WO2020120576A1 - p38α AUTOPHOSPHORYLATION INHIBITORS - Google Patents

p38α AUTOPHOSPHORYLATION INHIBITORS Download PDF

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WO2020120576A1
WO2020120576A1 PCT/EP2019/084646 EP2019084646W WO2020120576A1 WO 2020120576 A1 WO2020120576 A1 WO 2020120576A1 EP 2019084646 W EP2019084646 W EP 2019084646W WO 2020120576 A1 WO2020120576 A1 WO 2020120576A1
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ring
alkyl
group
compound
optionally substituted
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PCT/EP2019/084646
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Ángel RODRIGUEZ NEBREDA
Ana IGEA FERNÁNDEZ
Lorena GONZÁLEZ VELASCO
Modesto OROZCO LÓPEZ
Robert Soliva Soliva
Lucia DÍAZ BUENO
Daniel SOLER VILADRICH
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Fundació Institut De Recerca Biomèdica (Irb Barcelona)
Universitat De Barcelona
Institució Catalana De Recerca I Estudis Avançats
Barcelona Supercomputing Center - Centro Nacional De Supercomputación (Bsc-Cns)
Nostrum Biodiscovery, S.L.
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Publication of WO2020120576A1 publication Critical patent/WO2020120576A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D235/08—Radicals containing only hydrogen and carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D235/14—Radicals substituted by nitrogen atoms
    • 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
    • C07D401/04—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
    • 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
    • C07D403/04—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 directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur 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
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the present invention concerns inhibitors of p38a autophosphorylation, pharmaceutical compositions comprising them, and their use in the treatment of a number of diseases, such as myocardial ischemia reperfusion injury.
  • p38 mitogen-activated protein kinases are a class of protein kinases that are responsive to cytokines and stress stimuli.
  • MAPKs p38 mitogen-activated protein kinases
  • p38o MAPK14
  • r38b MAPK1 1
  • r38g MAPK12
  • r38d r38d
  • p38 MAPKs are activated by a variety of cellular stimuli including osmotic shock, inflammatory cytokines, lipopolysaccharide, ultraviolet light, and growth factors.
  • the most abundant p38 MAPK in many cell types is p38a.
  • the kinases MKK6 and MKK3 activate p38a by phosphorylation at Thr-180 and Tyr-182.
  • p38a Once p38a is active, it can in turn phosphorylate a range of downstream substrates such as the kinase MK2, the transcription factor ATF2 and the mRNA regulator TTP.
  • p38a has been the subject of intense study by the scientific community and also by the pharmaceutical industry. The latter has devoted huge resources over the last 20 years to the development of different series of clinical-stage inhibitors. Some notorious examples are VX-745, Pamapimod, AMG-548, BIRB-796, Losmapimod, PH-797804, Scio-469 and RWJ-67657 developed by Vertex, Roche, Amgen, Boehringer, GSK, Pfizer, Scios and Johnson&Johnson, respectively.
  • p38a has been studied for many years, important aspects of its biology are still being unveiled. This includes the capability of p38a to autophosphorylate via alternative (non-canonical) routes. One of them is induced by the kinase ZAP70 that can phosphorylate p38a at Tyr323, which in turn induces autophosphorylation and subsequent activation of p38a in T-cells (Salvador et al, 2005). A second non-canonical route has been described where p38a autophosphorylation is induced by binding to the protein TAB1 (Ge et al., 2002; De Nicola et al., 2013).
  • the present inventors have found compounds based on a nitrogenated bicyclic ring that inhibit the autophosphorylation of p38a and therefore can be used in therapy. These compounds are chemically different from the existing ATP competitors and with a different mechanism of action by specifically targeting the non-canonical p38a activation. Therefore, these compounds would inhibit only a subset of p38a regulated functions, which is expected to yield reduced undesired side-effects.
  • the invention relates to a compound A, optionally in combination with one or more pharmaceutically or veterinary acceptable carriers or excipients, for use in the prophylaxis or treatment of a disease associated with autophosphorylation of p38a kinase, wherein the compound A is either a compound of formula (I):
  • R 1 is an aliphatic carbocyclic ring or ring system having 4 to 10 ring atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C ⁇ alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 2 , -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 ; or alternatively
  • R 1 is an aliphatic heterocyclic or heteroaromatic monocyclic ring having 6 ring atoms selected from the group consisting of C, N, O, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR 15 R 16 , -C(0)CH 3I -C(0)NH 2I -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 ; or alternatively
  • R 1 is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 2 , -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 ; or alternatively
  • R 1 is -CONR 10 R 11 , wherein
  • R 10 is H or C alkyl; and R 11 is C ⁇ alkyl optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C ⁇ alkyl, C haloalkyl, C ⁇ alkoxy, Ci-4alkylthio, amino, halogen, oxo, -NHCi- 4 alkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 2 , -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 ; and optionally two hydrogen atoms attached to one carbon atom of the C ⁇ alkyl are replaced by two CH 2 moieties which form together a carbocyclic ring having 3 to 6 ring atoms; or alternatively
  • R 11 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C ⁇ alkyl, C haloalkyl, Ci. 4 alkoxy, Ci.
  • R 10 and R 11 form together a heterocyclic ring or ring system having 4 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci- 4 alkyl, C haloalkyl, Ci. 4 alkoxy, Ci- 4 alkylthio, amino, halogen, oxo, -NHCi- 4 alkyl, -NR 15 R 16 , -C(0)CH 3 ⁇ 4 -C(0)NH 3 ⁇ 4 -C(0)NR 15 R 16 , -NR 15 C(0)R 16 and -[NHC(0)] z R 9 ; or alternatively
  • R 1 is -Y-R 12 , wherein
  • Y is selected from the group consisting of NR 13 , 0, S, -C(0)-;
  • R 12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci- 4 alkyl, C haloalkyl, Ci- 4 alkoxy, Ci- 4 alkylthio, amino, halogen, oxo, -NHCi. 4 alkyl, -NR 15 R 16 , -C(0)CH 3 ⁇ 4 -C(0)NH 3 ⁇ 4 -C(0)NR 15 R 16 , -NR 15 C(0)R 16 and -[NHC(0)] z R 9 ; and
  • R 13 is H or Ci-4 alkyl; or alternatively
  • R 1 is -CH 2 -R 14 , wherein R 14 is an aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms , wherein the atoms of the heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR 15 R 16 , -C(0)CH 3I -C(0)NH 2I -C(0)NR 15 R 16 , -NR 15 C(0)R 16 and -[NHC(0)] z R 9 ; or alternatively
  • R 14 is an aliphatic heterocyclic ring having 5 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR 15 R 16 , -C(0)CH 3I -C(0)NH 2I -C(0)NR 15 R 16 , -NR 15 C(0)R 16 and -[NHC(0)] z R 9 ;
  • R 3 is -R 7 -R 8 ;
  • R 7 is -(CH 2 ) X -, with x being 0 to 3;
  • R 8 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH 2 ; z is O or 1;
  • R 9 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH 2 ;
  • R 15 is H or C alkyl
  • R 16 is R 9 or alternatively is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH 2 ; wherein R 4 , R 5 , and R have one of the following meanings:
  • R 4 is N; R 5 is CH; and R is CH; (ii) R 4 is N; R 5 is N; and R is CH;
  • R 4 is CH; R 5 is N; and R is N;
  • R 4 is CH; R 5 is CH; and R is N;
  • R 4 is CH; R 5 is N; and R is CH; or
  • R 4 is CH; R 5 is CH; and R is CH; an isomer thereof, particularly a tautomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, particularly a pharmaceutically acceptable salt thereof; or alternatively the compound A for use in the prophylaxis or treatment of a disease associated with autophosphorylation of p38a kinase is one of the following:
  • This aspect may also be formulated as the use of a compound A as defined above, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, for the manufacture of a medicament for the prophylaxis or treatment of a disease associated with autophosphorylation of p38a kinase.
  • the present invention also relates to method for the treatment and/or prophylaxis of a disease associated with autophosphorylation of p38a kinase, comprising administering an effective amount of the previously defined compound A, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, and one or more pharmaceutically or veterinary acceptable excipients or carriers, in a subject in need thereof, including a human.
  • the invention also provides the compounds A as defined above for use in the prophylaxis or treatment of a disease selected from the group consisting of cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer, in particular wherein the disease is associated with autophosphorylation of p38a kinase; for example cardiovascular diseases such as myocardial ischemia-reperfusion injury, cardiac amyloidosis, or atherosclerosis/hypercoagulability; inflammatory diseases such as skin inflammation or toxoplasmosis; t-cell senescence, cancer such as pancreatic cancer, and inflammatory autoimmune diseases.
  • a disease selected from the group consisting of cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer, in particular wherein the disease is associated with autophosphorylation of p38a kinase
  • cardiovascular diseases such as myocardial ischemia-reperfusion injury, cardiac amyloidosis, or atherosclerosis/hypercoagulability
  • inflammatory diseases such as skin inflammation or tox
  • This aspect may also be formulated as the use of a compound A as defined above, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, for the manufacture of a medicament for the prophylaxis or treatment of a disease selected from the group consisting of cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer, in particular wherein the disease is associated with autophosphorylation of p38a kinase.
  • a disease selected from the group consisting of cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer, in particular wherein the disease is associated with autophosphorylation of p38a kinase.
  • the present invention also relates to method for the treatment and/or prophylaxis of a disease selected from the group consisting of cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer, in particular wherein the disease is associated with autophosphorylation of p38a kinase, comprising administering an effective amount of the previously defined compound A, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, and one or more pharmaceutically or veterinary acceptable excipients or carriers, in a subject in need thereof, including a human.
  • a disease selected from the group consisting of cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer, in particular wherein the disease is associated with autophosphorylation of p38a kinase, comprising administering an effective amount of the previously defined compound A, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, and one or more pharmaceutically or veterinary acceptable excipients or carriers, in a subject in need thereof, including a human.
  • Another aspect of the invention relates to a compound B which is either a compound of formula (la):
  • R 1 is an aliphatic carbocyclic ring or ring system having 5 to 10 ring atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, amino, halogen, oxo, -NHCualkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 2 , -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 ; or alternatively
  • R 1 is an aliphatic heterocyclic or heteroaromatic monocyclic ring having 6 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, Ci-4alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR 15 R 16 , -C(0)CH 3I -C(0)NH 2I -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 ; or alternatively
  • R 1 is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCi. 4 alkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 2 , -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 ; or alternatively
  • R 1 is -CONR 10 R 11 , wherein
  • R 10 is H or C alkyl; and R 11 is Ci- 6 alkyl optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, Ci-4alkoxy, Ci-4alkylthio, amino, halogen, oxo, -NHCi- 4 alkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 3 ⁇ 4 -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 ; and optionally two hydrogen atoms attached to one carbon atom of the Ci- 6 alkyl are replaced by two CH 2 moieties which form together a carbocyclic ring having 3 to 6 ring atoms; or alternatively
  • R 11 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 3 ⁇ 4 -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 ; or alternatively
  • R 10 and R 11 form together a heterocyclic ring or ring system having 4 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCi- 4 alkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 3 ⁇ 4 -C(0)NR 15 R 16 , -NR 15 C(0)R 16 and -[NHC(0)] z R 9 ; or alternatively
  • R 1 is -Y-R 12 , wherein
  • Y is selected from the group consisting of NR 13 , 0, S, -C(O)-;
  • R 12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 2 , -C(0)NR 15 R 16 , -NR 15 C(0)R 16 and -[NHC(0)] z R 9 ; and
  • R 13 is H or C1-4 alkyl; or alternatively
  • R 1 is -CH2-R 14 , wherein
  • R 14 is an aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms , wherein the atoms of the heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C ⁇ alkyl, C haloalkyl, C ⁇ alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR 15 R 16 , -C(0)CH 3I -C(0)NH 2I -C(0)NR 15 R 16 , -NR 15 C(0)R 16 and -[NHC(0)] z R 9 ; or alternatively
  • R 14 is an aliphatic heterocyclic ring having 5 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR 15 R 16 , -C(0)CH 3I -C(0)NH 2 , -C(0)NR 15 R 16 , -NR 15 C(0)R 16 and -[NHC(0)] z R 9 ;
  • R 3 is -R 7 -R 8 ;
  • R 7 is -(CH 2 ) X -, with x being 0 to 3;
  • R 8 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, and 0, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, and -NHC alkyl; z is 0 or 1;
  • R 9 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH 2 ;
  • R 15 is H or C alkyl
  • R 16 is R 9 or alternatively is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH 2 ;
  • R 4 , R 5 , and R have one of the following meanings:
  • R 4 is N; R 5 is CH; and R is CH;
  • R 4 is CH; R 5 is N; and R is N;
  • R 4 is CH; R 5 is CH; and R is N;
  • R 4 is CH; R 5 is N; and R is CH; or
  • R 4 is CH; R 5 is CH; and R is CH; an isomer thereof, particularly a tautomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, particularly a pharmaceutically acceptable salt thereof; wherein the compound of formula (la) is other than:
  • compound B is a compound which is one of the following:
  • the invention concerns a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising a therapeutically effective amount of a compound A or B according to the invention as well as one or more pharmaceutically or veterinary acceptable carriers or excipients.
  • FIG. 1 shows IC50 curves for VX-702 (ATP-competitive inhibitor) and the compounds 1 and 10 (NanoBret Ratio vs Compound Log [M],
  • FIG 2. shows cleaved caspase-3 levels (normalized to DMSO) levels for SB203580 (ATP-competitor) and some compounds of the invention. Detailed description of the invention
  • alkyl refers to a saturated branched or linear hydrocarbon group which contains the number of carbon atoms herein indicated and only single bonds.
  • C alkyl is intended to mean a linear or branched hydrocarbon group having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso- butyl, sec-butyl, and tert-butyl.
  • Ci-2alkyl is intended to mean a linear hydrocarbon group having 1 to 2 carbon atoms, such as methyl and ethyl.
  • alkoxy relates to an alkyl group as defined above linked to the rest of the molecule through an oxygen atom, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and t-butoxy.
  • alkylthio relates to an alkyl group as defined above linked to the rest of the molecule through a sulfur atom, such as methylthio, ethylthio, n-propylthio, isopropylthio, and n-butylthio.
  • haloalkyl relates to an alkyl group as defined above wherein on or more hydrogen atoms have been replaced by halogen atoms, such as fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, and dichloroethyl.
  • C3-6cycloalkyl is intended to mean a cyclic hydrocarbon group having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • halogen includes fluoro, chloro, bromo, and iodo, more particularly, fluoro, chloro and bromo.
  • aromatic ring or ring system is intended to mean a fully or partially aromatic carbocyclic ring or ring system wherein all the ring system members are carbon atoms, such as phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl.
  • heteroaromatic ring or ring system groups examples include oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, coumaryl, furyl, thienyl, quinolyl, benzothiazolyl, benzotriazolyl, benzodiazolyl, benzooxozolyl, phthalazinyl, phthalanyl, triazolyl, tetrazolyl, isoquinolyl, acridinyl, carbazolyl, dibenzazepinyl, indolyl, benzopyrazolyl and phenoxazonyl.
  • heterocyclic groups examples include imidazolidine, piperazine, hexahydropyridazine, hexahydropyrimidine, diazepane, diazocane, pyrrolidine, piperidine, azepane, azocane, aziridine, azirine, azetidine, pyroline, tropane, oxazinane (morpholine), azepine, dihydroazepine, tetrahydroazepine, hexahydroazepine, oxazolane, oxazepane, oxazocane, thiazolane, thiazinane, thiazepane, thiazocane, oxazetane, diazetane, thiazetane, tetrahydrofuran, tetrahydropyran, oxepane, tetrahydrothiophene, tetrahydrothi
  • aliphatic cyclic and "aliphatic carbocyclic” ring or ring system are indistinctly used and mean an aliphatic ring or ring system wherein all the ring system members are carbon atoms, and which can be saturated or partially unsaturated.
  • Non-limiting examples of aliphatic carbocyclic rings include cyclobutane, cyclopentane, cyclohexane, cyclohexene, and the like.
  • ring atoms are to be understood as ring members, that is a ring or ring system having 3 to 6 ring atoms is equivalent to a 3- to 6-membered ring or ring system.
  • the term "optionally substituted” is intended to mean that the group in question may be substituted one or more times, preferably 1-2 times.
  • Preferred substituents include -OH, OCH3, -NH2; -NHCH3, and halogen.
  • the term “optionally substituted” may also mean that the group in question is unsubstituted.
  • the compounds of the present invention can be in a free form or in the form of a pharmaceutically or veterinary acceptable salt.
  • pharmaceutically or veterinary acceptable salt is to be understood as a salt formed with either a base or an acid, wherein the resulting counter-ion does not significantly add to the toxicity of the compound of the present invention.
  • the invention relates to the compounds B or the compounds A for use as defined herein or their pharmaceutically acceptable salts.
  • Examples of pharmaceutically or veterinary acceptable salts include inorganic acid salts such as hydrochloride, sulfate, bisulfate, nitrate, phosphate or hydrobromide, etc., organic acid salts such as acetate, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, maleate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, citrate, fumarate, succinate, tartrate, naphthylate, glucoheptonate, and laurylsulphonate salts, and the like.
  • a salt with a base for example, alkali metal salt such as sodium salt, potassium salt, etc. or alkaline earth metal salt such as calcium salt, etc.
  • the term “isomer” refers to individual compounds with identical formulas but distinct structures.
  • the term isomer includes enantiomers, racemates, racemic mixtures, geometric isomers (cis/trans or syn/anti or E/Z), diastereoisomers, tautomers.
  • the present invention relates to each of these isomers and mixtures thereof.
  • tautomers refers to isomers, which differ in the position of an atom, generally a hydrogen atom, and one or more multiple bonds, which are capable to change reversibly from one to another. All tautomers are to be considered equivalent for the purposes of the invention.
  • the compound A is a compound of formula (I),
  • R 4 , R 5 , and R have the following meanings:
  • R 4 is N; R 5 is CH; and R is CH.
  • the compound A is a compound of formula (I), wherein R 4 , R 5 , and R have the following meanings:
  • R 4 is N; R 5 is N; and R is CH.
  • the compound A is a compound of formula (I), wherein R 4 , R 5 , and R have the following meanings:
  • R 4 is CH; R 5 is CH; and R is CH.
  • the compound A is a compound of formula (I), wherein R 1 is an optionally substituted aliphatic carbocyclic ring or ring system as defined above having 4 to 10 ring atoms, more particularly 5 to 10 ring atoms, even more particularly 4 to 6 ring atoms.
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 2 , -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 .
  • substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 2 , -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 .
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , -C(0)NH 2 , and -[NHC(0)] z R 9 as previously defined; even more particularly R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C ⁇ alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , and -C(0)NH 2 ; even more particularly, R 1 is optionally substituted with one or more substituents selected from the group consisting of C alkyl, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , and -C(0)NH 2 .
  • R 1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH 3 , and -[NHC(0)] z R 9 , wherein z is 0 or 1, and R 9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
  • the compound A is a compound of formula (I), wherein R 1 is an optionally substituted cyclobutyl, an optionally substituted cyclopentyl, or an optionally substituted cyclohexyl.
  • R 1 is methylaminocyclopentyl, cyclobutyl, cyclopentyl, oxocyclopentyl (particularly 3-oxocyclopentyl), methylcyclopentyl (particularly methylclopent-2-yl), and methylcyclohexyl (particularly methylclohex-2-yl).
  • the compound A is a compound of formula (I), wherein R 1 is an optionally substituted aliphatic heterocyclic ring having 6 ring atoms as defined above; more particularly having 6 ring atoms selected from the group consisting of C, N; even more particularly one of the ring atoms is N. More particularly, R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci- 4 alkyl, C haloalkyl, Ci.
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci- 4 alkyl, Ci.
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci- 4 alkyl, Ci_ 4alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , and -C(0)NH 2 ; even more particularly, R 1 is optionally substituted with one or more substituents selected from the group consisting of Ci- 4 alkyl, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , and -C(0)NH 2 . In another particular embodiment, R 1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH 3 , and
  • the compound A is a compound of formula (I), wherein R 1 is N-methylpyrrolidinyl (particularly N-methylpyrrolidin-3-yl), and 1-acetylpiperidyl (particularly 1 -acetylpiperidin-4-yl).
  • the compound A is a compound of formula (I), wherein R 1 is an optionally substituted heteroaromatic monocyclic ring having 6 ring atoms as defined above; more particularly having 6 ring atoms selected from the group consisting of C, N, S; even more particularly having 6 ring atoms selected from the group consisting of C, N; even more particularly one of the ring atoms is N. More particularly, R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci- 4 alkyl, C haloalkyl, Ci- 4 alkoxy, Ci.
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci- 4 alkyl, Ci.
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci- 4 alkyl, Ci- 4 alkoxy, amino, halogen, oxo, -NHCi- 4 alkyl, -C(0)CH 3 , and -C(0)NH 2 ; even more particularly, R 1 is optionally substituted with one or more substituents selected from the group consisting of Ci- 4 alkyl, amino, halogen, oxo, -NHCi- 4 alkyl, -C(0)CH 3 , and -C(0)NH 2 .
  • R 1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH 3 , and -[NHC(0)] z R 9 , wherein z is 0 or 1, and R 9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
  • the compound A is a compound of formula (I), wherein R 1 is quinolinyl (particularly quinolin-6-yl), pyridinyl (particularly pyridin-4-yl), and indolyl (particularly indol-5-yl).
