WO2020142745A1 - Methods of treating graft versus host disease and neoplastic disease with amide compounds - Google Patents

Methods of treating graft versus host disease and neoplastic disease with amide compounds Download PDF

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WO2020142745A1
WO2020142745A1 PCT/US2020/012263 US2020012263W WO2020142745A1 WO 2020142745 A1 WO2020142745 A1 WO 2020142745A1 US 2020012263 W US2020012263 W US 2020012263W WO 2020142745 A1 WO2020142745 A1 WO 2020142745A1
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alkyl
cycloalkyl
nhc
aryl
substituted
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PCT/US2020/012263
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French (fr)
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Tong Wang
Stephen Gately
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Translational Drug Development, Llc
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Priority claimed from PCT/US2019/029003 external-priority patent/WO2019210008A1/en
Application filed by Translational Drug Development, Llc filed Critical Translational Drug Development, Llc
Publication of WO2020142745A1 publication Critical patent/WO2020142745A1/en
Priority to US17/080,425 priority Critical patent/US11555039B2/en
Priority to US18/147,450 priority patent/US20230143583A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/56—Amides
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/26—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by nitrogen atoms
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/75—Amino or imino radicals, acylated by carboxylic or carbonic acids, or by sulfur or nitrogen analogues thereof, e.g. carbamates
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/22—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the nitrogen-containing ring
    • C—CHEMISTRY; METALLURGY
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    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
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    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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    • C07—ORGANIC CHEMISTRY
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    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings

Definitions

  • the present invention relates to amide compounds, their compositions, and medicaments containing the same, as well as processes for the preparation and use of such compounds, compositions, and medicaments.
  • Such compounds are potentially useful in the treatment of graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • this disclosure is concerned with compounds and compositions inhibiting Rho-kinases, methods of treating diseases associated with Rho-kinases, and methods of synthesizing these compounds.
  • Rho-associated protein kinase is a key intracellular regulator of cytoskeletal dynamics and cell motility. Rho-kinase regulates a number of downstream targets of RhoA through phosphorylation, including, for example, myosin light chain, the myosin light chain phosphatase binding subunit and LIM-kinase 2. These substrates regulate actin filament organization and contractility. In smooth muscle cells Rho-kinase mediates calcium sensitization and smooth muscle contraction. 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. Rho kinase participates in a variety of cellular processes, including but not limited to cell adhesion, cell motility and migration, growth control, cell contraction, and cytokinesis. Rho kinase is also involved in Na/H exchange transport system activation, stress fiber formation, adducin activation, and physiological processes such as vasoconstriction, bronchial smooth muscle constriction, vascular smooth muscle and endothelial cell proliferation, platelet aggregation, and others.
  • Rho-kinase activity in animal models has demonstrated a number of benefits of Rho-kinase inhibition for the treatment of human diseases.
  • cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy, ocular hypertension, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, central nervous system disorders such as neuronal degeneration and spinal cord injury, and in neoplasias.
  • Inhibition of Rho-kinase activity has been shown to inhibit tumor cell growth and metastasis, angiogenesis, arterial thrombotic disorders such as platelet aggregation and leukocyte aggregation, asthma, regulation of intraoccular pressure, and bone resorption.
  • Rho-kinase activity in patients has benefits for controlling cerebral vasospasms and ischemia following subarachnoid hemorrhage, reduction of intraocular pressure, increase in ocular aqueous outflow by relaxation of trabecular meshwork tissue, improving blood flow to the optic nerve, and protection of healthy ganglion cells.
  • Rho-kinase In mammals, Rho-kinase consists of two isoforms, ROCK1 (ROCKP; pi 60-ROCK) and ROCK2 (ROCKa). ROCK1 and ROCK2 are differentially expressed and regulated in specific tissues. For example, ROCK1 is ubiquitously expressed at relatively high levels, whereas ROCK2 is preferentially expressed in cardiac and brain and skeletal muscle. The isoforms are also expressed in some tissues and in a developmental stage specific manner. ROCK1 is a substrate for cleavage by caspase-3 during apoptosis, whereas ROCK2 is not. Smooth muscle specific basic calponin is phosphorylated only by ROCK2.
  • the present invention relates to treatment of graft versus host disease (GVHD), including chronic GVHD (cGVHD), and neoplastic disease using compounds having the formulae I-IV, as set forth herein.
  • GVHD graft versus host disease
  • cGVHD chronic GVHD
  • neoplastic disease using compounds having the formulae I-IV, as set forth herein.
  • the invention provides a compound of formula I.
  • Zi is -H, -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the -phenyl, -naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R ⁇ -NHR’, -NR’R”, -NHC(0)R’, -
  • Z 2 is -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the - phenyl, -naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -
  • R is -H or -Ci-C 6 alkyl, wherein the -Ci-C 6 alkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R’, -S(0) 2 NR’R”, and -S(0) 2 R’;
  • Ri is -H, -C1-C6 alkyl, or -C3-C7 cycloalkyl, wherein the -C1-C6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, - COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR'R”, - NS(0) 2 R’, -S(0) 2 NR’R”, and -S(0) 2 R’;
  • X is a bond or -0(Ci-C 6 alkyl);
  • R 2 and R3 are independently -H, -C1-C6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle
  • the -Ci-C 6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, - CN, -CNR’R”, -COOR’, -OR’, -SR’, -0C(0)R ⁇ -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R’, -S(0) 2 NR’R”, -S(0) 2 R’, -guanidino, -nitro, - nitroso, -Ci-C 6 alkyl, aryl, -C3-C7 cycloalkyl, and 3- to 1 1-membered heterocycle,
  • the -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, - CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl,
  • the R 5 is -Ci-C 6 alkyl, -OCH 2 CH 2 -, -NR 6 CH 2 CH 2 -, or -NC(0)CH 2 CH 2 -, and the 5 is -H, -Ci-C 6 alkyl, or -C3-C7 cycloalkyl;
  • the R’ and R” are independently -H or -Ci-C 6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4 to 8 membered cyclic structure.
  • the invention provides a compound of Formula (II):
  • Zi is -H, -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole or - tetrazole, wherein the -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole or -tetrazole is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -
  • Z 2 is -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the -phenyl, - naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, - OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R ⁇ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0) 2 R’, -S(0) 2 NR’R”, -S(0) 2 R’, -guanidino, -nitro, -nitroso, -C 1 -C 6 alkyl, -aryl, -C 3 -C 7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -Ci-C 6 alkyl, -aryl, -C 3 -
  • R is -H or -Ci-C 6 alkyl, wherein the -Ci-C 6 alkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, - NHC(0)R’, -NHC(0)NR’R”, -C(0)NR'R”, -NS(0) 2 R’, -S(0) 2 NR’R”, and -S(0) 2 R’;
  • Ri is -H, -Ci-C 6 alkyl or -C 3 -C 7 cycloalkyl, wherein the -Ci-C 6 alkyl or -C 3 -C 7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R’, - S(0) 2 NR’R”, and -S(0) 2 R’;
  • R 2 and R 3 are independently -H, -Ci-C 6 alkyl, -C 3 -C 7 cycloalkyl, -aryl, a heterocycle comprising
  • the -C 1 -C 6 alkyl, -C 3 -C 7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, - CN, -CNR’R”, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R’, -S(0)2NR’R”, -S(0) 2 R’, -guanidino, -nitro, - nitroso, -Ci-C 6 alkyl, aryl, -C3-C7 cycloalkyl, and 3- to 1 1-membered heterocycle,
  • the -Ci-C 6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, - CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl,
  • the R 5 is -Ci-C 6 alkyl, -OCH 2 CH 2 -, -NR 6 CH 2 CH 2 -, or -NC(0)CH 2 CH 2 -, and the 5 is -H, -C 1 -C 6 alkyl, or -C 3 -C 7 cycloalkyl;
  • R’ and R are independently -H or -Ci-C 6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4- to 8- membered cyclic structure;
  • R4 is H, -C 1 -C 6 alkyl or -C 3 -C 7 cycloalkyl.
  • the invention provides a compound of Formula (III):
  • Zi is -H, -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole, or - tetrazole, wherein the -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, - indazole, or -tetrazole is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R’, - S(0) 2 NR’R”, -S(0) 2 R’, -guanidino, -nitro, -nitroso, -Ci-C 6 alkyl,
  • Z 2 is -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the -phenyl, - naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, -
  • Ri is -H, -Ci-C 6 alkyl, or -C3-C7 cycloalkyl, wherein the -Ci-C 6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R’, - S(0) 2 NR’R”, and -S(0) 2 R’;
  • R 2 and R 3 are independently -H, -Ci-C 6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle comprising
  • the -Ci-C 6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -CNR’R”, - COOR’, -OR’, -SR’, -0C(0)R ⁇ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, - NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, and 3- to 11-membered heterocycle,
  • the -Ci-C 6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl, the R 5 is -Ci-C 6 alkyl, -OCH 2 CH 2 -, -NR 6 CH 2 CH 2 -,
  • the 5 is -H, -C1-C6 alkyl, or -C3-C7 cycloalkyl;
  • the R’ and R are independently -H or -C1-C6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4- to 8- membered cyclic structure.
  • the invention provides a compound of Formula (IV):
  • Zi is -H, -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole, or - tetrazole, wherein the -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, - indazole, or -tetrazole is unsubstituted or substituted with one or more of the following: -halo, - OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R ⁇ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0) 2 R ⁇ -S(0) 2 NR’R”, -S(0) 2 R’, -guanidino, -nitro, -nitroso, -C1-
  • Ri is -H, -Ci-C 6 alkyl, or -C3-C7 cycloalkyl, wherein the -Ci-C 6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR'R”, -NS(0) 2 R’, - S(0) 2 NR’R”, and -S(0) 2 R’;
  • R 2 and R 3 are independently -H, -Ci-C 6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle comprising
  • the -C1-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -CNR’R”, - COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -C(0)NR’R”, -
  • NS(0) 2 R’ -S(0) 2 NR’R”, -S(0) 2 R’, -guanidino, -nitro, -nitroso, -Ci-C 6 alkyl, aryl, -C3-C7 cycloalkyl, and 3- to 11-membered heterocycle,
  • the -Ci-C 6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R ⁇ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl, the R 5 is -Ci-C 6 alkyl, -OCH 2 CH 2 -, -NR 6 CH 2 CH 2 -, or
  • the 5 is -H, -Ci-C 6 alkyl, or -C3-C7 cycloalkyl;
  • R’ and R are independently -H or -Ci-C 6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4- to 8- membered cyclic structure;
  • R v is -H, -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R ⁇ -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R’, -S(0) 2 NR’R”, -S(0) 2 R’, -Ci-C 6 alkyl, -C3-C7 cycloalkyl, -aryl, or a heterocycle comprising 5 to 10 carbons, wherein the -Ci-C 6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with - -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R
  • R is hydroxymethyl with S configuration. In other embodiments, wherein Zi is pyridine.
  • R 2 is -H.
  • R3 is -C1-C6 alkyl or a heterocycle comprising 5 to 10 carbons substituted with one or more of the following: -halo, -OH, -CN, -CNR’R”, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R’, -S(0) 2 NR’R”, -S(0) 2 R’, - guanidino, -nitro, -nitroso, -Ci-C 6 alkyl, aryl, -C3-C7 cycloalkyl, and a 3- to 11-membered heterocycle; and
  • the -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is substituted with one or more of the following: -halo, -CNR’R”, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -OR’OR”, - S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl .
  • R3 is -C1-C6 alkyl substituted with a 3- to 11-membered heterocycle and the 3- to 11-membered heterocycle is substituted with -COOR’.
  • R3 is a heterocycle comprising 5 to 10 carbons substituted with - COOR’ or -OR’.
  • R3 is a heterocycle comprising 5 to 10 carbons substituted with -Ci-C 6 alkyl and the -Ci-C 6 alkyl is substituted with one or more -halo.
  • the invention provides at least one compound selected from the group consisting of:
  • the compound is N-(2-aminoethyl)-2-aminoethyl
  • the compound is selected from the group consisting of:
  • R is methyl with R configuration.
  • R is hydroxymethyl with S configuration.
  • R 2 and R3 are independently -H, -C3-C7 cycloalkyl, or -C3-C7 cycloalkyl methyl, wherein the -C3-C7 cycloalkyl or -C3-C7 cycloalkyl methyl is unsubstituted or substituted with one or more of the following: -H, -halo, -OH, -CN, -COOR’, -OR’, -SR’, - OC(0)R ⁇ -NHR’, -NR’R”, -CNR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR'R”, -NS(0) 2 R’, -S(0) 2 NR’R”, -S(0) 2 R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl methyl, wherein the
  • R 2 and R 3 are independently -H, -cyclohexyl, - cyclopentyl, -cyclobutyl, -cyclopropyl, -cyclohexyl methyl, -cyclopentyl methyl, -cyclobutyl methyl, or -cyclopropyl methyl, wherein the -cyclohexyl, -cyclopentyl, -cyclobutyl, - cyclopropyl, -cyclohexyl methyl, -cyclopentyl methyl, -cyclobutyl methyl, or -cyclopropyl methyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, - COOR’, OR’, -SR’, -OC(0)R ⁇ -NHR’, -NR’R”, -CNR’R”, -NHC(0)R’, -NH
  • R 2 and R3 are independently -H, -phenyl, or -benzyl, wherein the -phenyl or -benzyl is unsubstituted or substituted with one or more of the following: -halo, -
  • Zi is -pyridine unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R ⁇ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R ⁇ -S(0) 2 NR’R”, -S(0) 2 R’, -guanidino, -nitro, -nitroso, -Ci-C 6 alkyl, - aryl, and -C3-C7 cycloalkyl;
  • Z 2 is a heterocycle comprising 5 to 10 carbons, wherein the heterocycle is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R ⁇ -NHR’, -NR’R”, - NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R’, -S(0) 2 NR’R”, -S(0) 2 R’, -guanidino, - nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle; and
  • R 2 and R3 are independently -H, -phenyl, -benzyl, -C 3 -C 7 cycloalkyl, or -C 3 -C 7 cycloalkyl methyl, wherein the -phenyl, -benzyl, -C 3 -C 7 cycloalkyl, or -C 3 -C 7 cycloalkyl methyl is optionally substituted with -H, -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0) 2 R’, -S(0) 2 NR’R”, -S(0) 2 R’, - guanidino, -nitro, -nitroso, -C 1 -C 6 alkyl, -aryl, -C 3 -
  • Z 2 is -pyridine or -pyrazole.
  • Ri, R 2 , or both are -H.
  • the invention provides a pharmaceutical composition, comprising any of the presently disclosed compounds and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition comprising any of the presently disclosed compounds and a pharmaceutically acceptable carrier for the treatment of a disease associated with Rho-kinase modulation, graft versus host disease (GVHD), or a neoplastic disease.
  • a pharmaceutical composition comprising any of the presently disclosed compounds and a pharmaceutically acceptable carrier for the treatment of a disease associated with Rho-kinase modulation, graft versus host disease (GVHD), or a neoplastic disease.
  • the present invention provides the use of a compound disclosed herein for the manufacture of a medicament for the treatment of a disease associated with Rho-kinase modulation, graft versus host disease (GVHD), or a neoplastic disease.
  • GVHD graft versus host disease
  • the invention provides a method of treating a disease associated with Rho-kinase modulation in a subject in need thereof, comprising administering to the subject an effective amount of a rho kinase inhibitor or pharmaceutically acceptable salt thereof, wherein the rho kinase inhibitor comprises at least one presently disclosed compound or pharmaceutical composition.
  • the invention provides a method of treating a graft versus host disease (GVHD) in a subject in need thereof, comprising administering to the subject an effective amount of a rho kinase inhibitor or pharmaceutically acceptable salt thereof, wherein the rho kinase inhibitor comprises at least one presently disclosed compound or pharmaceutical composition.
  • GVHD graft versus host disease
  • the GVHD is a chronic GVHD disease (cGVHD).
  • the present invention provides a method of treating a neoplastic disease selected from the group consisting of lymphoma, carcinoma, leukemia, sarcoma, and blastoma in a subject in need thereof, the method comprising administering to the subject an effective amount of a rho kinase inhibitor or pharmaceutically acceptable salt thereof, wherein the rho kinase inhibitor comprises at least one compound disclosed herein or a pharmaceutical composition disclosed herein.
