
    25 HPLC/MS: (M + H)+ m/z 421.4. Rf = 0.13 (1/1, EtOAc/Hex). 1H NMR (CD3OD): δ 9.0 (s, 1H), 8.8 (s, 1H), 8.7 (t, J = 1.8 Hz, 1H), 8.5-8.6 (m, 1H), 8.4 (m, 1H), 8.3 (d, J = 6.0 Hz, 1H), 8.1 (m, 1H), 8.0 (m, 1H), 7.6-7.9 (m, 4H), 7.5-7.6 (d, J = 5.7 Hz, 2H)
    26 Rf = 0.33 (CH2Cl2/MeOH, 95/5). 1H NMR (CD3OD) δ 8.2 (1H, s), 8.2 (1H, s), 8.1 (1H, s), 8.0 (1H, dd, J = 1.8, 6.0 Hz), 7.9-7.8 (1H, m), 7.8-7.7 (1H, d, J = 9.3 Hz), 7.7-7.6 (2H, m), 7.6-7.5 (1H, m), 7.4 (1H, dd, J = 5,4, 2.1 Hz), 7.4-7.3 (1H, m).
    27 Rf = 0.33 (CH2Cl2/MeOH, 95/5). 1H NMR (CD3OD) δ 9.1 (1H, d, J = 2.4 Hz), 8.6 (1H, dd, J = 2.4, 8.7 Hz), 8.1-8.0 (2H, m), 8.0-7.9 (1H, d, J = 5.4 Hz), 7.7 (1H, dd, J = 2.2, 8.7 Hz), 7.6 (1H, d, J = 8.7 Hz), 7.42 (1H, d, J = 5.4 Hz), 6.8 (1H, d, J = 9.3 Hz).
    28 Rf = 0.47 (Hexane/EtOAc = 1/1). 1H NMR (CD3OD) δ 9.7 (1H, d, j = 1.4 Hz), 9.2 (1H, d, J = 8.7 Hz), 8.24-8.20 (2H, m), 8.1 (1H, d, J = 2.2 Hz), 7.8 (1H, d, J = 2.8 Hz), 7.71 (1Hf, d, J = 1.4 Hz), 7.66-7.56 (4H, m), 7.21-7.19 (2H, m), 709-7.07 (1H, m).
    29 Rf = 0.28 (Hexane/EtOAc, 1/2). 1H NMR (CD3OD) δ 8.60 (2H, d, J = 5.7 Hz), 8.53 (2H, dd, J = 1.5, 6.9 Hz), 8.15 (1H, d, J = 1.0 Hz), 8.10 (1H, d, J = 2.2 Hz), 8.01 (1H, d, J = 5.4 Hz), 7.88 (2H, dd, J = 1.8, 6.6 Hz), 7.79 (2H, dd, J = 1.8, 4.5 Hz), 7.74 (1H, dd, J = 2.1, 9.0 Hz), 7.76 (1H, d, J = 8.7 Hz), 7.48 (1H, d, J = 6.5 Hz).
    30 Rf = 0.38 (Hexane/EtOAc, 1/1). 1H NMR (DMSO-d6) δ 13.08 (1H, s), 9.02 (1H, s), 8.40 (2H, dd, J = 2.4, 5.7 Hz), 8.15 (1H, d, J = 6.0 Hz), 8.18.-8.09 (2H, m), 7.68 (1H, dd, J = 2.1, 9.3 Hz), 7.60 (1H, d, J = 9,0 Hz), 7.46 (1H, d, J = 5.7 Hz), 7.29 (2H, t, J = 7.2 Hz)
    31 1H NMR (DMSO-d6) δ 11.01 (1H, s), 9.34 (1H, s), 8.72 (1H, d. J = 5.7 Hz), 8.18 (1H, s), 8.12 (1H, J = 6.6 Hz), 7.97-7.90 (4H, m), 7.87 (1H, s), 7.56 (1H, d, J = 3.0 Hz), 7.47 (1H, d, J = 6.0 Hz), 7.36-7.34 (1H, m)
    32 Rf = 0.42 (Hexane/EtOAc, 1/1). (CD3OD) δ 8.91 (1H, d, J = 6.3 Hz), 8.37 (1H d, J = 6.3 Hz), 8.30 (1H, dd, J = 1.2, 9.0 Hz), 8.25 (1H, dd, J = 1.5, 6.3 Hz), 8.20 (1H, d, J = 0.60 Hz), 8.12 (1H, d, J =1.0 Hz), 7.86 (1H, t, J = 6.2 Hz), 7.80 (1H, s), 7.80-7.78 (1H, m), 7.76 (1H, d, J = 1.8 Hz), 7.71 (1H, d, J = 6.0 Hz), 7.65-7.62 (1H, m), 7.54-7.46 (2H, m).
    33 HPLC/MS: (M + H) m/z 462, Rf = 0.28 (EtOAc/Hexanes 10:90).
    34 Purified by silica gel column chromatography (gradient, EtOAc in hexanes from 20% to 70%); Rf = 0.22 (EtOAc/hexanes, 1/1); (3.18 g. 68% yield); LCMS m/z 374 (M + H)+; 1H NMR (DMSO-d6) δ 13.0 (s, 1H), 9.67 (s, 1H), 8.3 (d, 2H), 8.1 (m, 3H), 7.62 (d, 1H), 7.55 (d, 1H), 7.4 (d, 1H), 6.94 (d, 2H), 3.78 (s, 3H).
    35 Purified by silica gel column chromatography (gradient, EtOAc in hexanes from 20% to 70%) (2.8 g, 70 % yield); LCMS m/z 344 (M + H)+; Rf = 0.24 (EtOAc/hexanes, 1/1); 1H NMR (300 MHz, CD3OD) δ 8.35 (d, 2H), 8.15 (s, 1H), 8.05 (s, 1H), 7.95 (d, 1H), 7.7 (d, 1H), 7.55 (d, 1H), 7.46 (s, 4H).
    36 Purified by silica gel column chromatography (gradient, EtOAc in hexanes from 10% to 70%); Rf = 0.19 (EtOAc/hexanes, 3/2); (55 mg, 15% yield); LCMS m/z 429 (M+ H)+; 1H NMR (DMSO-d6) δ 13.07 (s, 1H), 9.65 (s, 1H), 8.24 (d, 2H), 8.1 (m, 2H), 7.66 (d, 1H), 7.56 (d, 1H), 7.4 (d, 1H), 3.73 (s, 4H), 3.21 (4H).
    37 Purified by silica gel column chromatography (gradient, EtOAc in hexanes from 10% to 50%); Rf = 0.3 (EtOAc/hexanes, 1/1); (194 mg, 70% yield); LCMS m/z 420 (M + H)+ ; 1H NMR (DMSO-d6) δ 13.1 (s, 1H), 9.8 (s, 1H), 8.44 (d, 2H), 8.15 (m, 3H), 7.8 (m, 5H), 7.6 (d, 1H), 7.47 (m, 3H), 7.36 (m, 1H).
    38 Crude 7e was purified by filtration and washed with methanol; Rf = 0.16 (MeOH/CH2Cl2, 5/95); (0.36 g, 61% yield); LCMS m/z 401 (M + H)+; 1H NMR (DMSO-d6) δ 13.09 (s, 1H), 9,97 (s, 1H), 9.17 (s, 1H), 8.42 (d, 1H), 8.3 (s, 1H), 8.18 (m, 2H), 8.14 (s, 1H), 8.06 (d, 1H), 7.73 (d, 1H), 7.62 (d, 1H), 7.49 (d, 1H).
    39 N-BOC protected boronic acid used in step 3; BOC group was removed by TFA in CH2CL2 at rt. Purified by silica gel column chromatography (gradient, EtOAc in hexanes from 10% to 75%); (45 mg, 19% yield); LCMS m/z 528 (M + M)+).
    40 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 428.2. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 3.27.
    41 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 380.1. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 3.26
    42 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 388.2. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.52.
    43 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 362.1. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.69.
    44 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 374.1. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.43.
    45 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 358.2. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.46.
    46 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 358.2. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.50.
    47 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 372.2. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.61.
    48 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 370.2. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.67.
    49 The residue was purified by colunm chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 400.2. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.81.
    50 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 386.2. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.73.
    51 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 386.2. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.79.
    52 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 374.2. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.06.
    53 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 374.3. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 2.17.