  • the compound A is a compound of formula (I), wherein when R 1 is substituted with -[NHC(0)] z R 9 z is 0 or 1, and R 9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
  • the compound A is a compound of formula (I), wherein R 1 is -CONR 10 R 11 , even more particularly R 10 is H or C ⁇ alkyl; and R 11 is C ⁇ alkyl optionally substituted as previously defined and optionally two hydrogen atoms attached to one carbon atom of the C ⁇ alkyl are replaced by two Chh moieties which form together a carbocyclic ring having 3 to 6 ring atoms.
  • R 10 is H or Ci-2alkyl
  • R 11 is Ci-3alkyl optionally substituted as previously defined and optionally two hydrogen atoms attached to one carbon atom of the Ci-3alkyl are replaced by two Chh moieties which form together a carbocyclic ring having 3 to 6 ring atoms.
  • the compound A is a compound of formula (I), wherein R 1 is -CONR 10 R 11 , wherein R 10 and R 11 form together a heterocyclic ring or ring system having 4 to 6 ring atoms selected from the group consisting of C, and N, this ring being optionally substituted as previously defined.
  • the compound A is a compound of formula (I), wherein R 1 is -CONR 10 R 11 as previously defined and R 4 , R 5 , and R have the following meaning:
  • R 4 is CH; R 5 is CH; and R is CH.
  • the compound A is a compound of formula (I), wherein R 1 is -Y-R 12 , and Y is selected from the group consisting of NR 13 , 0, and -C(0)-, and R 12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, more particularly 4 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, more particularly C, N.
  • R 1 is -Y-R 12
  • Y is selected from the group consisting of NR 13 , 0, and -C(0)-
  • R 12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, more particularly 4 to 6 ring atoms, wherein the atoms of the
  • R 12 is an aliphatic carbocyclic, or aliphatic heterocyclic ring or ring system as previously defined, even more particularly R 12 is cyclobutyl, piperidinyl (particularly piperidin-3-yl) or piperazinyl (particularly piperazin-4-yl).
  • the compound A is a compound of formula (I), wherein R 1 is -Y-R 12 , and Y is NR 13 ; R 13 is H or C alkyl; more particularly R 13 is H or Ci-2alkyl; more particularly R 13 is Ci-2alkyl; and
  • the compound A is a compound of formula (I), wherein R 1 is -CH2-R 14 , wherein R 14 is an aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, more particularly 4 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, more particularly C, N. Even more particularly, R 14 is an aliphatic carbocyclic ring or ring system as previously defined, even more particularly R 14 is cyclobutyl.
  • the compound A is a compound of formula (I), wherein R 1 is -CH2-R 14 , wherein R 14 is an aliphatic heterocyclic ring having 5 ring atoms selected from the group consisting of C, N, 0, and S, more particularly C, N. Even more particularly, R 14 is an aliphatic heterocyclic ring or ring system as previously defined.
  • the compound A is a compound of formula (I), wherein R 3 is -R 7 -R 8 , wherein R 7 is— (CH2) X -, with x being 0-3, more particularly 0 or 1; and R 8 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system, more particularly an aromatic or heteroaromatic ring or ring system, wherein the ring or ring system has 5 to 10 ring atoms, more particularly 5 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, S, more particularly selected from the group consisting of C, N, 0, more particularly wherein the ring or ring system has no or one atom other than C as ring atom.
  • R 3 is -R 7 -R 8 , wherein R 7 is— (CH2) X -, with x being 0-3,
  • R 8 is an aromatic or heteroaromatic ring or ring system having 5 to 6 ring atoms, wherein the atoms of the heteroaromatic ring or ring system are selected from the group consisting of of C, N, 0, S, more particularly selected from the group consisting of C, N, and 0, more particularly C, N, even more particularly wherein the ring or ring system has no or one atom other than C as ring atom.
  • R 8 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C1-4 alkyl, halogen, oxo, -NHC alkyl and -C(0)NH 2 ; even more particularly R 8 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C1-4 alkyl, halogen, oxo, and -NHC alkyl; even more particularly, R 8 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C1-4 alkyl, halogen, oxo, -NHC alkyl; even more particularly, R 8 is optionally substituted with one, two, or three substituents selected from the group consisting of C alkyl, halogen, and -C(0)NH 2 . Alternatively, R 8 is optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
  • the compound A is a compound of formula (I), wherein R 3 is selected from the group consisting of benzyl, fluorobenzyl, phenyl, tolyl, optionally substituted phenyl, pyridinyl (particularly pyridin-4- yl), optionally substituted pyridinyl, tiophenyl (particularly thiophen-2-yl or thiophen-3-yl), optionally substituted thiophenyl, pyrazolyl (particularly pyrazol-5-yl), and optionally substituted pyrazolyl.
  • R 3 is selected from the group consisting of benzyl, fluorobenzyl, phenyl, tolyl, optionally substituted phenyl, pyridinyl (particularly pyridin-4- yl), optionally substituted pyridinyl, tiophenyl (particularly thiophen-2-yl or thiophen-3-yl), optionally substituted thiophen
  • the compound A is a compound of formula (I), wherein R 3 is phenyl, optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
  • Particularly preferred compounds A are the following compounds, 1 to 36: or an isomer thereof, particularly a tautomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, particularly a pharmaceutically acceptable salt thereof.
  • Compounds 9 (CAS RN: 1384795-21-4), 10 (CAS RN: 2094667-81-7), 1 1 (CAS RN: 2094511-69-8), 12 (CAS RN: 2094380-73-9), 13 (CAS RN: 1384790-95-7), 16 (CAS RN: 1808772-10-2), 17 (CAS RN: 1797537-02-0), and 18 (CAS RN: 1197756-53-8) were purchased from the company Enamine.
  • Compound 14 (CAS RN: 1324076-13-2) was purchased from Ambinter, and Compound 15 (CAS RN: 1795657-46-3), was purchased from Otava. These commercial products have not been described in any bibliographic references to the best of inventors' knowledge.
  • the present invention also relates to compounds B, which can be a compound of formula (la)
  • the compound B is a compound of formula (la), wherein R 4 , R 5 , and R have one of the following meanings:
  • R 4 is N; R 5 is CH; and R is CH.
  • the compound B is a compound of formula (la), wherein R 4 , R 5 , and R have the following meanings:
  • R 4 is N; R 5 is N; and R is CH.
  • the compound A is a compound of formula (I), wherein R 4 , R 5 , and R have the following meanings:
  • R 4 is CH; R 5 is CH; and R is CH.
  • the compound B is a compound of formula (la), wherein R 1 is an optionally substituted aliphatic carbocyclic ring or ring system as defined above having 5 to 10 ring atoms, more particularly 5 to 6 ring atoms.
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR 15 R 16 , -C(0)CH 3I -C(0)NH 2I -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 .
  • substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR 15 R 16 , -C(0)CH 3I -C(0)NH 2I -C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 .
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , -C(0)NH2, and -[NHC(0)] z R 9 as previously defined; even more particularly R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , and -C(0)NH 2 ; even more particularly, R 1 is optionally substituted with one or more substituents selected from the group consisting of C alkyl, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , and -C(0)NH 2 .
  • R 1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH 3 , and - [NHC(0)] z R 9 , wherein z is 0 or 1, and R 9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
  • the compound B is a compound of formula (la), wherein R 1 is an optionally substituted cyclopentyl, or an optionally substituted cyclohexyl.
  • R 1 is methylaminocyclopentyl, cyclopentyl, oxocyclopentyl (particularly 3-oxocyclopentyl), methylcyclopentyl (particularly 2-methylclopentyl), and methylcyclohexyl (particularly 2-methylclohexyl).
  • the compound B is a compound of formula (la), wherein R 1 is an optionally substituted aliphatic heterocyclic ring having 6 ring atoms as defined above; more particularly having 6 ring atoms selected from the group consisting of C, N; even more particularly one of the ring atoms is N.
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, Ci_ 4haloalkyl, C ⁇ alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 2 , - C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 .
  • substituents selected from the group consisting of hydroxyl, C alkyl, Ci_ 4haloalkyl, C ⁇ alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR 15 R 16 , -C(0)CH 3 , -C(0)NH 2 , - C(0)NR 15 R 16 , -NR 15 C(0)R 16 , and -[NHC(0)] z R 9 .
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , -C(0)NH 2 , and -[NHC(0)] z R 9 as previously defined; even more particularly R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, Ci_ 4alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , and -C(0)NH 2 ; even more particularly, R 1 is optionally substituted with one or more substituents selected from the group consisting of C alkyl, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3 , and -C(0)NH 2 .
  • R 1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH 3 , and - [NHC(0)] z R 9 , wherein z is 0 or 1, and R 9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
  • the compound B is a compound of formula (la), wherein R 1 is N-methylpyrrolidinyl (particularly N-methylpyrrolidin-3-yl), and 1-acetylpiperidyl (particularly 1 -acetylpiperidin-4-yl).
  • the compound B is a compound of formula (la), wherein R 1 is an optionally substituted heteroaromatic monocyclic ring having 6 ring atoms as defined above; more particularly having 6 ring atoms selected from the group consisting of C, N, S; even more particularly having 6 ring atoms selected from the group consisting of C, N; even more particularly one of the ring atoms is N. More particularly, R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci- 4 alkyl, C haloalkyl, Ci_ 4 alkoxy, Ci. 4 alkylthio, amino, halogen, oxo, -NHCi.
  • R 1 is an optionally substituted heteroaromatic monocyclic ring having 6 ring atoms as defined above; more particularly having 6 ring atoms selected from the group consisting of C, N, S; even more particularly having 6 ring atoms selected from the group consisting of C, N; even more particularly one of the
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci. 4 alkyl, Ci. 4 alkoxy, amino, halogen, oxo, -NHCi- 4 alkyl, -C(0)CH 3 , -C(0)NH 2 , and -[NHC(0)] z R 9 as previously defined; even more particularly R 1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci.
  • R 1 is optionally substituted with one or more substituents selected from the group consisting of Ci. 4 alkyl, amino, halogen, oxo, -NHCi- 4 alkyl, -C(0)CH 3 , and -C(0)NH 2 .
  • R 1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH 3 , and - [NHC(0)] z R 9 , wherein z is 0 or 1, and R 9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
  • the compound B is a compound of formula (la), wherein R 1 is quinolinyl (particularly quinolin-6-yl), pyridinyl (particularly pyridin-4-yl), and indolyl (particularly indol-5-yl).
  • the compound B is a compound of formula (la), wherein when R 1 is substituted with -[NHC(0)] z R 9 z is 0 or 1, and R 9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
  • the compound B is a compound of formula (la), wherein R 1 is -CONR 10 R 11 , even more particularly R 10 is H or Ci- 4 alkyl; and R 11 is C ⁇ alkyl optionally substituted as previously defined and optionally two hydrogen atoms attached to one carbon atom of the C ⁇ alkyl are replaced by two CH2 moieties which form together a carbocyclic ring having 3 to 6 ring atoms.
  • R 10 is H or Ci-2alkyl
  • R 11 is Ci-3alkyl optionally substituted as previously defined and optionally two hydrogen atoms attached to one carbon atom of the Ci-3alkyl are replaced by two CH2 moieties which form together a carbocyclic ring having 3 to 6 ring atoms.
  • the compound B is a compound of formula (la), wherein R 1 is -CONR 10 R 11 , wherein R 10 and R 11 form together a heterocyclic ring or ring system having 4 to 6 ring atoms selected from the group consisting of C, and N, this ring being optionally substituted as previously defined.
  • the compound B is a compound of formula (la), wherein R 1 is -CONR 10 R 11 as previously defined and R 4 , R 5 , and R have the following meaning:
  • R 4 is CH; R 5 is CH; and R is CH.
  • the compound B is a compound of formula (la), wherein R 1 is -Y-R 12 , and Y is selected from the group consisting of NR 13 , 0, and -C(O)-, and R 12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, more particularly 4 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, more particularly C, N.
  • R 12 is an aliphatic carbocyclic, or aliphatic heterocyclic ring or ring system as previously defined, even more particularly R 12 is cyclobutyl, piperidinyl (particularly piperidin-3-yl) or piperazinyl (particularly piperazin-4-yl).
  • the compound B is a compound of formula (la), wherein R 1 is -Y-R 12 , and Y is NR 13 ; R 13 is H or C alkyl; more particularly R 13 is H or Ci-2alkyl; more particularly R 13 is Ci-2alkyl; and
  • the compound A is a compound of formula (I), wherein R 1 is -CH2-R 14 , wherein R 14 is an aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, more particularly 4 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, more particularly C, N. Even more particularly, R 14 is an aliphatic carbocyclic ring or ring system as previously defined, even more particularly R 14 is cyclobutyl.
  • the compound A is a compound of formula (I), wherein R 1 is -CH2-R 14 , wherein R 14 is an aliphatic heterocyclic ring having 5 ring atoms selected from the group consisting of C, N, 0, and S, more particularly C, N. Even more particularly, R 14 is an aliphatic heterocyclic ring or ring system as previously defined.
  • the compound B is a compound of formula (la), wherein R 3 is -R 7 -R 8 , wherein R 7 is— (CH2) X -, with x being 0-3, more particularly 0 or 1, and R 8 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system, more particularly an aromatic or heteroaromatic ring or ring system, wherein the ring or ring system has 5 to 10 ring atoms, more particularly 5 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, and 0, more particularly C, N, even more particularly wherein the ring or ring system has no or one atom other than C as ring atom.
  • R 8 is an aromatic or heteroaromatic ring or ring system having 5 to 6 ring atoms, wherein the atoms of the heteroaromatic ring or ring system are selected from the group consisting of C, N, and 0, more particularly C, N, even more particularly wherein the ring or ring system has no or one atom other than C as ring atom.
  • the compound B is a compound of formula (la), R 8 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C1-4 alkyl, halogen, oxo, and -NHC alkyl; even more particularly, R 8 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C1-4 alkyl, halogen, oxo, -NHC alkyl; even more particularly, R 8 is optionally substituted with one, two, or three substituents selected from the group consisting of C1-4 alkyl, and halogen. Alternatively, R 8 is optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
  • the compound B is a compound of formula (la), wherein R 3 is selected from the group consisting of benzyl, fluorobenzyl, phenyl, tolyl, optionally substituted phenyl, pyridinyl (particularly pyridin-4- yl), optionally substituted pyridinyl, tiophenyl (particularly thiophen-2-yl or thiophen-3-yl), optionally substituted thiophenyl, pyrazolyl (particularly pyrazol-5-yl), and optionally substituted pyrazolyl.
  • R 3 is selected from the group consisting of benzyl, fluorobenzyl, phenyl, tolyl, optionally substituted phenyl, pyridinyl (particularly pyridin-4- yl), optionally substituted pyridinyl, tiophenyl (particularly thiophen-2-yl or thiophen-3-yl), optionally substituted thiophen
  • the compound B is a compound of formula (la), wherein R 3 is phenyl, optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
  • the compound B is selected from the group consisting of compounds 1-8, 19-36 as defined above, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof.
  • the compounds of the present invention preferably inhibit p38a autophosphorylation. This can be demonstrated with the assays described in Examples 28 and 29.
  • Particularly preferred compounds are those that show inhibition of p38a autophosphorylation of at least 40% at 5 mM and/or at least 50% at 10 mM and/or at least 80% at 30 mM in the assay described in Example 28, and/or those that show inhibition of TAB1 -induced p38a autophosphorylation of at least 20% at 5 mM and/or at least 60% at 10 mM and/or at least 90% at 30 mM in the assay described in Example 29.
  • the preferred compounds of the present invention show no inhibition (particularly the inhibition is equal to or lower than 20% in the assays described herein) of the canonical p38a phosphorylation at 2 mM, more preferably at 10 mM, and even more preferably at 30 mM. This can be demonstrated with the assays described in Examples 28 and 29.
  • Particularly preferred compounds are thus those compounds that inhibit p38a autophosphorylation by at least 70 % in the assays described in Examples 28 and 29, but show no inhibition of MKK6-induced p38a phosphorylation at the same concentration (e.g. at 1 mM or 10 mM), in the assay described in Example 26, and/or compounds that show no inhibition of p38a kinase activity on MK2 at the same concentration (e.g. at 1 mM or 10 mM), in the assay described in Example 27.
  • Process for preparing compounds are thus those compounds that inhibit p38a autophosphorylation by at least 70 % in the assays described in Examples 28 and 29, but show no inhibition of MKK6-induced p38a phosphorylation at the same concentration (e.g. at 1 mM or 10 mM), in the assay described in Example 26, and/or compounds that show no inhibition of p38a kinase activity on MK2 at the same concentration (e.g. at 1 mM
  • the compounds of the present invention are intended for use as a medicament.
  • the compounds of the invention may in principle be administered on their own, but they are preferably formulated with a pharmaceutically or veterinary acceptable carrier.
  • a pharmaceutically or veterinary acceptable carrier is an inert carrier suitable for each administration method, and can be formulated into conventional pharmaceutical preparation (tablets, granules, capsules, powder, solution, suspension, emulsion, injection, infusion, etc.).
  • a carrier there may be mentioned, for example, a binder, an excipient, a lubricant, a disintegrant and the like, which are pharmaceutically or veterinary acceptable.
  • a pharmaceutical or veterinary composition comprising a compound according to any one of the preceding aspects as well as one or more pharmaceutically acceptable carriers or excipients.
  • the present invention also relates to a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising a therapeutically effective amount of a compound A or a compound B as previously defined, as well as one or more pharmaceutically or veterinary acceptable carriers or excipients.
  • therapeutically effective amount refers to the amount of a compound which is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed.
  • pharmaceutically or veterinary acceptable excipients or carriers refers to pharmaceutically or veterinary acceptable materials, compositions or vehicles which are compatible with the other ingredients of the pharmaceutical or veterinary composition and are also suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • a further aspect of the present invention includes pharmaceutical or veterinary compositions comprising as one or more compounds of the invention disclosed above, associated with a pharmaceutically acceptable carrier.
  • the compounds are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
  • the administration method of the compounds of the present invention is not particularly limited, and a usual oral or parenteral administration method (intravenous, intramuscular, subcutaneous, percutaneous, intranasal, transdermal, transmucosal, enteral, etc.) can be applied.
  • a usual oral or parenteral administration method intravenous, intramuscular, subcutaneous, percutaneous, intranasal, transdermal, transmucosal, enteral, etc.
  • the compounds may be administered alone or in combination with one or more other compounds of the invention or one or more other drugs, for example in combination with Beta-blockers or thrombolytic agents (e.g. for the treatment of Ischemia-reperfusion); corticosteroids or NSAIDs (e.g. in the treatment of skin inflammation), or in combination with chemotherapeutic agents (e.g. in the treatment of pancreatic cancer).
  • Beta-blockers or thrombolytic agents e.g. for the treatment of Ischemia-reperfusion
  • corticosteroids or NSAIDs e.g. in the treatment of skin inflammation
  • chemotherapeutic agents e.g. in the treatment of pancreatic cancer
  • the dosage of the compounds of the present invention may optionally be set in a range of an effective amount sufficient for showing a pharmacological effect, in accordance with the potency or characteristics of the compound to be used as an effective ingredient.
  • the dosage may vary depending on administration method, age, body weight or conditions of a patient.
  • the compounds of the invention inhibit the autophosphorylation of p38a.
  • the present invention concerns a compound of the invention for use as a medicament.
  • the invention concerns a compound or composition according to the invention for use in the prophylaxis or treatment of a disease associated with autophosphorylation of p38a kinase.
  • Autophosphorylation of p38a has been associated with a number of diseases, such as cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer.
  • the diseases that can be treated with the compounds and formulations of the present invention thus include myocardial ischemia reperfusion injury (De Nicola et al., 2018; Tanno et al., 2003; Wang et al., 2013), cardiac amyloidosis (Mishra et al., 2013; Shi et al., 2010), atherosclerosis/hypercoagulability (Grimsey et al., 2015), skin inflammation (Theivanthiran et al., 2015), impaired immune cell responses owing to T-cell senescence (Lanna et al., 2014), Toxoplasmosis (Kim et al., 2005; Braun et al., 2013; Pellegrini et al., 2017), pancreatic cancer (Alam et al., 2015), inflammatory autoimmune diseases such as arthritis and encephalomyelitis (Jirmanova et al., 201 1 ); and pre-term labor and premature birth (Richard
  • treatment refers to stopping or delaying of the disease progress, when the compound is used in the subject exhibiting symptoms of disease onset.
  • prophylaxis refers to stopping or delaying of symptoms of disease onset, when the compound is used in the subject exhibiting no symptoms of disease onset but having high risk of disease onset.
  • cardiovascular diseases include arteriosclerosis, peripheral vascular disease, hyperlipidemia, mixed dyslipidemia betalipoproteinemia, hypoalphalipoproteinemia, hypercholesteremia, hypertriglyceridemia, familial- hypercholesteremia, angina, ischemia, cardiac ischemia, stroke, myocardial infarction, reperfusion injury, restenosis after angioplasty, hypertension, cerebral infarction and cerebral stroke.
  • autoimmune diseases include arthritis, including rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis; inflammatory hyperproliferative skin diseases; psoriasis, such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails; atopy, including atopic diseases such as hay fever and Job's syndrome; dermatitis, including contact dermatitis, chronic contact derma
  • inflammatory diseases include allergic inflammation, arthritis (to include osteoarthritis and rheumatoid arthritis), fibromyalgia, chronic fatigue syndrome, inflammatory bowel disease, interstitial cystitis, irritable bowel syndrome, migraines, atherosclerosis, coronary inflammation, ischemia, chronic prostatitis, eczema, multiple sclerosis, psoriasis, sun burn, periodontal disease of the gums, superficial vasodilator flush syndromes, hormonally dependent cancers, and endometriosis.