  • the neoplastic disease is ITO-FLT3 + acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the rho kinase inhibitor or pharmaceutically acceptable salt thereof is ROCK2 selective.
  • the rho kinase inhibitor is selected from the group consisting of:
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.
  • alkyl refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups and branched-chain alkyl groups.
  • a straight chain or branched chain alkyl has 10 or fewer carbon atoms in its backbone (e.g., Ci-Ci 0 for straight chain, C 3 -C 10 for branched chain).
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3 to 6 carbons in the ring structure.
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to six carbons, and more preferably from one to four carbon atoms.
  • lower alkenyl and “lower alkynyl” have similar chain lengths (C 2 -C 6 ).
  • Preferred alkyl groups are lower alkyls.
  • a substituent designated herein as alkyl is a lower alkyl.
  • cycloalkyl refers to saturated, carbocyclic groups having from 3 to 7 carbons in the ring.
  • Preferred cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • aralkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
  • alkenyl and“alkynyl” refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • aryl as used herein includes 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as“aryl heterocycles,”“heteroaromatics,” or“heteroaryl.”
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF 3 , -CN, or the like.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are“fused rings”) wherein at least one of the rings is aromatic, e.g ., the other cyclic rings can be cycloalkyls, cycloalkenyls, aryls and/or heterocyclic groups.
  • heterocyclyl or“heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 5- or 6-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles.
  • Heterocyclic groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, ox
  • the heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF 3 , -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxy
  • polycyclyl or“polycyclic group” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, aryls and/or heterocyclyl s) in which two or more carbons are common to two adjoining rings, e.g, the rings are“fused rings.” Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • rings e.g., cycloalkyls, cycloalkenyls, aryls and/or heterocyclyl s
  • Each of the rings of the polycyclic group can be substituted with such substituents as described above, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF 3 , -CN, or the like.
  • substituents as described above, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, si
  • nitro means -NO2.
  • halogen or “halo” designates -F, -Cl, -Br or -F
  • hydroxyl means -OH.
  • alkoxyl refers to an alkyl group, as defined above, having an oxygen radical attached thereto.
  • Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
  • lower alkoxy refers to an alkoxy group having from 1 to 6 carbon atoms.
  • oxo refers to an oxygen atom that has a double bond to another atom, particularly to carbon or sulfur.
  • each expression e.g ., alkyl, m, n, R, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
  • “substituted,”“substitution,” or“substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g. , which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • the term“substituted” is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non aromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described herein above.
  • Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis-and trans-isomers, R- and S-enantiomers, diastereomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are included in this invention.
  • Certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids.
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention.
  • Representative salts include the hydrochloride, hydrobromide, sulfate, bisulfate, phosphate, nitrate, acetate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, and mesylate salts and the like. See, for example, Berge et al.“Pharmaceutical Salts,” J. Pharm. Sci. (1977) 66: 1-19.
  • the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases.
  • Representative salts include alkali or alkaline earth salts such as lithium, sodium, potassium, calcium, magnesium salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethyl amine, diethyl amine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. See, for example, Berge et al. , supra.
  • the present invention provides compounds of Formula I that are inhibitors of Rho-kinase.
  • Rho kinase a serine/threonine kinase
  • Rho serves as a target protein for small GTP -binding protein Rho
  • Rho a serine/threonine kinase
  • ROCK plays an important role in Ca sensitization and the control of vascular tone. It modulates the level of phosphorylation of the myosin II light chain of myosin II, mainly through inhibition of myosin phosphatase, and contributes to agonist-induced Ca sensitization in smooth muscle contraction.
  • Rho kinase is found in two forms, ROCK 1 (ROCKP; p 160-ROCK) and ROCK 2 (ROCKa).
  • the compound of Formula I is selectively inhibits ROCK1.
  • the compound of Formula I selectively inhibits ROCK2.
  • the compound of Formula I is non-selective with respect to inhibition of ROCK1 and ROCK2.
  • a method of treating a patient suffering from a disease comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of the present invention.
  • therapeutically-effective amount means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment, e.g ., reasonable side effects applicable to any medical treatment.
  • Rho-kinase and or Rho-kinase mediated phosphorylation are useful for treatment of patients suffering from cardiovascular and non- cardiovascular diseases involving Rho-kinase function, such as hypertension, pulmonary hypertension, atherosclerosis, restenosis, coronary heart disease, cardiac hypertrophy, ocular hypertension, retinopathy, ischemic diseases, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, peripheral circulatory disorder, peripheral artery occlusive disease, glaucoma, (e.g, regulating intraoccular pressure), fibroid lung, fibroid liver, fibroid kidney, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome, central nervous system disorders such as neuronal degeneration and spinal cord injury.
  • Rho- kinase inhibiters of the invention can be used to treat arterial thrombotic disorders such as platelet aggregation and leukocyte aggregation, and bone thrombotic disorders such as platelet aggregati
  • a Rho-kinase inhibitor of the invention is used to treat inflammation, including, but not limited to asthma, cardiovascular inflammation, renal inflammation, and arteriosclerosis.
  • Neoplastic diseases include any malignant growth or tumor caused by abnormal or uncontrolled cell division, and may spread to other parts of the body through the lymphatic system or the blood stream.
  • Neoplastic disease includes, without limitation, lymphoma (a neoplasm of lymph tissue that is usually malignant), carcinoma (any malignant tumor derived from epithelial tissue), leukemia (malignant neoplasm of blood-forming tissues; characterized by abnormal proliferation of leukocytes), sarcoma (a usually malignant tumor arising from connective tissue (bone or muscle etc.), and blastoma (malignancy in precursor cells).
  • Nonlimiting examples include squamous cell cancer, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer, testis cancer, cholangiocarcinoma, gallbladder carcinoma, gastric cancer, melanoma, and various types of head and neck cancer.
  • ROCK inhibitors are used to effect weight loss and/or limit weight gain.
  • the ROCK inhibitor is ROCK2 selective.
  • ROCK-2 inhibitors promote weight loss in normal subjects, and limit weight gain in subjects prone to obesity.
  • a ROCK inhibitor is used to reduce or prevent insulin resistance or restore insulin sensitivity. Accordingly, in one embodiment, the compounds of the invention are used to promote or restore insulin-dependent glucose uptake. In another embodiment of the invention, a ROCK-inhibitors of the invention is used to promote or restore glucose tolerance. In another embodiment of the invention, a ROCK inhibitor of the invention is used to treat metabolic syndrome. In another embodiment, a ROCK- inhibitors of the invention is used to reduce or prevent hyperinsulinemia. In an embodiment of the invention, a ROCK inhibitor is used to treat diabetes (particularly type 2 diabetes). ROCK inhibitors of the invention may also be used to promote or restore insulin-mediated relaxation of vascular smooth muscle cells (VSMCs). In preferred embodiments, the ROCK inhibitor is ROCK2 selective.
  • VSMCs vascular smooth muscle cells
  • compounds of the invention are used for treatment of central nervous system disorders. Such disorders may involve neuronal degeneration or physical injury to neural tissue, including without limitation, Huntington's disease, Parkinson's Disease, Alzheimer's, Amyotrophic lateral sclerosis (ALS), or multiple sclerosis.
  • compounds of the invention have properties particularly useful for treatment of such disorders, such as beneficial tissue distribution to tissues of the central nervous system, and ability to cross the blood brain barrier.
  • the invention provides pan-ROCK inhibitors (i.e., compounds that inhibit ROCK1 and ROCK1) as well as ROCK inhibitors that are isoform selective.
  • a ROCK2-selective inhibitor may be preferred.
  • ROCK2 is frequently over expressed in hepatocellular cancer compared to non-timorous livers while ROCK1 expression is unaltered.
  • Other cancers which may benefit from treatment with a ROCK2 selective inhibitor include, but are not limited to, colon and bladder cancer.
  • ROCK1 expression levels have been observed to be higher in mammary tumors. Any cancer may be tested to determine whether there is overexpression of ROCK1 and/or ROCK2 and treated accordingly.
  • ROCK 1 and ROCK2 isoforms show similarity in regulating certain downstream targets.
  • the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the compounds of Formula I, Formula II, Formula III, or Formula IV formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals with toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydro
  • Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 percent to about 30 percent.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • the preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred.
  • parenteral administration and“administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day.
  • a dose of a compound or a composition is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • a dose(s) of a compound or a composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days.
  • a dose of a compound or a composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.
  • composition While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
  • the patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
  • the compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides.
  • Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
  • Microemulsification technology may be employed to improve bioavailability of lipophilic (water insoluble) pharmaceutical agents.
  • examples include Trimetrine (Dordunoo, S. K., et al, Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991) and REV 5901 (Sheen, P. C, et al, J Pharm Sci 80(7), 712-714, 1991).
  • microemulsification provides enhanced bioavailability by preferentially directing absorption to the lymphatic system instead of the circulatory system, which thereby bypasses the liver, and prevents destruction of the compounds in the hepatobiliary circulation.
  • the formulations contain micelles formed from a compound of the present invention and at least one amphiphilic carrier, in which the micelles have an average diameter of less than about 100 nm. More preferred embodiments provide micelles having an average diameter less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter less than about 30 nm, or even less than about 20 nm.
  • amphiphilic carriers While all suitable amphiphilic carriers are contemplated, the presently preferred carriers are generally those that have Generally-Recognized-as-Safe (GRAS) status, and that can both solubilize the compound of the present invention and microemulsify it at a later stage when the solution comes into a contact with a complex water phase (such as one found in human gastro-intestinal tract).
  • GRAS Generally-Recognized-as-Safe
  • amphiphilic ingredients that satisfy these requirements have HLB (hydrophilic to lipophilic balance) values of 2-20, and their structures contain straight chain aliphatic radicals in the range of C-6 to C-20. Examples are polyethylene-glycolized fatty glycerides and polyethylene glycols.
  • Particularly preferred amphiphilic carriers are saturated and monounsaturated polyethyleneglycolyzed fatty acid glycerides, such as those obtained from fully or partially hydrogenated various vegetable oils.
  • oils may advantageously consist of tri-. di- and mono fatty acid glycerides and di- and mono-polyethyleneglycol esters of the corresponding fatty acids, with a particularly preferred fatty acid composition including capric acid 4-10, capric acid 3-9, lauric acid 40-50, myristic acid 14-24, palmitic acid 4-14 and stearic acid 5-15%.
  • amphiphilic carriers includes partially esterified sorbitan and/or sorbitol, with saturated or mono-unsaturated fatty acids (SPAN-series) or corresponding ethoxylated analogs (TWEEN-series).
  • SPAN-series saturated or mono-unsaturated fatty acids
  • TWEEN-series corresponding ethoxylated analogs
  • amphiphilic carriers are particularly contemplated, including Gelucire-series, Labrafil, Labrasol, or Lauroglycol (all manufactured and distributed by Gattefosse Corporation, Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono- laurate and di-laurate, Lecithin, Polysorbate 80, etc. (produced and distributed by a number of companies in USA and worldwide).
  • Hydrophilic polymers suitable for use in the present invention are those which are readily water-soluble, can be covalently attached to a vesicle-forming lipid, and which are tolerated in vivo without toxic effects (i.e., are biocompatible).
  • Suitable polymers include polyethylene glycol (PEG), polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolic acid copolymer, and polyvinyl alcohol.
  • PEG polyethylene glycol
  • polylactic also termed polylactide
  • polyglycolic acid also termed polyglycolide
  • polyvinyl alcohol polyvinyl alcohol.
  • Preferred polymers are those having a molecular weight of from about 100 or 120 daltons up to about 5,000 or 10,000 daltons, and more preferably from about 300 daltons to about 5,000 daltons.
  • the polymer is polyethyleneglycol having a molecular weight of from about 100 to about 5,000 daltons, and more preferably having a molecular weight of from about 300 to about 5,000 daltons.
  • the polymer is polyethyleneglycol of 750 daltons (PEG(750)).
  • PEG(750) polyethyleneglycol of 750 daltons
  • the polymers used in the present invention have a significantly smaller molecular weight, approximately 100 daltons, compared to the large MW of 5000 daltons or greater that used in standard pegylation techniques. Polymers may also be defined by the number of monomers therein; a preferred embodiment of the present invention utilizes polymers of at least about three monomers, such PEG polymers consisting of three monomers (approximately 150 daltons).
  • hydrophilic polymers which may be suitable for use in the present invention include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • a formulation of the present invention comprises a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses, polypropylene, polyethylenes, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.
  • a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and
  • the release characteristics of a formulation of the present invention depend on the encapsulating material, the concentration of encapsulated drug, and the presence of release modifiers.
  • release can be manipulated to be pH dependent, for example, using a pH sensitive coating that releases only at a low pH, as in the stomach, or a higher pH, as in the intestine.
  • An enteric coating can be used to prevent release from occurring until after passage through the stomach.
  • Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach, followed by later release in the intestine.
  • Release can also be manipulated by inclusion of salts or pore forming agents, which can increase water uptake or release of drug by diffusion from the capsule.
  • Excipients which modify the solubility of the drug can also be used to control the release rate.
  • Agents which enhance degradation of the matrix or release from the matrix can also be incorporated. They can be added to the drug, added as a separate phase (i.e., as particulates), or can be co-dissolved in the polymer phase depending on the compound. In all cases the amount should be between 0.1 and thirty percent (w/w polymer).
  • Types of degradation enhancers include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine and surfactants such as Tween® and Pluronic®.
  • Pore forming agents which add microstructure to the matrices i.e., water soluble compounds such as inorganic salts and sugars
  • the range should be between one and thirty percent (w/w polymer).
  • Uptake can also be manipulated by altering residence time of the particles in the gut. This can be achieved, for example, by coating the particle with, or selecting as the encapsulating material, a mucosal adhesive polymer.
  • a mucosal adhesive polymer examples include most polymers with free carboxyl groups, such as chitosan, celluloses, and especially polyacrylates (as used herein, polyacrylates refers to polymers including acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates).
  • Compounds of the invention can be advantageously administered with second agents to patients in need thereof.
  • a rho-kinase inhibitor is administered with a second agent
  • the rho-kinase inhibitor and the second agent can be administered sequentially or concomitantly.
  • Sequentially means that one agent is administered for a time followed by administration of the second agent, which may be followed by administration of the first agent.
  • agents are administered sequentially, the level or one agent may not be maintained at a therapeutically effective level when the second agent is administered, and vice versa.
  • Concomitantly means that the first and second agent are administered according to a schedule that maintains both agents at an substantially therapeutically effective level, even though the agents are not administered simultaneously.
  • Each agent can be administered in single or multiple doses, and the doses can be administered on any schedule, including, without limitation, twice daily, daily, weekly, every two weeks, and monthly.
  • the invention also includes adjunctive administration.
  • Adjunctive administration means that a second agent is administered to a patient in addition to a first agent that is already being administered to treat a disease or disease symptom.
  • adjunctive administration involves administering a second agent to a patient in which administration of the first agent did not sufficiently treat a disease or disease symptom.
  • adjunctive administration involves administration of the second agent to a patient whose disease has been effectively treated by administration of the first agent, with the expectation that the adjunctive treatment improves the outcome of the treatment.
  • the effect of administering the first and second agents is synergistic.
  • administration of the first and second agents prevents or lengthens the time until relapse, compared to administration of either of the agents alone. In some embodiments, administration of the first and second agents allows for reduced dosage and/or frequency of administration of the first and second agent.
  • a rho-kinase inhibitor of the invention is administered and an anti -neoplastic agent are administered to a subject in need thereof.
  • a rho-kinase inhibitor of the invention and an angiogenesis inhibitor are administered to a subject in need thereof.
  • a rho-kinase inhibitor of the invention and an anti -inflammatory agent are administered to a subject in need thereof.
  • a rho-kinase inhibitor of the invention and an immunosuppressant are administered.
  • the second agent can be, without limitation, a small molecule, an antibody or antigen binding fragment thereof, or radiation.
  • Antineoplastic agents include, without limitation, cytotoxic chemotherapeutic agents, targeted small molecules and biological molecules, and radiation.