    54 The residue was purified by column chromatography (dry mount, gradient from 20% to 75% EtOAc/Hex) to afford product. HPLC/MS: (M + H)+ m/z 412.1. Retention time (HPLC/MS, CH3CN/H2O/0.1% TFA): Rt = 3.28. HPLC/MS: (M + H)+ 412.1 m/z. 1H NMR (DMSO-d6): 13.12 (s, 1H, NH); 9.95 (s, 1H, NH); 8.71 (s, 1H); 8.66 (d, 1H); 8.21 (d, 1H); 8.16 (s, 1H); 8.07 (s, 1H); 7.85 (s, 1H); 7.74 (t, 1H); 7.65 (d, 1H); 7.60 (s, 1H); 7.55 (d, 1H).
    55 The residue was purified by column chromatography (gradient from 35-50% EtoAc/Hexane) to afford the pure product. HPLC/MS: (M + H)+ 427. Rf = 0.64 (EtOAc/Hexanes, 80/20). 1H-NMR (300 MHz, CD3OD) δ = 8.23 (d, 2H), 8.16-8.14 (m, 1H), 8.08 (s, 1H), 7.95 (d, 1H), 7.73 (dd, 1H), 7.61 (d, 1H), 7.40 (d, 1H), 7.01 (d, 2H), 3.32-3.23 (m, 4H), 1.72-1.60 (m, 6H).
    56 HPLC/MS: (M + H)+ m/z 426.4, Retention time (HPLC): Rt = 2.55, Rf = 0.68 in (EtOAc/Hex, 80/20). 1H-NMR (300 MHz, DMSO d6) δ =8.17-7.43 (m, 13H).
    57 HPLC/MS: (M + H)+ m/z 421.4, Rf = 0.46 in (MeOH/EtOAc, 07/93). 1H-NMR (300 MHz, CD3OD) δ = 7.5-6.25 (m, 14H).
    • Example 59 Preparation of 4-(N-5-aminoindazole)-2-(4-phenol)-thieno[3,2-d]pyrimidine
  • Figure US20100216789A1-20100826-C00074
  • To a slurry of (N-5-aminoindazole)-2-(4-methoxyphenyl)thieno[3,2-d]-pyrimidine (Example 35, 0.86 g, 2.3 mmol) in anhyd CH2Cl2 was added BBr3 at −78° C. The mixture was stirred overnight and warmed to room temperature. The reaction was cooled to 78° C. and treated with aqueous NH4Cl slowly to form precipitate. The solvent was concentrated by rotary evaporation and co-evaporated with toluene. The residue was treated with mixed solvent of MeOH and CH2Cl2, and then filtered. The filtrate was added to 6 g of silica gel and the solvent was removed by rotary evaporation. The residue was poured onto a silica gel column and eluted with a gradient mobile phase containing EtOAc and hexane to afford the product (0.42 g, 1.17 mmol, 51% yield). LC/MS m/z 360 (M+H)+, 1H NMR (DMSO-d6): δ 13.15 (s, 1H), 9.8 (s, 1H), 9.7 (s, 1H), 8.2 (d, 2H), 8.1 (d, 3H), 7.7 (dd, 1H), 7.6 (dd, 1H), 7.4 (d, 1H), 6.8 (d, 2H); mp 297-299° C.
    • Example 60 Preparation of N-(1H-indazol-5-yl)-2-{4-[2-(4-morpholinyl)ethoxy]-phenyl}thieno[3,2-d]pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00075
  • To a mixture of (Example 56, 62 mg, 0.17 g), CsCO3 (100 mg, 0.3 mmol) in acetone (3 mL) was added N-(2-bromoethylene)-morpholine (34 mg in 1 mL of acetone). The solution was stirred at room temperature for 4 h and was filtered. The filtrate was added to 0.4 g of silica gel and the solvent was removed by rotary evaporation. The residue was poured onto a silica gel column and eluted with a gradient of EtOAc in hexane (from 50% to 100%) to afford a white precipitate (30 mg, 37% yield). Rf=0.18 (MeOH/CH2Cl2, 5/95). HPLC/MS: m/z 473 (M+H)+; 1H NMR (DMSO-d6): δ13.07 (s, 1H), 9.71 (s, 1H), 8.30 (d, 2H), 8.09 (m, 3H), 7.70 (d, 1H), 7.57 (d, 1H), 7.43 (d, 1H), 7.02 (d, 2H), 4.14 (t, 2H), 3.57 (t, 4H), 2.68 (t, 2H), 2.46 (t, 4H).
    • Example 61 Preparation of N-(1H-indazol-5-yl)-N-(5-{4-[(2-pyridinylamino)methyl]phenyl}-1-benzothien-7-yl)amine
  • Figure US20100216789A1-20100826-C00076
    • Step 1: Preparation of 4-[(2-pyridinylamino)methyl]phenylboronic acid
  • Figure US20100216789A1-20100826-C00077
  • A mixture of 4-formylphenylboronic acid (1 g, 6.67 mmol) and 2-pyridinylamine (2.51 g, 26.7 mmol) in dichloroethane (25 mL) was stirred under argon at room temperature. To this mixture was added NaB(OAc)3 (1.84 g, 8.67 mmol). The reaction mixture was quenched with H2O after 3 h. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc and washed with water. The organic layer was then dried and concentrated in vacuo. The crude product was then purified by column chromatography (gradient from 20% to 80% EtOAc/hexane) to give product as colorless liquid (0.39 g, 30%). HPLC/MS: (M+H)+ 229.1 m/z. Retention time (LC-MS)=4.54 min. 1H NMR (CD3OD): 7.90 (1H, d); 7.59 (2H, s); 7.43 (1H, m); 7.30 (2H, d); 6.55 (2H, m); 4.47 (2H
    • Step 2: Preparation of N-(1H-indazol-5-yl)-N-(5-{4-[(2-pyridinylamino)methyl]-phenyl}-1-benzothien-7-yl)amine
  • Figure US20100216789A1-20100826-C00078
  • To a mixture of N-(2-chlorothieno[3,2-d]pyrimidin-4-yl)-N-(1H-indazol-5-yl)amine (50 mg, 0.16 mmol), Na2CO3(aq) (2 M, 1.0 mL) in butanol/toluene (1:1, 4 mL) a stream of argon was bubbled through for 15 min. To the mixture was added 4-[(2-pyridinylamino)methyl]phenylboronic acid (0.15 g, 0.66 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.06 g, 0.08 mmol) in a single portion. The resulting reaction mixture was heated to reflux for 24 h. On cooling, the solution was concentrated, taken up in EtOAc and washed with water. The organic layers were dried over anhyd sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel column chromatography (gradient from 20% to 80% EtOAc/hexane) to give the product (25 mg). HPLC/MS: (M+H)+ 450.1 m/z. Retention time (LC-MS)=min. 1H NMR (CD3OD): 8.31 (2H, d); 8.11 (1H, s); 8.05 (1H, s); 7.96 (1H, d); 7.91 (1H, d); 7.71 (1H, d); 7.58 (1H, d); 7.47 (4H, m); 6.61 (2H, m); 4.55 (2H, s).
    • Example 62
  • Figure US20100216789A1-20100826-C00079
    • Step 1: Preparation of methyl 3-[(2-quinoxalinylcarbonyl)amino]-2-thiophenecarboxylate
  • Figure US20100216789A1-20100826-C00080
  • To a solution of methyl 3-amino-2-thiophenecarboxylate (0.82 g, 5.2 mmol) and 2-quinoxalinecarbonyl chloride at 0° C. was added pyridine (1 mL) dropwise. The resulting solution was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc, washed with saturated NH4Cl, and NaHCO3. The organic layer was the dried and concentrated in vacuo. The crude product was used directly in the next step without further purification.
    • Step 2: Preparation of 3-[(2-quinoxalinylcarbonyl)amino]-2-thiophenecarboxylic acid
  • Figure US20100216789A1-20100826-C00081
  • Methyl 3-[(2-quinoxalinylcarbonyl)amino]-2-thiophenecarboxylate (0.5 g, 1.6 mmol) was dissolved in CH2Cl2 (100 mL) at 0° C. and BBr3 (4.8 mL, 1M solution in CH2Cl2) was added dropwise. The solution was stirred at room temperature overnight. The reaction was quenched with NH4Cl at 0° C. The organic layer was washed with NaHCO3 and separated. The resulting organic layer was treated slowly with dil HCl until the solution became acidic. The mixture was concentrated and redissolved in EtOAc. This solution was washed with water, The solvent was removed under reduced pressure and the crude product was re-crystallized from methanol to give the desired product (0.2 g, 42%). HPLC/MS: (M+H)+ 300.1 m/z. Retention time (LC-MS)=2.65 min. 1H NMR (DMSO-D6): 13.75 (s, 1H, broad); 12.43 (s, 1H); 9.61 (s, 1H); 8.26 (m, 2H); 8.17 (m, 1H); 8.07 (m, 2H); 7.99 (d, 1H).