  • arthritis to include osteoarthritis and rheumatoid arthritis
  • fibromyalgia chronic fatigue syndrome
  • inflammatory bowel disease interstitial cystitis
  • irritable bowel syndrome migraines
  • atherosclerosis coronary inflammation
  • ischemia chronic prostatitis
  • eczema multiple sclerosis
  • psoriasis sun burn
  • periodontal disease of the gums superficial vasodilator flush syndromes
  • Particular neurodegenerative diseases include diabetic neuropathy, senile dementias, Alzheimer's disease, Mild Cognitive Impairment (MCI), dementia, Lewy Body Dementia, Frontal Temporal Lobe dementia, Parkinson's Disease, facial nerve (Bell's) palsy, glaucoma, Huntington's chorea, amyotrophic lateral sclerosis (ALS), status epilepticus, non-arteritic optic neuropathy, intervertebral disc herniation, vitamin deficiency, prion diseases such as Creutzfeldt-Jakob disease, carpal tunnel syndrome, peripheral neuropathies associated with various diseases, including but not limited to, uremia, porphyria, hypoglycemia, Sjorgren Larsson syndrome, acute sensory neuropathy, chronic ataxic neuropathy, biliary cirrhosis, primary amyloidosis, obstructive lung diseases, acromegaly, malabsorption syndromes, polycythemia vera, IgA and IgG
  • the compounds of the present invention are used for treating said disease associated with autophosphorylation of p38a kinase, such as one of the diseases listed above.
  • the compounds of the invention selectively inhibit the autophosphorylation of p38a.
  • the compound of the present invention inhibits autophosphorylation of p38a induced by ZAP70- mediated Tyr phosphorylation.
  • the compound of the present invention inhibits TAB1 -induced autophosphorylation.
  • TAB1 stimulates the autophosphorylation of p38a by binding onto two different clefts.
  • TAB1 The protein TAB1 stimulates the autophosphorylation of p38a by binding onto two different clefts.
  • p38a binding partners other than TAB1 such as upstream and downstream kinases, as well as phosphatases.
  • the other one is thought to bind only to TAB1 (De Nicola et al., 2013).
  • the activity of upstream kinases such as MKK3 and MKK6 should not be affected and the p38a kinase activity is not shut down in all cellular contexts. This is deemed to reduce general toxicity in comparison with classical p38a inhibitors that interfere with ATP binding.
  • the compounds of the present invention therefore preferably inhibit autophosphorylation such as TAB1 -induced autophosphorylation, without showing an inhibitory effect on MKK6- induced p38a phosphorylation or the kinase activity of canonically-activated p38a.
  • Step 1 Synthesis of N6-[(4-methoxyphenyl) methyl]-3-nitro-pyhdine-2, 6-diamine (3)
  • Step 4 Synthesis of 2-cyclopentyl-3H-imidazo[4,5-b] pyridin-5-amine (6); [J. Heterocyclic Chem., 17, 1757, 1980]
  • a mixture of 5 (1 g, 8.05 mmol) and cyclopentane carboxylic acid (0.91 g, 8.05 mmol) in polyphosphoric acid (20 g) was heated at 180°C for 6 hours. After completion of reaction, the reaction mixture was cooled down to room temperature and basified with 10% aqueous sodium carbonate solution.
  • the product was extracted with ethyl acetate (3 X 30 mL). The organic layer was washed with water followed by brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • the crude product so obtained was purified by column chromatography using 0-10% methanol in dichloromethane as eluent to give the desired product 6 (800 mg, 50%) as a brown color solid.
  • Step 5 N-(2-cyclopentyl-3H-imidazo[4,5-b] pyridin-5-yl) benzenesulfonamide
  • Step 5 Synthesis of N-[2-[3-(methylamino) cyclopentyl]-3H-benzimidazol-5-yl] benzenesulfonamide
  • N-1 H-Benzo[d]imidazol-6-yl)benzenesulfonamide from the previous step (66 mg, 0.24 mmol), Pd(OAc)2 (2.7 mg, 0.012 mmol), 6-iodoquinoline (123 mg, 0.483) and Cul (92 mg, 0.48 mmol) were added to a sealed vial.
  • Previously degassed DMF (1.2 mL) was added under a l ⁇ atmosphere and the reaction heated to 140 °C and stirred at this temperature for 4 days. At this stage a saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc.
  • N-1 H-Benzo[d]imidazol-6-yl)benzenesulfonamide from the previous step (258 mg, 0.94 mmol), Pd(OAc)2 (10.6 mg, 0.047 mmol), 5-iodo-1 H-indole (344 mg, 1.42 mmol) and Cul (360 mg, 1.88 mmol) were added to a sealed vial.
  • Previously degassed DMF (4.0 mL) was added under a N2 atmosphere and the reaction heated to 140 °C and stirred at this temperature for 4 days. At this stage a saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc.
  • reaction was then filtered through a short pad of Celite and concentrated to deliver 101 mg (quantitative yield) of 1 -(4-(6-amino-1 H-benzo[d]imidazol-2-yl)piperidin-1 -yl)ethan-1 - one, wich was used without further purification.
  • Step 1 Synthesis ofN-(2-amino-4-nitro-phenyl)-1-methyl-pyrrolidine-3-carboxamide (5)
  • Step 3 Synthesis of tert-butyl 2-(1-methyipyrroiidin-3-yi)-8-nitro-benz/ ⁇ midazoie-1-carboxyiate (7)
  • Step 4 Synthesis of tert-butyl 6-amino-2-(1-methylpyrrolidin-3-yl) benz/ ⁇ midazoie-1-carboxyiate (8)
  • Step 5 Synthesis of tert-butyl 6-(benzenesulfonamido)-2-(1-methylpyrrolidin-3-yl) benz/ ⁇ midazoie-1-carboxyiate (11)
  • benzene sulfonyl chloride (0.03 mL, 0.2 mmol).
  • the resulting reaction mixture was stirred at room temperature for 3 hours. After the completion of reaction, the reaction mixture was quenched with water (5 mL) and the product was extracted with dichloromethane (2 X 15 mL).
  • Step 6 Synthesis of N-[2-(1-methylpyrrolidin-3-yl)-3H-benzimidazol-5-yl] benzene sulfonamide
  • Step 3 Synthesis o ⁇ N-[2-(2-methylcyclopentyl)-3H-benzimidazol-5-yl] benzenesulfonamide
  • Step 1 Synthesis of tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3)
  • Step 2 Synthesis of tert-butyl (4-(b-nitro-1H-benzo[d] ⁇ midazoi-2-yi)cyciohexyi)carbamate (4)
  • tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3) (1.3 g, 3.4 mmol) in glacial acetic acid (20 mL) was heated to 65 °C and stirred at the same temperature for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated to get a residue. The residue was diluted with ethyl acetate (250 mL) and washed with 10% aqueous sodium bicarbonate solution (200 mL). The ethyl acetate layer was separated, dried over sodium sulphate and concentrated.
  • Step 4 Synthesis of2-(4-(4-methylpiperazin-1-yl)cyclohexyl)-6-nitro-1H-benzo[d]imidazole (7)
  • Step 5 Synthesis of tert-butyl 2-(4-(4-methyipiperaz ⁇ n-1-yi)cyciohexyi)-%-nitro-1H-benzo[d] ⁇ midazoie-1-carboxyiate
  • Step 6 Synthesis of tert-butyl 6-amino-2-(4-(4-methylpiperazin-1-yl)cyclohexyl)-1H-benzo[d]imidazole-1 -carboxylate
  • Step 7 Synthesis of tert-butyl 2-(4-(4-methylpiperazin-1-yl)cyclohexyl)-6-(phenylsulfonamido)-1H- benzo[d]imidazole-1 -carboxylate (11)
  • Step 8 Synthesis ofN-(2-(4-(4-methy/piperazin-1-y/)cyc/ohexy/)-1H-benzo[d]imidazo/-6-y/)benzenesu/fonamide
  • tert-butyl 2-(4-(4-methylpiperazin-1 -yl)cyclohexyl)-6-(phenylsulfonamido)-1 H-benzo[d]imidazole-1- carboxylate (11) (0.15 g, crude) in dry DCM (10 mL) was added 4N HCL in dioxane (3 mL) at 0 °C. The reaction mixture was stirred at RT for 6 h.
  • Step 1 Synthesis of tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3)
  • Step 2 Synthesis of tert-butyl (4-(b-nitro-1H-benzo[0] ⁇ m ⁇ 0azoi-2-yi)cyciohexyi)carbamate (4)
  • tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3) (1.3 g, 3.4 mmol) in glacial acetic acid (20 mL) was heated to 65 °C and stirred at the same temperature for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated to get a residue. The residue was diluted with ethyl acetate (250 mL) and washed with 10% aqueous sodium bicarbonate solution (200 mL). The ethyl acetate layer was separated, dried over sodium sulphate and concentrated.
  • Step 4 and 5 Synthesis of tert-butyl 4-((4-(6-nitro-1H-benzo[d]imidazol-2-yl)cyclohexyl)carbamoyl)piperazine-1- carboxylate (7)
  • the DCM layer was separated, dried over sodium sulphate and concentrated under reduced pressure to get the crude phenyl (4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamate (0.32 g, crude).
  • the crude was washed with hexane (50 mL) and dried.
  • Step 6 Synthesis of tert-butyl 2-(4-(4-(ted-butoxycarbonyi)piperaz ⁇ ne-1-carboxamido)cyciohexyi)-b-nitro-1H- benzo[d]imidazole- 1 -carboxylate
  • Step 8 Synthesis of tert-butyl 2-(4-(4-(ted-butoxycarbonyi)piperaz ⁇ ne-1-carboxamido)cyciohexyi)-8- (phenylsulfonamido)-l H-benzo[d]imidazole- 1 -carboxylate
  • Step 9 Synthesis of N-(4-(6-(phenylsulfonamido)-1H-benzo[d]imidazol-2-yl)cyclohexyl)piperazine-1 -carboxamide
  • tert-butyl 2-(4-(4-(tert-butoxycarbonyl)piperazine-1-carboxamido)cyclohexyl)-6- (phenylsulfonamido)-1 H-benzo[d]imidazole-1-carboxylate (0.15 g, 0.22 mmol) in dry DCM (5 mL) was added 4N HCL in dioxane (5 mL). The reaction mixture was stirred at RT for 6 h.
  • Step 2 Synthesis of tert-butyl (4-(b-nitro-1H-benzo[d] ⁇ midazoi-2-yi)cyciohexyi)carbamate (4)
  • tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3) (1.3 g, 3.4 mmol) in glacial acetic acid (20 mL) was heated to 65 °C and stirred at the same temperature for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated to get a residue. The residue was diluted with ethyl acetate (250 mL) and washed with 10% aqueous sodium bicarbonate solution (200 mL). The ethyl acetate layer was separated, dried over sodium sulphate and concentrated.
  • Step 4 Synthesis o ⁇ 3-methyl-N-(4-(6-nitro-1H-benzo[d]imidazol-2-yl)cyclohexyl)benzamide (7)
  • Step 5 Synthesis of tert-butyl 2-(4-(3-methylbenzamido)cyclohexyl)-8-nitro-1H-benzo[d]imidazole-1-carboxylate (8) 3-Methyl-N-(4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)benzamide (7) (0.25 g, 0.66 mmol) in DCM (10 mL) was taken in a RB flask (50 mL) under N2 at 0 °C.
  • Step 6 Synthesis of tert-butyl 8-amino-2-(4-(3-methyibenzamido)cyciohexyi)-1H-benzo[d] ⁇ midazoie-1-carboxyiate
  • Step 7 and Step 8 Synthesis of 3-methyl-N-(4-(6-(phenylsulfonamido)-1H-benzo[d]imidazol-2- yl)cyclohexyl)benzamide
  • N-(2-Amino-4-nitrophenyl)cyclopentanecarboxamide (3) (0.25 g, 0.1 mmol) in glacial acetic acid (10 mL) was heated at 65 °C for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated to get a residue which was diluted with ethyl acetate (150 mL) and washed with 10% aqueous sodium bicarbonate solution (75 mL). The ethyl acetate layer was separated, dried over sodium sulphate and concentrated. The crude was triturated with diethyl ether (25 mL) to get (4) (0.125 g, yield: 54%) as a pale yellow solid. This solid was taken to the next step without further purification. LCMS: 232.5 (M+1 ).
  • Step 4a Synthesis ofN-(2-cyclopentyl-1H-benzo[d]imidazol-6-yl)-1-phenylmethanesulfonamide
  • Step 4b Synthesis of N-benzhydryl-2-cyclopentyl-1 H-benzo[d]imidazol-6-amine
  • Step 5 Synthesis of tert-butyl %-bromo-2-cyciopenfyi-1H-pyrroio[3,2-b]pyridine-1-ca ⁇ boxyiate (7)
  • Step 6 and Step 7 Synthesis of N-(2-cyclopentyl-1H-pyrrolo[3,2-b]pyridin-6-yl)benzenesulfonamide
  • Step 3 N-(2-amino-4-(phenylsulfonamido) phenyl)-3-methoxy-4-(1H-pyrazol-4-yl)benzamide (5)
  • N-(3, 4-diaminophenyl)benzenesulfonamide 3 200 mg, 0.760 mmol
  • 3-methoxy-4-(1 H-pyrazol-4-yl)benzoic acid 4 132 mg, 0.60 mmol
  • HATU 346 mg, 0.912 mmol
  • DIPEA 0.24 mL, 1.52 mmol
  • Step 2 N-(2-(3-methoxy-4-(1H-pyrazol-4-yl)phenyl)-1H-benzo[d]imidazol-6-yl)benzene sulfonamide
  • N-(2-amino-4-nitrophenyl) cyclopentane carboxamide (2) (1.3 g, 5.21 mmol) in glacial acetic acid (40 mL) was heated to 60 °C for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated to get a residue. The residue was diluted with ethyl acetate (100 mL) and washed with 10% aqueous sodium bicarbonate solution (50 mL). The ethyl acetate layer was separated, dried over anhydrous sodium sulphate, filtered and concentrated to afford 2-cyclopentyl-6-nitro-1 H-benzo[d]imidazole (3) (1.15 g, yield: 96%).
  • Step-3 tert-butyl 2-cyclopent/l-5-nitro-1H-benzo[d] ⁇ midazole-1-carboxylate (4)
  • Step-4 tert-butyl 5-amino-2-cyclopent/l-1H-benzo[d] ⁇ midazole-1-carboxylate (5)
  • Step 3 tert-butyl((1r,3r)-3-(2-((2-amino-4-(phenylsulfonamido) phenyl) amino)-2-oxoethyl) cyclobutyl) carbamate (4)
  • N-(3, 4-diaminophenyl) benzenesulfonamide (3) 0.1 g, 0.379 mmol
  • 2-(trans-3-((tert-Butoxycarbonyl) amino) cyclobutyl) acetic acid 96 mg, 0.418 mmol
  • HATU 216 mg, 0.569 mmol
  • DIPEA 0.13 mL, 0.759 mmol
  • Step 4 tert-butyl ((1r,3r)-3-((6-(phenylsulfonamido)-1H-benzo[cl]imiclazol-2-yl) methyl) cyclobutyl) carbamate (5) tert-butyl ((1 r,3r)-3-(2-((2-amino-4-(phenylsulfonamido) phenyl) amino)-2-oxoethyl) cyclobutyl) carbamate (4) (50 mg, 0.105 mmol) in glacial acetic acid (2 mL) was heated at 60 °C for 16 h. The reaction was monitored by TLC.
  • Step 5 N-(2-(((1r,3r)-3-aminocyclobutyl) methyl)-1H-benzo[d]imidazol-6-yl) benzenesulfonamide
  • Example 21 Preparation of Compound 33: N-((1 r,3r)-3-((6-(phenylsulfonamido)-1 FI-benzo[d1imidazol-2-yl)methyl) cyclobutyl) acetamide Step 1: Synthesis of2-((1r,3r)-3-acetamidocyclobutyl)acetic acid (2):
  • trans-(3-aminocyclobutyl)acetic acid hydrochloride (1) (0.05 g, 0.302 mmol) in dioxane/water (3 mL, 2: 1 ) was added NaHC03 (126 mg, 1.51 mmol).
  • Acetic anhydride (0.06 mL, 0.604 mmol) was added and the mixture was stirred at RT for 16 h.
  • the mixture was diluted with water (5 mL) and washed with EtOAc (2 x 5 mL).
  • the aqueous layer was acidified using 1.5N HCI and extracted with 10% MeOH in DCM mixture (2 x 10 mL).
  • Step 2 2-((1r,3r)-3-acetamidocyclobutyl)-N-(2-amino-4-(phenylsulfonamido)phenyl) acetamide (4)
  • Step 3 N-((1r,3r)-3-((6-(phenylsulfonamido)-1H-benzo[d]imidazol-2-yl)methyl)cyclobutyl) acetamide
  • Example 22 Preparation of Compound 34 and 35: 4: 4-(6-(phenylsulfonamido)-1 H-indole-2-carbonyl) piperazin-1 - ium and N-(3-aminopropyl)-N-methyl-6-(phenylsulfonamido)-1 H-indole-2-carboxamide
  • Step 1 1 -(tert-butyl) 2-methyl 6-(phenylsulfonamido)-1 H-indole-1 ,2-dicarboxylate (2)
  • Step 3 tert-butyl 4-(6-(phenylsulfonamido)-1 H-indole-2-carbonyl) piperazine-1 -carboxylate (4)
  • 1 -(tert-butoxycarbonyl)-6-(phenylsulfonamido)-1 H-indole-2-carboxylic acid (3) 75 mg, 0.072 mmol
  • 1 -Boc piperazine 37 mg, 0.079 mmol
  • HATU 103 mg, 0.108 mmol
  • DIPEA 0.06 mL, 0.144 mmol
  • Step 4 4-(6-(phenylsulfonamido)-1H-indole-2-carbonyl) piperazin-1-ium
  • Step 5 tert-butyl (3-(N-methyl-6-(phenylsulfonamido)-1H-indole-2-carboxamido) propyl) carbamate (4)
  • Step 6 N-( 3-aminopropyl) -N-methyl-6-(phenylsulfonamido) - 1 H-indole-2-carboxamide
  • Step 2 methyl 1-(((tert-butoxycarbonyl)(methyl)amino)methyl)cyclopropane-1-carboxylate (3)
  • Step 3 1-(((tert-butoxycarbonyl)(methyl)amino)methyl)cyclopropane-1-carboxylic acid (4)
  • Step 4 tert-butyl ((1-carbamoylcyclopropyl)methyl)(methyl)carbamate (5)
  • Step 6 tert-butyl 2-(((1-carbamoylcyclopropyl)methyl)(methyl)carbamoyl)-8-(phenylsulfonamido)-1H-indole-1- carboxylate (8)
  • Human p38a with a N-terminal GST tag (64 kDa) was produced in BL21 (BL21-DE3 or BL21 -DE3-pLysS) E. Coli.
  • Bacteria was grown in 250-1000 ml of LB medium containing ampicillin (50 pg/ml) at 37°C (25 °C with BL21-DE3- pLysS) until an O.D. of 0.5-0.8 and induced for 3 h at 18°C with IPTG (0.05 mM).
  • cells were harvested by centrifugation (4000 rpm for 10 min at 4 °C), resuspended in 13.5 ml of cold PBS containing 1 mg/ml lysozyme, 5 mM EDTA, 1 complete EDTA-free protease inhibitor cocktail tablet (Roche, 1 1873580001 ), and sonicated. Then, Triton X-100 was added to a final concentration of 1 % (v/v) and cell debris was cleared by centrifugation (10000 rpm for 20 min at 4 °C).
  • Glutathione-SepharoseTM 4 Fast Flow beads (GE Healthcare, GE17-5132-01) were washed in cold PBS buffer, resuspended in PBS (50:50), and 70 mI of the bead slurry were added per 1 ml of supernatant.
  • Protein elution was repeated three times. To remove glutathione, eluted proteins were put into a dialysis bag (12-14 MWCO, SpectralPor) and dialyzed against 2 1 of buffer containing 20 mM Tris pH 8.0, 50 mM NaCI, 0, 1 mM EDTA, 0.5 mM DTT and 5% glycerol, overnight at 4°C. Following dialysis, samples were collected, aliquoted and stored at -80°C. An aliquot was analyzed in 10% SDS-PAGE gel and stained with Coomassie blue to quantify the GST-p38a using BSA as a reference.
  • Purified and dialyzed recombinant GST-p38a protein (4 pg) was incubated with purified MBP-MKK6 (1 pg) in 20 mI of buffer containing 50 mM Tris-HCI pH 7.5, 2 mM DTT, 10 mM MgCh, 100 pM NaaVC , 1 mM PMSF, 10 pg/ml aprotinin, 10 pg/ml leupeptin and 200 pM ATP for 30 min at 37 °C. Reactions were stopped by the addition of 5x sample loading buffer and boiling at 95 °C for 5 min.
  • Phosphorylation of GST-p38a was detected by immunoblotting using phospho antibodies against the activation loop residues Thr180 and Tyr182 of p38a (BD Biosciences, 612288). Particularly preferred are compounds that show no inhibition of MKK6-induced p38a phosphorylation at 10 mM, more preferably 30 mM.