  • Compounds and agents that can be administered for oncological treatment, in addition to a rho kinase inhibitor of the invention include the following: irinotecan, etoposide, camptothecin, 5-fluorouracil, hydroxyurea, tamoxifen, paclitaxel, capcitabine, carboplatin, cisplatin, bleomycin, dactomycin, gemcitabine, doxorubicin, danorubicin, cyclophosphamide, and radiotherapy, which can be external (e.g ., external beam radiation therapy (EBRT)) or internal (e.g., brachytherapy (BT)).
  • EBRT external beam radiation therapy
  • BT brachytherapy
  • Targeted small molecules and biological molecules include, without limitation, inhibitors of components of signal transduction pathways, such as modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor-specfic antigens.
  • Examples include inhibitors of epidermal growth factor receptor (EGFR), including gefitinib, erlotinib, and cetuximab, inhibitors of HER2 (e.g., trastuzumab, trastuzumab emtansine (trastuzumab-DMl; T-DM1) and pertuzumab), anti-VEGF antibodies and fragments (e.g., bevacizumab), antibodies that inhibit CD20 (e.g., rituximab, ibritumomab), anti-VEGFR antibodies (e.g., ramucirumab (IMC- 112 IB), IMC-1C1 1, and CDP791), anti-PDGFR antibodies, and imatinib.
  • EGFR epidermal growth factor receptor
  • HER2 e.g., trastuzumab, trastuzumab emtansine (trastuzumab-DMl; T-DM1) and pertuzum
  • Small molecule kinase inhibitors can be specific for a particular tyrosine kinase or be inhibitors of two or more kinases.
  • the compound N-(3,4-dichloro-2- fluorophenyl)-7-( ⁇ [(3aR,6aS)-2-methyloctahydrocyclopenta[c] pyrrol-5-yl]methyl ⁇ oxy)-6- (methyloxy)quinazolin-4-amine also known as XL647, EXEL-7647 and KD-019
  • RTKs receptor tyrosine kinases
  • EGFR epigallocate
  • EphB4 KDR
  • Flt4 Flt4
  • ErbB2 ErbB2
  • treatment of a subject in need comprises administration of a rho-kinas
  • Dasatinib (BMS-354825; Bristol-Myers Squibb, New York) is another orally bioavailable, ATP-site competitive Src inhibitor. Dasatanib also targets Bcr-Abl (FDA-approved for use in patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL)) as well as c-Kit, PDGFR, c-FMS, EpbA2, and Src family kinases. Two other oral tyrosine kinase inhibitor of Src and Bcr-Abl are bosutinib (SKI-606) and saracatinib (AZD0530).
  • angiogenesis inhibitors can be administered to a subject in conjunction with compounds of the invention.
  • Angiogenesis inhibitors include any substance that inhibits the growth of new blood vessels.
  • angiogenesis inhibitors include antagonists of VEGF, P1GF, and VEGF receptors, including the antibodies disclosed herein.
  • inhibitor is meant an inhibitor of a biological process or inhibitor of a target.
  • an angiogenesis inhibitor is an agent that reduces angiogenesis.
  • a Rho-kinase inhibitor is an agent, such as a competitive inhibitor of ATP binding, that inhibits an intrinsic activity or blocks an interaction of Rho-kinase.
  • antagonist is meant a substance that reduces or inhibits an activity or function in a cell associated with a target.
  • a VEGF antagonist reduces or blocks a function in a cell that is associated with VEGF.
  • a VEGF antagonist may act on VEGF, by binding to VEGF and blocking binding to its receptors and/or may act on another cellular component involved in VEGF-mediated signal transduction.
  • a VEGFR2 antagonist is an agent that reduces or blocks VEGFR2 -mediated signal transduction by binding to VEGFR2 and blocking ligand binding or interaction with a VEGFR2 substrate, or acts on another cellular component to reduce or block VEGFR2 -mediated signal transduction.
  • angiogenesis inhibitors include antagonists of, without limitation, VEGF, VEGFRl, VEGFR2, PDGF, PDGFR-b, neuropilin-1 (NRP1), and complement.
  • Non-limiting examples of VEGF -binding agents include VEGF antibodies and VEGF traps (i.e., ligand binding domains of VEGF receptors.
  • VEGF traps i.e., ligand binding domains of VEGF receptors.
  • a VEGF trap is a protein that comprises VEGF binding domains of one or more VEGF receptor protein.
  • VEGF- traps include, without limitation, soluble VEGFR- 1, soluble neuropilin 1 (NRPl), soluble VEGFR-3 (which binds VEGF-C and VEGF-D), and aflibercept (Zaltrap; Eyelea; VEGF Trap R1R2), comprised of segments of the extracellular domains of human vascular endothelial growth factor receptors VEGFRl and VEGFR2 fused to the constant region (Fc) of human IgGl .
  • Conbercept (KH902) is a fusion protein which contains the extracellular domain 2 of VEGFR- 1 (Fit- 1 ) and extracellular domain 3, 4 of VEGFR-2 (KDR) fused to the Fc portion of human IgGl.
  • DARPins an acronym for designed ankyrin repeat proteins
  • DARPin® MP0112 is a vascular endothelial growth factor (VEGF) inhibitor and has entered clinical trials for the treatment of wet macular degeneration and diabetic macular edema.
  • VEGF expression can be targeted.
  • VEGF inhibitor PTC299 targets VEGF post-transcriptionally by selectively binding the 5'- and 3 '- untranslated regions (UTR) of VEGF messenger RNA (mRNA), thereby preventing translation of VEGF.
  • UTR 5'- and 3 '- untranslated regions
  • mRNA VEGF messenger RNA
  • Pegaptanib is an RNA aptamer directed against VEGF- 165.
  • Placental growth factor has been implicated in pathological angiogenesis.
  • PIGF is structurally related to VEGF and is also a ligand for VEGFR-l. Consequently, VEGF traps comprising the extracellular domain of VEGFRl (see above) are useful for targeting PIGF.
  • PDGF is composed of four polypeptide chains that form homodimers PDGF-AA, BB, CC, and DD as well as the heterodimer PDGF-AB.
  • the PDGF receptors (PDGFR) -a and -b mediate PDGF functions. Specifically, PDGFRa binds to PDGF-AA, -BB, -AB, and -CC, whereas PDGFRP interacts with -BB and -DD.
  • Non- limiting examples of PDGF -binding agents include anti-PDGF antibodies and PDGF traps.
  • Agents that target PDGF include FovistaTM (E10030, Ophthotech), a pegylated aptamer targeting PDGF-B, and AX102 (Sennino et al., 2007, Cancer Res. 75(15):7359-67), a DNA oligonucleotide aptamer that binds PDGF-B.
  • ramucirumab IMC-3G3, human IgGi
  • crenolanib CP-868596
  • nilotinib Tasigna®
  • Angiogenesis inhibitors include intracellular agents that block signal transduction mediated by, for example, VEGF, PDGF, ligands of VEGF or PDGF receptors, or complement.
  • Intracellular agents that inhibit angiogenesis inhibitors include the following, without limitation.
  • Sunitinib (Sutent; SU1 1248) is a panspecific small-molecule inhibitor of VEGFRl- VEGFR3, PDGFRa and PDGFRp, stem cell factor receptor (cKIT), Flt-3, and colony- stimulating factor- 1 receptor (CSF-1R).
  • Axitinib (AG013736; Inlyta) is another small molecule tyrosine kinase inhibitor that inhibits VEGFR- 1 - VEGFR-3, PDGFR, and cKIT.
  • Cediranib (AZD2171) is an inhibitor of VEGFR- 1- VEGFR-3, PDGFRp, and cKIT.
  • Sorafenib (Nexavar) is another small molecular inhibitor of several tyrosine protein kinases, including VEGFR, PDGFR, and Raf kinases.
  • Pazopanib (Votrient; (GW786034) inhibits VEGFR- 1, -2 and -3, cKIT and PDGFR.
  • Foretinib (GSK1363089; XL880) inhibits VEGFR2 and MET.
  • CP-547632 is as a potent inhibitor of the VEGFR-2 and basic fibroblast growth factor (FGF) kinases.
  • FGF basic fibroblast growth factor
  • E-3810 ((6-(7-((l-aminocyclopropyl) methoxy)-6-methoxyquinolin-4-yloxy)-N-methyl- 1 -naphthamide) inhibits VEGFR- 1, -2, and -3 and FGFR-1 and -2 kinases in the nanomolar range.
  • Brivanib (BMS-582664) is a VEGFR-2 inhibitor that also inhibits FGF receptor signaling.
  • CT-322 (Adnectin) is a small protein based on a human fibronectin domain and binds to and inhibits activation of VEGFR2.
  • Vandetanib (Caprelas; Zactima; ZD6474) is an inhibitor of VEGFR2, EGFR, and RET tyrosine kinases.
  • X-82 (Xcovery) is a small molecule indolinone inhibitor of signaling through the growth factor receptors VEGFR and PDGFR.
  • Immune checkpoint antagonist therapies have been developed to enable a patient's own immune system to fight tumors by facilitating an existing immune response or allowing for the initiation of an immune response. These therapies have been shown to be effective at treating some cancers in some subjects.
  • Three immune checkpoint targets are the subject of most on going clinical work: Programmed cell death protein 1 (PD-1), its ligands, PD-L1, cytotoxic T- lymphocyte-associated protein 4 (CTLA-4), lymphocyte-activation gene 3 (LAG-3), and cluster of differentiation 276 gene (CD276 also known as B7-H3). More recently, a new checkpoint target, GARP, has been investigated. Currently, a number of checkpoint antagonists are being evaluated in clinical trials.
  • Non-limiting examples of these antagonists include fully human and humanized anti-PD-1 monoclonal antibodies (e.g., nivolumab, pembrolizumab), an anti-PD-Ll antibody (e.g., durvalumab, atezolizumab, MEDI4736), a fusion protein comprising PD-L2 extracellular domain and lgGl, an anti-CTLA-4 antibody (e.g., tremelimumab, ipilimumab), a CD276 inhibitor (e.g., enoblituzumab; pidilizumab, MGD009) and an antibody to GARP (ARGX-1 15).
  • nivolumab, pembrolizumab an anti-PD-Ll antibody
  • durvalumab e.g., durvalumab, atezolizumab, MEDI4736
  • a fusion protein comprising PD-L2 extracellular domain and lgGl
  • the method comprises administering one or more checkpoint antagonist(s), the one or more checkpoint antagonist(s) is a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, and/or a PD-L2 antagonist.
  • the method comprises administering more than one checkpoint antagonist, each checkpoint antagonist is independently selected from a PD-1 antagonist, a PD- L1 antagonist, a CTLA-4 antagonist, and/or a PD-L2 antagonist.
  • the administration of the checkpoint antagonist comprises administering:
  • a first checkpoint antagonist selected from a PD-1 antagonists, a PD- LI antagonists, or PD-L2 antagonists; and a second checkpoint antagonist selected from CTLA-4 antagonists; or
  • a first checkpoint antagonist selected from a PD-1 antagonists and a second checkpoint antagonist selected from a PD-L1 and PD-L2 antagonists.
  • the PD-1 antagonist is selected from nivolumab, pembrolizumab, and AMP-224.
  • the PD-L1 antagonist is selected from MDX-1 105, atezolizumab, and durvalumab.
  • the CTLA-4 antagonist is selected from ipulimumab and tremelimumab.
  • the checkpoint antagonist is pidilizumab.
  • Anti-inflammatories and immunosuppressants include steroid drugs such as glucocorticoids (e.g., dexamethasone), FK506 (tacrolimus), ciclosporin, fmgolimod, interferon, such as IFNP or IFNy, a tumor necrosis factor-alpha (TNF-a) binding protein such as infliximab (Remicade), etanercept (Enbrel), or adalimumab (Humira), and mycophenolic acid.
  • glucocorticoids e.g., dexamethasone
  • FK506 tacrolimus
  • ciclosporin fmgolimod
  • interferon such as IFNP or IFNy
  • TNF-a binding protein such as infliximab (Remicade), etanercept (Enbrel), or adalimumab (Humira)
  • TNF-a tumor necrosis
  • ROCK inhibitors of the invention are coadministered with agents used to treat metabolic disorders.
  • the ROCK inhibitors may be combined with weight loss drugs such as, but not limited to, phentermine, fat adsorption inhibitors (e.g., Xenical), appetite suppressants, and the like.
  • Weight loss drugs such as, but not limited to, phentermine, fat adsorption inhibitors (e.g., Xenical), appetite suppressants, and the like.
  • Procedures used to assist weight loss include, for example, stomach bands, stomach bypass or stapling.
  • ROCK inhibitors of the invention can be coadministered with compounds that lower cholesterol levels, for example, one or more medicines such as statins, fibrates, or nicotinic acid.
  • ROCK inhibitors of the invention can be coadministered with, for example, one or more antihypertensive medicines such as diuretics or angiotensin-converting enzyme (ACE) inhibitors.
  • ROCK inhibitors of the invention can be administered in a treatment program that includes lifestyle changes such as increased physical activity, an improved diet, and/or quitting smoking.
  • the ROCK inhibitor is ROCK2 selective.
  • Thl7 cells are novel subset of helper CD4 + T cells that secrete IL-17, IL-21 and IL- 22.
  • the pro-inflammatory activity of Thl7 cells can be beneficial to the host during infection, but uncontrolled Thl7 function has been linked and actively involved in several autoimmune pathologies. Indeed, high levels of IL-17 are detected in the sera and biopsies of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients which correlates with destruction of synovial tissue and disease activity.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • the pathological role of IL-17 in arthritic joints is associated with its stimulation of pro -inflammatory cytokine production and increased recruitment of T cells and innate immune cells.
  • Thl7 cells are significantly increased in the peripheral blood of RA patients as well as elevated concentrations of IL-17 were seen in supernatants of their PBMCs after stimulation with anti-CD3/CD28 antibodies ex vivo.
  • MS multiple sclerosis
  • myelin reactive Thl7 cells are also enriched and produce high amounts of IL-22 and IFN-g.
  • a significantly higher number of IL-17 + cells is detected in disease-affected gut areas compared to healthy areas of the same subjects with Crohn's disease (CD).
  • Thl7 cells The development and function of Thl7 cells depends on activation of specific intracellular signaling pathways.
  • the steroid receptor-type nuclear receptor RORyt is selectively expressed in Thl7 cells and appears to be required for IL-17 production.
  • the induction of RORyt has been observed to be mediated by IL-6, IL-21 and IL-23 via a STAT3 -dependent mechanism.
  • STAT3 also binds directly to the IL-17 and IL-21 promoters.
  • the interferon regulatory factor 4 (IRF4) is required for the differentiation of Thl7 cells since IRF4 KO mice failed to mount Thl7 response and were resistant to development of autoimmune responses.
  • IRF4 interferon regulatory factor 4
  • Recent studies have demonstrated that phosphorylation of IRF4 by Rho-kinase 2 (ROCK2) regulates IL-17 and IL-21 production and development of autoimmunity in mice.
  • targeting Thl7 (IL-17-secreting) cells by rho-kinase inhibition provides a method for treating Thl7 cell-mediated diseases, including but not limited to autoimmune disorders such as rheumatoid arthritis (RA) multiple sclerosis (MS), systemic lupus erythematosus (SLE), psoriasis, Crohn's disease, atopic dermatitis, eczema, and GVHD in humans.
  • the Rho-kinase inhibitor is a compound of Formula I.
  • the rho-kinase inhibitor inhibits ROCK1 and ROCK2.
  • Tregs Regulatory T cells
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • Tregs depend on activation of specific signaling transduction pathways.
  • TGF-b and IL-2 activate expression of Foxp3 and STAT5 transcription factors that both play an essential role in the control of Treg suppressive function.
  • pro-inflammatory cytokines inhibit Foxp3 expression via up-regulation of STAT3 phosphorylation.
  • ROCK2 pharmacological inhibition of ROCK2 (e.g., with selective ROCK2 inhibitors such as KD025, ROCK2-specific siRNA-mediated inhibition of ROCK2), but not ROCK1, leads to down-regulation of STAT3 phosphorylation, interferon regulatory factor 4 (IRF4) and steroid receptor-type nuclear receptor RORyt protein levels in human T cells.
  • IRF4 interferon regulatory factor 4
  • ROCK2 inhibition leads to an increased proportion of Foxp3 + T cells via a STAT5 -dependent mechanism and positively regulates their suppressive activity towards autoreactive lymphocytes.