    • Step 3: Preparation of N-[2-(aminocarbonyl)-3-thienyl]-2-quinoxalinecarboxamide
  • Figure US20100216789A1-20100826-C00082
  • A mixture of 3-[(2-quinoxalinylcarbonyl)amino]-2-thiophenecarboxylic acid (0.70 g, 2.3 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC, 2.24 g, 11.5 mmol), and 4-(dimethylamino)pyridine (DMAP, 1.43 g, 11.5 mmol) in CH2Cl2 (100 mL) was stirred at room temperature for 2 h. To the reaction mixture was added aqueous ammonia (40%, 5 mL), and the stirring continued for another 12 h. The solvent was evaporated to dryness and the resulting solid was washed with satd NH4Cl solution (3×), NaHCO3 solution and H2O. The organic phase was evaporated to dryness resulting in a white powder (0.32 g, 46%).
  • HPLC/MS: (M+H)+ 299.4 m/z
  • Retention time (LC-MS)=2.74 min.
    • Step 4: Preparation of 2-(2-quinoxalinyl)thieno[3,2-d]pyrimidin-4-ol
  • Figure US20100216789A1-20100826-C00083
  • To a solution of N-[2-(aminocarbonyl)-3-thienyl]-2-quinoxalinecarboxamide (0.24 g, 0.8 mmol) in ethanol (15 mL) was added aqueous NaOH (1.0 M, 2.4 mL). The resulting mixture was stirred at reflux temperature overnight. The mixture cooled to rt and the solvent was removed under reduced pressure. The residue was dissolved in water and acidified with HCl. The white precipitate was filtered and washed thoroughly with water to give product as yellow powder (0.17 g, 77%). HPLC/MS: (M+H)+ 281.1 m/z
  • Retention time (LC-MS)=2.78 min. 1H NMR (DMSO-D6): 12.65 (s, 1H, broad); 9.77 (s, 1H); 8.32 (d, 1H); 8.28 (m, 1H); 8.23 (m, 1H); 8.01 (m, 2H); 7.63 (d, 1H).
    • Step 5: Preparation of 2 (4-chlorothieno[3,2-d]pyrimidin-2-yl)quinoxaline
  • Figure US20100216789A1-20100826-C00084
  • A solution of 2-(2-quinoxalinyl)thieno[3,2-c]pyrimidin-4-ol (150 mg, 0.54 mmol) in POCl3 (5 mL) was heated to reflux and maintained at reflux overnight. After cooling to rt, excess POCK was removed under reduced pressure to give the crude product which was used in the next step without further purification.
    • Step 6: Preparation of N-(1H-indazol-5-yl)-2-(2-quinoxalinyl)thieno[3,2-d]-pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00085
  • Bg9 procedure similar to Example 1, step 2,2-(4-chlorothieno[3,2-d]pyrimidin-2-yl)quinoxaline and 5-aminoindoazole were allowed to react in THF. After the removal of the solvent, the crude product was purified through column (gradient from 20% to 80% EtOAc/hexane) to afford the product. HPLC/MS: (M+H)+ 396.1 m/z. Retention time (LC-MS)=2.42 min.
  • Utilizing the method described above for Example 62 and using the appropriate starting materials, the examples shown below in Table 2 were prepared.
  • TABLE 2
    MS
    Ex. RT (min) [M +
    No. Structure (LC-MS) H]+
    63
    Figure US20100216789A1-20100826-C00086
         		2.76 395
    64
    Figure US20100216789A1-20100826-C00087
         		3.42 350
    65
    Figure US20100216789A1-20100826-C00088
         		3.42 344
    • Example 66 Preparation of N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-5-methylfuro[2,3-d]-pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00089
    • Step 1: Preparation of 2-amino-4-methyl-3-furonitrile
  • Figure US20100216789A1-20100826-C00090
  • To a mixture of malononitrile (0.96 g, 14.6 mmol), acetol (1.08 g, 14.6 mmol) in methanol (10 mL) at 0° C. was added, dropwise, triethylamine (2.0 mL). The reaction mixture was stirred at room temperature overnight. After removal of solvent under reduced pressure the crude solid was washed with cold isopropanol to give product as white powder (0.54 g, 30%). HPLC/MS: (M+H)+ 123.3 m/z. Retention time (LC-MS)=1.81 min. 1H NMR (CD3OD): 6.58 (s, 1H); 1.95 (s, 3H).
    • Step 2: Preparation of 4-chloro-2-(3-methoxyphenyl)-5-methylfuro[2,3-d]-pyrimidine
  • Figure US20100216789A1-20100826-C00091
  • The Vilsmeier Reagent was prepared by stirring of 3-methoxy-N,N-dimethylbenzamide (1.17 g, 7.9 mmol) and POCl3 (3.0 g, 19.7 mmol) at 0° C. for 30 minutes. To this reagent was added 2-amino-4-methyl-3-furonitrile (1.0 g, 6.6 mmol) and dry dichloroethane (5.0 ml). The reaction mixture was heated to 40° C. and stirred at this temperature for 18 h. The mixture was then poured into ice water. After adjusting the pH of the solution to 9 via treatment with NaHCO3 solution, the solution was extracted with dichloromethane. The organic layer was then dried and concentrated. The crude product was purified by silica gel column chromatography (10/90, ethyl acetate/hexane). HPLC/MS: (M+H)+ 275.1 m/z. Retention time (LC-MS)=3.99 min. 1H NMR (DMSO-D6): 8.10 (s, 1H); 7.98 (d, 1H); 7.86 (m, 1H); 7.49 (t, 1H); 7.15 (m, 1H); 3.85 (s, 3H).
    • Step 3: Preparation of N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-5-methylfuro[2,3-d]pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00092
  • A mixture of 4-chloro-2-(3-methoxyphenyl)-5-methylfuro[2,3-d]pyrimidine and 5-Aminoindazole in butanol (2.0 ml) was heated to 100° C. overnight. After the removal of the solvent, the crude product was purified through column (gradient from 20% to 80% ethyl acetate/hexane) to give product Bay 59-8843 (15.2 mg). HPLC/MS: (M+H)+ 372.4 m/z. Retention time (LC-MS)=2.53 min. 1H NMR (DMSO-D6): 13.10 (s, 1H, NH); 8.70 (s, 1H, NH); 8.11 (s, 2H); 7.90 (m, 2H); 7.75 (m, 2H); 7.63 (d, 1H); 7.41 (t, 1H); 7.05 (m, 1H); 3.80 (s, 3H).
  • Utilizing a similar procedure to that described above and substituting the appropriate 4-chloro-2-substituted phenyl 5-methylfluro[2,3 mL]pyrimidine, the following compounds were prepared.
  • TABLE 3
    Figure US20100216789A1-20100826-C00093
    RT (min)
    Example (from LC- Mass Spec
    No R1 R2 MS) [electrospray]
    67 5-Me H 3.31 MH+ 342.1
    68 5-Me 4-OMe 3.42 MH+ 372.2
    69 5.6-Di- H 3.65 MH+ 356.3
    Me
    70 5.6-Di- 3-OCH3 3.49 MH+ 386.1
    Me
    LC-MS system: Acetonitrile/Water/0.1% TFA
    LC-MS Detector: UV and ELSD
    • Example 71 Preparation of 3-[4-(1H-indazol-5-ylamino)-5-methylfuro[2,3-d]pyrimidin-2-yl]phenol
  • Figure US20100216789A1-20100826-C00094
  • To a solution of N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-5-methylfuro[2,3-d]pyrimidin-4-amine (50.0 mg, 0.13 mmol) in CH2Cl2 (20 mL) cooled to −78° C. was added BBr3 (0.7 mL, 2.83 mmol, 1M solution in CH2Cl2) drop-wise. The mixture warmed to it and was stirred overnight. The reaction was quenched with water and the organic and aqueous phases were separated. The organic layer was dried and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient from 20% to 80% EtOAc/hexane) to afford are product (8.4 mg, 17.5%). HPLC/MS: (M+H)+ 358.1 m/z. Retention time (LC-MS) 3.01 min. 1H NMR (DMSO-D6): 8.54 (s, NH); 8.11 (d, 1H); 8.07 (s, 1H); 7.75 (m, 3H); 7.60 (d, 1H); 7.47 (d, 1H); 7.20 (t, 1H); 6.83 (m, 1H).