  • Active GST-p38a 200 ng was prepared by incubation with MKK6 as describe above, and then was incubated with purified GST-MK2 protein (500 ng) in 20 mI of buffer containing 50 mM Tris-HCI pH 7.5, 2 mM DTT, 10 mM MgCh, 100 pM Na 3 V04, 1 mM PMSF, 10 pg/ml aprotinin, 10 pg/ml leupeptin and 200 pM ATP for 30 min at 30 °C. Reactions were stopped by the addition of 5x sample loading buffer and boiling at 95 °C for 5 min. Phosphorylation of MK2 on Thr334 was detected by immunoblotting using phospho-specific antibodies (Cell Signalling, 3007S).
  • a peptide corresponding to amino acids 386-414 of TAB1 [RVYPVSVPYSSAQSTSKTSVTLSLVMPSQ] (GenScript) was resuspended in 50 mM Tris-base adjusted to pH 7.5, at a concentration of 100-300 mM and stored in aliquots at -80°C.
  • Dephosphorylated GST-p38a protein (2 pg) was incubated with 15 pM TAB1 peptide in 20 mI of buffer containing 100 mM NaCI, 20 mM Tris-HCI pH 7.5, 2 mM DTT, 2 mM MgCh and 600 pM ATP for 2 h at 37°C.
  • Caco-2 cells were grown as monolayers for 21 days in 96-well plates and used as a model to study transport across the intestinal barrier. Compounds were assayed at 10 mM (in triplicates) in HBSS-Hepes buffer, pH 7.4 and added to the donor side, while the receiver side was filled only with HBSS-Hepes buffer, pH 7.4. Teer and Lucifer yellow Assay were used as markers of Integrity of the cell monolayer. After incubation at 37 °C for 2 h, media was removed from the receiver side and samples were analyzed by HPLC-MS to calculate, the Papp values. Metoprolol was used as reference compound. In the table below, the apparent permeability (Papp) values for the indicated compounds in Caco-2 cells is shown.
  • Papp apparent permeability
  • the compounds of the invention showed high permeability in Caco-2 cells.
  • Example 32 Stability in human liver microsomes in vitro
  • the compounds of the invention 10, 11 and 15 showed high stability in human liver microsomes in vitro.
  • CHO-hERG DUO cells were cultured in Ham's Nutrient Mixture F-12 (Lonza, BE12-615F), supplemented with 10% Fetal Bovine Serum (Sigma, F7524), 5% Penicillin/Streptomycin (Lonza, DE17-602E), G418 (Sigma, G8168; 1 mL of 50mg/mL stock solution) and Hygromycine B (Invivogen, ant-hm-5; 0.5 mL of 100 mg/mL stock solution). 2x10 6 CHO-hERG DUO cells were seeded 48 h before the experiment onto a T225 flask.
  • Intracellular solution 120 mM KCI, 5.37 mM CaCI2, 1.75 mM MgCI2, 10 mM EGTA, 10 mM HEPES, 4 mM Na2- ATP (pH 7.2 with KOH).
  • Extracellular solution 145 mM NaCI, 4 mM KCI, 1 mM MgCI2, 2 mM CaCI2, 10 mM HEPES, 10 mM Glucose (pH 7.4 with NaOH).
  • the compounds of the invention 1 , 10, 11 , 12, 15 and 23 showed no inhibition of human hERG potassium channel. This means that the compounds of the invention are less likely to be cardiotoxic.
  • HEK293 cells were grown in DMEM (Life Technologies, 11995) supplemented with 10 % Fetal Bovine Serum (HyClone, SH30070.03) (growth medium) and detached from the plate with 0.05 % Trypsin/EDTA (Life Technologies, 11995) supplemented with 10 % Fetal Bovine Serum (HyClone, SH30070.03) (growth medium) and detached from the plate with 0.05 % Trypsin/EDTA (Life
  • cell transfection reagents were prepared as follows:
  • lipid:DNA complex was added to 20 ml HEK293T cells at 2x10 5 cells/ml and mixed by inversion. 100 pl/well was seeded into 96-well plates (Corning, 3917) and cultured for 20 h to allow NanoLuc® fusion p38a expression.
  • 3X Complete NanoBRETTM Nano-Glo® Substrate was prepared in OptiMEM without serum or phenol red (1 : 166 dilution of NanoBRETTM Nano-Glo® Substrate plus a 1 :500 dilution of Extracellular NanoLuc Inhibitor in OptiMEM without serum or phenol red) and mixed gently by inversion 5-10 times in a conical tube. 50pL were added per well of 3X Complete NanoBRETTM Nano-Glo® Substrate for a 96-well plate and incubated 2-3 min at room temperature. Measurements were done within 10 min of substrate activation. Following addition of NanoBRETTM Nano-Glo® Substrate, donor emission (e.g.
  • acceptor emission e.g. 610 nm or 630 nm
  • acceptor emission e.g. 610 nm or 630 nm
  • acceptor emission values e.g. 610 nm
  • donor emission value e.g. 450 nm
  • FIG. 1 shows IC50 curves for VX-702 (ATP-competitive inhibitor) and the compounds 1 and 10.
  • the lower table shows the NanoBRET inhibition (%) for the indicated compounds at 50 mM, 15 pM and 5 pM (in duplicates). According to the results, the compounds of the invention 3, 5, 10 and 15 were able to interact with p38a in cells.
  • Example 35 Inhibition of ischemia-reperfusion induced cell death
  • H9c2 cells were cultured in high-glucose DMEM medium (Sigma, D5796), supplemented with 10% Fetal Bovine Serum (LifeTechnologies, 10500064), 1 % Penicillin/Streptomycin (LabClinics, L0022-100, 5 mL of 100x Solution) and 1 % L-glutamine (LabClinics, X0550-100)] in 10 cm plates at 37°C with 5% C02 until they reach 80-90% confluence. Then, cells were washed with 1X DPBS (Sigma, D1408), detached from the plates with trypsin-EDTA (Sigma, T3924) and counted with an automated cell counter (Bio-Rad T20). 250,000 cells in 3 ml of medium were seeded in 60 mm plates and grown for 24 h.
  • the medium was changed to 3 ml of Ischemic Buffer (137 mM NaCI, 12 mM KCI, 0.5 mM MgCh, 0.9 mM CaCh, 4 mM HEPES, 10 mM 2-deoxy-glucose, and 20 mM sodium lactate (pH 6.2) containing either DMSO, SB203580 or the compounds.
  • Cells were kept in H35-hypoxistation chamber flushed with 0.1 % O2 5% CO2 and 95 % N2 (65% HR) for 2 h.
  • cells were switched back to the original medium and kept for 4 h in a normal incubator with 5%C02. Non-treated cells were maintained in a normal incubator with 5%C02 (normoxia).
  • the cell suspension was centrifuged 5 min at 1500 rpm and the cell pellet was lysed in RIPA buffer (150 mM NaCI, 50 mM Tris-HCI pH 7.3, 1 % NP-40, 5mM EDTA, 20 mM NaF, 1 mM Na3V04, 1 mM PMSF, 1 mM DTT, 2.5 mM benzamide, 10 pg/ml pepstatin, 10 pg/ml aprotinin, 10 pg/ml leupeptin and 1 pM microcystin) for 30 min on ice.
  • RIPA buffer 150 mM NaCI, 50 mM Tris-HCI pH 7.3, 1 % NP-40, 5mM EDTA, 20 mM NaF, 1 mM Na3V04, 1 mM PMSF, 1 mM DTT, 2.5 mM benzamide, 10 pg/ml pepstatin, 10 pg/ml a
  • FIG 2 shows cleaved caspase-3 levels as a measure of cell death in H9c2 cardiomyocytes pre-treated with the indicated compounds and subjected to simulated ischemia-reperfusion. Values were normalized to DMSO treated cells. Each dot represents an independent experiment. According to these results the compounds of the invention 10, 11 , 12 and 15 were able to reduce simulated ischemia-reperfusion induced cell death in H9c2 cells.
  • Neonatal rat ventricular myocytes were isolated from newborn (1-2 days) Wistar rats.
  • Hearts (11-15) were collected in ice-cold CBFHH buffer (137 mM NaCI, 20 mM Hepes, 0.81 mM Mg 2 S0 4 , 0.44 mM K 2 PO 4 , 0.33 mM Na2HP04, 5.6 mM glucose, 5.4 mM KCI, pH 7.3) and atria, blood vessels and tissues from other organs were existed.
  • Minced ventricles were put into a 100 ml Erlenmeyer flask and NRVMs were purified by serial enzymatic digestions of tissue with 0.6 mg/ml of collagenase Type II (Worthington, LS004174) and 0.4 mg/ml pancreatin (Sigma, P3292) in CBFHH buffer shaking at 37°C. First digestion (20 min) was discarded. After the second (20 min), third (25 min), fourth (25 min) and fifth (15 min) digestions, the cell suspension was placed into 4 ml FBS (Pan Biotech, P30-3302) at 37°C.
  • FBS Pan Biotech, P30-3302
  • NRVMs/well in a final volume of 100 pi were seeded onto 1 % gelatin-coated 96-well plates. After 24 h, media was changed and cells were kept at 37°C and 5% CO2 for 3 days, when a confluent monolayer of spontaneously beating myocytes was observed. At this point, compounds were added to the cells.
  • fibroblasts were maintained with high-glucose DMEM medium (Sigma, D5796), supplemented with 10% Fetal Bovine Serum (LifeT echnologies, 10500064), 1 % Penicillin/Streptomycin (LabClinics, L0022-100, 5 mL of 100x Solution) and 1 % L-glutamine (LabClinics, X0550-100).
  • DMEM medium Sigma, D5796
  • Fetal Bovine Serum LifeT echnologies, 10500064
  • Penicillin/Streptomycin (LabClinics, L0022-100, 5 mL of 100x Solution)
  • 1 % L-glutamine LabClinics, X0550-100
  • Stocks of the compounds 1 , 10 and 15 were prepared at 10 mM in 100% DMSO. Compounds were diluted serially in 100% DMSO to obtain solutions at 100X of the final concentrations used for cells. These solutions were diluted 1 :100 in 100 pi of media to obtain final concentrations of 100 pM, 30 pM, 10 pM, 5 pM and 1 pM, which all contain 1 % final DMSO concentration, and 100 pi were added per well.
  • Cell viability was determined after 48 h and 72 h using an MTT kit (Roche, 11465007001), by adding first 10 pi of reagent A to every well and 4 h later 100 pi of reagent B. Next day, absorbance was read at 570 nm using an Epoch 2 plate reader. Absorbance was proportional to the viable number of cells and values were normalized to the DMSO control.
  • the compounds of the invention 1 , 10 and 15 showed no toxicity on cardiomyocytes or fibroblasts, neither at 48 h nor at 72 h.
  • a Toxoplasma dense granule protein, GRA24 modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation. J Exp Med 210 , 2071-2086.
  • Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes. J. Cell Biol.,
  • Amyloidogenic light chains induce cardiomyocyte contractile dysfunction and apoptosis via a non-canonical p38alpha MAPK pathway. Proc Natl Acad Sci U S A 107, 4188-4193.
  • R 1 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms selected from the group consisting of C, N, O, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C ⁇ alkyl, C ⁇ alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and— [NHC(0)] z R 9 , wherein z is 0 or 1 , and wherein R 9 is aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -
  • R 1 is an aliphatic acyclic group with 4 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl,
  • R 3 is -R 7 -R 8 , wherein R 7 is -(CH2) X -, with x being 0 to 3, and R 8 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH2;
  • R 6 is hydrogen, an alkyl group with 1 to 3 carbon atoms, or an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH2;
  • R 4 , R 5 , and R have one of the following meanings:
  • R 4 is N; R 5 is CH; and R is CH;
  • R 4 is CH; R 5 is N; and R is N;
  • R 4 is CH; R 5 is CH; and R is N;
  • R 4 is CH; R 5 is N; and R is CH; or
  • R 4 is N; R 5 is CH; and R is CH; or
  • R 4 is N; R 5 is N; and R is CH.
  • R 1 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 4 to 6 ring atoms selected from the group consisting of C and N, wherein the ring has no or one N as ring atom, and which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)] z R 9 , wherein z is 0 or 1 , and wherein R 9 is aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino,
  • R 3 is -R 7 -R 8 , wherein R 7 is -(CH2) X -, with x being 0 or 1
  • R 8 is an aromatic or heteroaromatic ring or ring system having 5 to 6 ring atoms selected from the group consisting of C, N, and 0, wherein the ring or ring system has no or one atom other than C as ring atom, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, and -NHC alkyl.
  • Clause 7 The compound according to one or more of the preceding clauses, wherein said compound is selected from the group consisting of compounds 1 to 29, a tautomer thereof, or a pharmaceutically acceptable salt thereof.
  • Clause 8 The compound according to any one of clauses 1 to 7 in combination with one or more pharmaceutically acceptable carriers or excipients.
  • R 1 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, and -C(0)NH2; or R 1 is an aliphatic acyclic group with 4 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)] z R 9 , wherein z is 0 or 1 , and wherein R 9 is aliphatic cyclic, alipha
  • R 1 is an aliphatic acyclic group with 4 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH 3I and -C(0)NH 2 ;
  • R 3 is -R 7 -R 8 , wherein R 7 is -(CH2) X -, with x being 0 to 3, and R 8 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, and 0, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, and -NHC alkyl;
  • R 6 is hydrogen, an alkyl group with 1 to 3 carbon atoms, or an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH2;
  • R 4 , R 5 , and R have one of the following meanings:
  • R 4 is N; R 5 is CH; and R is CH;
  • R 4 is CH; R 5 is N; and R is N;
  • R 4 is CH; R 5 is CH; and R is N;
  • R 4 is CH; R 5 is N; and R is CH; or
  • R 4 is CH; R 5 is CH; and R is CH; a tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (la) is not:
  • R 4 is N; R 5 is N; and R is CH.
  • R 1 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C and N, wherein the ring or ring system has no or one N as ring atom, and which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, Ci_ alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)] z R 9 , wherein z is 0 or 1 , and wherein R 9 is aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl,
  • R 3 is -R 7 -R 8 , wherein R 7 is -(CH2) X -, with x being 0 or 1 , and R 8 is an aromatic or heteroaromatic ring or ring system having 5 to 6 ring atoms selected from the group consisting of C, N, O, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH 2 .
  • Clause 14 The compound according to one or more of clauses 10 to 13, wherein R 3 is phenyl, optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
  • Clause 15 The compound according to one or more of clauses 10 to 14, wherein said compound is selected from the group consisting of compounds 1 to 8, and 19 to 29, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

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Abstract

The present invention concerns inhibitors of p38α autophosphorylation, pharmaceutical compositions comprising them, and their use in the treatment of a number of diseases, such as myocardial ischemia reperfusion injury. The inhibitors satisfy the following general formula: wherein R, R1, R2, R4, and R5 may have different meanings.

Description

r38a AUTOPHOSPHORYLATION INHIBITORS
This application claims the benefit of the European Patent Application EP1838291 1.8 filed on December 1 1th,
2018.
Field of the invention
The present invention concerns inhibitors of p38a autophosphorylation, pharmaceutical compositions comprising them, and their use in the treatment of a number of diseases, such as myocardial ischemia reperfusion injury.
Background of the invention
p38 mitogen-activated protein kinases (MAPKs) are a class of protein kinases that are responsive to cytokines and stress stimuli. Four p38 MAPKs, p38o (MAPK14), r38b (MAPK1 1 ), r38g (MAPK12), and r38d (MAPK13), have been identified. p38 MAPKs are activated by a variety of cellular stimuli including osmotic shock, inflammatory cytokines, lipopolysaccharide, ultraviolet light, and growth factors. The most abundant p38 MAPK in many cell types is p38a. The kinases MKK6 and MKK3 activate p38a by phosphorylation at Thr-180 and Tyr-182. This is the so- called canonical route of activation. Once p38a is active, it can in turn phosphorylate a range of downstream substrates such as the kinase MK2, the transcription factor ATF2 and the mRNA regulator TTP. p38a has been the subject of intense study by the scientific community and also by the pharmaceutical industry. The latter has devoted huge resources over the last 20 years to the development of different series of clinical-stage inhibitors. Some notorious examples are VX-745, Pamapimod, AMG-548, BIRB-796, Losmapimod, PH-797804, Scio-469 and RWJ-67657 developed by Vertex, Roche, Amgen, Boehringer, GSK, Pfizer, Scios and Johnson&Johnson, respectively. Most of the indications tested in clinical stages involved inflammatory and/or autoimmune diseases such as Rheumatoid arthritis, Crohn's or Chronic obstructive pulmonary disease. The rationale behind all these inhibitors was to antagonize p38a activity by interfering with ATP binding, either directly (for instance VX-745) or indirectly (for instance Doramapimod), thus supressing downstream phosphorylation of all p38a substrates in all cellular contexts (Yong et al., 2009).
Despite these tremendous efforts, not a single p38a inhibitor has reached late clinical stages, all having been discontinued due either to undesirable adverse events or to lack of efficacy at the doses tested.
Although p38a has been studied for many years, important aspects of its biology are still being unveiled. This includes the capability of p38a to autophosphorylate via alternative (non-canonical) routes. One of them is induced by the kinase ZAP70 that can phosphorylate p38a at Tyr323, which in turn induces autophosphorylation and subsequent activation of p38a in T-cells (Salvador et al, 2005). A second non-canonical route has been described where p38a autophosphorylation is induced by binding to the protein TAB1 (Ge et al., 2002; De Nicola et al., 2013). These non- canonical routes have been proposed to be relevant in the development of a range of conditions, such as ischemia reperfusion injury (Denise Martin et al., 2012), pancreatic cancer (Alam et al., 2015), and skin inflammation (Theivanthiran et al., 2015), to name a few. In spite of the potential importance of this mechanism, very little is known on how to inhibit p38a autophosphorylation (De Nicola et al. 2018).
There is still a need of developing compounds that inhibit the autophosphorylation of p38a and that can be used in therapy.
Summary of the invention
The present inventors have found compounds based on a nitrogenated bicyclic ring that inhibit the autophosphorylation of p38a and therefore can be used in therapy. These compounds are chemically different from the existing ATP competitors and with a different mechanism of action by specifically targeting the non-canonical p38a activation. Therefore, these compounds would inhibit only a subset of p38a regulated functions, which is expected to yield reduced undesired side-effects.
Thus, in a first aspect the invention relates to a compound A, optionally in combination with one or more pharmaceutically or veterinary acceptable carriers or excipients, for use in the prophylaxis or treatment of a disease associated with autophosphorylation of p38a kinase, wherein the compound A is either a compound of formula (I):
Figure imgf000003_0001
wherein
R1 is an aliphatic carbocyclic ring or ring system having 4 to 10 ring atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C^alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is an aliphatic heterocyclic or heteroaromatic monocyclic ring having 6 ring atoms selected from the group consisting of C, N, O, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is -CONR10R11, wherein
R10 is H or C alkyl; and R11 is C^alkyl optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C^alkyl, C haloalkyl, C^alkoxy, Ci-4alkylthio, amino, halogen, oxo, -NHCi-4alkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; and optionally two hydrogen atoms attached to one carbon atom of the C^alkyl are replaced by two CH2 moieties which form together a carbocyclic ring having 3 to 6 ring atoms; or alternatively
R11 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C^alkyl, C haloalkyl, Ci.4alkoxy, Ci.4alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH¾ -C(0)NH¾ -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R10 and R11 form together a heterocyclic ring or ring system having 4 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci-4alkyl, C haloalkyl, Ci.4alkoxy, Ci-4alkylthio, amino, halogen, oxo, -NHCi-4alkyl, -NR15R16, -C(0)CH¾ -C(0)NH¾ -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; or alternatively
R1 is -Y-R12, wherein
Y is selected from the group consisting of NR13, 0, S, -C(0)-;
R12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci-4alkyl, C haloalkyl, Ci-4alkoxy, Ci-4alkylthio, amino, halogen, oxo, -NHCi.4alkyl, -NR15R16, -C(0)CH¾ -C(0)NH¾ -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; and
R13 is H or Ci-4 alkyl; or alternatively
R1 is -CH2-R14, wherein R14 is an aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms , wherein the atoms of the heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; or alternatively
R14 is an aliphatic heterocyclic ring having 5 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9;
Figure imgf000005_0001
R3 is -R7-R8;
R7 is -(CH2)X-, with x being 0 to 3;
R8 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH2; z is O or 1;
R9 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2;
R15 is H or C alkyl;
R16 is R9 or alternatively is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2; wherein R4, R5, and R have one of the following meanings:
(i) R4 is N; R5 is CH; and R is CH; (ii) R4 is N; R5 is N; and R is CH;
(iii) R4 is N; R5 is N; and R is N;
(iv) R4 is CH; R5 is N; and R is N;
(v) R4 is CH; R5 is CH; and R is N;
(vi) R4 is CH; R5 is N; and R is CH; or
(vii) R4 is CH; R5 is CH; and R is CH; an isomer thereof, particularly a tautomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, particularly a pharmaceutically acceptable salt thereof; or alternatively the compound A for use in the prophylaxis or treatment of a disease associated with autophosphorylation of p38a kinase is one of the following:
Figure imgf000006_0001
or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof. This aspect may also be formulated as the use of a compound A as defined above, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, for the manufacture of a medicament for the prophylaxis or treatment of a disease associated with autophosphorylation of p38a kinase. The present invention also relates to method for the treatment and/or prophylaxis of a disease associated with autophosphorylation of p38a kinase, comprising administering an effective amount of the previously defined compound A, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, and one or more pharmaceutically or veterinary acceptable excipients or carriers, in a subject in need thereof, including a human.