  • This effect of ROCK2 inhibition on Tregs is critical to limiting or preventing the onset of aberrant self-immune responses. This effect may be shown, for example, by assaying the ability of Tregs treated with a ROCK2 inhibitor to inhibit proliferation and cytokine secretion in target cells in vitro.
  • ROCK2 inhibitors of the invention effectively reduce or prevent chronic GVHD pathologies.
  • the compounds of the invention reduce or prevent cGVHD pathologies in target organs.
  • a selective ROCK2 inhibitor administered to a transplant patient maintains or restores pulmonary function.
  • the maintenance or restoration of pulmonary function correlates with reduced germinal center activity which would otherwise lead to production of autoreactive antibodies, and with reduced collagen deposition and antibody deposition in affected tissues.
  • pathologies are common to other targets of cGVHD as well, e.g, skin, gut, and liver, which would be similarly reduced or prevented.
  • Table 1 Non-limiting examples of kinase inhibitor compounds. Unless specified, example compounds with a chiral center represent racemic mixture of the corresponding R and S enantiomers and all racemates and isolated enantiomers are within the scope of the invention.
  • the following assay protocol is for measuring the phosphorylation of a peptide substrate (FAM-KKLRRTLSVA-OH wherein FAM is carboxyfluorescein).
  • the peptide is >98% purity by Capillary Electrophoresis.
  • the peptide is phosphorylated by the protein kinase ROCK1 or ROCK2.
  • the ROCK1 or ROCK2 enzyme, substrate, and cofactors are combined in a well of a microtiter plate and incubated for 3 hours at 25°C. At the end of the incubation, the reaction is quenched by the addition of an EDTA-containing buffer.
  • the substrate and product are separated and quantified electrophoretically using the microfluidic-based LABCHIP ® 3000 Drug Discovery System from Caliper Life Sciences (Hopkinton, Massachusetts).
  • the components of the assay mixture are:
  • Substrate and product peptides present in each sample are separated electrophoretically using the LABCHIP ® 3000 capillary electrophoresis instrument. As substrate and product peptides are separated two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks is the parameter measured reflecting enzyme activity. Capillary electrophoregramms (RDA acquisition files) are analyzed using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Massachusetts). The kinase activity in each sample is determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product peptide and S is the peak height of the substrate peptide.
  • PSR product to sum ratio
  • Pinh (PSR0% - PSRinh)/(PSR0% - PSR100%)* 100
  • PSRinh is the product sum ratio in the presence of inhibitor
  • PSR0% is the average product sum ratio in the absence of inhibitor
  • PSR100% is the average product sum ratio in 100%-inhibition control samples.
  • the IC50 values of inhibitors are determined by fitting the inhibition curves (Pinh versus inhibitor concentration) by 4 parameter sigmoidal dose- response model using XLfit 4 software (IBDS).
  • This assay can be used to test the activity of each of the exemplary compounds identified in Table 2. It is expected that each of these compounds will demonstrate inhibition of the protein kinase ROCK1 and/or ROCK2.
  • Cell Viability Assay- Cell viability was measured by the CELLTITER-GIO ® cell viability assay from Promega (Madison, WI).
  • the CELLTITER-GIO ® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. Following treatment, CELLTITER-GIO ® is added to treatment wells and incubated at 37° C. luminescence values were measured at using a Molecular Devices Spectramax microplate reader.
  • f test is the luminescence of the tested sample
  • f ve hicie is the luminescence of the vehicle in which the drug is dissolved.
  • the experiments also evaluated the selectivity of the compounds for inhibiting growth of cancer cells carrying a mutation in the Flt3 gene.
  • the MV411 cell line expresses the mutant allele of Flt3 with internal tandem duplications (ITD) of the gene.
  • ITD internal tandem duplications
  • K562 is a chronic myeloid leukemia cell line that does not express FLT3 protein.
  • Rho-associated kinase may be manipulated for the treatment of YYD-FLT3 + AML as reported in Onish et al. ,“Internal Tandem Duplication Mutations in FLT3 Gene Augment Chemotaxis to Cxcll2 Protein by Blocking the Down-regulation of the Rho-associated Kinase via the Cxcll2/Cxcr4 Signaling Axis,” J. Biol. Chem. 289 (45), 2014, 31053-31065.
  • DMSO Dimethyl-sulfoxide
  • a working stock was prepared by diluting it in Methanol -Water 50:50 (v/v) at 20 pg/mL for calibration curve preparation and stored at 4°C.
  • Standards used for quantitation and quality control samples (QC’s) were prepared on the same day of sample processing using blank plasma obtained from non-treated mice.
  • Eluent was then transferred into a LCMS plate for analysis using suitable column for separation and MRM detection.
  • Concentration of analytes was calculated based on the calibration curve and analyzed for pharmacokinetic parameters by using Non- compartmental analysis (NCA). Parameters such as Cmax, Tmax, half-life, AUC (0-last), AUC (0-co), volume of distribution (Vss) and clearance (Cl/F) were reported.
  • NCA Non- compartmental analysis

Abstract

The invention relates to treatment of graft versus host disease (GVHD) or neoplastic disease using compounds that inhibit Rho-associated protein kinases. In preferred aspects, the present invention provides methods for the treatment of chronic GVHD (cGVHD) or ITD-FLT3 + acute myeloid leukemia (AML) using compounds as set forth herein.

Description

METHODS OF TREATING GRAFT VERSUS HOST DISEASE AND NEOPLASTIC DISEASE WITH AMIDE COMPOUNDS
CROSS REFERENCE TO RELATED APPLICATIONS
[001] This application claims priority to U.S. Provisional Patent Application No. 62/788,461, filed on January 4, 2019, and to International Patent Application No. PCT/US2019/029003, filed on April 24, 2019, the contents of each of which are incorporated herein by reference in their entireties.
FIELD
[002] The present invention relates to amide compounds, their compositions, and medicaments containing the same, as well as processes for the preparation and use of such compounds, compositions, and medicaments. Such compounds are potentially useful in the treatment of graft versus host disease (GVHD). Specifically, this disclosure is concerned with compounds and compositions inhibiting Rho-kinases, methods of treating diseases associated with Rho-kinases, and methods of synthesizing these compounds.
BACKGROUND
[003] Rho-associated protein kinase (ROCK) is a key intracellular regulator of cytoskeletal dynamics and cell motility. Rho-kinase regulates a number of downstream targets of RhoA through phosphorylation, including, for example, myosin light chain, the myosin light chain phosphatase binding subunit and LIM-kinase 2. These substrates regulate actin filament organization and contractility. In smooth muscle cells Rho-kinase mediates calcium sensitization and smooth muscle contraction. 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. Rho kinase participates in a variety of cellular processes, including but not limited to cell adhesion, cell motility and migration, growth control, cell contraction, and cytokinesis. Rho kinase is also involved in Na/H exchange transport system activation, stress fiber formation, adducin activation, and physiological processes such as vasoconstriction, bronchial smooth muscle constriction, vascular smooth muscle and endothelial cell proliferation, platelet aggregation, and others.
[004] Inhibition of Rho-kinase activity in animal models has demonstrated a number of benefits of Rho-kinase inhibition for the treatment of human diseases. These include models of cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy, ocular hypertension, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, central nervous system disorders such as neuronal degeneration and spinal cord injury, and in neoplasias. Inhibition of Rho-kinase activity has been shown to inhibit tumor cell growth and metastasis, angiogenesis, arterial thrombotic disorders such as platelet aggregation and leukocyte aggregation, asthma, regulation of intraoccular pressure, and bone resorption. The inhibition of Rho-kinase activity in patients has benefits for controlling cerebral vasospasms and ischemia following subarachnoid hemorrhage, reduction of intraocular pressure, increase in ocular aqueous outflow by relaxation of trabecular meshwork tissue, improving blood flow to the optic nerve, and protection of healthy ganglion cells.
[005] In mammals, Rho-kinase consists of two isoforms, ROCK1 (ROCKP; pi 60-ROCK) and ROCK2 (ROCKa). ROCK1 and ROCK2 are differentially expressed and regulated in specific tissues. For example, ROCK1 is ubiquitously expressed at relatively high levels, whereas ROCK2 is preferentially expressed in cardiac and brain and skeletal muscle. The isoforms are also expressed in some tissues and in a developmental stage specific manner. ROCK1 is a substrate for cleavage by caspase-3 during apoptosis, whereas ROCK2 is not. Smooth muscle specific basic calponin is phosphorylated only by ROCK2.
[006] Given the extent of involved cellular processes and diseases, compounds that selectively inhibit one rho kinase, or inhibit ROCK1 and ROCK2, are desired.
SUMMARY
[007] The present invention relates to treatment of graft versus host disease (GVHD), including chronic GVHD (cGVHD), and neoplastic disease using compounds having the formulae I-IV, as set forth herein.
[008] In one aspect, the invention provides a compound of formula I.
Figure imgf000004_0001
[009] wherein:
[0010] Zi is -H, -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the -phenyl, -naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -
NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, - Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -C1-C6 alkyl, - aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, - guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
[0011] Z2 is -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the - phenyl, -naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -
NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, - C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -C1-C6 alkyl, - aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, - guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
[0012] R is -H or -Ci-C6 alkyl, wherein the -Ci-C6 alkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, and -S(0)2R’;
[0013] Ri is -H, -C1-C6 alkyl, or -C3-C7 cycloalkyl, wherein the -C1-C6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, - COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR'R”, - NS(0)2R’, -S(0)2NR’R”, and -S(0)2R’;
[0014] X is a bond or -0(Ci-C6 alkyl); [0015] R2 and R3 are independently -H, -C1-C6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle
comprising 5 to 10 carbons,
Figure imgf000005_0001
Figure imgf000005_0002
wherein
the -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, - CN, -CNR’R”, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, and 3- to 1 1-membered heterocycle,
the -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, - CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl,
the R5 is -Ci-C6 alkyl, -OCH2CH2-, -NR6CH2CH2-, or -NC(0)CH2CH2-, and the 5 is -H, -Ci-C6 alkyl, or -C3-C7 cycloalkyl; and
[0016] the R’ and R” are independently -H or -Ci-C6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4 to 8 membered cyclic structure.
[0017] In another aspect, the invention provides a compound of Formula (II):
Figure imgf000005_0003
wherein:
Zi is -H, -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole or - tetrazole, wherein the -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole or -tetrazole is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -
OC(0)R, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, - S(0)2NR’R”, -S(0)2R\ -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or 3- to 10- membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, - OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
Z2 is -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the -phenyl, - naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, - OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -Ci-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
R is -H or -Ci-C6 alkyl, wherein the -Ci-C6 alkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, - NHC(0)R’, -NHC(0)NR’R”, -C(0)NR'R”, -NS(0)2R’, -S(0)2NR’R”, and -S(0)2R’;
Ri is -H, -Ci-C6 alkyl or -C3-C7 cycloalkyl, wherein the -Ci-C6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, - S(0)2NR’R”, and -S(0)2R’;
R2 and R3 are independently -H, -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle comprising
Figure imgf000006_0001
the -C1-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, - CN, -CNR’R”, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, and 3- to 1 1-membered heterocycle,
the -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, - CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl,
the R5 is -Ci-C6 alkyl, -OCH2CH2-, -NR6CH2CH2-, or -NC(0)CH2CH2-, and the 5 is -H, -C1-C6 alkyl, or -C3-C7 cycloalkyl;
the R’ and R” are independently -H or -Ci-C6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4- to 8- membered cyclic structure; and
R4 is H, -C1-C6 alkyl or -C3-C7 cycloalkyl.
[0018] In one aspect, the invention provides a compound of Formula (III):
Figure imgf000007_0001
wherein:
Zi is -H, -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole, or - tetrazole, wherein the -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, - indazole, or -tetrazole is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, - S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, - COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, - NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -Ci-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
Z2 is -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the -phenyl, - naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, -
Figure imgf000008_0001
C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -Ci-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
Ri is -H, -Ci-C6 alkyl, or -C3-C7 cycloalkyl, wherein the -Ci-C6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, - S(0)2NR’R”, and -S(0)2R’;
R2 and R3 are independently -H, -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle comprising
Figure imgf000008_0002
the -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -CNR’R”, - COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, - NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, and 3- to 11-membered heterocycle,
the -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl, the R5 is -Ci-C6 alkyl, -OCH2CH2-, -NR6CH2CH2-, or -NC(0)CH2CH2-, and
the 5 is -H, -C1-C6 alkyl, or -C3-C7 cycloalkyl; and
the R’ and R” are independently -H or -C1-C6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4- to 8- membered cyclic structure. [0019] In one aspect, the invention provides a compound of Formula (IV):
Figure imgf000009_0002
, wherein:
Zi is -H, -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole, or - tetrazole, wherein the -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, - indazole, or -tetrazole is unsubstituted or substituted with one or more of the following: -halo, - OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R\ -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, and a 3- to 10-membered heterocycle, and the -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
Ri is -H, -Ci-C6 alkyl, or -C3-C7 cycloalkyl, wherein the -Ci-C6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR'R”, -NS(0)2R’, - S(0)2NR’R”, and -S(0)2R’;
R2 and R3 are independently -H, -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle comprising
Figure imgf000009_0001
the -C1-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -CNR’R”, - COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -
NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, and 3- to 11-membered heterocycle,
the -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl, the R5 is -Ci-C6 alkyl, -OCH2CH2-, -NR6CH2CH2-, or -NC(0)CH2CH2-, and
the 5 is -H, -Ci-C6 alkyl, or -C3-C7 cycloalkyl;
the R’ and R” are independently -H or -Ci-C6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4- to 8- membered cyclic structure; and
Rv is -H, -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or a heterocycle comprising 5 to 10 carbons, wherein the -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with - -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, - Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle.
[0020] In some embodiments, R is hydroxymethyl with S configuration. In other embodiments, wherein Zi is pyridine.
[0021] In one implementation, R2 is -H.
[0022] In other implementations,
R3 is -C1-C6 alkyl or a heterocycle comprising 5 to 10 carbons substituted with one or more of the following: -halo, -OH, -CN, -CNR’R”, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, - guanidino, -nitro, -nitroso, -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, and a 3- to 11-membered heterocycle; and
the -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is substituted with one or more of the following: -halo, -CNR’R”, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -OR’OR”, - S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl .
[0023] In one aspect, R3 is -C1-C6 alkyl substituted with a 3- to 11-membered heterocycle and the 3- to 11-membered heterocycle is substituted with -COOR’. [0024] In another aspect, R3 is a heterocycle comprising 5 to 10 carbons substituted with - COOR’ or -OR’. In yet another aspect, R3 is a heterocycle comprising 5 to 10 carbons substituted with -Ci-C6 alkyl and the -Ci-C6 alkyl is substituted with one or more -halo.
[0025] In one embodiment, the invention provides at least one compound selected from the group consisting of:
Figure imgf000011_0001
Figure imgf000012_0001
[0026] In certain aspects, the compound is
Figure imgf000012_0002
[0027] In other aspects, the compound is selected from the group consisting of:
Figure imgf000013_0001
[0028] In one embodiment, R is methyl with R configuration.
[0029] In another embodiment, R is hydroxymethyl with S configuration.
[0030] In further embodiment, R2 and R3 are independently -H, -C3-C7 cycloalkyl, or -C3-C7 cycloalkyl methyl, wherein the -C3-C7 cycloalkyl or -C3-C7 cycloalkyl methyl is unsubstituted or substituted with one or more of the following: -H, -halo, -OH, -CN, -COOR’, -OR’, -SR’, - OC(0)R\ -NHR’, -NR’R”, -CNR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR'R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or -3 to 10-membered heterocycle, and wherein R2 and R3 are not both -H.
[0031] In yet further embodiment, R2 and R3 are independently -H, -cyclohexyl, - cyclopentyl, -cyclobutyl, -cyclopropyl, -cyclohexyl methyl, -cyclopentyl methyl, -cyclobutyl methyl, or -cyclopropyl methyl, wherein the -cyclohexyl, -cyclopentyl, -cyclobutyl, - cyclopropyl, -cyclohexyl methyl, -cyclopentyl methyl, -cyclobutyl methyl, or -cyclopropyl methyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, - COOR’, OR’, -SR’, -OC(0)R\ -NHR’, -NR’R”, -CNR’R”, -NHC(0)R’, -NHC(0)NR’R”, C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6, -alkyl, - aryl, -C3-C7 cycloalkyl, and 3- to 10-membered heterocycle.