    • Example 72 Preparation of N-{3-[4-(1H-indazol-5-ylamino)thieno[3,2-d]pyrimidin-2-yl}isonicotinamide
  • Figure US20100216789A1-20100826-C00095
    • Step 1: Preparation of 2-(3-aminophenyl)-N-(1H-indazol-5-yl)thieno[3,2-d]-pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00096
  • 2-Chloro-N-(1H-indazol-5-yl)thieno[3,2-d]pyrimidin-4-amine (10 mmol, 1 equivalent) was suspended in dimethyl ethylene glycol (60 mL) and Na2CO3 solution (2M, 10 mL) and flushed with argon for 20 min. To this suspension 3-aminophenyl boronic acid (25 mmol, 2.5 eq) and Pd(PPh3)4 (2.5 mmol, 0.25 eq) were added. The reaction mixture was refluxed under AR at 100° C. for 48 h. The solvent was evaporated off in vacuo. The residue was taken into THF (100 mL) plus EtOAc/water. (100 mL, 1:1). The organic layer was evaporated to dryness. The residue was separated by silica gel column chromatography (0-5% MeOH in CH2Cl2). The product was obtained as yellow powder (2.04 g, 57%). (M+H)+=359, RT (LC-MS)=1.93.
    • Step 2: Preparation of 2-(3-aminophenyl)-N-(1H-indazol-5-yl)thieno[3,2-d]-pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00097
  • To a cold solution of the product of step 1 (3.35 mmol, 1 eq) in dry pyridine (50 mL) was added isonicotonic anhydride (6.7 mmol, 2 eq) in two portions. A precipitate formed shortly after the addition. The reaction mixture was stirred at room temperature for 4 h and poured into ice and was stirred continued. The solid was collected by filtration and washed with water. This crude product was further purified by silica gel column chromatography (0-4% MeOH/CH2Cl2) to afford a pale yellow solid (0.8 g, 50%). (M+H)+=464, RT (LC-MS)=2.12.
    • Example 73
  • Figure US20100216789A1-20100826-C00098
    • Preparation of N-(1H-indazol-5-yl)-N-[2-(2-naphthylamino)thieno[3,2-d]pyrimidin-4-yl]amine
  • To a solution of 2-chloro-N-(1-H-indazol-5-yl)thieno[3,2-d]pyrimidin-4-amine (0.166 mmol) in n-BuOH (1.5 mL) was added 2-aminonaphthylene (0.5 mmol, 3 equivalent) and was stirred at 100° C. for two days. The resulted solid was collected on funnel, washed with isopropanol and ether to give a pale gray crystalline product (74%). (M+H)+=409, RT (LC-MS)=2.56.
  • Utilizing this method and substituting the appropriate starting materials, the compounds shown in Table 5 were also prepared.
  • TABLE 5
    Figure US20100216789A1-20100826-C00099
    Mass
    LC-MS Spec
    Ex. No Ar RT (min) (source)
    73 3-aminophenyl 1.93 359
    74 3- 2.12 464
    isonicotinamido-
    phenyl
    75 5-(1H- 2.34 398
    indolyl)amino
    76 4- 2.78 451
    phenoxyanilino
    • Example 77 Preparation of 2-(1,1′-biphenyl-3-yl)-N-(3-methyl-1H-indazol-5-yl)thieno[3,2-d]-pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00100
  • Step 1: Preparation of 3-methyl-5-nitro-1H-indazole
  • Figure US20100216789A1-20100826-C00101
  • A solution of 2-fluoro-5-nitroacetophenone (1.57 g, 8.6 mmol) in ethylene glycol (50 mL) was added hydrazine (029 g, 9.0 mmol) was stirred for 2 h at room temperature and then heated at 165° C. for 24 h. The reaction mixture was cooled to room temperature, poured over EtOAc (100 mL), and extracted with H2O (2×100 mL). The organic layers was dried over Na2SO4 and the solvent was removed in vacuo. The crude product was purified by silica gel chromatography to afford a light yellow solid (0.8 g, 53%) Rf=0.2 (EtOAc/hexane, 1/3). 1H NMR CDCl3 δ 8.63 (s, 1H), 8.23 (d, 2H, J=3 Hz), 7.46 (d, 1H, J=3 Hz), 2.60 (s, 3H).
    • Step 2: Preparation of 3-methyl-1H-indazol-5-amine
  • Figure US20100216789A1-20100826-C00102
  • To a stirred solution of the compound prepared in step 1 (0.8 g, 4 mmol) in methanol was added Pd/C catalyst (0.1 g) The resulting reaction mixture was stirred under a hydrogen atmosphere (1 atm) at room temperature overnight. The mixture was filtered and the filtrate was concentrated in vacuo to afford crude product (0.7 g) as light yellow solid that was not further purified. Rf=0.50 (EtOAc/hexane, 1/1). 1H NMR CDCl3 δ 7.20 (s, 1H), 6.90-6.80 (m, 2H), 2.41 (s, 3H).
    • Step 3: Preparation of 2-chloro-N-(3-methyl-1H-indazol-5-yl)thieno[3,2-d]-pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00103
  • A mixture of the compound in step 2 (0.7 g, 4.8 mmol), Example 1, step 1, (0.98 g, 4.8 mmol) and potassium acetate (0.66 g, 6.7 mmol) in THF/H2O (75 mL, 2/1) was stirred at room temperature for 16 h. The mixture was extracted with EtOAc, washed with brine, and dried over NaSO4. The solvent was filtered and evaporated to dryness. The residue was purified by silica gel column chromatography to afford a yellow solid (1.2 g, 80% yield). Rf=0.3 (EtOAc/hexane, 1/1). 1H NMR (Methanol-d4) δ 7.95-7.80 (m, 2H), 7.63-7.50 (m, 2H), 7.19 (d, 1H, J=3 Hz), 2.40 (s, 3H).
    • Step 4: Preparation of 2-(1,1′-biphenyl-3-yl)-N-(3-methyl-1H-indazol-5-yl)thieno[3,2-d]pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00104
  • A procedure analogous for that of Example 1, step 3 was followed. A mixture of the step 3 product (0.4 g, 1.3 mmol), 3-phenylphenylboronic acid, (0.3 g, 1.5 mmol) and sodium bicarbonate (0.33 g, 3.9 mmol) in DME/H2O (3/1, 56 mL) was flushed with Ar for 1 h, and Pd(dppf)Cl2 was added. The solution was heated to reflux for 48 h at 100° C. After removal of solvent in vacuo, the crude product was purified by silica gel chromatography to afford a white solid (0.35 g, 65%). Rf=0.2 (EtOAc/hexane, 1/1). 1H NMR (Methanol-d4) δ 8.69 (s, 1H), 8.35 (d, 1H, J=3 Hz), 8.25 (s, 1H), 8.00 (d, 1H, J=3 Hz), 7.70-7.60 (m, 4H), 7.51-7.31 (m, 6H), 2.42 (s, 3H).
    • Example 78 Preparation of 2-(1-benzothien-2-yl)-N-(1H-indazol-5-yl)period[2,3-d]pyrimidin-4-amine
  • Figure US20100216789A1-20100826-C00105
    • Step 1: Preparation of 2-[(1-benzothien-2-ylcarbonyl)amino]nicotinic acid
  • Figure US20100216789A1-20100826-C00106
  • To a solution of 2-aminonicotinic acid (91.1 g, 8.0 mmol), triethylamine (2.7 mL, 19.1 mmol) and acetone/H2O (3:1, 100 mL) was added 1-benzothiophene-2-carbonyl chloride (2.04 g, 10.4 mmol) at 0° C. The resulting solution was allowed to warm to rt and stir overnight. The volatile solvent was removed under reduced pressure and the aqueous solution was acidified with HCl (conc.). The resulting precipitate was filtered and then washed with EtOAc and dried under reduced pressure to afford the product. (1.2 g, 52%). NMR (DMSO): 11.7 (1H, broad), 8.60 (1H, m); 8.36 (1H, s); 8.25 (1H, d); 8.07 (1H, d); 8.02 (1H, d); 7.51 (2H, m); 7.36 (1H, m).