The invention also provides the compounds A as defined above for use in the prophylaxis or treatment of a disease selected from the group consisting of cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer, in particular wherein the disease is associated with autophosphorylation of p38a kinase; for example cardiovascular diseases such as myocardial ischemia-reperfusion injury, cardiac amyloidosis, or atherosclerosis/hypercoagulability; inflammatory diseases such as skin inflammation or toxoplasmosis; t-cell senescence, cancer such as pancreatic cancer, and inflammatory autoimmune diseases.
This aspect may also be formulated as the use of a compound A as defined above, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, for the manufacture of a medicament for the prophylaxis or treatment of a disease selected from the group consisting of cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer, in particular wherein the disease is associated with autophosphorylation of p38a kinase. The present invention also relates to method for the treatment and/or prophylaxis of a disease selected from the group consisting of cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer, in particular wherein the disease is associated with autophosphorylation of p38a kinase, comprising administering an effective amount of the previously defined compound A, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, and one or more pharmaceutically or veterinary acceptable excipients or carriers, in a subject in need thereof, including a human.
Another aspect of the invention relates to a compound B which is either a compound of formula (la):
Figure imgf000007_0001
wherein
R1 is an aliphatic carbocyclic ring or ring system having 5 to 10 ring atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is an aliphatic heterocyclic or heteroaromatic monocyclic ring having 6 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, Ci-4alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCi.4alkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is -CONR10R11, wherein
R10 is H or C alkyl; and R11 is Ci-6alkyl optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, Ci-4alkoxy, Ci-4alkylthio, amino, halogen, oxo, -NHCi-4alkyl, -NR15R16, -C(0)CH3, -C(0)NH¾ -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; and optionally two hydrogen atoms attached to one carbon atom of the Ci-6alkyl are replaced by two CH2 moieties which form together a carbocyclic ring having 3 to 6 ring atoms; or alternatively
R11 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH¾ -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R10 and R11 form together a heterocyclic ring or ring system having 4 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCi-4alkyl, -NR15R16, -C(0)CH3, -C(0)NH¾ -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; or alternatively
R1 is -Y-R12, wherein
Y is selected from the group consisting of NR13, 0, S, -C(O)-;
R12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; and
R13 is H or C1-4 alkyl; or alternatively
R1 is -CH2-R14, wherein
R14 is an aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms , wherein the atoms of the heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C^alkyl, C haloalkyl, C^alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; or alternatively
R14 is an aliphatic heterocyclic ring having 5 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9;
Figure imgf000009_0001
R3 is -R7-R8;
R7 is -(CH2)X-, with x being 0 to 3;
R8 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, and 0, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, and -NHC alkyl; z is 0 or 1;
R9 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2;
R15 is H or C alkyl; R16 is R9 or alternatively is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2;
wherein R4, R5, and R have one of the following meanings:
(i) R4 is N; R5 is CH; and R is CH;
(ii) R4 is N; R5 is N; and R is CH;
(iii) R4 is N; R5 is N; and R is N;
(iv) R4 is CH; R5 is N; and R is N;
(v) R4 is CH; R5 is CH; and R is N;
(vi) R4 is CH; R5 is N; and R is CH; or
(vii) R4 is CH; R5 is CH; and R is CH; an isomer thereof, particularly a tautomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, particularly a pharmaceutically acceptable salt thereof; wherein the compound of formula (la) is other than:
Figure imgf000010_0001
or alternatively compound B is a compound which is one of the following:
Figure imgf000011_0001
or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof. In a further aspect of the present invention, the invention concerns a pharmaceutical or veterinary composition comprising a therapeutically effective amount of a compound A or B according to the invention as well as one or more pharmaceutically or veterinary acceptable carriers or excipients.
Brief Description of Drawings
FIG. 1 shows IC50 curves for VX-702 (ATP-competitive inhibitor) and the compounds 1 and 10 (NanoBret Ratio vs Compound Log [M],
FIG 2. shows cleaved caspase-3 levels (normalized to DMSO) levels for SB203580 (ATP-competitor) and some compounds of the invention. Detailed description of the invention
Definitions
In the present context, the term "alkyl” refers to a saturated branched or linear hydrocarbon group which contains the number of carbon atoms herein indicated and only single bonds. The term "C alkyl" is intended to mean a linear or branched hydrocarbon group having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso- butyl, sec-butyl, and tert-butyl. In the present context, the term "Ci-2alkyl" is intended to mean a linear hydrocarbon group having 1 to 2 carbon atoms, such as methyl and ethyl. In the present context, the term "alkoxy” relates to an alkyl group as defined above linked to the rest of the molecule through an oxygen atom, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and t-butoxy.
The term "alkylthio” relates to an alkyl group as defined above linked to the rest of the molecule through a sulfur atom, such as methylthio, ethylthio, n-propylthio, isopropylthio, and n-butylthio.
The term "haloalkyl” relates to an alkyl group as defined above wherein on or more hydrogen atoms have been replaced by halogen atoms, such as fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, and dichloroethyl.
When used herein, the term "C3-6cycloalkyl" is intended to mean a cyclic hydrocarbon group having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
Herein, the term "halogen" includes fluoro, chloro, bromo, and iodo, more particularly, fluoro, chloro and bromo.
In the present context the term "aromatic ring or ring system" is intended to mean a fully or partially aromatic carbocyclic ring or ring system wherein all the ring system members are carbon atoms, such as phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl.
The term "heteroaromatic ring or ring system" is intended to mean a fully or partially aromatic carbocyclic ring or ring system where one or more of the carbon atoms have been replaced with heteroatoms, e.g. nitrogen (=N- or -NH-), sulphur, and/or oxygen atoms. Examples of such heteroaromatic ring or ring system groups are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, coumaryl, furyl, thienyl, quinolyl, benzothiazolyl, benzotriazolyl, benzodiazolyl, benzooxozolyl, phthalazinyl, phthalanyl, triazolyl, tetrazolyl, isoquinolyl, acridinyl, carbazolyl, dibenzazepinyl, indolyl, benzopyrazolyl and phenoxazonyl.
In the present context, the term "heterocyclic ring or ring system" is intended to mean a non-aromatic carbocyclic ring or ring system where one or more of the carbon atoms have been replaced with heteroatoms, e.g. nitrogen (=N- or -NH-), sulphur, and/or oxygen atoms. Examples of such heterocyclic groups are imidazolidine, piperazine, hexahydropyridazine, hexahydropyrimidine, diazepane, diazocane, pyrrolidine, piperidine, azepane, azocane, aziridine, azirine, azetidine, pyroline, tropane, oxazinane (morpholine), azepine, dihydroazepine, tetrahydroazepine, hexahydroazepine, oxazolane, oxazepane, oxazocane, thiazolane, thiazinane, thiazepane, thiazocane, oxazetane, diazetane, thiazetane, tetrahydrofuran, tetrahydropyran, oxepane, tetrahydrothiophene, tetrahydrothiopyrane, thiepane, dithiane, dithiepane, dioxane, dioxepane, oxathiane and oxathiepane.
In the present context, the term "aliphatic cyclic” and "aliphatic carbocyclic” ring or ring system are indistinctly used and mean an aliphatic ring or ring system wherein all the ring system members are carbon atoms, and which can be saturated or partially unsaturated. Non-limiting examples of aliphatic carbocyclic rings include cyclobutane, cyclopentane, cyclohexane, cyclohexene, and the like.
For the purposes of the invention ring atoms are to be understood as ring members, that is a ring or ring system having 3 to 6 ring atoms is equivalent to a 3- to 6-membered ring or ring system.
In the present context, the term "optionally substituted" is intended to mean that the group in question may be substituted one or more times, preferably 1-2 times. Preferred substituents include -OH, OCH3, -NH2; -NHCH3, and halogen. Furthermore, the term "optionally substituted" may also mean that the group in question is unsubstituted.
The compounds of the present invention can be in a free form or in the form of a pharmaceutically or veterinary acceptable salt. In the context of the present invention, the term "pharmaceutically or veterinary acceptable salt” is to be understood as a salt formed with either a base or an acid, wherein the resulting counter-ion does not significantly add to the toxicity of the compound of the present invention. In particular embodiments, the invention relates to the compounds B or the compounds A for use as defined herein or their pharmaceutically acceptable salts.
Examples of pharmaceutically or veterinary acceptable salts include inorganic acid salts such as hydrochloride, sulfate, bisulfate, nitrate, phosphate or hydrobromide, etc., organic acid salts such as acetate, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, maleate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, citrate, fumarate, succinate, tartrate, naphthylate, glucoheptonate, and laurylsulphonate salts, and the like. Also, there may be mentioned a salt with a base (for example, alkali metal salt such as sodium salt, potassium salt, etc. or alkaline earth metal salt such as calcium salt, etc.).
Some of the compounds of the invention may have chiral centres that can give rise to various isomers. As used herein, the term "isomer" refers to individual compounds with identical formulas but distinct structures. The term isomer includes enantiomers, racemates, racemic mixtures, geometric isomers (cis/trans or syn/anti or E/Z), diastereoisomers, tautomers. The present invention relates to each of these isomers and mixtures thereof. The term "tautomers" refers to isomers, which differ in the position of an atom, generally a hydrogen atom, and one or more multiple bonds, which are capable to change reversibly from one to another. All tautomers are to be considered equivalent for the purposes of the invention.
It is to be understood that where the substituents of an R group of a formula described herein are described as being preferred, most preferred or having no level of preference, these substituents can be combined with any of the substituents of a different R group which are described as being preferred, most preferred, or having no level of preference. The compounds of the present invention
In a particular embodiment, the compound A is a compound of formula (I),
Figure imgf000014_0001
wherein R4, R5, and R have the following meanings:
(i) R4 is N; R5 is CH; and R is CH.
In another particular embodiment, the compound A is a compound of formula (I), wherein R4, R5, and R have the following meanings:
(ii) R4 is N; R5 is N; and R is CH.
In another particular embodiment, the compound A is a compound of formula (I), wherein R4, R5, and R have the following meanings:
(vii) R4 is CH; R5 is CH; and R is CH.
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is an optionally substituted aliphatic carbocyclic ring or ring system as defined above having 4 to 10 ring atoms, more particularly 5 to 10 ring atoms, even more particularly 4 to 6 ring atoms. More particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9. Even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)]zR9 as previously defined; even more particularly R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C^alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, and -C(0)NH2; even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of C alkyl, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, and -C(0)NH2. In another particular embodiment, R1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH3, and -[NHC(0)]zR9, wherein z is 0 or 1, and R9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is an optionally substituted cyclobutyl, an optionally substituted cyclopentyl, or an optionally substituted cyclohexyl. Even more particularly, R1 is methylaminocyclopentyl, cyclobutyl, cyclopentyl, oxocyclopentyl (particularly 3-oxocyclopentyl), methylcyclopentyl (particularly methylclopent-2-yl), and methylcyclohexyl (particularly methylclohex-2-yl).
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is an optionally substituted aliphatic heterocyclic ring having 6 ring atoms as defined above; more particularly having 6 ring atoms selected from the group consisting of C, N; even more particularly one of the ring atoms is N. More particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci-4alkyl, C haloalkyl, Ci.4alkoxy, Ci-4alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9. Even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci-4alkyl, Ci.4alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)]zR9 as previously defined; even more particularly R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci-4alkyl, Ci_ 4alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, and -C(0)NH2; even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of Ci-4alkyl, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, and -C(0)NH2. In another particular embodiment, R1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH3, and
-[NHC(0)]zR9, wherein z is 0 or 1, and R9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is N-methylpyrrolidinyl (particularly N-methylpyrrolidin-3-yl), and 1-acetylpiperidyl (particularly 1 -acetylpiperidin-4-yl).
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is an optionally substituted heteroaromatic monocyclic ring having 6 ring atoms as defined above; more particularly having 6 ring atoms selected from the group consisting of C, N, S; even more particularly having 6 ring atoms selected from the group consisting of C, N; even more particularly one of the ring atoms is N. More particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci-4alkyl, C haloalkyl, Ci-4alkoxy, Ci.4alkylthio, amino, halogen, oxo, -NHC^alkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9. Even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci-4alkyl, Ci.4alkoxy, amino, halogen, oxo, -NHCi-4alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)]zR9 as previously defined; even more particularly R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci-4alkyl, Ci-4alkoxy, amino, halogen, oxo, -NHCi-4alkyl, -C(0)CH3, and -C(0)NH2; even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of Ci-4alkyl, amino, halogen, oxo, -NHCi-4alkyl, -C(0)CH3, and -C(0)NH2. In another particular embodiment, R1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH3, and -[NHC(0)]zR9, wherein z is 0 or 1, and R9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is quinolinyl (particularly quinolin-6-yl), pyridinyl (particularly pyridin-4-yl), and indolyl (particularly indol-5-yl).
In another particular embodiment, the compound A is a compound of formula (I), wherein when R1 is substituted with -[NHC(0)]zR9 z is 0 or 1, and R9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is -CONR10R11, even more particularly R10 is H or C^alkyl; and R11 is C^alkyl optionally substituted as previously defined and optionally two hydrogen atoms attached to one carbon atom of the C^alkyl are replaced by two Chh moieties which form together a carbocyclic ring having 3 to 6 ring atoms. Even more particularly R10 is H or Ci-2alkyl; and R11 is Ci-3alkyl optionally substituted as previously defined and optionally two hydrogen atoms attached to one carbon atom of the Ci-3alkyl are replaced by two Chh moieties which form together a carbocyclic ring having 3 to 6 ring atoms.
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is -CONR10R11, wherein R10 and R11 form together a heterocyclic ring or ring system having 4 to 6 ring atoms selected from the group consisting of C, and N, this ring being optionally substituted as previously defined.
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is -CONR10R11 as previously defined and R4, R5, and R have the following meaning:
(vii) R4 is CH; R5 is CH; and R is CH.
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is -Y-R12, and Y is selected from the group consisting of NR13, 0, and -C(0)-, and R12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, more particularly 4 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, more particularly C, N. Even more particularly, R12 is an aliphatic carbocyclic, or aliphatic heterocyclic ring or ring system as previously defined, even more particularly R12 is cyclobutyl, piperidinyl (particularly piperidin-3-yl) or piperazinyl (particularly piperazin-4-yl).
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is -Y-R12, and Y is NR13; R13 is H or C alkyl; more particularly R13 is H or Ci-2alkyl; more particularly R13 is Ci-2alkyl; and
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is -CH2-R14, wherein R14 is an aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, more particularly 4 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, more particularly C, N. Even more particularly, R14 is an aliphatic carbocyclic ring or ring system as previously defined, even more particularly R14 is cyclobutyl.
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is -CH2-R14, wherein R14 is an aliphatic heterocyclic ring having 5 ring atoms selected from the group consisting of C, N, 0, and S, more particularly C, N. Even more particularly, R14 is an aliphatic heterocyclic ring or ring system as previously defined.
In another particular embodiment, the compound A is a compound of formula (I), wherein R3 is -R7-R8, wherein R7 is— (CH2)X-, with x being 0-3, more particularly 0 or 1; and R8 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system, more particularly an aromatic or heteroaromatic ring or ring system, wherein the ring or ring system has 5 to 10 ring atoms, more particularly 5 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, S, more particularly selected from the group consisting of C, N, 0, more particularly wherein the ring or ring system has no or one atom other than C as ring atom. Even more particularly, R8 is an aromatic or heteroaromatic ring or ring system having 5 to 6 ring atoms, wherein the atoms of the heteroaromatic ring or ring system are selected from the group consisting of of C, N, 0, S, more particularly selected from the group consisting of C, N, and 0, more particularly C, N, even more particularly wherein the ring or ring system has no or one atom other than C as ring atom.
In another particular embodiment, R8 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C1-4 alkyl, halogen, oxo, -NHC alkyl and -C(0)NH2; even more particularly R8 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C1-4 alkyl, halogen, oxo, and -NHC alkyl; even more particularly, R8 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C1-4 alkyl, halogen, oxo, -NHC alkyl; even more particularly, R8 is optionally substituted with one, two, or three substituents selected from the group consisting of C alkyl, halogen, and -C(0)NH2. Alternatively, R8 is optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
In another particular embodiment, the compound A is a compound of formula (I), wherein R3 is selected from the group consisting of benzyl, fluorobenzyl, phenyl, tolyl, optionally substituted phenyl, pyridinyl (particularly pyridin-4- yl), optionally substituted pyridinyl, tiophenyl (particularly thiophen-2-yl or thiophen-3-yl), optionally substituted thiophenyl, pyrazolyl (particularly pyrazol-5-yl), and optionally substituted pyrazolyl.
In another embodiment, the compound A is a compound of formula (I), wherein R3 is phenyl, optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
Particularly preferred compounds A are the following compounds, 1 to 36:
Figure imgf000018_0001
Figure imgf000019_0001
or an isomer thereof, particularly a tautomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, particularly a pharmaceutically acceptable salt thereof. Compounds 9 (CAS RN: 1384795-21-4), 10 (CAS RN: 2094667-81-7), 1 1 (CAS RN: 2094511-69-8), 12 (CAS RN: 2094380-73-9), 13 (CAS RN: 1384790-95-7), 16 (CAS RN: 1808772-10-2), 17 (CAS RN: 1797537-02-0), and 18 (CAS RN: 1197756-53-8) were purchased from the company Enamine. Compound 14 (CAS RN: 1324076-13-2) was purchased from Ambinter, and Compound 15 (CAS RN: 1795657-46-3), was purchased from Otava. These commercial products have not been described in any bibliographic references to the best of inventors' knowledge.
The present invention also relates to compounds B, which can be a compound of formula (la)
Figure imgf000020_0001
as previously defined. The compound of formula (la) is other than the following ones:
Figure imgf000020_0002
Figure imgf000021_0001
The compounds included in the table above are commercial products and to the best of inventors' knowledge they have no associated bibliographic reference. In a particular embodiment, the compound B is a compound of formula (la), wherein R4, R5, and R have one of the following meanings:
(i) R4 is N; R5 is CH; and R is CH.
In another particular embodiment, the compound B is a compound of formula (la), wherein R4, R5, and R have the following meanings:
(ii) R4 is N; R5 is N; and R is CH.
In another particular embodiment, the compound A is a compound of formula (I), wherein R4, R5, and R have the following meanings:
(vii) R4 is CH; R5 is CH; and R is CH.
In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is an optionally substituted aliphatic carbocyclic ring or ring system as defined above having 5 to 10 ring atoms, more particularly 5 to 6 ring atoms. More particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9. Even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)]zR9 as previously defined; even more particularly R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, and -C(0)NH2; even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of C alkyl, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, and -C(0)NH2. In another particular embodiment, R1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH3, and - [NHC(0)]zR9, wherein z is 0 or 1, and R9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is an optionally substituted cyclopentyl, or an optionally substituted cyclohexyl. Even more particularly, R1 is methylaminocyclopentyl, cyclopentyl, oxocyclopentyl (particularly 3-oxocyclopentyl), methylcyclopentyl (particularly 2-methylclopentyl), and methylcyclohexyl (particularly 2-methylclohexyl).
In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is an optionally substituted aliphatic heterocyclic ring having 6 ring atoms as defined above; more particularly having 6 ring atoms selected from the group consisting of C, N; even more particularly one of the ring atoms is N. More particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, Ci_ 4haloalkyl, C^alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3, -C(0)NH2, - C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9. Even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)]zR9 as previously defined; even more particularly R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, Ci_ 4alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, and -C(0)NH2; even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of C alkyl, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, and -C(0)NH2. In another particular embodiment, R1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH3, and - [NHC(0)]zR9, wherein z is 0 or 1, and R9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is N-methylpyrrolidinyl (particularly N-methylpyrrolidin-3-yl), and 1-acetylpiperidyl (particularly 1 -acetylpiperidin-4-yl).
In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is an optionally substituted heteroaromatic monocyclic ring having 6 ring atoms as defined above; more particularly having 6 ring atoms selected from the group consisting of C, N, S; even more particularly having 6 ring atoms selected from the group consisting of C, N; even more particularly one of the ring atoms is N. More particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci-4alkyl, C haloalkyl, Ci_ 4alkoxy, Ci.4alkylthio, amino, halogen, oxo, -NHCi.4alkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, - NR15C(0)R16, and -[NHC(0)]zR9. Even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci.4alkyl, Ci.4alkoxy, amino, halogen, oxo, -NHCi-4alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)]zR9 as previously defined; even more particularly R1 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, Ci.4alkyl, Ci_ 4alkoxy, amino, halogen, oxo, -NHCi.4alkyl, -C(0)CH3, and -C(0)NH2; even more particularly, R1 is optionally substituted with one or more substituents selected from the group consisting of Ci.4alkyl, amino, halogen, oxo, -NHCi-4alkyl, -C(0)CH3, and -C(0)NH2. In another particular embodiment, R1 is optionally substituted with one or two substituents selected from the group consisting of methyl, halogen, oxo, -NHCH3, -C(0)CH3, and - [NHC(0)]zR9, wherein z is 0 or 1, and R9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is quinolinyl (particularly quinolin-6-yl), pyridinyl (particularly pyridin-4-yl), and indolyl (particularly indol-5-yl).
In another particular embodiment, the compound B is a compound of formula (la), wherein when R1 is substituted with -[NHC(0)]zR9 z is 0 or 1, and R9 is phenyl or piperazinyl, which may be substituted with one or two methyl groups.