[0032] In one implementation, R2 and R3 are independently -H, -phenyl, or -benzyl, wherein the -phenyl or -benzyl is unsubstituted or substituted with one or more of the following: -halo, -
OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -CNR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, - C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, and 3- to 10-membered heterocycle, and wherein R2 and R3 are not both -H.
[0033] In another implementation,
Zi is -pyridine unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R\ -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -Ci-C6 alkyl, - aryl, and -C3-C7 cycloalkyl;
Z2 is a heterocycle comprising 5 to 10 carbons, wherein the heterocycle is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R\ -NHR’, -NR’R”, - NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, - nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle; and
R2 and R3 are independently -H, -phenyl, -benzyl, -C3-C7 cycloalkyl, or -C3-C7 cycloalkyl methyl, wherein the -phenyl, -benzyl, -C3-C7 cycloalkyl, or -C3-C7 cycloalkyl methyl is optionally substituted with -H, -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, - guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and wherein R2 and R3 are not both -H.
[0034] In further implementation, Z2 is -pyridine or -pyrazole.
[0035] In yet further implementation, Ri, R2, or both are -H.
[0036] In another aspect, the invention provides a pharmaceutical composition, comprising any of the presently disclosed compounds and a pharmaceutically acceptable carrier.
[0037] In certain implementations, the present invention provides a pharmaceutical composition comprising any of the presently disclosed compounds and a pharmaceutically acceptable carrier for the treatment of a disease associated with Rho-kinase modulation, graft versus host disease (GVHD), or a neoplastic disease.
[0038] In other implementations, the present invention provides the use of a compound disclosed herein for the manufacture of a medicament for the treatment of a disease associated with Rho-kinase modulation, graft versus host disease (GVHD), or a neoplastic disease.
[0039] In another aspect, the invention provides a method of treating a disease associated with Rho-kinase modulation in a subject in need thereof, comprising administering to the subject an effective amount of a rho kinase inhibitor or pharmaceutically acceptable salt thereof, wherein the rho kinase inhibitor comprises at least one presently disclosed compound or pharmaceutical composition.
[0040] In another aspect, the invention provides a method of treating a graft versus host disease (GVHD) in a subject in need thereof, comprising administering to the subject an effective amount of a rho kinase inhibitor or pharmaceutically acceptable salt thereof, wherein the rho kinase inhibitor comprises at least one presently disclosed compound or pharmaceutical composition.
[0041] In one embodiment, the GVHD is a chronic GVHD disease (cGVHD).
[0042] In another embodiment, the present invention provides a method of treating a neoplastic disease selected from the group consisting of lymphoma, carcinoma, leukemia, sarcoma, and blastoma in a subject in need thereof, the method comprising administering to the subject an effective amount of a rho kinase inhibitor or pharmaceutically acceptable salt thereof, wherein the rho kinase inhibitor comprises at least one compound disclosed herein or a pharmaceutical composition disclosed herein.
[0043] In one aspect, the neoplastic disease is ITO-FLT3+ acute myeloid leukemia (AML).
[0044] In a further embodiment, the rho kinase inhibitor or pharmaceutically acceptable salt thereof is ROCK2 selective.
[0045] In one implementation, the rho kinase inhibitor is selected from the group consisting of:
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000020_0001
Figure imgf000021_0001
DETAILED DESCRIPTION
[0046] The term“heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.
[0047] The term“alkyl” refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups and branched-chain alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 10 or fewer carbon atoms in its backbone (e.g., Ci-Ci0 for straight chain, C3-C10 for branched chain). Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3 to 6 carbons in the ring structure.
[0048] Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to six carbons, and more preferably from one to four carbon atoms. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths (C2-C6). Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.
[0049] The term“cycloalkyl” refers to saturated, carbocyclic groups having from 3 to 7 carbons in the ring. Preferred cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
[0050] The term“aralkyl,” as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
[0051] The terms“alkenyl” and“alkynyl” refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
[0052] The term“aryl” as used herein includes 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as“aryl heterocycles,”“heteroaromatics,” or“heteroaryl.” The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are“fused rings”) wherein at least one of the rings is aromatic, e.g ., the other cyclic rings can be cycloalkyls, cycloalkenyls, aryls and/or heterocyclic groups.
[0053] The terms“heterocyclyl” or“heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 5- or 6-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclic groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
[0054] The terms“polycyclyl” or“polycyclic group” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, aryls and/or heterocyclyl s) in which two or more carbons are common to two adjoining rings, e.g, the rings are“fused rings.” Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycyclic group can be substituted with such substituents as described above, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
[0055] As used herein, the term “nitro” means -NO2. The term “halogen” or “halo” designates -F, -Cl, -Br or -F The term“hydroxyl” means -OH.
[0056] The terms“alkoxyl” or“alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. The term lower alkoxy refers to an alkoxy group having from 1 to 6 carbon atoms.
[0057] The term“oxo” as used herein refers to an oxygen atom that has a double bond to another atom, particularly to carbon or sulfur.
[0058] As used herein, the definition of each expression, e.g ., alkyl, m, n, R, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
[0059] It will be understood that“substituted,”“substitution,” or“substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g. , which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
[0060] As used herein, the term“substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non aromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above.
[0061] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis-and trans-isomers, R- and S-enantiomers, diastereomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are included in this invention. [0062] Certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term“pharmaceutically-acceptable salts” in this context, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. Representative salts include the hydrochloride, hydrobromide, sulfate, bisulfate, phosphate, nitrate, acetate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, and mesylate salts and the like. See, for example, Berge et al.“Pharmaceutical Salts,” J. Pharm. Sci. (1977) 66: 1-19.
[0063] In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. Representative salts include alkali or alkaline earth salts such as lithium, sodium, potassium, calcium, magnesium salts and the like. Representative organic amines useful for the formation of base addition salts include ethyl amine, diethyl amine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. See, for example, Berge et al. , supra.
[0064] In one aspect, the present invention provides compounds of Formula I that are inhibitors of Rho-kinase. Rho kinase (ROCK), a serine/threonine kinase, serves as a target protein for small GTP -binding protein Rho, and is an important mediator of numerous cellular functions, including focal adhesions, motility, smooth muscle contraction, and cytokinesis. In smooth muscle, ROCK plays an important role in Ca sensitization and the control of vascular tone. It modulates the level of phosphorylation of the myosin II light chain of myosin II, mainly through inhibition of myosin phosphatase, and contributes to agonist-induced Ca sensitization in smooth muscle contraction.
[0065] Rho kinase is found in two forms, ROCK 1 (ROCKP; p 160-ROCK) and ROCK 2 (ROCKa). In some embodiments, the compound of Formula I is selectively inhibits ROCK1. In some embodiments, the compound of Formula I selectively inhibits ROCK2. In some embodiments, the compound of Formula I is non-selective with respect to inhibition of ROCK1 and ROCK2.
[0066] In another aspect of the present invention there is provided a method of treating a patient suffering from a disease comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of the present invention. The phrase“therapeutically-effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment, e.g ., reasonable side effects applicable to any medical treatment.
[0067] Compounds of the invention that inhibit Rho-kinase and or Rho-kinase mediated phosphorylation are useful for treatment of patients suffering from cardiovascular and non- cardiovascular diseases involving Rho-kinase function, such as hypertension, pulmonary hypertension, atherosclerosis, restenosis, coronary heart disease, cardiac hypertrophy, ocular hypertension, retinopathy, ischemic diseases, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, peripheral circulatory disorder, peripheral artery occlusive disease, glaucoma, (e.g, regulating intraoccular pressure), fibroid lung, fibroid liver, fibroid kidney, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome, central nervous system disorders such as neuronal degeneration and spinal cord injury. Further, Rho- kinase inhibiters of the invention can be used to treat arterial thrombotic disorders such as platelet aggregation and leukocyte aggregation, and bone resorption.
[0068] In certain embodiments, a Rho-kinase inhibitor of the invention is used to treat inflammation, including, but not limited to asthma, cardiovascular inflammation, renal inflammation, and arteriosclerosis.
[0069] Rho-kinase inhibitors of the invention inhibit tumor cell growth and metastasis, and angiogenesis, and are useful for treating neoplastic diseases. Neoplastic diseases include any malignant growth or tumor caused by abnormal or uncontrolled cell division, and may spread to other parts of the body through the lymphatic system or the blood stream. Neoplastic disease includes, without limitation, lymphoma (a neoplasm of lymph tissue that is usually malignant), carcinoma (any malignant tumor derived from epithelial tissue), leukemia (malignant neoplasm of blood-forming tissues; characterized by abnormal proliferation of leukocytes), sarcoma (a usually malignant tumor arising from connective tissue (bone or muscle etc.), and blastoma (malignancy in precursor cells). Nonlimiting examples include squamous cell cancer, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer, testis cancer, cholangiocarcinoma, gallbladder carcinoma, gastric cancer, melanoma, and various types of head and neck cancer.
[0070] According to the invention, ROCK inhibitors are used to effect weight loss and/or limit weight gain. In a preferred embodiment, the ROCK inhibitor is ROCK2 selective. ROCK-2 inhibitors promote weight loss in normal subjects, and limit weight gain in subjects prone to obesity.
[0071] In an embodiment of the invention, a ROCK inhibitor is used to reduce or prevent insulin resistance or restore insulin sensitivity. Accordingly, in one embodiment, the compounds of the invention are used to promote or restore insulin-dependent glucose uptake. In another embodiment of the invention, a ROCK-inhibitors of the invention is used to promote or restore glucose tolerance. In another embodiment of the invention, a ROCK inhibitor of the invention is used to treat metabolic syndrome. In another embodiment, a ROCK- inhibitors of the invention is used to reduce or prevent hyperinsulinemia. In an embodiment of the invention, a ROCK inhibitor is used to treat diabetes (particularly type 2 diabetes). ROCK inhibitors of the invention may also be used to promote or restore insulin-mediated relaxation of vascular smooth muscle cells (VSMCs). In preferred embodiments, the ROCK inhibitor is ROCK2 selective.
[0072] In certain embodiments, compounds of the invention are used for treatment of central nervous system disorders. Such disorders may involve neuronal degeneration or physical injury to neural tissue, including without limitation, Huntington's disease, Parkinson's Disease, Alzheimer's, Amyotrophic lateral sclerosis (ALS), or multiple sclerosis. In certain embodiments, compounds of the invention have properties particularly useful for treatment of such disorders, such as beneficial tissue distribution to tissues of the central nervous system, and ability to cross the blood brain barrier.
[0073] The invention provides pan-ROCK inhibitors (i.e., compounds that inhibit ROCK1 and ROCK1) as well as ROCK inhibitors that are isoform selective. As discussed above, in certain embodiments of the invention, a ROCK2-selective inhibitor may be preferred. For example, one study observed that ROCK2 is frequently over expressed in hepatocellular cancer compared to non-timorous livers while ROCK1 expression is unaltered. Other cancers which may benefit from treatment with a ROCK2 selective inhibitor include, but are not limited to, colon and bladder cancer. In contrast, ROCK1 expression levels have been observed to be higher in mammary tumors. Any cancer may be tested to determine whether there is overexpression of ROCK1 and/or ROCK2 and treated accordingly. In certain circumstances, ROCK 1 and ROCK2 isoforms show similarity in regulating certain downstream targets.
[0074] In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the compounds of Formula I, Formula II, Formula III, or Formula IV formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.
[0075] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals with toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
[0076] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.
[0077] Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 percent to about 30 percent.
[0078] Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
[0079] When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier. [0080] The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred.
[0081] The phrases“parenteral administration” and“administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
[0082] The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and“administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
[0083] These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
[0084] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
[0085] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
[0086] In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day.
[0087] In certain embodiments, a dose of a compound or a composition is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, a dose(s) of a compound or a composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days. In certain embodiments, a dose of a compound or a composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.
[0088] The above-described administration schedules are provided for illustrative purposes only and should not be considered limiting. A person of ordinary skill in the art will readily understand that all doses are within the scope of the invention.
[0089] While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
[0090] The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
[0091] The compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
[0092] Microemulsification technology may be employed to improve bioavailability of lipophilic (water insoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo, S. K., et al, Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991) and REV 5901 (Sheen, P. C, et al, J Pharm Sci 80(7), 712-714, 1991). Among other things, microemulsification provides enhanced bioavailability by preferentially directing absorption to the lymphatic system instead of the circulatory system, which thereby bypasses the liver, and prevents destruction of the compounds in the hepatobiliary circulation.
[0093] In one aspect of invention, the formulations contain micelles formed from a compound of the present invention and at least one amphiphilic carrier, in which the micelles have an average diameter of less than about 100 nm. More preferred embodiments provide micelles having an average diameter less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter less than about 30 nm, or even less than about 20 nm.
[0094] While all suitable amphiphilic carriers are contemplated, the presently preferred carriers are generally those that have Generally-Recognized-as-Safe (GRAS) status, and that can both solubilize the compound of the present invention and microemulsify it at a later stage when the solution comes into a contact with a complex water phase (such as one found in human gastro-intestinal tract). Usually, amphiphilic ingredients that satisfy these requirements have HLB (hydrophilic to lipophilic balance) values of 2-20, and their structures contain straight chain aliphatic radicals in the range of C-6 to C-20. Examples are polyethylene-glycolized fatty glycerides and polyethylene glycols.
[0095] Particularly preferred amphiphilic carriers are saturated and monounsaturated polyethyleneglycolyzed fatty acid glycerides, such as those obtained from fully or partially hydrogenated various vegetable oils. Such oils may advantageously consist of tri-. di- and mono fatty acid glycerides and di- and mono-polyethyleneglycol esters of the corresponding fatty acids, with a particularly preferred fatty acid composition including capric acid 4-10, capric acid 3-9, lauric acid 40-50, myristic acid 14-24, palmitic acid 4-14 and stearic acid 5-15%. Another useful class of amphiphilic carriers includes partially esterified sorbitan and/or sorbitol, with saturated or mono-unsaturated fatty acids (SPAN-series) or corresponding ethoxylated analogs (TWEEN-series).
[0096] Commercially available amphiphilic carriers are particularly contemplated, including Gelucire-series, Labrafil, Labrasol, or Lauroglycol (all manufactured and distributed by Gattefosse Corporation, Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono- laurate and di-laurate, Lecithin, Polysorbate 80, etc. (produced and distributed by a number of companies in USA and worldwide). [0097] Hydrophilic polymers suitable for use in the present invention are those which are readily water-soluble, can be covalently attached to a vesicle-forming lipid, and which are tolerated in vivo without toxic effects (i.e., are biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolic acid copolymer, and polyvinyl alcohol. Preferred polymers are those having a molecular weight of from about 100 or 120 daltons up to about 5,000 or 10,000 daltons, and more preferably from about 300 daltons to about 5,000 daltons. In a particularly preferred embodiment, the polymer is polyethyleneglycol having a molecular weight of from about 100 to about 5,000 daltons, and more preferably having a molecular weight of from about 300 to about 5,000 daltons. In a particularly preferred embodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)). The polymers used in the present invention have a significantly smaller molecular weight, approximately 100 daltons, compared to the large MW of 5000 daltons or greater that used in standard pegylation techniques. Polymers may also be defined by the number of monomers therein; a preferred embodiment of the present invention utilizes polymers of at least about three monomers, such PEG polymers consisting of three monomers (approximately 150 daltons).
[0098] Other hydrophilic polymers which may be suitable for use in the present invention include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
[0099] In certain embodiments, a formulation of the present invention comprises a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses, polypropylene, polyethylenes, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.