    • Step 2: Preparation of 2-[(1-benzothien-2-ylcarbonyl)amino]nicotinamide
  • Figure US20100216789A1-20100826-C00107
  • A mixture of 2-[(1-benzothien-2-ylcarbonyl)amino]nicotinic acid (1.2 g, 4.0 mmol), EDC (2.3 g, 12.0 mmol) and DMAP (1.46 g, 12.0 mmol) in anhyd dichloromethane (50 mL) was stirred at room temperature for 2 h. To this mixture was added 40% aqueous ammonia (4.0 mL), and the resulting solution stirred at room temperature overnight. The solvent was removed in vacuo and the resulting solid was washed with said NH4Cl solution, NaHCO3 solution, and water. The solid was dried to give product as white powder (0.92 g, 77%). HPLC/MS: (M+H)+ 298.15 m/z Retention time (LC-MS)=2.44 min. 1H NMR (DMSO-D6): 12.25 (1H, s); 8.54 (1H, m); 8.21 (1H, s); 8.18 (1H, d); 8.07 (2H, m); 7.52 (2H, m); 7.32 (1H, m).
    • Step 3: Preparation of 2-(1-benzothien-2-yl)pyrido[2,3-d]pyrimidin-4-ol
  • Figure US20100216789A1-20100826-C00108
  • To a solution of 2-[(1-benzothien-2-ylcarbonyl)amino]nicotinamide (0.3 g, 1.0 mmol) in ethanol (20 mL) was added NaOH (10 N, 0.3 mL, 3.0 mmol). The resulting solution was heated to reflux overnight. On cooling to rt the solvent was removed under reduced pressure. The residue was dissolved in excess of water and acidified with HCl. The resulting precipitate was filtered and washed with water to afford the product as yellow powder (0.16 g, 57%). HPLC/MS: (M+H)+ 280.0 m/z; Retention time (LC-MS)=2.66 min; 1H NMR (DMSO-D6): 13.17 (1H, s); 8.94 (1H, m); 8.61 (1H, s); 8.50 (1H, d); 8.08 (1H, d); 7.97 (1H, d); 7.52 (3H, m).
    • Step 4: Preparation of 2-(1-benzothien-2-yl)-4-chloropyrido[2,3-d]pyrimidine
  • Figure US20100216789A1-20100826-C00109
  • To a suspension of 2-(1-benzothien-2-yl)pyrido[2,3-d]pyrimidin-4-ol (150 mg, 0.54 mmol) in chloroform (10 mL) was added thionyl chloride (0.47 mL, 6.4 mmol) and a catalytic amount of DMF. The reaction was heated to reflux for 4 h and allowed to cool to rt. The solvent was removed under reduced pressure and the resultant product was (18.2 mg, 12%) used directly in the next step without further purification. HPLC/MS: (M+H)+ 298.2 m/z; Retention time (LC-MS)=3.76 min; 1H NMR (DMSO-D6): 9.24 (1H, m); 8.73 (1H, d); 8.55 (1H, s); 8.10 (2H, t); 7.86 (1H, m); 7.50 (2H, m).
    • Step 5: Preparation of N-[2-(1-benzothien-2-yl)pyrido[2,3-d]pyrimidin-4-yl]-N-(1H-indazol-5-yl)amine
  • Figure US20100216789A1-20100826-C00110
  • A mixture of 2-(1-benzothien-2-yl)-4-chloropyrido[2,3-d]pyrimidine (18.2 mg, 0.06 mmol), 5-aminoindazole (40 mg, 0.3 mmol) and potassium carbonate (83 mg, 0.6 mmol) in dioxane (20 mL) was heated to 100° C. and continued for 24 h. After removal of the solvent, the crude product was purified by silica gel column chromatography (gradient from 20% to 80% EtOAc/hexane) to give the product (1.0 mg, 4.0%); HPLC/MS: (M+H)+ 395.2 m/z; Retention time (LC-MS)=2.75 min.
  • The following compounds are prepared analogously:
  • MS
    Example (Exact MW
    No. Structure Mass) (calcd)
    79
    Figure US20100216789A1-20100826-C00111
         		372.12 372.45
    80
    Figure US20100216789A1-20100826-C00112
         		406.08 406.90
    81
    Figure US20100216789A1-20100826-C00113
         		386.13 386.48
    82
    Figure US20100216789A1-20100826-C00114
         		386.13 386.48
    83
    Figure US20100216789A1-20100826-C00115
         		418.14 416.51
    84
    Figure US20100216789A1-20100826-C00116
         		386.13 386.48
    85
    Figure US20100216789A1-20100826-C00117
         		420.09 420.93
    86
    Figure US20100216789A1-20100826-C00118
         		390.11 390.44
    87
    Figure US20100216789A1-20100826-C00119
         		390.11 390.44
    88
    Figure US20100216789A1-20100826-C00120
         		390.11 390.44
    89
    Figure US20100216789A1-20100826-C00121
         		408.10 408.43
    90
    Figure US20100216789A1-20100826-C00122
         		404.12 404.47
    91
    Figure US20100216789A1-20100826-C00123
         		440.10 440.45
    92
    Figure US20100216789A1-20100826-C00124
         		386.33 386.48
    93
    Figure US20100216789A1-20100826-C00125
         		400.15 400.51
    94
    Figure US20100216789A1-20100826-C00126
         		428.18 428.56
    95
    Figure US20100216789A1-20100826-C00127
         		432.14 432.51
    96
    Figure US20100216789A1-20100826-C00128
         		390.11 390.44
    97
    Figure US20100216789A1-20100826-C00129
         		465.14 465.54
    98
    Figure US20100216789A1-20100826-C00130
         		452.12 452.50
    99
    Figure US20100216789A1-20100826-C00131
         		467.10 467.58
    100
    Figure US20100216789A1-20100826-C00132
         		467.10 467.58
    101
    Figure US20100216789A1-20100826-C00133
         		435.13 435.51
    102
    Figure US20100216789A1-20100826-C00134
         		465.14 465.54
    103
    Figure US20100216789A1-20100826-C00135
         		467.10 467.58
    104
    Figure US20100216789A1-20100826-C00136
         		412.12 412.48
    105
    Figure US20100216789A1-20100826-C00137
         		429.08 429.53
    106
    Figure US20100216789A1-20100826-C00138
         		433.05 433.88
    107
    Figure US20100216789A1-20100826-C00139
         		438.14 438.52
    108
    Figure US20100216789A1-20100826-C00140
         		442.08 442.53
    109
    Figure US20100216789A1-20100826-C00141
         		470.14 470.53
    110
    Figure US20100216789A1-20100826-C00142
         		492.98 494.40
    111
    Figure US20100216789A1-20100826-C00143
         		409.11 409.47
    112
    Figure US20100216789A1-20100826-C00144
         		409.11 409.47
    113
    Figure US20100216789A1-20100826-C00145
         		418.10 418.44
    114
    Figure US20100216789A1-20100826-C00146
         		425.11 425.47
    115
    Figure US20100216789A1-20100826-C00147
         		457.17 457.56
    116
    Figure US20100216789A1-20100826-C00148
         		441.17 441.56
    117
    Figure US20100216789A1-20100826-C00149
         		463.14 463.59
    118
    Figure US20100216789A1-20100826-C00150
         		471.18 471.59
    119
    Figure US20100216789A1-20100826-C00151
         		483.11 483.48
    120
    Figure US20100216789A1-20100826-C00152
         		492.11 492.49
    121
    Figure US20100216789A1-20100826-C00153
         		443.19 443.58
    122
    Figure US20100216789A1-20100826-C00154
         		463.13 463.99
    123
    Figure US20100216789A1-20100826-C00155
         		443.15 443.53
    124
    Figure US20100216789A1-20100826-C00156
         		373.11 373.44
    125
    Figure US20100216789A1-20100826-C00157
         		373.11 373.44
    126
    Figure US20100216789A1-20100826-C00158
         		373.11 373.44
    127
    Figure US20100216789A1-20100826-C00159
         		387.13 387.47
    128
    Figure US20100216789A1-20100826-C00160
         		402.13 402.48
    129
    Figure US20100216789A1-20100826-C00161
         		392.06 392.87
    130
    Figure US20100216789A1-20100826-C00162
         		417.10 417.45
    131
    Figure US20100216789A1-20100826-C00163
         		417.10 417.45
    132
    Figure US20100216789A1-20100826-C00164
         		440.10 440.45
    133
    Figure US20100216789A1-20100826-C00165
         		456.10 456.45
    134
    Figure US20100216789A1-20100826-C00166
         		408.10 408.43
    135
    Figure US20100216789A1-20100826-C00167
         		408.10 408.43
    136
    Figure US20100216789A1-20100826-C00168
         		458.09 458.44
    137
    Figure US20100216789A1-20100826-C00169
         		425.12 425.48
    138
    Figure US20100216789A1-20100826-C00170
         		398.11 398.45
    139
    Figure US20100216789A1-20100826-C00171
         		362.09 362.41
    140
    Figure US20100216789A1-20100826-C00172
         		395.15 395.49
    141
    Figure US20100216789A1-20100826-C00173
         		386.13 386.48
    142
    Figure US20100216789A1-20100826-C00174
         		506.20 506.64
    143
    Figure US20100216789A1-20100826-C00175
         		484.09 484.97