In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is -CONR10R11, even more particularly R10 is H or Ci-4alkyl; and R11 is C^alkyl optionally substituted as previously defined and optionally two hydrogen atoms attached to one carbon atom of the C^alkyl are replaced by two CH2 moieties which form together a carbocyclic ring having 3 to 6 ring atoms. Even more particularly R10 is H or Ci-2alkyl; and R11 is Ci-3alkyl optionally substituted as previously defined and optionally two hydrogen atoms attached to one carbon atom of the Ci-3alkyl are replaced by two CH2 moieties which form together a carbocyclic ring having 3 to 6 ring atoms.
In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is -CONR10R11, wherein R10 and R11 form together a heterocyclic ring or ring system having 4 to 6 ring atoms selected from the group consisting of C, and N, this ring being optionally substituted as previously defined.
In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is -CONR10R11 as previously defined and R4, R5, and R have the following meaning:
(viii) R4 is CH; R5 is CH; and R is CH. In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is -Y-R12, and Y is selected from the group consisting of NR13, 0, and -C(O)-, and R12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, more particularly 4 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, more particularly C, N. Even more particularly, R12 is an aliphatic carbocyclic, or aliphatic heterocyclic ring or ring system as previously defined, even more particularly R12 is cyclobutyl, piperidinyl (particularly piperidin-3-yl) or piperazinyl (particularly piperazin-4-yl).
In another particular embodiment, the compound B is a compound of formula (la), wherein R1 is -Y-R12, and Y is NR13; R13 is H or C alkyl; more particularly R13 is H or Ci-2alkyl; more particularly R13 is Ci-2alkyl; and
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is -CH2-R14, wherein R14 is an aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, more particularly 4 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, more particularly C, N. Even more particularly, R14 is an aliphatic carbocyclic ring or ring system as previously defined, even more particularly R14 is cyclobutyl.
In another particular embodiment, the compound A is a compound of formula (I), wherein R1 is -CH2-R14, wherein R14 is an aliphatic heterocyclic ring having 5 ring atoms selected from the group consisting of C, N, 0, and S, more particularly C, N. Even more particularly, R14 is an aliphatic heterocyclic ring or ring system as previously defined.
In another particular embodiment, the compound B is a compound of formula (la), wherein R3 is -R7-R8, wherein R7 is— (CH2)X-, with x being 0-3, more particularly 0 or 1, and R8 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system, more particularly an aromatic or heteroaromatic ring or ring system, wherein the ring or ring system has 5 to 10 ring atoms, more particularly 5 to 6 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, and 0, more particularly C, N, even more particularly wherein the ring or ring system has no or one atom other than C as ring atom. Even more particularly, R8 is an aromatic or heteroaromatic ring or ring system having 5 to 6 ring atoms, wherein the atoms of the heteroaromatic ring or ring system are selected from the group consisting of C, N, and 0, more particularly C, N, even more particularly wherein the ring or ring system has no or one atom other than C as ring atom.
In another particular embodiment, the compound B is a compound of formula (la), R8 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C1-4 alkyl, halogen, oxo, and -NHC alkyl; even more particularly, R8 is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C1-4 alkyl, halogen, oxo, -NHC alkyl; even more particularly, R8 is optionally substituted with one, two, or three substituents selected from the group consisting of C1-4 alkyl, and halogen. Alternatively, R8 is optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
In another particular embodiment, the compound B is a compound of formula (la), wherein R3 is selected from the group consisting of benzyl, fluorobenzyl, phenyl, tolyl, optionally substituted phenyl, pyridinyl (particularly pyridin-4- yl), optionally substituted pyridinyl, tiophenyl (particularly thiophen-2-yl or thiophen-3-yl), optionally substituted thiophenyl, pyrazolyl (particularly pyrazol-5-yl), and optionally substituted pyrazolyl.
In another embodiment, the compound B is a compound of formula (la), wherein R3 is phenyl, optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
In another embodiment, the compound B is selected from the group consisting of compounds 1-8, 19-36 as defined above, or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof.
The compounds of the present invention preferably inhibit p38a autophosphorylation. This can be demonstrated with the assays described in Examples 28 and 29.
Particularly preferred compounds are those that show inhibition of p38a autophosphorylation of at least 40% at 5 mM and/or at least 50% at 10 mM and/or at least 80% at 30 mM in the assay described in Example 28, and/or those that show inhibition of TAB1 -induced p38a autophosphorylation of at least 20% at 5 mM and/or at least 60% at 10 mM and/or at least 90% at 30 mM in the assay described in Example 29. Most preferred are those that show inhibition of p38a autophosphorylation of at least 50% at 1 mM in the assay described in Example 28, and/or those that show inhibition of TAB1 -induced p38a autophosphorylation of at least 60% at 1 mM in the assay described in Example 29.
Moreover, the preferred compounds of the present invention show no inhibition (particularly the inhibition is equal to or lower than 20% in the assays described herein) of the canonical p38a phosphorylation at 2 mM, more preferably at 10 mM, and even more preferably at 30 mM. This can be demonstrated with the assays described in Examples 28 and 29.
Particularly preferred compounds are thus those compounds that inhibit p38a autophosphorylation by at least 70 % in the assays described in Examples 28 and 29, but show no inhibition of MKK6-induced p38a phosphorylation at the same concentration (e.g. at 1 mM or 10 mM), in the assay described in Example 26, and/or compounds that show no inhibition of p38a kinase activity on MK2 at the same concentration (e.g. at 1 mM or 10 mM), in the assay described in Example 27. Process for preparing compounds
The compounds of formulae (I) and (la), wherein R4 is N and R5 and R are CH, may, in one embodiment, be prepared according to the following scheme:
Cl
VRl
Figure imgf000026_0002
8 Step 3
An alternative to this scheme for preparing the compounds is a reaction according to the scheme below. Hence, in another embodiment, the compounds of formulae (I) and (la), wherein R4 is N and R5 and R are CH, may be prepared according to the following scheme:
Figure imgf000026_0001
Another alternative for preparing these compounds is a reaction according to the scheme below. Hence, in still another embodiment, the compounds of formulae (I) and (la), wherein R4 is N and R5 and R are CH may be prepared according to the following scheme:
Figure imgf000026_0003
For compounds of formulae (I) or (la), wherein R4 and R5 are N and R is CH may in one embodiment be prepared according to the scheme:
Figure imgf000027_0001
In a further embodiment, the compounds of formulae (I) and (la), wherein each of R4, R5, and R is CH, may be prepared according to this scheme:
Figure imgf000027_0002
The skilled person will know how to adjust the reaction conditions based on these schemes to achieve maximum yield, purity etc. The skilled person will also know how to apply protecting groups if the R1 and R2 groups contain functional groups likely to be reactive under the reaction conditions employed in the schemes.
Pharmaceutical formulation
The compounds of the present invention are intended for use as a medicament. The compounds of the invention may in principle be administered on their own, but they are preferably formulated with a pharmaceutically or veterinary acceptable carrier. A pharmaceutically or veterinary acceptable carrier is an inert carrier suitable for each administration method, and can be formulated into conventional pharmaceutical preparation (tablets, granules, capsules, powder, solution, suspension, emulsion, injection, infusion, etc.). As such a carrier there may be mentioned, for example, a binder, an excipient, a lubricant, a disintegrant and the like, which are pharmaceutically or veterinary acceptable. When they are used as an injection solution or an infusion solution, they can be formulated by using distilled water for injection, physiological saline, an aqueous glucose solution. In one aspect of the invention, it concerns a pharmaceutical or veterinary composition comprising a compound according to any one of the preceding aspects as well as one or more pharmaceutically acceptable carriers or excipients.
The present invention also relates to a pharmaceutical or veterinary composition comprising a therapeutically effective amount of a compound A or a compound B as previously defined, as well as one or more pharmaceutically or veterinary acceptable carriers or excipients.
In the context of the present invention "therapeutically effective amount" refers to the amount of a compound which is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed.
The expression "pharmaceutically or veterinary acceptable excipients or carriers" refers to pharmaceutically or veterinary acceptable materials, compositions or vehicles which are compatible with the other ingredients of the pharmaceutical or veterinary composition and are also suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
Accordingly, a further aspect of the present invention includes pharmaceutical or veterinary compositions comprising as one or more compounds of the invention disclosed above, associated with a pharmaceutically acceptable carrier. For administration, the compounds are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
The administration method of the compounds of the present invention is not particularly limited, and a usual oral or parenteral administration method (intravenous, intramuscular, subcutaneous, percutaneous, intranasal, transdermal, transmucosal, enteral, etc.) can be applied.
The compounds may be administered alone or in combination with one or more other compounds of the invention or one or more other drugs, for example in combination with Beta-blockers or thrombolytic agents (e.g. for the treatment of Ischemia-reperfusion); corticosteroids or NSAIDs (e.g. in the treatment of skin inflammation), or in combination with chemotherapeutic agents (e.g. in the treatment of pancreatic cancer).
The dosage of the compounds of the present invention may optionally be set in a range of an effective amount sufficient for showing a pharmacological effect, in accordance with the potency or characteristics of the compound to be used as an effective ingredient. The dosage may vary depending on administration method, age, body weight or conditions of a patient.
Pharmaceutical utility
The compounds of the invention inhibit the autophosphorylation of p38a.
Hence, in one aspect, the present invention concerns a compound of the invention for use as a medicament. In a further aspect, the invention concerns a compound or composition according to the invention for use in the prophylaxis or treatment of a disease associated with autophosphorylation of p38a kinase. Autophosphorylation of p38a has been associated with a number of diseases, such as cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer. The diseases that can be treated with the compounds and formulations of the present invention thus include myocardial ischemia reperfusion injury (De Nicola et al., 2018; Tanno et al., 2003; Wang et al., 2013), cardiac amyloidosis (Mishra et al., 2013; Shi et al., 2010), atherosclerosis/hypercoagulability (Grimsey et al., 2015), skin inflammation (Theivanthiran et al., 2015), impaired immune cell responses owing to T-cell senescence (Lanna et al., 2014), Toxoplasmosis (Kim et al., 2005; Braun et al., 2013; Pellegrini et al., 2017), pancreatic cancer (Alam et al., 2015), inflammatory autoimmune diseases such as arthritis and encephalomyelitis (Jirmanova et al., 201 1 ); and pre-term labor and premature birth (Richardson et al., 2018).
For the purposes of the invention, the term "treatment” of the disease refers to stopping or delaying of the disease progress, when the compound is used in the subject exhibiting symptoms of disease onset. The term "prophylaxis” refers to stopping or delaying of symptoms of disease onset, when the compound is used in the subject exhibiting no symptoms of disease onset but having high risk of disease onset.
Particular cardiovascular diseases include arteriosclerosis, peripheral vascular disease, hyperlipidemia, mixed dyslipidemia betalipoproteinemia, hypoalphalipoproteinemia, hypercholesteremia, hypertriglyceridemia, familial- hypercholesteremia, angina, ischemia, cardiac ischemia, stroke, myocardial infarction, reperfusion injury, restenosis after angioplasty, hypertension, cerebral infarction and cerebral stroke.
Particular autoimmune diseases include arthritis, including rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis; inflammatory hyperproliferative skin diseases; psoriasis, such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails; atopy, including atopic diseases such as hay fever and Job's syndrome; dermatitis, including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis; x-linked hyper IgM syndrome; allergic intraocular inflammatory diseases; urticaria, such as chronic allergic urticaria, chronic idiopathic urticaria, and chronic autoimmune urticaria; myositis; polymyositis/dermatomyositis; juvenile dermatomyositis; toxic epidermal necrolysis; scleroderma, including systemic scleroderma; sclerosis, such as systemic sclerosis, multiple sclerosis (MS), spino- optical MS, primary progressive MS (PPMS), relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis; neuromyelitis optica (NMO); inflammatory bowel disease (IBD), including Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, and autoimmune inflammatory bowel disease; bowel inflammation; pyoderma gangrenosum; erythema nodosum; primary sclerosing cholangitis; respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS); meningitis; inflammation of all or part of the uvea; iritis; choroiditis; an autoimmune hematological disorder; rheumatoid spondylitis; rheumatoid synovitis; hereditary angioedema; cranial nerve damage, as in meningitis; herpes gestationis; pemphigoid gestationis; pruritis scroti; autoimmune premature ovarian failure; sudden hearing loss due to an autoimmune condition; IgE-mediated diseases, such as anaphylaxis and allergic and atopic rhinitis; encephalitis, such as Rasmussen's encephalitis and limbic and/or brainstem encephalitis; uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis; glomerulonephritis (GN) with and without nephrotic syndrome, such as chronic or acute glomerulonephritis, primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN; proliferative nephritis; autoimmune polyglandular endocrine failure; balanitis, including balanitis circumscripta plasmacellularis; balanoposthitis; erythema annulare centrifugum; erythema dyschromicum perstans; eythema multiform; granuloma annulare; lichen nitidus; lichen sclerosus et atrophicus; lichen simplex chronicus; lichen spinulosus; lichen planus; lamellar ichthyosis; epidermolytic hyperkeratosis; premalignant keratosis; pyoderma gangrenosum; allergic conditions and responses; allergic reaction; eczema, including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema; asthma, such as asthma bronchiale, bronchial asthma, and auto-immune asthma; conditions involving infiltration of T cells and chronic inflammatory responses; immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy; chronic pulmonary inflammatory disease; autoimmune myocarditis; leukocyte adhesion deficiency; lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE), cutaneous SLE, subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus; juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, and diabetic large-artery disorder; immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes; tuberculosis; sarcoidosis; granulomatosis, including lymphomatoid granulomatosis; Wegener's granulomatosis; agranulocytosis; vasculitides, including vasculitis, large-vessel vasculitis, polymyalgia rheumatica and giant-cell (Takayasu's) arteritis, medium-vessel vasculitis, Kawasaki's disease, polyarteritis nodosa/periarteritis nodosa, microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis, systemic necrotizing vasculitis, ANCA-associated vasculitis, Churg-Strauss vasculitis or syndrome (CSS), and ANCA-associated small-vessel vasculitis; temporal arteritis; aplastic anemia; autoimmune aplastic anemia; Coombs positive anemia; Diamond Blackfan anemia; hemolytic anemia or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa); Addison's disease; pure red cell anemia or aplasia (PRCA); Factor VIII deficiency; hemophilia A; autoimmune neutropenia; pancytopenia; leukopenia; diseases involving leukocyte diapedesis; CNS inflammatory disorders; multiple organ injury syndrome, such as those secondary to septicemia, trauma or hemorrhage; antigen-antibody complex- mediated diseases; anti-glomerular basement membrane disease; anti-phospholipid antibody syndrome; allergic neuritis; Behget's disease/syndrome; Castleman's syndrome; Goodpasture's syndrome; Reynaud's syndrome; Sjogren's syndrome; Stevens-Johnson syndrome; pemphigoid, such as pemphigoid bullous and skin pemphigoid, pemphigus, pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus; autoimmune polyendocrinopathies; Reiter's disease or syndrome; thermal injury; preeclampsia; an immune complex disorder, such as immune complex nephritis, and antibody-mediated nephritis; polyneuropathies; chronic neuropathy, such as IgM polyneuropathies and IgM-mediated neuropathy; thrombocytopenia (as developed by myocardial infarction patients, for example), including thrombotic thrombocytopenic purpura (TTP), post transfusion purpura (PTP), heparin-induced thrombocytopenia, autoimmune or immune-mediated thrombocytopenia, idiopathic thrombocytopenic purpura (ITP), and chronic or acute ITP; scleritis, such as idiopathic cerato-scleritis, and episcleritis; autoimmune disease of the testis and ovary including, autoimmune orchitis and oophoritis; primary hypothyroidism; hypoparathyroidism; autoimmune endocrine diseases, including thyroiditis, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes, autoimmune polyglandular syndromes, and polyglandular endocrinopathy syndromes; paraneoplastic syndromes, including neurologic paraneoplastic syndromes; Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome; stiff-man or stiff-person syndrome; encephalomyelitis, such as allergic encephalomyelitis, encephalomyelitis allergica, and experimental allergic encephalomyelitis (EAE); myasthenia gravis, such as thymoma-associated myasthenia gravis; cerebellar degeneration; neuromyotonia; opsoclonus or opsoclonus myoclonus syndrome (QMS); sensory neuropathy; multifocal motor neuropathy; Sheehan's syndrome; hepatitis, including autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis, and autoimmune chronic active hepatitis; lymphoid interstitial pneumonitis (LIP); bronchiolitis obliterans (non-transplant) vs NSIP; Guillain-Barre syndrome; Berger's disease (IgA nephropathy); idiopathic IgA nephropathy; linear IgA dermatosis; acute febrile neutrophilic dermatosis; subcorneal pustular dermatosis; transient acantholytic dermatosis; cirrhosis, such as primary biliary cirrhosis and pneumonocirrhosis; autoimmune enteropathy syndrome; Celiac or Coeliac disease; celiac sprue (gluten enteropathy); refractory sprue; idiopathic sprue; cryoglobulinemia; amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease); coronary artery disease; autoimmune ear disease, such as autoimmune inner ear disease (AIED); autoimmune hearing loss; polychondritis, such as refractory or relapsed or relapsing polychondritis; pulmonary alveolar proteinosis; Cogan's syndrome/nonsyphilitic interstitial keratitis; Bell's palsy; Sweet's disease/syndrome; rosacea autoimmune; zoster-associated pain; amyloidosis; a non-cancerous lymphocytosis; a primary lymphocytosis, including monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS); peripheral neuropathy; channelopathies, such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS; autism; inflammatory myopathy; focal or segmental or focal segmental glomerulosclerosis (FSGS); endocrine opthalmopathy; uveoretinitis; chorioretinitis; autoimmune hepatological disorder; fibromyalgia; multiple endocrine failure; Schmidt's syndrome; adrenalitis; gastric atrophy; presenile dementia; demyelinating diseases, such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy; Dressler's syndrome; alopecia areata; alopecia totalis; CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia); male and female autoimmune infertility (e.g., due to anti-spermatozoan antibodies); mixed connective tissue disease; Chagas' disease; rheumatic fever; recurrent abortion; farmer's lung; erythema multiforme; post-cardiotomy syndrome; Cushing's syndrome; bird-fancier's lung; allergic granulomatous angiitis; benign lymphocytic angiitis; Alport's syndrome; alveolitis, such as allergic alveolitis and fibrosing alveolitis; interstitial lung disease; transfusion reaction; leprosy; malaria; Samter's syndrome; Caplan's syndrome; endocarditis; endomyocardial fibrosis; diffuse interstitial pulmonary fibrosis; interstitial lung fibrosis; pulmonary fibrosis; idiopathic pulmonary fibrosis; cystic fibrosis; endophthalmitis; erythema elevatum et diutinum; erythroblastosis fetalis; eosinophilic faciitis; Shulman's syndrome; Felty's syndrome; flariasis; cyclitis, such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis; Henoch-Schonlein purpura; sepsis; endotoxemia; pancreatitis; thyroxicosis; Evan's syndrome; autoimmune gonadal failure; Sydenham's chorea; post-streptococcal nephritis; thromboangitis ubiterans; thyrotoxicosis; tabes dorsalis; chorioiditis; giant-cell polymyalgia; chronic hypersensitivity pneumonitis; keratoconjunctivitis sicca; epidemic keratoconjunctivitis; idiopathic nephritic syndrome; minimal change nephropathy; benign familial and ischemia-reperfusion injury; transplant organ reperfusion; retinal autoimmunity; joint inflammation; bronchitis; chronic obstructive airway/pulmonary disease; silicosis; aphthae; aphthous stomatitis; arteriosclerotic disorders; aspermiogenese; autoimmune hemolysis; Boeck's disease; cryoglobulinemia; Dupuytren's contracture; endophthalmia phacoanaphylactica; enteritis allergica; erythema nodosum leprosum; idiopathic facial paralysis; febris rheumatica; Hamman-Rich's disease; sensoneural hearing loss; haemoglobinuria paroxysmatica; hypogonadism; ileitis regionalis; leucopenia; mononucleosis infectiosa; traverse myelitis; primary idiopathic myxedema; nephrosis; ophthalmia symphatica; orchitis granulomatosa; pancreatitis; polyradiculitis acuta; pyoderma gangrenosum; Quervain's thyreoiditis; acquired spenic atrophy; non-malignant thymoma; vitiligo; toxic-shock syndrome; food poisoning; conditions involving infiltration of T cells; leukocyte- adhesion deficiency; immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes; diseases involving leukocyte diapedesis; multiple organ injury syndrome; antigen-antibody complex-mediated diseases; antiglomerular basement membrane disease; allergic neuritis; autoimmune polyendocrinopathies; oophoritis; primary myxedema; autoimmune atrophic gastritis; sympathetic ophthalmia; rheumatic diseases; mixed connective tissue disease; nephrotic syndrome; insulitis; polyendocrine failure; autoimmune polyglandular syndrome type I; adult-onset idiopathic hypoparathyroidism (AOIH); cardiomyopathy such as dilated cardiomyopathy; epidermolisis bullosa acquisita (EBA); hemochromatosis; myocarditis; nephrotic syndrome; primary sclerosing cholangitis; purulent or nonpurulent sinusitis; acute or chronic sinusitis; ethmoid, frontal, maxillary, or sphenoid sinusitis; an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils; anaphylaxis; seronegative spondyloarthritides; polyendocrine autoimmune disease; sclerosing cholangitis; chronic mucocutaneous candidiasis; Bruton's syndrome; transient hypogammaglobulinemia of infancy; Wiskott-Aldrich syndrome; ataxia telangiectasia syndrome; angiectasis; autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization; allergic hypersensitivity disorders; glomerulonephritides; reperfusion injury; ischemic re-perfusion disorder; reperfusion injury of myocardial or other tissues; lymphomatous tracheobronchitis; inflammatory dermatoses; dermatoses with acute inflammatory components; multiple organ failure; bullous diseases; renal cortical necrosis; acute purulent meningitis or other central nervous system inflammatory disorders; ocular and orbital inflammatory disorders; granulocyte transfusion-associated syndromes; cytokine-induced toxicity; narcolepsy; acute serious inflammation; chronic intractable inflammation; pyelitis; endarterial hyperplasia; peptic ulcer; valvulitis; and endometriosis.