[00100] The release characteristics of a formulation of the present invention depend on the encapsulating material, the concentration of encapsulated drug, and the presence of release modifiers. For example, release can be manipulated to be pH dependent, for example, using a pH sensitive coating that releases only at a low pH, as in the stomach, or a higher pH, as in the intestine. An enteric coating can be used to prevent release from occurring until after passage through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach, followed by later release in the intestine. Release can also be manipulated by inclusion of salts or pore forming agents, which can increase water uptake or release of drug by diffusion from the capsule. Excipients which modify the solubility of the drug can also be used to control the release rate. Agents which enhance degradation of the matrix or release from the matrix can also be incorporated. They can be added to the drug, added as a separate phase (i.e., as particulates), or can be co-dissolved in the polymer phase depending on the compound. In all cases the amount should be between 0.1 and thirty percent (w/w polymer). Types of degradation enhancers include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine and surfactants such as Tween® and Pluronic®. Pore forming agents which add microstructure to the matrices (i.e., water soluble compounds such as inorganic salts and sugars) are added as particulates. The range should be between one and thirty percent (w/w polymer).
[00101] Uptake can also be manipulated by altering residence time of the particles in the gut. This can be achieved, for example, by coating the particle with, or selecting as the encapsulating material, a mucosal adhesive polymer. Examples include most polymers with free carboxyl groups, such as chitosan, celluloses, and especially polyacrylates (as used herein, polyacrylates refers to polymers including acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates).
[00102] The above-described administration schedules are provided for illustrative purposes only and should not be considered limiting. A person of ordinary skill in the art will readily understand that all doses are within the scope of the invention.
[00103] Compounds of the invention can be advantageously administered with second agents to patients in need thereof. When a rho-kinase inhibitor is administered with a second agent, the rho-kinase inhibitor and the second agent can be administered sequentially or concomitantly. Sequentially means that one agent is administered for a time followed by administration of the second agent, which may be followed by administration of the first agent. When agents are administered sequentially, the level or one agent may not be maintained at a therapeutically effective level when the second agent is administered, and vice versa. Concomitantly means that the first and second agent are administered according to a schedule that maintains both agents at an substantially therapeutically effective level, even though the agents are not administered simultaneously. Each agent can be administered in single or multiple doses, and the doses can be administered on any schedule, including, without limitation, twice daily, daily, weekly, every two weeks, and monthly.
[00104] The invention also includes adjunctive administration. Adjunctive administration means that a second agent is administered to a patient in addition to a first agent that is already being administered to treat a disease or disease symptom. In some embodiments, adjunctive administration involves administering a second agent to a patient in which administration of the first agent did not sufficiently treat a disease or disease symptom. In other embodiments, adjunctive administration involves administration of the second agent to a patient whose disease has been effectively treated by administration of the first agent, with the expectation that the adjunctive treatment improves the outcome of the treatment. In some embodiments, the effect of administering the first and second agents is synergistic. In some embodiments, administration of the first and second agents prevents or lengthens the time until relapse, compared to administration of either of the agents alone. In some embodiments, administration of the first and second agents allows for reduced dosage and/or frequency of administration of the first and second agent.
[00105] In an embodiment of the invention, a rho-kinase inhibitor of the invention is administered and an anti -neoplastic agent are administered to a subject in need thereof. In another embodiment, a rho-kinase inhibitor of the invention and an angiogenesis inhibitor are administered to a subject in need thereof. In another embodiment, a rho-kinase inhibitor of the invention and an anti -inflammatory agent are administered to a subject in need thereof. In yet another embodiment, a rho-kinase inhibitor of the invention and an immunosuppressant are administered. The second agent can be, without limitation, a small molecule, an antibody or antigen binding fragment thereof, or radiation.
[00106] Antineoplastic agents include, without limitation, cytotoxic chemotherapeutic agents, targeted small molecules and biological molecules, and radiation. Compounds and agents that can be administered for oncological treatment, in addition to a rho kinase inhibitor of the invention, include the following: irinotecan, etoposide, camptothecin, 5-fluorouracil, hydroxyurea, tamoxifen, paclitaxel, capcitabine, carboplatin, cisplatin, bleomycin, dactomycin, gemcitabine, doxorubicin, danorubicin, cyclophosphamide, and radiotherapy, which can be external ( e.g ., external beam radiation therapy (EBRT)) or internal (e.g., brachytherapy (BT)).
[00107] Targeted small molecules and biological molecules include, without limitation, inhibitors of components of signal transduction pathways, such as modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor-specfic antigens. Examples include inhibitors of epidermal growth factor receptor (EGFR), including gefitinib, erlotinib, and cetuximab, inhibitors of HER2 (e.g., trastuzumab, trastuzumab emtansine (trastuzumab-DMl; T-DM1) and pertuzumab), anti-VEGF antibodies and fragments (e.g., bevacizumab), antibodies that inhibit CD20 (e.g., rituximab, ibritumomab), anti-VEGFR antibodies (e.g., ramucirumab (IMC- 112 IB), IMC-1C1 1, and CDP791), anti-PDGFR antibodies, and imatinib. Small molecule kinase inhibitors can be specific for a particular tyrosine kinase or be inhibitors of two or more kinases. For example, the compound N-(3,4-dichloro-2- fluorophenyl)-7-({[(3aR,6aS)-2-methyloctahydrocyclopenta[c] pyrrol-5-yl]methyl}oxy)-6- (methyloxy)quinazolin-4-amine (also known as XL647, EXEL-7647 and KD-019) is an in vitro inhibitor of several receptor tyrosine kinases (RTKs), including EGFR, EphB4, KDR (VEGFR), Flt4 (VEGFR3) and ErbB2, and is also an inhibitor of the SRC kinase, which is involved in pathways that result in nonresponsiveness of tumors to certain TKIs. In an embodiment of the invention, treatment of a subject in need comprises administration of a rho-kinase inhibitor of Formula I and administration of KD-019.
[00108] Dasatinib (BMS-354825; Bristol-Myers Squibb, New York) is another orally bioavailable, ATP-site competitive Src inhibitor. Dasatanib also targets Bcr-Abl (FDA-approved for use in patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL)) as well as c-Kit, PDGFR, c-FMS, EpbA2, and Src family kinases. Two other oral tyrosine kinase inhibitor of Src and Bcr-Abl are bosutinib (SKI-606) and saracatinib (AZD0530).
[00109] According to the invention, angiogenesis inhibitors can be administered to a subject in conjunction with compounds of the invention. Angiogenesis inhibitors include any substance that inhibits the growth of new blood vessels. For example, angiogenesis inhibitors include antagonists of VEGF, P1GF, and VEGF receptors, including the antibodies disclosed herein. By inhibitor is meant an inhibitor of a biological process or inhibitor of a target. In this regard, an angiogenesis inhibitor is an agent that reduces angiogenesis. A Rho-kinase inhibitor is an agent, such as a competitive inhibitor of ATP binding, that inhibits an intrinsic activity or blocks an interaction of Rho-kinase. By antagonist is meant a substance that reduces or inhibits an activity or function in a cell associated with a target. For example, a VEGF antagonist reduces or blocks a function in a cell that is associated with VEGF. A VEGF antagonist may act on VEGF, by binding to VEGF and blocking binding to its receptors and/or may act on another cellular component involved in VEGF-mediated signal transduction. Similarly, a VEGFR2 antagonist is an agent that reduces or blocks VEGFR2 -mediated signal transduction by binding to VEGFR2 and blocking ligand binding or interaction with a VEGFR2 substrate, or acts on another cellular component to reduce or block VEGFR2 -mediated signal transduction. Thus, angiogenesis inhibitors include antagonists of, without limitation, VEGF, VEGFRl, VEGFR2, PDGF, PDGFR-b, neuropilin-1 (NRP1), and complement.
[00110] Non-limiting examples of VEGF -binding agents include VEGF antibodies and VEGF traps (i.e., ligand binding domains of VEGF receptors. In general, a VEGF trap is a protein that comprises VEGF binding domains of one or more VEGF receptor protein. VEGF- traps include, without limitation, soluble VEGFR- 1, soluble neuropilin 1 (NRPl), soluble VEGFR-3 (which binds VEGF-C and VEGF-D), and aflibercept (Zaltrap; Eyelea; VEGF Trap R1R2), comprised of segments of the extracellular domains of human vascular endothelial growth factor receptors VEGFRl and VEGFR2 fused to the constant region (Fc) of human IgGl . Conbercept (KH902) is a fusion protein which contains the extracellular domain 2 of VEGFR- 1 (Fit- 1 ) and extracellular domain 3, 4 of VEGFR-2 (KDR) fused to the Fc portion of human IgGl. Several VEGF traps containing KDR and FLT-1 Ig-like domains in various combinations are disclosed in U.S. Patent 8,216,575. DARPins (an acronym for designed ankyrin repeat proteins) are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. DARPin® MP0112 is a vascular endothelial growth factor (VEGF) inhibitor and has entered clinical trials for the treatment of wet macular degeneration and diabetic macular edema.
[00111] According to the invention, VEGF expression can be targeted. For example, VEGF inhibitor PTC299 targets VEGF post-transcriptionally by selectively binding the 5'- and 3 '- untranslated regions (UTR) of VEGF messenger RNA (mRNA), thereby preventing translation of VEGF. Pegaptanib (Macugen) is an RNA aptamer directed against VEGF- 165.
[00112] Placental growth factor (PIGF) has been implicated in pathological angiogenesis. PIGF is structurally related to VEGF and is also a ligand for VEGFR-l. Consequently, VEGF traps comprising the extracellular domain of VEGFRl (see above) are useful for targeting PIGF.
[00113] PDGF is composed of four polypeptide chains that form homodimers PDGF-AA, BB, CC, and DD as well as the heterodimer PDGF-AB. The PDGF receptors (PDGFR) -a and -b mediate PDGF functions. Specifically, PDGFRa binds to PDGF-AA, -BB, -AB, and -CC, whereas PDGFRP interacts with -BB and -DD. Non- limiting examples of PDGF -binding agents include anti-PDGF antibodies and PDGF traps. Agents that target PDGF include Fovista™ (E10030, Ophthotech), a pegylated aptamer targeting PDGF-B, and AX102 (Sennino et al., 2007, Cancer Res. 75(15):7359-67), a DNA oligonucleotide aptamer that binds PDGF-B.
[00114] Agents that target PDGF receptors include ramucirumab (IMC-3G3, human IgGi) an anti-PDGFRa antibody, crenolanib (CP-868596), a selective inihibitor of PDGFRa (IC50 = 0.9 nM) and PDGFRp (IC5o =1.8 nM), and nilotinib (Tasigna®), an inhibitor of PDGFRa and PDGFRP and other tyrosine kinases.
[00115] Angiogenesis inhibitors include intracellular agents that block signal transduction mediated by, for example, VEGF, PDGF, ligands of VEGF or PDGF receptors, or complement. Intracellular agents that inhibit angiogenesis inhibitors include the following, without limitation. Sunitinib (Sutent; SU1 1248) is a panspecific small-molecule inhibitor of VEGFRl- VEGFR3, PDGFRa and PDGFRp, stem cell factor receptor (cKIT), Flt-3, and colony- stimulating factor- 1 receptor (CSF-1R). Axitinib (AG013736; Inlyta) is another small molecule tyrosine kinase inhibitor that inhibits VEGFR- 1 - VEGFR-3, PDGFR, and cKIT. Cediranib (AZD2171) is an inhibitor of VEGFR- 1- VEGFR-3, PDGFRp, and cKIT. Sorafenib (Nexavar) is another small molecular inhibitor of several tyrosine protein kinases, including VEGFR, PDGFR, and Raf kinases. Pazopanib (Votrient; (GW786034) inhibits VEGFR- 1, -2 and -3, cKIT and PDGFR. Foretinib (GSK1363089; XL880) inhibits VEGFR2 and MET. CP-547632 is as a potent inhibitor of the VEGFR-2 and basic fibroblast growth factor (FGF) kinases. E-3810 ((6-(7-((l-aminocyclopropyl) methoxy)-6-methoxyquinolin-4-yloxy)-N-methyl- 1 -naphthamide) inhibits VEGFR- 1, -2, and -3 and FGFR-1 and -2 kinases in the nanomolar range. Brivanib (BMS-582664) is a VEGFR-2 inhibitor that also inhibits FGF receptor signaling. CT-322 (Adnectin) is a small protein based on a human fibronectin domain and binds to and inhibits activation of VEGFR2. Vandetanib (Caprelas; Zactima; ZD6474) is an inhibitor of VEGFR2, EGFR, and RET tyrosine kinases. X-82 (Xcovery) is a small molecule indolinone inhibitor of signaling through the growth factor receptors VEGFR and PDGFR.
[00116] Immune checkpoint antagonist therapies have been developed to enable a patient's own immune system to fight tumors by facilitating an existing immune response or allowing for the initiation of an immune response. These therapies have been shown to be effective at treating some cancers in some subjects. Three immune checkpoint targets are the subject of most on going clinical work: Programmed cell death protein 1 (PD-1), its ligands, PD-L1, cytotoxic T- lymphocyte-associated protein 4 (CTLA-4), lymphocyte-activation gene 3 (LAG-3), and cluster of differentiation 276 gene (CD276 also known as B7-H3). More recently, a new checkpoint target, GARP, has been investigated. Currently, a number of checkpoint antagonists are being evaluated in clinical trials. Non-limiting examples of these antagonists include fully human and humanized anti-PD-1 monoclonal antibodies (e.g., nivolumab, pembrolizumab), an anti-PD-Ll antibody (e.g., durvalumab, atezolizumab, MEDI4736), a fusion protein comprising PD-L2 extracellular domain and lgGl, an anti-CTLA-4 antibody (e.g., tremelimumab, ipilimumab), a CD276 inhibitor (e.g., enoblituzumab; pidilizumab, MGD009) and an antibody to GARP (ARGX-1 15).
[00117] In some aspects, the method comprises administering one or more checkpoint antagonist(s), the one or more checkpoint antagonist(s) is a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, and/or a PD-L2 antagonist.
[00118] In some aspects, the method comprises administering more than one checkpoint antagonist, each checkpoint antagonist is independently selected from a PD-1 antagonist, a PD- L1 antagonist, a CTLA-4 antagonist, and/or a PD-L2 antagonist.
[00119] In some aspects, the administration of the checkpoint antagonist comprises administering:
[00120] a. a first checkpoint antagonist selected from a PD-1 antagonists, a PD- LI antagonists, or PD-L2 antagonists; and a second checkpoint antagonist selected from CTLA-4 antagonists; or
[00121] b. a first checkpoint antagonist selected from a PD-1 antagonists, and a second checkpoint antagonist selected from a PD-L1 and PD-L2 antagonists. [00122] In some aspects, the PD-1 antagonist is selected from nivolumab, pembrolizumab, and AMP-224.
[00123] In some aspects, the PD-L1 antagonist is selected from MDX-1 105, atezolizumab, and durvalumab.
[00124] In some aspects, the CTLA-4 antagonist is selected from ipulimumab and tremelimumab.
[00125] In some aspects, the checkpoint antagonist is pidilizumab.
[00126] Anti-inflammatories and immunosuppressants include steroid drugs such as glucocorticoids (e.g., dexamethasone), FK506 (tacrolimus), ciclosporin, fmgolimod, interferon, such as IFNP or IFNy, a tumor necrosis factor-alpha (TNF-a) binding protein such as infliximab (Remicade), etanercept (Enbrel), or adalimumab (Humira), and mycophenolic acid.
[00127] In certain embodiments, ROCK inhibitors of the invention are coadministered with agents used to treat metabolic disorders. For example, for treatment of obesity, the ROCK inhibitors may be combined with weight loss drugs such as, but not limited to, phentermine, fat adsorption inhibitors (e.g., Xenical), appetite suppressants, and the like. Procedures used to assist weight loss include, for example, stomach bands, stomach bypass or stapling. For insulin resistance or metabolic syndrome or hyperinsulinemia, ROCK inhibitors of the invention can be coadministered with compounds that lower cholesterol levels, for example, one or more medicines such as statins, fibrates, or nicotinic acid. For high blood pressure associated with such diseases, ROCK inhibitors of the invention can be coadministered with, for example, one or more antihypertensive medicines such as diuretics or angiotensin-converting enzyme (ACE) inhibitors. ROCK inhibitors of the invention can be administered in a treatment program that includes lifestyle changes such as increased physical activity, an improved diet, and/or quitting smoking. In certain embodiments, the ROCK inhibitor is ROCK2 selective.