    144
    Figure US20100216789A1-20100826-C00176
         		378.16 378.50
    146
    Figure US20100216789A1-20100826-C00177
         		359.10 359.41
    146
    Figure US20100216789A1-20100826-C00178
         		373.11 373.44
    147
    Figure US20100216789A1-20100826-C00179
         		393.06 393.86
    148
    Figure US20100216789A1-20100826-C00180
         		409.11 409.47
    149
    Figure US20100216789A1-20100826-C00181
         		409.11 409.47
    150
    Figure US20100216789A1-20100826-C00182
         		409.11 409.47
    151
    Figure US20100216789A1-20100826-C00183
         		392.06 392.87
    152
    Figure US20100216789A1-20100826-C00184
         		388.11 388.45
    153
    Figure US20100216789A1-20100826-C00185
         		402.09 402.44
    154
    Figure US20100216789A1-20100826-C00186
         		372.12 372.45
    155
    Figure US20100216789A1-20100826-C00187
         		386.13 386.48
    156
    Figure US20100216789A1-20100826-C00188
         		383.10 383.44
    157
    Figure US20100216789A1-20100826-C00189
         		372.12 372.45
    158
    Figure US20100216789A1-20100826-C00190
         		402.13 402.48
    159
    Figure US20100216789A1-20100826-C00191
         		402.13 402.48
    160
    Figure US20100216789A1-20100826-C00192
         		418.12 418.48
    161
    Figure US20100216789A1-20100826-C00193
         		404.11 404.45
    162
    Figure US20100216789A1-20100826-C00194
         		380.14 380.47
    163
    Figure US20100216789A1-20100826-C00195
         		401.14 401.50
    164
    Figure US20100216789A1-20100826-C00196
         		442.08 442.42
    165
    Figure US20100216789A1-20100826-C00197
         		336.12 336.42
    166
    Figure US20100216789A1-20100826-C00198
         		409.17 409.52
    167
    Figure US20100216789A1-20100826-C00199
         		376.09 376.42
    168
    Figure US20100216789A1-20100826-C00200
         		326.09 326.38
    169
    Figure US20100216789A1-20100826-C00201
         		322.10 322.39
    170
    Figure US20100216789A1-20100826-C00202
         		380.11 380.43
    171
    Figure US20100216789A1-20100826-C00203
         		350.13 350.45
    172
    Figure US20100216789A1-20100826-C00204
         		378.16 378.50
    173
    Figure US20100216789A1-20100826-C00205
         		365.14 365.46
    174
    Figure US20100216789A1-20100826-C00206
         		364.15 364.47
    176
    Figure US20100216789A1-20100826-C00207
         		366.13 366.45
    176
    Figure US20100216789A1-20100826-C00208
         		455.19 455.59
    177
    Figure US20100216789A1-20100826-C00209
         		414.16 414.53
    178
    Figure US20100216789A1-20100826-C00210
         		361.11 361.43
    179
    Figure US20100216789A1-20100826-C00211
         		402.13 402.48
    180
    Figure US20100216789A1-20100826-C00212
         		445.08 445.53
    181
    Figure US20100216789A1-20100826-C00213
         		436.01 437.33
    182
    Figure US20100216789A1-20100826-C00214
         		398.13 398.49
    183
    Figure US20100216789A1-20100826-C00215
         		433.06 433.49
    184
    Figure US20100216789A1-20100826-C00216
         		475.04 475.98
    185
    Figure US20100216789A1-20100826-C00217
         		379.07 379.47
    186
    Figure US20100216789A1-20100826-C00218
         		418.17 418.53
    187
    Figure US20100216789A1-20100826-C00219
         		434.03 434.43
    188
    Figure US20100216789A1-20100826-C00220
         		402.14 402.48
    189
    Figure US20100216789A1-20100826-C00221
         		450.13 450.52
    190
    Figure US20100216789A1-20100826-C00222
         		467.10 467.58
    191
    Figure US20100216789A1-20100826-C00223
         		388.11 388.45
    192
    Figure US20100216789A1-20100826-C00224
         		386.13 386.48
    193
    Figure US20100216789A1-20100826-C00225
         		386.13 386.48
    194
    Figure US20100216789A1-20100826-C00226
         		408,12 408.49
    195
    Figure US20100216789A1-20100826-C00227
         		389.11 389.44
    196
    Figure US20100216789A1-20100826-C00228
         		426.09 426.42
    197
    Figure US20100216789A1-20100826-C00229
         		402.13 402.48
    198
    Figure US20100216789A1-20100826-C00230
         		426.09 426.42
    199
    Figure US20100216789A1-20100826-C00231
         		388.11 388.45
    200
    Figure US20100216789A1-20100826-C00232
         		422.13 422.51
    201
    Figure US20100216789A1-20100826-C00233
         		378.07 378.48
    202
    Figure US20100216789A1-20100826-C00234
         		440.04 441.34
    203
    Figure US20100216789A1-20100826-C00235
         		448.15 448.55
    204
    Figure US20100216789A1-20100826-C00236
         		416.14 416.51
    205
    Figure US20100216789A1-20100826-C00237
         		359.10 359.41
    206
    Figure US20100216789A1-20100826-C00238
         		398.13 398.49
    207
    Figure US20100216789A1-20100826-C00239
         		412.15 412.52
    208
    Figure US20100216789A1-20100826-C00240
         		456.18 456.58
    209
    Figure US20100216789A1-20100826-C00241
         		437.01 438.32
    210
    Figure US20100216789A1-20100826-C00242
         		450.03 451.35
    211
    Figure US20100216789A1-20100826-C00243
         		443.13 443.51
    212
    Figure US20100216789A1-20100826-C00244
         		404.18 404.54
    213
    Figure US20100216789A1-20100826-C00245
         		409.11 409.47
    214
    Figure US20100216789A1-20100826-C00246
         		436.12 436.50
    215
    Figure US20100216789A1-20100826-C00247
         		441.14 441.52
    216
    Figure US20100216789A1-20100826-C00248
         		444.09 444.54
    217
    Figure US20100216789A1-20100826-C00249
         		446.07 446.61
    218
    Figure US20100216789A1-20100826-C00250
         		446.11 446.56
    219
    Figure US20100216789A1-20100826-C00251
         		451.05 451.48
    220
    Figure US20100216789A1-20100826-C00252
         		453.14 453.53
    221
    Figure US20100216789A1-20100826-C00253
         		466.13 466.53
    222
    Figure US20100216789A1-20100826-C00254
         		477.11 477.98
    223
    Figure US20100216789A1-20100826-C00255
         		484.10 484.57
    224
    Figure US20100216789A1-20100826-C00256
         		465.14 465.54
    225
    Figure US20100216789A1-20100826-C00257
         		404.08 404.41
    226
    Figure US20100216789A1-20100826-C00258
         		393.06 393.86
    227
    Figure US20100216789A1-20100826-C00259
         		434.13 434.52
    228
    Figure US20100216789A1-20100826-C00260
         		423.13 423.50
    229
    Figure US20100216789A1-20100826-C00261
         		393.17 393.52
    230
    Figure US20100216789A1-20100826-C00262
         		451.09 451.53
    231
    Figure US20100216789A1-20100826-C00263
         		398.13 398.49
    232
    Figure US20100216789A1-20100826-C00264
         		462.03 463.37
    233
    Figure US20100216789A1-20100826-C00265
         		399.09 399.44
    234
    Figure US20100216789A1-20100826-C00266
         		433.20 433.58
    235
    Figure US20100216789A1-20100826-C00267
         		426.16 426.55
    236
    Figure US20100216789A1-20100826-C00268
         		427.16 427.53
    237
    Figure US20100216789A1-20100826-C00269
         		467.16 467.56
    238
    Figure US20100216789A1-20100826-C00270
         		457.17 457.56
    • INTERMEDIATES Intermediate A Preparation of thieno[3,2-c]pyridin-2-ylboronic acid
  • Figure US20100216789A1-20100826-C00271
    • Step 1: Preparation of 2,2-dimethoxy-N-(3-thienylmethyl)ethanamine
  • Figure US20100216789A1-20100826-C00272
  • To 3-thiophenecarboxaldehyde (8.75 mL) cooled to 0° C. was added aminoacetaldehyde dimethylacetal (14.2 mL). The clear solution was stirred at room temperature for 60 h. The solution was diluted with ethanol and hydrogenated at 56 psi in the presence of 10% Pd/C (5 g) overnight. The catalyst was filtered off and washed with MeOH. The solvent was removed by rotary evaporation and co-evaporated with toluene to afford an oil (19 g, 94% yield).