Particular inflammatory diseases include allergic inflammation, arthritis (to include osteoarthritis and rheumatoid arthritis), fibromyalgia, chronic fatigue syndrome, inflammatory bowel disease, interstitial cystitis, irritable bowel syndrome, migraines, atherosclerosis, coronary inflammation, ischemia, chronic prostatitis, eczema, multiple sclerosis, psoriasis, sun burn, periodontal disease of the gums, superficial vasodilator flush syndromes, hormonally dependent cancers, and endometriosis.
Particular neurodegenerative diseases include diabetic neuropathy, senile dementias, Alzheimer's disease, Mild Cognitive Impairment (MCI), dementia, Lewy Body Dementia, Frontal Temporal Lobe dementia, Parkinson's Disease, facial nerve (Bell's) palsy, glaucoma, Huntington's chorea, amyotrophic lateral sclerosis (ALS), status epilepticus, non-arteritic optic neuropathy, intervertebral disc herniation, vitamin deficiency, prion diseases such as Creutzfeldt-Jakob disease, carpal tunnel syndrome, peripheral neuropathies associated with various diseases, including but not limited to, uremia, porphyria, hypoglycemia, Sjorgren Larsson syndrome, acute sensory neuropathy, chronic ataxic neuropathy, biliary cirrhosis, primary amyloidosis, obstructive lung diseases, acromegaly, malabsorption syndromes, polycythemia vera, IgA and IgG gammapathies, complications of various drugs (e.g., metronidazole) and toxins (e.g., alcohol or organophosphates), Charcot-Marie-Tooth disease, ataxia telangectasia, Friedreich's ataxia, amyloid polyneuropathies, adrenomyeloneuropathy, Giant axonal neuropathy, Refsum's disease, Fabry's disease and lipoproteinemia.
According to the present invention, the compounds of the present invention are used for treating said disease associated with autophosphorylation of p38a kinase, such as one of the diseases listed above.
In one aspect of the present invention, the compounds of the invention selectively inhibit the autophosphorylation of p38a.
In one embodiment, the compound of the present invention inhibits autophosphorylation of p38a induced by ZAP70- mediated Tyr phosphorylation.
In another, and preferred embodiment, the compound of the present invention inhibits TAB1 -induced autophosphorylation.
The protein TAB1 stimulates the autophosphorylation of p38a by binding onto two different clefts. One of these is exploited by many p38a binding partners other than TAB1 such as upstream and downstream kinases, as well as phosphatases.
The other one is thought to bind only to TAB1 (De Nicola et al., 2013). By targeting the cleft that only binds TAB1 , the activity of upstream kinases such as MKK3 and MKK6 should not be affected and the p38a kinase activity is not shut down in all cellular contexts. This is deemed to reduce general toxicity in comparison with classical p38a inhibitors that interfere with ATP binding. The compounds of the present invention therefore preferably inhibit autophosphorylation such as TAB1 -induced autophosphorylation, without showing an inhibitory effect on MKK6- induced p38a phosphorylation or the kinase activity of canonically-activated p38a.
Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise” encompasses the case of "consisting of . Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.
Examples
Example 1 - Preparation of Compound 1 : N-(2-cvclopentyl-3H-imidazo[4,5-bl pyridin-5-yl) benzenesulfonamide
Figure imgf000035_0001
Step 1: Synthesis of N6-[(4-methoxyphenyl) methyl]-3-nitro-pyhdine-2, 6-diamine (3)
To a solution of 6-chloro-3-nitro-pyridin-2-amine (2 g, 1 1.5 mmol) in isopropanol (20 mL) was added p-methoxy benzyl amine (1.89 g, 13.8 mmol) followed by triethyl amine (2.3 mL, 16.6 mmol) and the resulting reaction mixture was refluxed for 12 hours. After completion of reaction, solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate and water was added. The organic layer was separated, washed with water followed by brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the crude product so obtained was washed with hexanes to give the desired product 3 (3.1 g, 98%) as a yellow colored solid.
Step 2: Synthesis of 3-nitropyridine-2, 6-diamine (4)
A solution of 3 (3.1 g, 1 1.7 mmol) in trifluoracetic acid (30 mL) was heated at 70°C for 16 hours. After completion of reaction, trifluoracetic acid was evaporated under reduced pressure. Water was added to the residue and the reaction mixture was neutralized with 10% aqueous sodium bicarbonate solution. The product was extracted with ethyl acetate (3 x 30 mL). Organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product 4 (1.6 g, 88%) so obtained was taken as such for next step without further purification.
Step 3: Synthesis of pyridine-2, 3, 6-triamine (5)
To a solution of 4 (1.6 g, 10.38 mmol) in methanol (20 mL) was added 10% Pd/C (160 mg) and the resulting reaction mixture was stirred under hydrogen atmosphere at room temperature for 2 hours. After completion of reaction, the reaction mixture was filtered over a pad of cealite and washed with methanol. Filtrate was evaporated under reduced pressure to give the desired product 5 (1 g, 78%) as a purple color solid that was pure enough to take ahead for next step
Step 4: Synthesis of 2-cyclopentyl-3H-imidazo[4,5-b] pyridin-5-amine (6); [J. Heterocyclic Chem., 17, 1757, 1980] A mixture of 5 (1 g, 8.05 mmol) and cyclopentane carboxylic acid (0.91 g, 8.05 mmol) in polyphosphoric acid (20 g) was heated at 180°C for 6 hours. After completion of reaction, the reaction mixture was cooled down to room temperature and basified with 10% aqueous sodium carbonate solution. The product was extracted with ethyl acetate (3 X 30 mL). The organic layer was washed with water followed by brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product so obtained was purified by column chromatography using 0-10% methanol in dichloromethane as eluent to give the desired product 6 (800 mg, 50%) as a brown color solid.
Step 5: N-(2-cyclopentyl-3H-imidazo[4,5-b] pyridin-5-yl) benzenesulfonamide
To a solution of 6 (130 mg, 0.6 mmol) in dichloromethane (5 mL) was added benzene sulfonyl chloride (0.08 mL, 0.6 mmol) followed by pyridine (0.05 mL, 0.6 mmol) and the resulting reaction mixture was stirred at room temperature for 24 hours. After completion of reaction, water was added to the reaction mixture and the product was extracted with dichloromethane (3 X 20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product so obtained was purified by flash chromatography using 50-60% ethyl acetate in hexanes as the eluent to give the desired product Compound 1 (0.06 mg, 27%) as a light brown solid. 1H NMR (400 MHz, CD3OD): d 1.71-1.73 (br. m, 2H), 1.82-1.92 (br., m, 4H), 2.10-2.17 (br., m, 2H), 3.24-3.30 (br. m, 1 H, merged in CD3OD peak), 7.04 (d, J = 8 Hz, 1 H), 7.47 (t like, J = 7.2 Hz, 2H), 7.53 (d, J = 7.6 Hz, 1 H), 7.73 (d, J
= 8.8 Hz, 1 H), 7.96 (d, J = 7.2 Hz, 2H)
LCMS: m/z 343.1 [M++1] HPLC Purity: 94.6% [Retention Time: 11.03]
Example 2 - Preparation of Compound 3 and Compound 21 : N-[2-[3-(methylamino) cvclopentyll-3H-benzimidazol-
5-yll benzenesulfonamide and N-[2-(3-oxocvclopentyl)-3H-benzimidazol-5-yl1 benzenesulfonamide, respectively
Figure imgf000037_0001
Step 1: Synthesis ofN-(2-amino-4-nitro-phenyl)-3-oxo-cyclopentanecarboxamide (10)
To a solution of 4-nitrobenzene-1 , 2-diamine (480 mg, 3.13 mmol) in DMF (10 mL) was added HATU (1.78 g, 4.7 mmol) and diisopropyl ethyl amine (1.6 mL, 9.41 mmol). The resulting reaction mixture was stirred at room temperature for 16 hours. After the completion of reaction, DMF was evaporated under reduced pressure. Water was added to the residue and the product was extracted with ethyl acetate (3 x 20 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product 10 (700 mg) so obtained was taken as such for the next step; LCMS: m/z 264.1 [M++1],
Step 2: Synthesis of 3-(6-nitro-1 H-benzimidazol-2-yl) cyclopentanone (11)
A solution of 10 (700 mg, 2.6 mmol) in acetic acid (10 mL) was heated at 100°C for 16 hours. After completion of reaction, acetic acid was evaporated under reduced pressure and the crude product so obtained was purified by flash chromatography using 50-70% hexanes in ethyl acetate as eluent to give the desired product 11 (500 mg, 65% over 2 steps) as a yellow solid.
Step 3: Synthesis of 3-(6-amino-1 H-benzimidazol-2-yl) cyclopentanone (12)
To a solution of 11 (500 mg, 2.04 mmol) in methanol (5 mL) was added Palladium on carbon (50 mg) and the reaction mixture was stirred for 2 hours under hydrogen atmosphere using hydrogen balloon at room temperature. After completion of reaction, reaction mixture was filtered on cealite bed and washed with methanol. The filtrate was concentrated under reduced pressure to get the desired product 12 (300 mg, 69%) as a brown solid; LCMS: m/z 214 [M+-1], Step 4: Synthesis ofN-[2-(3-oxocyclopentyl)-3H-benzimidazol-5-yl] benzenesulfonamide
To a solution of 12 (300 mg, 1.4 mmol) in pyridine (5 mL) at 0°C was added benzene sulfonyl chloride (0.27 g, 1.5 mmol) and the resulting reaction mixture was stirred at 0°C for 2 hours. After completion of reaction, water was added to the reaction mixture and the product was extracted with dichloromethane (2 x 25 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product so obtained was purified by flash chromatography using 60-70% ethyl acetate in hexanes to give the desired product (Compound 21 ) as a brown solid (150 mg, 30%).
Step 5: Synthesis of N-[2-[3-(methylamino) cyclopentyl]-3H-benzimidazol-5-yl] benzenesulfonamide
To a solution of the compound obtained in the previous step (140 mg, 0.4 mmol) in ethanol (10 mL) was added methyl amine hydrochloride (53 mg, 0.8 mmol) followed by triethyl amine (0.1 mL, 0.8 mmol) and titanium (IV) isopropoxide (0.22 g, 0.8 mmol). The resulting reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was then cooled to OoC and NaBH4 (22 mg, 0.6 mmol) was added. The resulting suspension was stirred at room temperature for another 8 hours. After the completion of reaction, the reaction mixture was quenched with ammonium hydroxide and the product was extracted with dichloromethane (3 X 25 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product (60 mg) so obtained was purified by crystallization to give desired product Compound 3 (15 mg) with 77% HPLC purity. 1HNMR (400 MHz, CD3OD): 6 1.70-1.77 (m, 1 H), 1.79-1.89 (m, 1 H), 1.93-2.08 (m, 1 H), 2.10-2.24 (m, 3H), 2.35-2.45 (m, 1 H), 2.51 (s, 3H), 3.34-3.48 (m, 1 H), 6.88 (dd, J = 2 Hz, 6.4 Hz, 1 H), 7.26 (d, J = 2 Hz, 1 H), 7.31 (d, J = 8.8 Hz, 1 H),7.43 (m, 2H), 7.52 (m, 1 H), 7.69 (d, J = 7.2 Hz, 2H)
LCMS: /z 371.1 [M++1] HPLC Purity: 76.9% [Retention Time: 9.556]
Example 3 - Preparation of Compound 4: N-(2-cvclopentyl-1 H-indol-6-yl) benzenesulfonamide
Figure imgf000038_0001
Step A
2-Bromo-5-nitroaniline (500 mg, 2.30 mmol), Pd(PPfi3)2Cl2 (48.5 mg, 0.069 mmol) and Cul (21 .9 mg , 0.1 15 mmol) were added to a flask. A previously degassed solution of NHEt2 (0.36 mL, 3.46 mmol) in DMF (4.5 mL) was then added to the flask under a N2 atmosphere. Cyclopentyl acetylene (0.40 mL, 3.46 mmol) was then added and the reaction stirred at 70 °C for 2 hours. At this point a saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgSC and concentrated. Purification by column chromatography (S1O2, cyclohexane/EtOAc) afforded 322 mg (61 % yield, 95% purity) of 2-(cyclopentylethynyl)-5-nitroaniline ).
Step B
2-(Cyclopentylethynyl)-5-nitroaniline from the previous step (322 mg, 1.40 mmol), PdCb (49.6 mg, 0.28 mmol) and Cul (53.3 mg, 0.28 mmol) were added to a flask and, under a N2 atmosphere, dissolved in acetonitrile (14 mL). The reaction was stirred at 80 °C for 2 hours and then allowed to cool down to rt and concentrated. Purification by column chromatography (S1O2, cyclohexane/EtOAc) afforded 191 mg (59% yield, 95% purity) of 2-cyclopentyl-6-nitro-1 IH- indole.
Step C
The resulting product from the previous step (137 mg, 0.60 mmol) was dissolved in EtOPH (3.0 mL) and SnCb^PpO (201 mg, 0.89 mmol) was added and the mixture stirred at 70 °C overnight. At this stage a saturated aqueous solution of NaPIC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgS04 and concentrated. Purification by column chromatography (S1O2, cyclohexane/EtOAc) afforded 30 mg (25% yield, 90-95% purity (1PINMR)) of 2-cyclopentyl- 1 PH-indole-6-amine.
Step D
To a solution of 2-cyclopentyl-1 PH-indole-6-amine from the previous step (30 mg, 0.15 mmol) in pyridine (0.2 mL) at 0 °C, benzensulfonyl chloride (17 mί, 0.14 mmol) was slowly added and the reaction allowed to reach rt and stirred overnight. Then the mixture was concentrated and purified by column chromatography (S1O2, cyclohexane/EtOAc) to deliver 27 mg (53% yield, 98% purity) of Compound 4. 1H NMR (400 MHz, DMSO-d6) 10.76 (s, 1 H), 9.85 (bs, 1 H), 7.67 (d, J = 7.2 Hz, 2 H), 7.55-7.45 (m, 3 H), 7.14 (d, J = 8.4 Hz, 1 H), 7.03 (s, 1 H), 6.62 (dd, , J = 8.3 and 1.8 Hz, 1 H), 5.99 (s, 1 H), 3.09-3.00 (m, 1 H), 2.01 -1.94 (m, 2 H), 1.72-1.56 (m, 6 H) ppm. LCMS: m/z (ESP+) 341 (MH+)
Example 4 - Preparation of Compound 5: N-[2-(2,6-dimethylphenyl)-1 H-benzimidazol-6-yl1 benzenesulfonamide
Figure imgf000039_0001
Step A
In a flask equipped with a reflux condenser was added 4-nitrobenzene-1 , 2-diamine (1.12 gr, 7.32 mmol), polyphosphoric acid (4.0 mL) and 2,6-dimethylbenzoic acid (1.0 gr, 6.66 mmol). The reaction was then stirred at 130 °C overnight. At this point, the solution was cooled down to 0 °C and basified carefully with a 1 M aqueous solution of NaOH, and the crude extracted twice with DCM and twice with EtOAc. The organic layers were combined, dried over MgSC and concentrated. Purification by column chromatography (S1O2, cyclohexane/EtOAc) afforded 82 mg (5% yield, 81 % purity) of 2-(2,6-dimethylphenyl)-6-nitro-1 H-benzo[d]imidazole.
Step B
2-(2,6-Dimethylphenyl)-6-nitro-1 H-benzo[d]imidazole from the previous step (82 mg, 0.37 mmol) was then dissolved in EtOH (1.2 mL) and SnCh^hbO (76.0 mg, 0.34 mmol) was added and the mixture stirred at 70 °C overnight. At this stage a saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgS04 and concentrated. Purification by column chromatography (S1O2, DCM/MeOH) afforded 50 mg (69% yield, 90-95% purity (1HNMR)) of 2-(2,6-dimethylphenyl)-1 H-benzo[d]imidazol-6-amine.
Step C
To a solution of 2-(2,6-dimethylphenyl)-1 H-benzo[d]imidazol-6-amine from the previous step (50 mg, 0.21 mmol) in pyridine (0.28 mL) at 0 °C, benzenesulfonyl chloride (24 mί, 0.19 mmol) was slowly added and the reaction stirred overnight and concentrated. Purification by (S1O2, cyclohexane/EtOAc) delivered 28 mg (35% yield, 94% purity) of Compound 5 (as mixture of stereoisomers). 1H NMR (400 MHz, DMSO-de) 12.45 (bs, 0.44 H), 12.37 (bs, 0.56 H), 10.16 (bs, 0.4 4H), 10.07 (bs, 0.56 H), 7.75 (d, 0 = 8.0 Hz, 2 H), 7.60-7.47 (m, 3.44 H), 7.32-7.26 (m, 2 H), 7.21 (d, J = 2.0 Hz , 0.56 H), 7.14 (d, J = 7.6 Hz, 2 H), 6.93 (ddd, J = 17.2, 8.6 and 1.9 Hz, 1 H), 2.04 (s, 3.3 H), 2.62 (s, 2.7 H) ppm. LCMS: m/z (ESP+) 378 (MH+)
Example 5 - Preparation of Compound 6: N-[2-(quinolin-6-yl)-1 H-benzimidazol-6-yl1 benzenesulfonamide
Figure imgf000040_0001
Step A
To a solution of 1 H-benzo[d]imidazol-6-amine (1.0 gr, 7.51 mmol) in acetone (25.0 mL) at 0 °C, pyridine (0.59 mL, 7.51 mmol) and benzenesulfonyl chloride (0.96 mL, 7.51 mmol) were slowly added and the reaction stirred for 3 days and concentrated. Purification by (S1O2, DCM/MeOH) delivered 406 mg (20% yield, 91 % purity) of N-1 H- benzo[d]imidazol-6-yl)benzenesulfonamide. Step B
N-1 H-Benzo[d]imidazol-6-yl)benzenesulfonamide from the previous step (66 mg, 0.24 mmol), Pd(OAc)2 (2.7 mg, 0.012 mmol), 6-iodoquinoline (123 mg, 0.483) and Cul (92 mg, 0.48 mmol) were added to a sealed vial. Previously degassed DMF (1.2 mL) was added under a l\ atmosphere and the reaction heated to 140 °C and stirred at this temperature for 4 days. At this stage a saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgS04 and concentrated. Purification by column chromatography (S1O2, DCM/MeOH) afforded 22 mg (21 % yield, 95% purity) of Compound 6 (as mixture of stereoisomers). 1H NMR (400 MHz, DMSO-de) 13.19 (bs, 0.43 H), 13.10 (bs, 0.57 H), 10.24 (bs, 1 H), 9.02 (dd, J = 4.1 and 1.4 Hz, 1 H), 8.79 (bs, 0.43 H), 8.75 (bs, 0.57 H), 8.53 (d, J = 7.8 Hz, 2 H), 8.21 (d, J = 8.8 Hz, 1 H), 7.81 (d, J = 7.7 Hz , 2 H), 7.69-7.57 (m, 5 H), 7.50-7.38 (m, 1 H), 7.08-7.06 (m, 1 H) ppm. LCMS: m/z (ESP+) 401 (MH+)
Example 6 - Preparation of Compound 7: N-f2-(1 H-indol-5-yl)-1 H-benzimidazol-6-yll benzenesulfonamide
Figure imgf000041_0001
Step A
To a solution of 1 H-benzo[d]imidazol-6-amine (1.0 gr, 7.51 mmol) in acetone (25.0 mL) at 0 °C, pyridine (0.59 mL, 7.51 mmol) and benzenesulfonyl chloride (0.96 mL, 7.51 mmol) were slowly added and the reaction stirred at rt overnight. Purification by (S1O2, DCM/MeOH) delivered 258 mg (13% yield, 86% purity) of N-1 H-benzo[d]imidazol-6- yl)benzenesulfonamide.