[00128] Thl7 cells are novel subset of helper CD4+ T cells that secrete IL-17, IL-21 and IL- 22. The pro-inflammatory activity of Thl7 cells can be beneficial to the host during infection, but uncontrolled Thl7 function has been linked and actively involved in several autoimmune pathologies. Indeed, high levels of IL-17 are detected in the sera and biopsies of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients which correlates with destruction of synovial tissue and disease activity. The pathological role of IL-17 in arthritic joints is associated with its stimulation of pro -inflammatory cytokine production and increased recruitment of T cells and innate immune cells. Moreover, numbers of Thl7 cells are significantly increased in the peripheral blood of RA patients as well as elevated concentrations of IL-17 were seen in supernatants of their PBMCs after stimulation with anti-CD3/CD28 antibodies ex vivo. In addition, in multiple sclerosis (MS) patients, myelin reactive Thl7 cells are also enriched and produce high amounts of IL-22 and IFN-g. Further, a significantly higher number of IL-17+ cells is detected in disease-affected gut areas compared to healthy areas of the same subjects with Crohn's disease (CD).
[00129] The development and function of Thl7 cells depends on activation of specific intracellular signaling pathways. The steroid receptor-type nuclear receptor RORyt is selectively expressed in Thl7 cells and appears to be required for IL-17 production. The induction of RORyt has been observed to be mediated by IL-6, IL-21 and IL-23 via a STAT3 -dependent mechanism. STAT3 also binds directly to the IL-17 and IL-21 promoters. In addition to RORyt and STAT3, the interferon regulatory factor 4 (IRF4) is required for the differentiation of Thl7 cells since IRF4 KO mice failed to mount Thl7 response and were resistant to development of autoimmune responses. Recent studies have demonstrated that phosphorylation of IRF4 by Rho-kinase 2 (ROCK2) regulates IL-17 and IL-21 production and development of autoimmunity in mice.
[00130] According to the invention, targeting Thl7 (IL-17-secreting) cells by rho-kinase inhibition provides a method for treating Thl7 cell-mediated diseases, including but not limited to autoimmune disorders such as rheumatoid arthritis (RA) multiple sclerosis (MS), systemic lupus erythematosus (SLE), psoriasis, Crohn's disease, atopic dermatitis, eczema, and GVHD in humans. In an embodiment of the invention, the Rho-kinase inhibitor is a compound of Formula I. In some embodiments, the rho-kinase inhibitor inhibits ROCK1 and ROCK2. In some embodiments, the rho-kinase inhibitor selectively inhibits ROCK2. Selective inhibition of ROCK2 provides for treatment of Thl7 cell-mediated diseases and reduces or prevents toxicities associated with complete inhibition of ROCK activity.
[00131] Regulatory T cells (Tregs) play a critical role in the maintenance of immunological tolerance to self-antigens and inhibition of autoimmune responses, but, at the same time, prevent an effective immune response against tumor cells. Indeed, Tregs isolated from the peripheral blood of patients with autoimmune disease, such as rheumatoid arthritis (RA) and multiple sclerosis (MS), show a defect in their ability to suppress effector T cell function, while increased accumulation of Tregs correlates with a poor prognosis in many cancers. Thus, the level of Treg function effects a balance between effective immunity and avoidance of pathological autoreactivity.
[00132] The development and function of Tregs depend on activation of specific signaling transduction pathways. TGF-b and IL-2 activate expression of Foxp3 and STAT5 transcription factors that both play an essential role in the control of Treg suppressive function. On the other hand, pro-inflammatory cytokines inhibit Foxp3 expression via up-regulation of STAT3 phosphorylation. As shown herein, pharmacological inhibition of ROCK2 (e.g., with selective ROCK2 inhibitors such as KD025, ROCK2-specific siRNA-mediated inhibition of ROCK2), but not ROCK1, leads to down-regulation of STAT3 phosphorylation, interferon regulatory factor 4 (IRF4) and steroid receptor-type nuclear receptor RORyt protein levels in human T cells.
[00133] Furthermore, as demonstrated herein, targeting ROCK2 with a selective inhibitor (e.g., KD025) leads to an increased proportion of Foxp3+ T cells via a STAT5 -dependent mechanism and positively regulates their suppressive activity towards autoreactive lymphocytes. This effect of ROCK2 inhibition on Tregs is critical to limiting or preventing the onset of aberrant self-immune responses. This effect may be shown, for example, by assaying the ability of Tregs treated with a ROCK2 inhibitor to inhibit proliferation and cytokine secretion in target cells in vitro.
[00134] Accordingly, ROCK2 inhibitors of the invention effectively reduce or prevent chronic GVHD pathologies. As shown herein, the compounds of the invention reduce or prevent cGVHD pathologies in target organs. For example, a selective ROCK2 inhibitor administered to a transplant patient maintains or restores pulmonary function. The maintenance or restoration of pulmonary function correlates with reduced germinal center activity which would otherwise lead to production of autoreactive antibodies, and with reduced collagen deposition and antibody deposition in affected tissues. These pathologies are common to other targets of cGVHD as well, e.g, skin, gut, and liver, which would be similarly reduced or prevented.
[00135] It is to be understood and expected that variations in the principles of invention herein disclosed may be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention.
[00136] Throughout this application, various publications are referenced. These publications are hereby incorporated into this application by reference in their entireties to more fully describe the state of the art to which this invention pertains. The following examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way.
EXAMPLES
[00137] Table 1. Non-limiting examples of kinase inhibitor compounds. Unless specified, example compounds with a chiral center represent racemic mixture of the corresponding R and S enantiomers and all racemates and isolated enantiomers are within the scope of the invention.
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
[00138] Additional non-limiting examples are:
[00139] The compounds of the present invention demonstrate improved ROCK enzyme inhibiting activity. In addition, the compounds demonstrate superior pharmacokinetic properties. As points of reference, two previously characterized compounds (i.e., Compounds A and B in Table 2) were analyzed. Additionally, the compounds disclosed herein generally demonstrate greater ROCK inhibition than Ripasudil, a clinical ROCK inhibitor, with relatively weak activity (ROCK1 IC50 = 51 nM and ROCK2 IC50 = 19 nM).
[00140] PREPARATION OF COMPOUNDS
[00141] EXAMPLES
[00142] The disclosure is further illustrated by the following examples and synthesis examples, that are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and/or scope of the appended claims.
[00143] Example 1.
Figure imgf000055_0001
[00146] 1A
[00147] To a solution of 4-(pyridin-4-yl)benzoic acid (100 mg, 0.50 mmol) and methyl 3- (aminomethyl)benzoate (99 mg, 0.6 mmol) in DMF were added diisopropylethylamine (DIEA,
O.26 mL, 1.5 mmol) and l-[Bis(dimethylamino)methylene]-li7-l,2,3-triazolo[4,5-/>]pyridinium 3-oxid hexafluorophosphate (HATU, 247 mg, 0.65 mmol). The reaction was stirred at room temperature for 2 hrs and diluted with water. The mixture was concentrated and purified by C-18 column chromatography to give 1A (141 mg).
[00148] Step 2.
Figure imgf000056_0001
[00149] IB
[00150] To a solution of 1A (141 mg, 0.41 mmol) in THF was added LiOH solution (1 M, 2.0 mL). The reaction was stirred at room temperature overnight and neutralized with HC1 solution. The mixture was concentrated and purified by C-18 column chromatography to give IB (97 mg).
[00151] Step 3.
[00152] To a solution of IB (7 mg, 0.02 mmol) and 5-methyl-4,5,6,7-tetrahydropyrazolo[l,5- a]pyrazin-2-amine (6 mg, 0.04 mmol) in DMF were added diisopropylethylamine (DIEA, 0.007 mL, 0.04 mmol) and HATU (15 mg, 0.04 mmol). The reaction was stirred at room temperature for 2 hrs and diluted with water. The mixture was concentrated and purified by C-18 column chromatography to give ID# 1 (8 mg).
[00153] Example 2.
Figure imgf000056_0002
[00156] 2A
[00157] To a solution of 4-(pyridin-4-yl)benzoic acid (50 mg, 0.25 mmol) and methyl 3-((lS)- 1 -amino-2 -hydroxy ethyl )benzoate HC1 salt (75 mg, 0.3 mmol) in DMF were added diisopropylethylamine (DIEA, 0.13 mL, 0.75 mmol) and HATU (123 mg, 0.32 mmol). The reaction was stirred at room temperature for 2 hrs and diluted with water. The mixture was concentrated and purified by C-18 column chromatography to give 2 A (68 mg).
[00158] Step 2.
Figure imgf000057_0001
[00159] 2B
[00160] To a solution of 1A (68 mg, 0.17 mmol) in THF was added LiOH solution (1 M, 0.85 mL). The reaction was stirred at room temperature overnight and neutralized with HC1 solution. The mixture was concentrated and purified by C-18 column chromatography to give 2B (40 mg).
[00161] Step 3.
[00162] To a solution of 2B (5 mg, 0.014 mmol) and cyclopentylamine (3 mg, 0.035 mmol) in DMF were added diisopropylethylamine (DIEA, 5 microL, 0.028 mmol) and HATU (7 mg, 0.018 mmol). The reaction was stirred at room temperature for 2 hrs and diluted with water. The mixture was concentrated and purified by C-18 column chromatography to give ID#2 (5 mg).
[00163] Example 3.
Figure imgf000057_0002
[00164] ID#3
[00165] Step 1.
Figure imgf000057_0003
[00166] 3A
[00167] To a solution of 4-(pyridin-4-yl)benzoic acid (50 mg, 0.25 mmol) and 3- (aminomethyl)phenol (37 mg, 0.3 mmol) in DMF were added diisopropylethylamine (DIEA, 0.13 mL, 0.75 mmol) and HATU (123 mg, 0.32 mmol). The reaction was stirred at room temperature for 2 hrs and diluted with water. The mixture was concentrated and purified by C-18 column chromatography to give 3 A (62 mg).
[00168] Step 2.
Figure imgf000058_0001
[00169] 3B
[00170] To a mixture of 3A (62 mg, 0.20 mmol) and ethyl chloroacetate (25 mg, 0.20 mmol) in acetonitrile was added potassium carbonate (33 mg, 0.24 mmol). The reaction was heated to 55 C, stirred overnight, filtered and concentrated. The residue was purified by C-18 column chromatography to give 3B (55 mg).
[00171] Step 3.
Figure imgf000058_0002
[00172] 3C
[00173] To a solution of 3B (55 mg, 0.14 mmol) in THF was added LiOH solution (1 M, 0.7 mL). The reaction was stirred at room temperature overnight and neutralized with HC1 solution. The mixture was concentrated and purified by C-18 column chromatography to give 3C (50 mg).
[00174] Step 4.
[00175] To a solution of 3C (10 mg, 0.028 mmol) and cyclopropylamine (5 mg, 0.084 mmol) in DMF was added HATU (13 mg, 0.034 mmol). The reaction was stirred at room temperature for 2 hrs and diluted with water. The mixture was concentrated and purified by C-18 column chromatography to give ID#3 (10 mg).
[00176] Example 4 - ROCK1 and ROCK2 Kinase Inhibition Assays
[00177] The following assay protocol is for measuring the phosphorylation of a peptide substrate (FAM-KKLRRTLSVA-OH wherein FAM is carboxyfluorescein). The peptide is >98% purity by Capillary Electrophoresis. The peptide is phosphorylated by the protein kinase ROCK1 or ROCK2. The ROCK1 or ROCK2 enzyme, substrate, and cofactors (ATP and Mg2+) are combined in a well of a microtiter plate and incubated for 3 hours at 25°C. At the end of the incubation, the reaction is quenched by the addition of an EDTA-containing buffer. The substrate and product are separated and quantified electrophoretically using the microfluidic-based LABCHIP® 3000 Drug Discovery System from Caliper Life Sciences (Hopkinton, Massachusetts).
[00178] The components of the assay mixture are:
[00179] 100 mM HEPES, pH 7.5
[00180] 0.1% BSA
[00181] 0.01% Triton X-100
[00182] 1 mM DTT
[00183] 10 mM MgCl2
[00184] 10 mM Sodium Orthovanadate
[00185] 10 mM Beta-Glycerophosphate
[00186] 5 pM ATP (for ROCK1) or 7 pM ATP (for ROCK2)
[00187] 1% DMSO (from compound)
[00188] 1.25 pM FAM-KKLRRTLSVA-OH
[00189] 3 nM ROCK1 or 2.5 nM ROCK2 enzyme
[00190] Substrate and product peptides present in each sample are separated electrophoretically using the LABCHIP® 3000 capillary electrophoresis instrument. As substrate and product peptides are separated two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks is the parameter measured reflecting enzyme activity. Capillary electrophoregramms (RDA acquisition files) are analyzed using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Massachusetts). The kinase activity in each sample is determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product peptide and S is the peak height of the substrate peptide. For each compound, enzyme activity is measured at various concentrations (12 concentrations of compound spaced by 3X dilution intervals). Negative control samples (0%- inhibition in the absence of inhibitor) and positive control samples (100%-inhibition in the presence of 20 mM EDTA) are assembled in replicates of four and are used to calculate %-inhibition values for each compound at each concentration. Percent inhibition (Pinh) is determined using the following equation:
[00191] Pinh = (PSR0% - PSRinh)/(PSR0% - PSR100%)* 100
[00192] where PSRinh is the product sum ratio in the presence of inhibitor, PSR0% is the average product sum ratio in the absence of inhibitor, and PSR100% is the average product sum ratio in 100%-inhibition control samples. The IC50 values of inhibitors are determined by fitting the inhibition curves (Pinh versus inhibitor concentration) by 4 parameter sigmoidal dose- response model using XLfit 4 software (IBDS).
[00193] This assay can be used to test the activity of each of the exemplary compounds identified in Table 2. It is expected that each of these compounds will demonstrate inhibition of the protein kinase ROCK1 and/or ROCK2.
[00194] Example 5. Cell Viability Assay
[00195] Cell viability in the presence of varying concentrations of the above listed compounds at different time points was used to assess cytotoxicity and the effect of the compounds on cell proliferation. IC50 (or percent activity) data for the compounds of the present invention in K562 or MV411 cell lines are summarized in Table 2.
[00196] Cell Viability Assay- Cell viability was measured by the CELLTITER-GIO® cell viability assay from Promega (Madison, WI). The CELLTITER-GIO® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. Following treatment, CELLTITER-GIO® is added to treatment wells and incubated at 37° C. luminescence values were measured at using a Molecular Devices Spectramax microplate reader.
[00197] Experimental Design
[00198] Single Agent Studies- Cells were grown to 70% confluency, trypsinized, counted, and seeded in 96 well flat-bottom plates at a final concentration of 2.5xl03-5xl03 cells/well (Day 0). Cells were allowed to incubate in growth media for 24 hours. Treatment with the test agents or standard agents began on Day 1 and continued for 72 hours. At the 72-hour time point, treatment containing media was removed. Viable cell numbers were quantified by the CELLTITER-GLO® cell viability assay as described above. Results from these studies were used to calculate an IC50 value (concentration of drug that inhibits cell growth by 50 percent of control) for each compound.
[00199] Data Collection- For single agent and combination studies, data from each experiment was collected and expressed as % Cell Growth using the following calculation:
[00200] % Cell Growth = ( fvehide) x 100
[00201] Where ftest is the luminescence of the tested sample, and fvehicie is the luminescence of the vehicle in which the drug is dissolved. Dose response graphs and IC50 values were generated using Prism 6 software (GraphPad) using the following equation:
[00202] Y = (Top-BottomV(l+l 0((logIC5° X) Hillslope))
[00203] Where X is the logarithm of the concentration and Y is the response. Y starts at the Bottom and goes to the Top with a sigmoid shape.
[00204] Example 7. ROCK1 and ROCK2 Kinase Inhibition and Cell Viability Assay Results
[00205] The protocols outlined in Examples 5 and 6 were followed to test ROCK1 and ROCK2 kinase inhibition and cancer cell viability with compounds from Table 1. As shown in Table 2, the compounds demonstrated inhibition of the ROCK1 and ROCK2 kinases and growth of cancer cells.