    • Step 2: Preparation of N-(2,2-dimethoxyethyl)-4-methyl-N-(3-thienylmethyl)-benzenesulfonamide
  • Figure US20100216789A1-20100826-C00273
  • To a solution of the compound prepared in step 1, (19 g, 95 mmol) in EtOAc (50 mL) at 15° C. was added triethylamine (12 mL, 85.5 mmol), followed by p-toluenesulfonyl chloride (16.3 g, 85.5 mmol). After the addition was complete, the solution was stirred at rt overnight. The reaction mixture was diluted with EtOAc and washed with water three times, HCl (1.0 M), and with said Na2CO3 The solution was dried over MgSO4, filtered, and concentrated to give a precipitate (31 g, 87 mmol, 91% yield). Rf=0.16 (EtOAc/hexane, 1/9).
    • Step 3: Preparation of thieno[3,2-e]pyridine
  • Figure US20100216789A1-20100826-C00274
  • To a solution of the compound from step 2 (31 g, 0.087 mol) in dioxane (80 mL) was added conc HCl (45 mL). The solution was heated to reflux temperature overnight. The mixture was cooled to rt and washed with CH2Cl2. The aqueous phase was basified with aqueous NH4OH while cooling with ice and extracted with CH2Cl2. The organic washings were combined and evaporated to dryness. The residue was purified by silica gel column chromatography (25% EtOAc in hexanes) to afford a white precipitate. The precipitate was dissolved with CH2Cl2, dried over MgSO4, filtered, and concentrated to give 5.97 g of white precipitate (50% yield).
    • Step 4: Preparation of
  • Figure US20100216789A1-20100826-C00275
  • To a solution of (0.3 g) in THF (15 mL) was added n-BuLi (1.4 mL, 1.6 M in hexane) at −78° C. The solution was stirred at −78° C. for 70 min and triisopropyl borate (0.52 mL, 2.27 mmol) was added. The solution was stirred for an additional 30 min at −78° C., and then at room temperature for 30 min. The solvent was concentrated under reduced pressure and the crude product was used without purification.
    • Intermediate B Preparation of 4-(4-morpholinyl)phenylboronic acid
  • Figure US20100216789A1-20100826-C00276
    • Step 1: Preparation of 4-(4-bromophenyl)morpholine
  • Figure US20100216789A1-20100826-C00277
  • A solution of 1-bromo-4-iodobenzene (5.0 g, 18 mmol), morpholine (1.85 mL, 21 mmol), sodium tert-butoxide (2.4 g, 25 mmol), 18-crown-6 (6.6 g, 25 mmol) in THF (150 mL) was purged with Ar for 20 min, then BINAP (0.11 g, 0.18 mmol) and Pd2(dba)3 (0.16 g, 0.18 mmol) was added. The mixture turned dark after stirring at room temperature overnight. The solvent was concentrated under reduced pressure and the residue was dissolved in diethyl ether and washed with water. The organic phase was mixed with silica gel, and the solvent was evaporated to dryness. The residue was purified by silica gel column chromatography (EtOAc in hexanes 5%) to of white precipitate (250 mg, 5% yield). 1H NMR (CDCl3): 7.3 (d, 2H), 6.85 (d, 4H), 3.1 (d, 4H).
    • Step 2: Preparation of 4-(4-morpholinyl)phenylboronic acid
  • Figure US20100216789A1-20100826-C00278
  • To a solution of the compound prepared in step 1 (0.3 g) in THF (15 mL) was added n-BuLi (0.74 mL, 2 M in pentane) at −78° C. After 1 h triisopropyl borate (0.86 mL) was added at −78° C. The solution was warmed up slowly from −78° C. to room temperature and was stirred overnight. The solution was added to aqueous NH4Cl, a white precipitate formed, and EtOAc was added. The organic solution was separated, concentrated and co-evaporated with toluene. The residue was washed twice with THF and filtered. The filtrate was concentrated and dried under high vacuum to give 0.25 g of light yellow precipitate. LC/MS m/z 208 (M+H)+.
  • The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

Claims (19)

1. A compound of formula I
Figure US20100216789A1-20100826-C00279
wherein X is —(CH2)x—, —O—(CH2)n—, —S—(CH2)n—, —NR7—CO—(CH2)n—, —NR7—SO2—(CH2)n—, —NR7—(CH2)n—, or —(O)C—NR7—,
each n is an integer which is independently 0, 1, 2 or 3,
x is 0-3
p is 0-3
a and c are each independently —CR5═, —N═, or —NR6—, wherein one of a or c is —NR6—, and b is —CR5═ or —N═;
A is H, halogen, —CO—OR8, —CO—R8, cyano, —OR8, —NR8R9, —CO—NR8R9, —NR8—CO—R9, —NR8—CO—OR9, —NR8—SO2—R9, —SR8, —SO2—R8, —SO2—NR8R9, NR8—CO—NHR9,
or
A is cyclohexyl; or C5-12-aryl or C5-12-heteroaryl each optionally independently substituted up to 3 times by (i) C1-C10 alkyl or C2-C10-alkenyl, each optionally substituted with halogen up to perhalo; (ii) C3-C10 cycloalkyl; (iii) aryl; (iv) heteroaryl; (v) halogen; (vi) —CO—OR8; (vii) —CO—R8; (viii) cyano; (ix) —OR8, (x) —NR8R13; (xi) nitro; (xii) —CO—NR8R9; (xiii) —C1-10-alkyl-NR8R9; (xiv) —NR8—CO—R12; (xv) —NR8—CO—OR9; (xvi) —NR8—SO2—R9; (xvii) —SR8; (xviii) —SO2—R8; (xix) —SO2—NR8R9; (xx) —NR8—CO—NHR9; or (xxi) aryl or heteroaryl substituted by halogen, C1-10-alkyl, C1-10-alkoxyphenyl, naphthyl, —OR10,
Figure US20100216789A1-20100826-C00280
wherein each Z independently is halogen, hydroxy, hydroxy-C1-10-alkyl, —CN, —NO2, C1-10-alkoxycarboxyl, —NR10—CO—R11 or —NR10—CO—OR11;
Y is 0-3;
Ring B represents a fused 5- or 6-membered heterocyclic ring containing 1-2 O,N, and/or S atoms and 1-5 C atoms;
R1, and R6-R11 are each independently H and C1-6 alkyl;
R2-R5 are each independently (i) C1-10 alkyl or C2-10-alkenyl each optionally substituted by amino, N-lower alkylamino, N,N-dilower alkylamino, N-lower alkanoylamino, hydroxy, cyano, —COOR10, —COR14, —OCOR14, —OR10, C5-10-heteroaryl, C5-10-heteroaryloxy, C5-10-heteroaryl-C1-10-alkoxy or halogen up to perhalo; (ii) C3-C10 cycloalkyl, in which 1-3 carbon atoms are optionally independently replaced by O, N or S; (iii) C3-10-cycloalkenyl; (iv) partially unsaturated C5-10-heterocyclyl; (v) aryl; (vi) heteroaryl; (vii) halogen; (viii) —CO—OR10; (ix) —OCOR10; (x) —OCO2R10; (xi) —CHO; (xii) cyano; (xiii) —OR16;
(xiv) —NR10R15; (xv) nitro; (xvi) —CO—NR10R11; (xvii) —NR10—CO—R12; (xviii) —NR10—CO—OR11; (xix) —NR10—SO2—R12; (xx) —SR16; (xxi) —SOR16; (xxii) —SO2—R16; (xxiii) —SO2—NR10R11; (xxiv) NR10—CO—NHR11; (xxv) amidino; (xxvi) guanidino; (xxvii) sulfo; (xxviii) —B(OH)2; (xxix) —OCON(R10)2; or (xxx) —NR10COON(R10)2;
R12 is H, C1-6-alkyl or C5-10-aryl,
R13 is H, C1-6-alkyl or C1-6-alkoxy,
R14 is lower alkyl or phenyl;
R15 is lower alkyl, halogen, amino, N-lower alkyl amino, N,N-dilower alkylamino, N-lower alkanoylamino, OH, CN, COOR10, —COR14 or —OCOR14;
R16 is hydrogen, C1-6-alkyl optionally substituted by halogen, up to perhalo, or C5-10-heteroaryl;
R15′ is H; phenyl optionally substituted by C1-10-alkyl, C1-10-alkoxy, C1-10-alkylcarboxyl, or halogen; benzyl; pyrimidyl or pyridyl; and R16′ is H, phenyl, —COOR10;
Figure US20100216789A1-20100826-C00281
or a pharmaceutically acceptable salt thereof,
with the provisos that A is not hydrogen when x is 0;
—X-A is not CH3 when B represents a thieno[3,2b]fused ring, and b and c are —CR5═, and a is NH;
and A is not phenyl when X is NH, B forms an imidazo fused ring, and -a-b-c- is —CR5═N—NR6— or —NR6═N—CR5—.