[00206] The experiments also evaluated the selectivity of the compounds for inhibiting growth of cancer cells carrying a mutation in the Flt3 gene. The MV411 cell line expresses the mutant allele of Flt3 with internal tandem duplications (ITD) of the gene. See Quentmeier el al ., “FLT3 Mutations in Acute Myeloid Leukemia Cell Lines,” Leukemia 17(1), 2003, 120-124. K562 is a chronic myeloid leukemia cell line that does not express FLT3 protein. See Grafone el al .,“Monitoring of FLT3 Phosphorylation Status and Its Response to Drugs By Flow Cytometry in AML Blast Cells,” Hematol Oncol. 26(3), 2008, 159-166. Patients with IYT)-FLT3+ acute myeloid leukemia (AML) experience an extremely poor prognosis. Surprisingly, many of the compounds demonstrated greater efficacy with the MV11 cells than with the K562 cells suggesting that these compounds could be used to effectively treat ITO-FLT3+ AML. [00207] Table 2. ROCK 1 and ROCK2 kinase inhibition and cell viability
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0002
* MV411 is TTO-FLT3+, and K562 does not express ITO-FLT3. Rho-associated kinase may be manipulated for the treatment of YYD-FLT3+ AML as reported in Onish et al. ,“Internal Tandem Duplication Mutations in FLT3 Gene Augment Chemotaxis to Cxcll2 Protein by Blocking the Down-regulation of the Rho-associated Kinase via the Cxcll2/Cxcr4 Signaling Axis,” J. Biol. Chem. 289 (45), 2014, 31053-31065.
** Compound A is shown below and is described by Schirok et al.,“Design and Synthesis of Potent and Selective Azaindole-Based Rho Kinase (ROCK) Inhibitors,” ChemMedChem 3, 2008, 1893 - 1904.
*** Compound B is shown below and is described by Cook et al,“RHO KINASE
INHIBITORS”.
Figure imgf000064_0001
[00208] Compound A
Figure imgf000065_0001
[00209] Compound B
[00210] Example 8. Pharmacokinetic activity
[00211] Compounds were individually and accurately weighed out to produce a stock solution in Dimethyl-sulfoxide (DMSO) at a concentration of 2 mg/mL and stored at -20°C. From the stock solution, a working stock was prepared by diluting it in Methanol -Water 50:50 (v/v) at 20 pg/mL for calibration curve preparation and stored at 4°C. Standards used for quantitation and quality control samples (QC’s) were prepared on the same day of sample processing using blank plasma obtained from non-treated mice. For each analyte, standards were prepared through serial dilution of the working stock at concentrations of 0.1, 0.5, 1.0, 10, 50, 100, 500 and 1000 ng/mL; QC’s were prepared at intermediate concentrations of 0.75, 7.5, 75 and 750 ng/mL. Plasma samples were stored at -80°C until ready for analysis and then placed on ice for thawing. An aliquot of 20 pL for samples, standards and QC’s, were transferred into a 96-well extraction plate according to a pre-defmed layout. Appropriate volume of Methanol -Formic Acid 99.9-0.1 containing internal standard (Verapamil at 25 ng/mL) was added to each well and samples were extracted under vacuum. Eluent was then transferred into a LCMS plate for analysis using suitable column for separation and MRM detection. Concentration of analytes was calculated based on the calibration curve and analyzed for pharmacokinetic parameters by using Non- compartmental analysis (NCA). Parameters such as Cmax, Tmax, half-life, AUC (0-last), AUC (0-co), volume of distribution (Vss) and clearance (Cl/F) were reported.
[00212] Table 3. Pharmacokinetic activity of 7, 10 and 36 in mice
Figure imgf000066_0001

Claims

CLAIMS What is claimed is:
1. A compound of Formula (IV):
Figure imgf000067_0001
wherein:
Zi is -H, -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole, or - tetrazole, wherein the -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, - indazole, or -tetrazole is unsubstituted or substituted with one or more of the following: -halo, -
Figure imgf000067_0002
C(0)NR’R”, -NS(0)2R\ -S(0)2NR’R”, -S(0)2R\ -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, and a 3- to 10-membered heterocycle, and the -Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -Ci-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
Ri is -H, -Ci-C6 alkyl, or -C3-C7 cycloalkyl, wherein the -Ci-C6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, - COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR'R”, - NS(0)2R’, -S(0)2NR’R”, and -S(0)2R’;
R2 and R3 are independently -H, -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle
comprising 5 to 10 carbons,
Figure imgf000067_0003
Figure imgf000067_0004
wherein the -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, - CN, -CNR’R”, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, and a 3- to 11-membered heterocycle,
the -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 11-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, - CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl,
the R5 is -Ci-C6 alkyl, -OCH2CH2-, -NR6CH2CH2-, or -NC(0)CH2CH2-, and the 5 is -H, -Ci-C6 alkyl, or -C3-C7 cycloalkyl;
the R’ and R” are independently -H or -C1-C6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4- to 8- membered cyclic structure; and
Rv is -H, -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, - NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -Ci-C6 alkyl, - C3-C7 cycloalkyl, -aryl, or a heterocycle comprising 5 to 10 carbons, wherein the -C1-C6 alkyl, - C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with - -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, - C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle.
2. A compound of Formula (III):
Figure imgf000068_0001
III
wherein:
Zi is -H, -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole, or - tetrazole, wherein the -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, - indazole, or -tetrazole is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, - S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, - COOR’, -OR’, -SR’, -0C(0)R', -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, - NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
Z2 is -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the - phenyl, -naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R, -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR'R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, - Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, wherein the -Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, - guanidino, -nitro, -nitroso, -Ci.C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
Ri is -H, -C1-C6 alkyl, or -C3-C7 cycloalkyl, wherein the -C1-C6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, - COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, - NS(0)2R’, -S(0)2NR’R”, and -S(0)2R’;
R2 and R3 are independently -H, -C1-C6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle
Figure imgf000069_0001
wherein:
the -C1-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, - CN, -CNR’R”, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, and a 3- to 1 1-membered heterocycle,
the -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, - CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl,
the R5 is -Ci-C6 alkyl, -OCH2CH2-, -NR6CH2CH2-, or -NC(0)CH2CH2-, and the 5 is -H, -C1-C6 alkyl, or -C3-C7 cycloalkyl; and
the R’ and R” are independently -H or -Ci-C6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4- to 8- membered cyclic structure.
3. A compound of Formula (II):
Figure imgf000070_0001
II
wherein:
Zi is -H, -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole or - tetrazole, wherein the -pyridine, -pyrimidine, -pyrazole, -imidazole, -oxazole, -thiazole, -indazole or -tetrazole is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, - 0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, - S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or 3- to 10- membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, - OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
Z2 is -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the - phenyl, -naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, - Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -Ci-C6 alkyl, - aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, - guanidino, -nitro, -nitroso, -Ci-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
R is -H or -Ci-C6 alkyl, wherein the -Ci-C6 alkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, - NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, and -S(0)2R’;
Ri is -H, -Ci-C6 alkyl or -C3-C7 cycloalkyl, wherein the -Ci-C6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, - OR’, -SR’, -OC(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, and -S(0)2R’;
R2 and R3 are independently -H, -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle
comprising 5 to 10 carbons,
Figure imgf000071_0001
Figure imgf000071_0002
wherein
the -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, - CN, -CNR’R”, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, and a 3- to 1 1-membered heterocycle,
the -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, - CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl, the R5 is -Ci-C6 alkyl, -OCH2CH2-, -NR6CH2CH2-, or -NC(0)CH2CH2-, and the R¾ is -H, -C1-C6 alkyl, or -C3-C7 cycloalkyl;
the R’ and R” are independently -H or -Ci-C6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4- to 8- membered cyclic structure; and
R4 is H, -C1-C6 alkyl or -C3-C7 cycloalkyl.
4. A compound of Formula I:
Figure imgf000072_0001
wherein:
Zi is -H, -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the - phenyl, -naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with
Figure imgf000072_0002
NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R\ -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, - C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -C1-C6 alkyl, - aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, - guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl;
Z2 is -phenyl, -naphthyl, or a heterocycle comprising 5 to 10 carbons, wherein the - phenyl, -naphthyl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R\ -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, - Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle, and the -Ci-C6 alkyl, - aryl, -C3-C7 cycloalkyl, or 3- to 10-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -OC(0)R’, -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, - guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl; R is -H or -C1-C6 alkyl, wherein the -C1-C6 alkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, - NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, and -S(0)2R’;
Ri is -H, -Ci-C6 alkyl, or -C3-C7 cycloalkyl, wherein the -Ci-C6 alkyl or -C3-C7 cycloalkyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, - COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NRR”, - NS(0)2R’, -S(0)2NR’R”, and -S(0)2R’;
X is a bond or -0(Ci-C6 alkyl);
R2 and R3 are independently -H, -C1-C6 alkyl, -C3-C7 cycloalkyl, -aryl, a heterocycle
comprising 5 to 10 carbons,
Figure imgf000073_0001
Figure imgf000073_0002
wherein
the -Ci-C6 alkyl, -C3-C7 cycloalkyl, -aryl, or heterocycle comprising 5 to 10 carbons is unsubstituted or substituted with one or more of the following: -halo, -OH, - CN, -CNR’R”, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, and a 3- to 1 1-membered heterocycle,
the -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 1 1-membered heterocycle is unsubstituted or substituted with one or more of the following: -halo, -CNR’R”, -OH, - CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, - C(0)NR’R”, -NS(0)2R’, -OR’OR”, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, - nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl,
the R5 is -Ci-C6 alkyl, -OCH2CH2-, -NR6CH2CH2-, or -NC(0)CH2CH2-, and the 5 is -H, -Ci-C6 alkyl, or -C3-C7 cycloalkyl; and
the R’ and R” are independently -H or -Ci-C6 alkyl, or the R’ and R” together, optionally attached to N or O atom, form a 4 to 8 membered cyclic structure.
5. The compound of any one of Claims 1 to 4, wherein R is hydroxymethyl with S configuration
6. The compound of any one of Claims 1 to 5, wherein Zi is pyridine.
7. The compound of any one of Claims 1 to 6, wherein R2 is -H.
8. The compound of any one of Claims 1 to 7, wherein
R3 is -C1-C6 alkyl or a heterocycle comprising 5 to 10 carbons substituted with one or more of the following: -halo, -OH, -CN, -CNR’R”, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, - NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, - guanidino, -nitro, -nitroso, -Ci-C6 alkyl, aryl, -C3-C7 cycloalkyl, and a 3- to 11-membered heterocycle; and
the -C1-C6 alkyl, aryl, -C3-C7 cycloalkyl, or 3- to 11-membered heterocycle is substituted with one or more of the following: -halo, -CNR’R”, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -OR’OR”, - S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, and -C3-C7 cycloalkyl .
9. The compound of Claim 8, wherein R3 is -Ci-C6 alkyl substituted with a 3- to 11- membered heterocycle and the 3- to 11-membered heterocycle is substituted with -COOR’.
10. The compound of Claim 8, wherein R3 is a heterocycle comprising 5 to 10 carbons substituted with -COOR’ or -OR’.
11. The compound of Claim 8, wherein R3 is a heterocycle comprising 5 to 10 carbons substituted with -C1-C6 alkyl and the -C1-C6 alkyl is substituted with one or more -halo.
12. The compound of any one of Claims 1 to 4 selected from the group consisting of:
Figure imgf000075_0001
13. The compound of any one of Claims 1, 2, and 4, wherein R2 and R3 are independently -H, -C3-C7 cycloalkyl, or -C3-C7 cycloalkyl methyl, wherein the -C3-C7 cycloalkyl or -C3-C7 cycloalkyl methyl is unsubstituted or substituted with one or more of the following: -H, -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -CNR’R”, -NHC(0)R’, - NHC(0)NR’R”, -C(0)NR'R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, - Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3 - to 11-membered heterocycle, and wherein R2 and R3 are not both -H.
14. The compound of Claim 13, wherein R2 and R3 are independently -H, - cyclohexyl, -cyclopentyl, -cyclobutyl, -cyclopropyl, -cyclohexyl methyl, -cyclopentyl methyl, - cyclobutyl methyl, or -cyclopropyl methyl, wherein the -cyclohexyl, -cyclopentyl, -cyclobutyl, - cyclopropyl, -cyclohexyl methyl, -cyclopentyl methyl, -cyclobutyl methyl, or -cyclopropyl methyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, - COOR’, OR’, -SR’, -0C(0)R\ -NHR’, -NR’R”, -CNR’R”, -NHC(0)R’, -NHC(0)NR’R”, C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -C1-C6, -alkyl, - aryl, -C3-C7 cycloalkyl, and a 3- to 1 1-membered heterocycle.
15. The compound of any one of Claims 1, 2, and 4, wherein R2 and R3 are independently -H, -phenyl, or -benzyl, wherein the -phenyl or -benzyl is unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -CNR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, - S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -Ci-C6 alkyl, -aryl, -C3-C7 cycloalkyl, and a 3- to 11-membered heterocycle, and wherein R2 and R3 are not both -H.
16. The compound of Claim 2 or 4, wherein:
Zi is -pyridine unsubstituted or substituted with one or more of the following: -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, -NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, -nitro, -nitroso, -Ci-C6 alkyl, - aryl, and -C3-C7 cycloalkyl;
Z2 is a heterocycle comprising 5 to 10 carbons, wherein the heterocycle is unsubstituted or substituted with -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, -NR’R”, - NHC(0)R’, -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R’, -S(0)2NR’R”, -S(0)2R’, -guanidino, - nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 10-membered heterocycle; and R2 and R3 are independently -H, -phenyl, -benzyl, -C3-C7 cycloalkyl, or -C3-C7 cycloalkyl methyl, wherein the -phenyl, -benzyl, -C3-C7 cycloalkyl, or -C3-C7 cycloalkyl methyl is optionally substituted with -H, -halo, -OH, -CN, -COOR’, -OR’, -SR’, -0C(0)R’, -NHR’, - NR’R”, -NHC(0)R\ -NHC(0)NR’R”, -C(0)NR’R”, -NS(0)2R\ -S(0)2NR’R”, -S(0)2R\ - guanidino, -nitro, -nitroso, -C1-C6 alkyl, -aryl, -C3-C7 cycloalkyl, or a 3- to 11-membered heterocycle, and wherein R2 and R3 are not both -H.
17. The compound of Claim 16, wherein Z2 is -pyridine or -pyrazole.
18. A pharmaceutical composition, comprising a compound of any one of Claims 1 to 17 and a pharmaceutically acceptable carrier.
19. The pharmaceutical composition of Claim 18 for the treatment of a disease associated with Rho-kinase modulation, graft versus host disease (GVHD), or a neoplastic disease.
20. The use of a compound of any one of Claims 1 to 17 for the manufacture of a medicament for the treatment of a disease associated with Rho-kinase modulation, graft versus host disease (GVHD), or a neoplastic disease.
21. A method of treating a disease associated with Rho-kinase modulation in a subject in need thereof, comprising administering to the subject an effective amount of a rho kinase inhibitor or pharmaceutically acceptable salt thereof, wherein the rho kinase inhibitor comprises at least one compound of any one of Claims 1 to 17 or the pharmaceutical composition of Claim 18.
22. A method of treating graft versus host disease (GVHD) in a subject in need thereof, comprising administering to the subject an effective amount of a rho kinase inhibitor or pharmaceutically acceptable salt thereof, wherein the rho kinase inhibitor comprises at least one compound of any one of Claims 1 to 17 or the pharmaceutical composition of Claim 18.
23. The method of Claim 22, wherein the GVHD is a chronic GVHD disease (cGVHD).
24. A method of treating a neoplastic disease selected from the group consisting of lymphoma, carcinoma, leukemia, sarcoma, and blastoma in a subject in need thereof, the method comprising administering to the subject an effective amount of a rho kinase inhibitor or pharmaceutically acceptable salt thereof, wherein the rho kinase inhibitor comprises at least one compound of any one of Claims 1 to 17 or the pharmaceutical composition of Claim 18.
25. The method of Claim 24, wherein the neoplastic disease is ITO-FLT3+ acute myeloid leukemia (AML).
26. The method of any one of Claims 21 to 25, wherein the rho kinase inhibitor or pharmaceutically acceptable salt thereof is ROCK2 selective.
27. The method of Claim 26, wherein the rho kinase inhibitor is selected from the group consisting of:
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
PCT/US2020/012263 2018-04-24 2020-01-03 Methods of treating graft versus host disease and neoplastic disease with amide compounds WO2020142745A1 (en)

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