2. The compound according to claim 1, wherein A is 2- or 3-furyl, 2- or 3-thienyl, 2- or 4-triazinyl, 1-, 2- or 3-pyrrolyl, 1-, 2-, 4- or 5-imidazolyl, 1-, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isoxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 2-, 3- or 4-pyridyl, 2-, 4-, 5- or 6-pyrimidinyl, 1,2,3-triazol-1-, -4- or 5-yl, 1,2,4-triazol-1-, -3- or B5-yl, 1- or 5-tetrazolyl, 1,2,3-oxadiazol-4- or 5-yl, 1,2,4-oxadiazol-3- or 5-yl, 1,3,4-thiadiazol-2- or 5-yl, 1,2,4-oxadiazol-3- or 5-yl, 1,3,4-thiadiazol-2- or 5-yl, 1,3,4-thiadiazol-3- or 5-yl, 1,2,3-thiadiazol-4- or 5-yl, 2-, 3-, 4-, 5- or 6-2H-thiopyranyl, 2-, 3- or 4-4H-thiopyranyl, 3- or 4-pyridazinyl, pyrazinyl, 2-, 3-, 4-, 5-, 6- or 7-benzofuryl, 2-, 3-, 4-, 5-, 6- or 7-benzothienyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-indolyl, 1-, 2-, 4- or 5-benzimidazolyl, 1-, 3-, 4-, 5-, 6- or 7-benzopyrazolyl, 2-, 4-, 5-, 6- or 7-benzoxazolyl, 3-, 4-, 5- 6- or 7-benzisoxazolyl, 1-, 3-, 4-, 5-, 6- or 7-benzothiazolyl, 2-, 4-, 5-, 6- or 7-benzisothiazolyl, 2-, 4-, 5-, 6- or 7-benz-1,3-oxadiazolyl, 2-, 3-, 4-, 5-, 6-, 7- or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-, 8-isoquinolinyl, 1-, 2-, 3-, 4- or 9-carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-acridinyl, or 2-, 4-, 5-, 6-, 7- or 8-quinazolinyl, or additionally optionally substituted phenyl, 2- or 3-thienyl, 1,3,4-thiadiazolyl, 3-pyrryl, 3-pyrazolyl, 2-thiazolyl or 5-thiazolyl.
3. The compound according to claim 1 wherein A is phenyl, pyridyl, pyrimidinyl, oxazolyl, furyl, thienyl, pyrrolyl, imidazolyl, isoxazolyl or pyrazinyl, each independently substituted up to three times by halogen, C1-10-alkyl, C1-10-alkoxyphenyl, naphthyl, —OR10,
Figure US20100216789A1-20100826-C00282
wherein each Z independently is halogen, hydroxy, hydroxy-C1-10-alkyl, —CN, —NO2, C1-10-alkoxycarboxyl, —NR10—CO—R11, or —NR10—CO—OR11, and
y is 0-3.
4. The compound according to claim 1 wherein A is
Figure US20100216789A1-20100826-C00283
wherein R15′ is H; phenyl optionally substituted by C1-10-alkyl, C1-10-alkoxy, C1-10-alkylcarboxyl, or halogen; benzyl; pyrimidyl or pyridyl; and R16′ is H, phenyl, —COOR10,
Figure US20100216789A1-20100826-C00284
5. A compound of the formula
Figure US20100216789A1-20100826-C00285
wherein Ar1 is
Figure US20100216789A1-20100826-C00286
Figure US20100216789A1-20100826-C00287
Figure US20100216789A1-20100826-C00288
Figure US20100216789A1-20100826-C00289
6. A compound of the formula
Figure US20100216789A1-20100826-C00290
Figure US20100216789A1-20100826-C00291
Figure US20100216789A1-20100826-C00292
Figure US20100216789A1-20100826-C00293
7. (canceled)
8. (canceled)
9. A compound of the formula
Figure US20100216789A1-20100826-C00294
wherein Ar is
3-aminophenyl, 3-isonicotinamido-phenyl, 5-(1H-indolyl)amino, or 4-phenoxyanilino.
10. (canceled)
11. A compound of the formula
Figure US20100216789A1-20100826-C00295
Figure US20100216789A1-20100826-C00296
Figure US20100216789A1-20100826-C00297
Figure US20100216789A1-20100826-C00298
Figure US20100216789A1-20100826-C00299
Figure US20100216789A1-20100826-C00300
Figure US20100216789A1-20100826-C00301
Figure US20100216789A1-20100826-C00302
Figure US20100216789A1-20100826-C00303
Figure US20100216789A1-20100826-C00304
Figure US20100216789A1-20100826-C00305
Figure US20100216789A1-20100826-C00306
Figure US20100216789A1-20100826-C00307
Figure US20100216789A1-20100826-C00308
Figure US20100216789A1-20100826-C00309
Figure US20100216789A1-20100826-C00310
Figure US20100216789A1-20100826-C00311
Figure US20100216789A1-20100826-C00312
Figure US20100216789A1-20100826-C00313
Figure US20100216789A1-20100826-C00314
Figure US20100216789A1-20100826-C00315
Figure US20100216789A1-20100826-C00316
Figure US20100216789A1-20100826-C00317
Figure US20100216789A1-20100826-C00318
Figure US20100216789A1-20100826-C00319
Figure US20100216789A1-20100826-C00320
Figure US20100216789A1-20100826-C00321
Figure US20100216789A1-20100826-C00322
Figure US20100216789A1-20100826-C00323
Figure US20100216789A1-20100826-C00324
Figure US20100216789A1-20100826-C00325
Figure US20100216789A1-20100826-C00326
Figure US20100216789A1-20100826-C00327
Figure US20100216789A1-20100826-C00328
Figure US20100216789A1-20100826-C00329
Figure US20100216789A1-20100826-C00330
Figure US20100216789A1-20100826-C00331
Figure US20100216789A1-20100826-C00332
Figure US20100216789A1-20100826-C00333
Figure US20100216789A1-20100826-C00334
Figure US20100216789A1-20100826-C00335
12. (canceled)
13. (canceled)
14. A method of treating hypertension, atherosclerosis, restenosis, cerebral ischemia, cerebral vasospasm, or erectile dysfunction, comprising administering to host in need thereof a compound according to claim 1.
15. A method of treating hypertension, atherosclerosis, restenosis, cerebral ischemia, cerebral vasospasm, or erectile dysfunction, comprising administering to a host in need thereof a compound according to claim 5.
16. (canceled)
17. (canceled)
18. A process according to claim 14, wherein the host is a human.
19. A process according to claim 15, wherein the host is a human.
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