Step B
N-1 H-Benzo[d]imidazol-6-yl)benzenesulfonamide from the previous step (258 mg, 0.94 mmol), Pd(OAc)2 (10.6 mg, 0.047 mmol), 5-iodo-1 H-indole (344 mg, 1.42 mmol) and Cul (360 mg, 1.88 mmol) were added to a sealed vial. Previously degassed DMF (4.0 mL) was added under a N2 atmosphere and the reaction heated to 140 °C and stirred at this temperature for 4 days. At this stage a saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgS04 and concentrated. Purification by column chromatography (S1O2, DCM/MeOH) afforded 24 mg (7% yield, 96% purity) of Compound 7 (as mixture of stereoisomers). 1H NMR (400 MHz, DMSO-de) 12.75 (bs, 0.43 H), 12.10 (bs, 0.57 H), 1 1.35 (s, 1 H), 10.12 (s, 0.57 H), 10.02 (s, 0.43 H), 8.37-8.33 (m, 1 H), 7.94- 7.89 (m, 1 H), 7.74 (dd, J = 8.5 and 1.7 Hz, 2 H), 7.62-7.51 (m, 5 H), 7.44 (t, J = 2.7 Hz, 1 H), 7.37-7.24 (m, 1 H), 6.92 (dd, J = 13.8 and 8.6 Hz, 1 H), 6.55 (s, 1 H) ppm. LCMS: m/z (ESP+) 389 (MH+) Example 7 - Preparation of Compound 8: N-[2-(1-acetylpiperidin-4-yl)-3H-benzimidazol-5-yl1 benzenesulfonamide
Figure imgf000042_0001
Step A
In a sealed vial was added 4-nitrobenzene-1 , 2-diamine (1.0 gr, 6.53 mmol), polyphosphoric acid (5.0 mL) and 1- (tert-butoxycarbonyl)piperidine-4-carboxylic acid (1.8 gr, 7.84 mmol). The reaction was then stirred at 100 °C for 3 days. At this point, the solution was cooled down to 0 °C and basified carefully with a 1 M aqueous solution of NaOH, and the crude extracted twice with DCM and twice with EtOAc. The organic layers were combined, dried over MgS04 and concentrated, affording 920 mg (57% yield, 62% purity) of 6-nitro-2-(piperidin-4-yl)-1 H-benzo[d]imidazole, which was used without further purification.
Step B
To a solution of 6-nitro-2-(piperidin-4-yl)-1 H-benzo[d]imidazole from the previous step (282 mg, 1.15 mmol) in DCM (2.0 mL) at 0 °C, NEΪ3 (0.16 mL, 1.15 mL) and acetic anhydride (0.1 1 mL, 1.20 mmol) were added and the reaction stirred at rt overnight. A saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgS04 and concentrated. Purification by column chromatography (S1O2, DCM/MeOH) afforded 102 mg (35% yield, 80% purity) of 1 -(4-(6-nitro-1 H-benzo[d]imidazol-2-yl)piperidin-1 -yl)ethan-1-one.
Step C
To a solution of 1 -(4-(6-Nitro-1 H-benzo[d]imidazol-2-yl)piperidin-1 -yl)ethan-1 -one from the previous step (102 mg, 0.35 mmol) in a 10: 1 solution of EtOH/DIPEA(containing 4% of thiophene) (1 mL), 10% Pt/C (20.0 mg, 0.35 mmol) was added and stirred under 2 atm of H2 at rt. The reaction was then filtered through a short pad of Celite and concentrated to deliver 101 mg (quantitative yield) of 1 -(4-(6-amino-1 H-benzo[d]imidazol-2-yl)piperidin-1 -yl)ethan-1 - one, wich was used without further purification.
Step D
To a solution of 1-(4-(6-amino-1 H-benzo[d]imidazol-2-yl)piperidin-1 -yl)ethan-1-one from the previous step (101 mg, 0.39 mmol) in pyridine (0.60 mL) at 0 °C, benzenesulfonyl chloride (45 mί, 0.35 mmol) was slowly added and the reaction stirred at rt for 3 days. A saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgS04 and concentrated. Purification by column chromatography (S1O2, DCM/MeOH) delivered 21 mg (15% yield, 99% purity) of Compound 8 (as mixture of stereoisomers). 1H NMR (400 MHz, DMSO- d6) 12.13 (s, 0.52 H), 12.03 (s, 0.48 H), 9.98 (bs, 1 H), 7.68 (dd, J = 7.0 and 1.5 Hz, 2 H), 7.56-7.46 (m, 3 H), 7.33- 7.13 (m, 2 H), 6.86-6.78 (m, 1 H), 4.36-4.30 (m, 1 H), 3.86-3.81 (m, 1 H), 3.08-2.97 (m, 1 H), 2.75-2.67 (m, 1 H), 1.99 (s, 3 H), 1.98-1.85 (m, 3 H), 1.75-1.51 (m, 2 H) ppm. LCMS: m/z (ESP+) 399 (MH+)
Example 8 - Preparation of Compound 2: N-[2-(1-methylpyrrolidin-3-yl)-3H-benzimidazol-5-yl1 benzenesulfonamide
Figure imgf000043_0001
Step 1: Synthesis ofN-(2-amino-4-nitro-phenyl)-1-methyl-pyrrolidine-3-carboxamide (5)
To a solution of 1 -methylpyrrolidine-3-carboxylic acid (0.35 g, 2.71 mmol) in DMF (10 mL) at 0°C was added diisopropyl ethylamine (1.18 mL, 6.8 mmol) followed by propylphosphonic anhydride solution (8.6 mL, 13.5 mmol). The resulting reaction mixture was stirred at 0°C for 10 minutes. 4-nitrobenzene-1 , 2-diamine (0.41 g, 2.71 mmol) was added to the reaction mixture and stirred at room temperature for 3 hours. After completion of reaction, the reaction mixture was quenched with water (5 mL) and washed with ethyl acetate to remove any organic bye products. The aqueous layer was then basified with 10% sodium bicarbonate solution and product was extracted with ethyl acetate. Organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give the desired product that was triturated with ether to give 5 (0.4 g crude) as a yellow solid; 1HNMR (400 MHz, DMSO-d6): d 1.99-2.02 (m, 2H), 2.25 (s, 3H), 2.38-2.42 (m, 1 H), 2.50-2.53 (m, 1 H, merged in DMSO peak), 2.56-2.60 (m, 1 H), 2.83 (t, J = 12 Hz, 1 H), 3.08-3.16 (m, 1 H), 6.44 (s, 2H), 6.76 (d, J = 9.2 Hz, 1 H), 7.84 (dd, J = 2.4, 6.8 Hz, 1 H), 8.24 (d, J = 2 Hz, 1 H), 9.16 (s, 1 H); LCMS: m/z 265.2 [M++1]
Step 2: Synthesis of 2-(1-methylpyrrolidin-3-yl)-6-nitro-1 H-benzimidazole (6)
A solution of 5 (0.4 g, 1.51 mmol) in acetic acid (10 mL) was heated at 100°C for 16 hours. After completion of reaction, acetic acid was evaporated under reduced pressure and the crude product so obtained was purified by silica gel column chromatography using 20% MeOH in dichloromethane as eluent to give the desired product 6 (0.26 g, 40% over 2 steps) as a yellow solid; 1HNMR (400 MHz, CD3OD): d 2.24-2.28 (m, 1 H), 2.43-2.48 (m, 1 H, merged in -NCHa peak), 2.47 (s, 3H), 2.76-2.80 (m, 1 H), 2.84-2.88 (m, 2H), 3.14-3.18 (m, 1 H), 3.74-3.78 (m, 1 H), 7.63 (d, J = 8.8 Hz, 1 H), 8.16 (dd, J = 2, 6.8 Hz, 1 H), 8.44 (d, J = 2.4 Hz, 1 H); LCMS: m/z 247.1 [M++1],
Step 3: Synthesis of tert-butyl 2-(1-methyipyrroiidin-3-yi)-8-nitro-benz/\midazoie-1-carboxyiate (7)
To a solution of 6 (0.15 g, 0.6 mmol) in dichloromethane (10 mL) at 0°C was added DMAP (0.007 g, 0.06 mmol) followed by triethyl amine (0.16 mL, 1.2 mmol) and Boc-anhydride (0.15 mL, 0.67 mmol). The resulting reaction mixture was stirred at room temperature for 24 hours. After the completion of reaction, residual solvents were concentrated under reduced pressure and the residue so obtained was purified by column chromatography using 0- 5% methanol in dichloromethane as eluent to give the desired product 7 (0.12 g, 57%) as a mixture of two compounds with same mass; LCMS: m/z 347.2 [M++1]
Step 4: Synthesis of tert-butyl 6-amino-2-(1-methylpyrrolidin-3-yl) benz/\midazoie-1-carboxyiate (8)
To a solution of 7 (0.12 g, 0.5 mmol) in methanol (10 mL) was added 10% Pd/C (12 mg) and the resulting reaction mixture was stirred under hydrogen atmosphere at room temperature for 2 hours. After completion of reaction, the reaction mixture was filtered over a pad of cealite and washed with methanol. Filtrate was evaporated under reduced pressure to give the desired product 8 (0.07 g, 64%) as a yellow color solid that was pure enough to take ahead for next step; LCMS: m/z 317.2 [M++1]
Step 5: Synthesis of tert-butyl 6-(benzenesulfonamido)-2-(1-methylpyrrolidin-3-yl) benz/\midazoie-1-carboxyiate (11) To a solution of 8 (0.07 g, 0.2 mmol) in pyridine (5 mL) at 0°C was added benzene sulfonyl chloride (0.03 mL, 0.2 mmol). The resulting reaction mixture was stirred at room temperature for 3 hours. After the completion of reaction, the reaction mixture was quenched with water (5 mL) and the product was extracted with dichloromethane (2 X 15 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the crude product that was purified by column chromatography using 20-30% ethyl acetate in hexanes as eluent to afford the desired product 9 (0.08 g, 80%) as a brown solid; LCMS: m/z 457.3 (M++1 )
Step 6: Synthesis of N-[2-(1-methylpyrrolidin-3-yl)-3H-benzimidazol-5-yl] benzene sulfonamide
To a solution of 9 (0.08 g, 0.17 mmol) in dioxane (1 mL) at 0°C was added 4 M HCI in dioxane (2 mL) and the resulting reaction mixture was stirred at room temperature for 3 hours. After completion of reaction, solvent was concentrated under reduced pressure. The residue was quenched with water (5 mL) and organic impurities were extracted with ethyl acetate (2 x 15 mL). Aqueous layer was then basified with saturated sodium bicarbonate solution and product was extracted with ethyl acetate (2 x 15 mL). Organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the desired product Compound 2 that was triturated with diethyl ether to give the product of desirable purity (0.04 g, 64%) as a yellow solid; 1HNMR (400 MHz, DMSO-de): d 2.01- 2.14 (m, 1 H), 2.16-2.20 (m, 1 H), 2.27 (s, 3H), 2.53-2.65 (m, 3H), 2.87 (t, J = 8 Hz, 1 H), 3.34-3-51 (m, 1 H), 6.84 (d, J = 8 Hz, 1 H), 7.17 (s, 1 H), 7.28 (d, J = 8Hz, 1 H), 7.48-7.58 (m, 3H), 7.68 (d, J = 8 Hz, 2H), 9.99 (br. s, 1 H), 12.05 (br. s, 1 H); LCMS: m/z 357.2 [M++1]; HPLC Purity: 92.22% [Retention time: 8.47],
Example 9: Preparation of Compound 19: N-[2-(3-bromo-2-methylphenyl)-1 H-benzimidazol-6-yl1
benzenesulfonamide
Figure imgf000045_0001
Step A
In a flask equipped with a reflux condenser was added 4-nitrobenzene-1 , 2-diamine (712 mg, 4.65 mmol), polyphosphoric acid (7.5 mL) and 2-bromo-2-methylbenzoic acid (1.0 gr, 4.65 mmol). The reaction was then stirred at 150 °C for 7 h. At this point, the solution was cooled down to 0 °C and basified carefully with a 1 M aqueous solution of NaOH, and the crude extracted twice with DCM and twice with EtOAc. The organic layers were combined, dried over MgS04 and concentrated. Purification by column chromatography (S1O2, cyclohexane/EtOAc) afforded 62 mg (4% yield, 34% purity) of 2-(3-bromo-2-methylphenyl)-5-nitro- 7/-/-benzo[o(]imidazole.
Step B
2-(3-bromo-2-methylphenyl)-5-nitro- 7/-/-benzo[d]imidazole (62 mg, 0.19 mmol) was then dissolved in EtOH (0.5 mL) and SnCl2'2H20 (46.3 mg, 0.21 mmol) was added and the mixture stirred at 70 °C for 2 h. At this stage a saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgS04 and concentrated. Purification by column chromatography (S1O2, DCM/MeOH) afforded 21 mg (37% yield, 90-95% purity (1HNMR)) of 2-(3-bromo-2-methylphenyl)- '//7-benzo[c/]imidazol-5-amine.
Step C
To a solution of 2-(3-bromo-2-methylphenyl)- '//7-benzo[c/|imidazol-5-amine (21 mg, 0.07 mmol) in pyridine (0.1 mL) at 0 °C, benzenesulfonyl chloride (12 mί, 0.07 mmol) was slowly added and the reaction stirred overnight and concentrated. Purification by (S1O2, cyclohexane/EtOAc) delivered 14.6 mg (48% yield, 91 % purity) of Compound 19. 1H RMN (400 MHz, DMSO-d6) 12.75 (bs, 0.30 H), 12.67 (bs, 0.70 H), 8.64 (dd, 0 = 5.8 and 1.7 Hz, 0.3 H), 7.81 (d, J = 7.4 Hz, 3 H), 7.70-7.57 (m, 4.7 H), 7.46-7.34 (m, 2 H), 7.09-6.99 (m, 1 H), 2.64 (s, 2.1 H), 2.62 (s, 0.9 H) ppm. LCMS: m/z (ESP+) 442 (MH+)
Example 10: Preparation of Compound 20: N -benzyl -2-cyclopentyl-3/7-benzo[c/|imidazol-5-amine
Figure imgf000046_0001
Step A
4-Nitrobenzene-1 , 2-diamine (789 mg, 5.15 mmol) and PTSA (89.0 mg, 0.47 mmol) were dissolved in EtOH (23 mL), and subsequently cyclopentanebarboxyaldehyde (0.5 mL, 0.46 mmol) was added. The reaction was then stirred at 95 °C overnight. A saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgS04 and concentrated Purification by column chromatography (S1O2, cyclohexane/EtOAc) afforded 452 mg (42% yield, 91 % purity) of 2-cyclopentyl-5-nitro- 7/-/-benzo[c/]imidazole.
Step B
2-Cyclopentyl-5-nitro- iH-benzo[d]imidazole (200 mg, 0.86 mmol) was then dissolved in EtOH (1.2 mL) and SnCl2'2H20 (215 mg, 0.95 mmol) was added and the mixture stirred at 70 °C for 6 h. At this stage the reaction was concentrated, and a saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgS04 and concentrated. Purification by column chromatography (S1O2, DCM/MeOH) afforded 70 mg (40% yield, 75% purity) of 2-cyclopentyl- f/7-benzo[c/]imidazol-5-amine.
Step C
To a solution of of 2-cyclopentyl- 7/-/-benzo[c/]imidazol-5-amine (55 mg, 0.27 mmol) in toluene (0.7 mL) benzaldehyde (31 mί, 0.30 mmol) were slowly added and the reaction stirred at 1 10 °C for 3 h.The reacion was then concentrated and the residue dissolved in EtOH (1.4 mL). Sodium borohydride (16 mg, 0.41 mmol) was then added at rt and the reaction heated to reflux for 3 h. At this stage the reaction was concentrated, and a saturated aqueous solution of NaHC03 and DCM were added. After layer separation, the aqueous phase was washed twice with DCM and twice with EtOAc. The organic layers were then combined, dried over MgSC and concentrated. Purification by column chromatography (S1O2, cyclohexane/EtOAc) delivered 42 mg (53% yield, 97% purity) of Compound 20. 1H RMN (400 MHz, DMSO-de) 1 1.56 (bs, 1 H), 7.36 (d, 0 = 7.0 Hz, 2 H), 7.28 (t, 0 = 7.5 Hz, 2 H), 7.18 (tt, 0 = 7.3 and 1.9 Hz, 1 H), 7.13 (d, J 8.6 Hz, 1 H), 6.51 (dd, J 8.6 and 2.2 Hz, 1 H), 6.42 (d, 0 = 1.8 Hz, 1 H), 5.94 (bs, 1 H), 4.24 (s, 2 H), 3.1 1 (quin, 0 = 8.1 Hz, 1 H), 1.99-1.91 (m, 2 H), 1.82-1.74 (m, 2 H), 1.72-1.65 (m, 2 H), 1.63-1.56 (m, 2 H) ppm. LCMS: m/z (ESP+) 292 (MH+)
Example 1 1 : Preparation of Compound 22: N-[2-(2-methylcvclopentyl)-3H-benzimidazol-5-yl1 benzenesulfonamide
Figure imgf000047_0001
2
Step 3
Step 1: Synthesis of 2-(2-methylcyclopentyl)-6-nitro-1H-benzimidazole (1)
To a solution of 2-methylcyclopentane carboxylic acid (0.25 g, 1.9 mmol) in DMF (10 mL) was added HATU (1.1 1 g, 2.92 mmol) and diisopropyl ethyl amine (1 mL, 5.85 mmol) followed by 4-nitrobenzene-1 , 2-diamine (0.3 g, 2.1 mmol). The resulting reaction mixture was stirred at room temperature for 16 hours. After the completion of reaction, DMF was evaporated under reduced pressure. Water (10 mL) was added to the residue and the product was extracted with ethyl acetate (3 x 20 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product (0.31 g) so obtained was dissolved in acetic acid (10 mL) and the reaction mixture was heated at 100°C for 16 hours. After completion of reaction, acetic acid was evaporated under reduced pressure and the crude product so obtained was purified by flash chromatography using 10-30% ethyl acetate in hexanes as eluent to give the desired product 1 (0.36 g, 76% over 2 steps) as a yellow solid; 1HNMR (400 MHz, CD3OD): d 1.07 (d, J = 6.8 Hz, 3H), 1.41-1.44 (m, 1 H), 1.86-2.00 (m, 2H), 2.03-2.09 (m, 2H), 2.22-2.31 (m, 2H), 2.86 (q, J = 8.8 Hz, 1 H), 7.62 (br. s, 1 H), 8.16 (dd, J = 2.0, 6.8 Hz, 1 H), 8.42 (s, 1 H); LCMS: m/z 246.1 [M++1]
Step 2: Synthesis of2-(2-methylcyclopentyl)-3H-benzimidazol-5-amine (2)
To a solution of 1 (0.36 g, 1.5 mmol) in methanol (10 mL) was added Palladium on carbon (36 mg) and the reaction mixture was stirred for 2 hours under hydrogen atmosphere using hydrogen balloon at room temperature. After completion of reaction, reaction mixture was filtered on cealite bed and washed with methanol. The filtrate was concentrated under reduced pressure to get the desired product 2 (0.29 g, 93%) as a yellow solid; LCMS: m/z 216.2 [M++1]
Step 3: Synthesis o†N-[2-(2-methylcyclopentyl)-3H-benzimidazol-5-yl] benzenesulfonamide
To a solution of 2 (0.15 g, 0.7 mmol) in dichloromethane (10 mL) was added pyridine (0.06 mL, 0.7 mmol) and at 0°C was added benzene sulfonyl chloride (0.09 mL, 0.7 mmol). The resulting reaction mixture was stirred at room temperature for 16 hours. After the completion of reaction, the reaction mixture was quenched with water (5 mL) and the product was extracted with dichloromethane (2 X 15 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the crude product that was purified by column chromatography using 0-5% methanol in dichloromethane as eluent to afford the desired product Compound 22 (0.06 g, 25%) as a light pink solid; 1HNMR (400 MHz, DMSO-d6): d 0.97 (d, J = 7.2 Hz, 3H), 1.26-1.30 (m, 1 H), 1.69-1.74 (m, 2H), 1.83-1.94 (m, 2H), 1.98-2.04 (m, 1 H), 2.06-2.17 (m, 1 H), 2.64-2.69 (distorted q, 1 H), 6.83 (d, J = 8.4 Hz, 1 H), 7.17 (s, 1 H), 7.29 (d, J = 8 Hz, 1 H), 7.49-7.59 (m, 3H), 7.69-7.72 (m, 2H), 10.01 (s, 1 H), 1 1.95-12.10 (Split br. s due to DMSO effect, 1 H); LCMS: m/z 356.2 [M++1]; HPLC Purity: 92.24% [Retention time: 1 1.375]
Example 12: Preparation of Compound 23: N-[2-(2-methylcvclohexyl)-3H-benzimidazol-5-yll benzenesulfonamide
Figure imgf000048_0001
Step 3
4
Step 1: Synthesis of 2-(2-methylcyclohexyl)-6-nitro-1 H-benzimidazole (3)
To a solution of 2-methylcyclohexanecarboxylic acid (0.25 g, 1.8 mmol) in DMF (10 mL) was added HATU (1.0 g, 2.64 mmol) and diisopropyl ethyl amine (0.92 mL, 5.27 mmol) followed by 4-nitrobenzene-1 , 2-diamine (0.29 g, 1.93 mmol). The resulting reaction mixture was stirred at room temperature for 16 hours. After the completion of reaction, DMF was evaporated under reduced pressure. Water (10 mL) was added to the residue and the product was extracted with ethyl acetate (3 x 20 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product (0.32 g) so obtained was dissolved in acetic acid (10 mL) and the reaction mixture was heated at 100°C for 16 hours. After completion of reaction, acetic acid was evaporated under reduced pressure and the crude product so obtained was purified by flash chromatography using 10-30% ethyl acetate in hexanes as eluent to give the desired product 3 (0.15 g, 33% over 2 steps) as a yellow solid; LCMS: m/z 260.2 [M++1]
Step 2: Synthesis o†2-(2-methylcyclohexyl)-3H-benzimidazol-5-amine (4)
To a solution of 3 (0.15 g, 0.58 mmol) in methanol (10 mL) was added 10% Pd/C (15 mg) and the resulting reaction mixture was stirred under hydrogen atmosphere at room temperature for 2 hours. After completion of reaction, the reaction mixture was filtered over a pad of cealite and washed with methanol. Filtrate was evaporated under reduced pressure to give the desired product 4 (0.12 g, 90%) as a brown color solid that was pure enough to take ahead for next step; LCMS: m/z 230.2 [M++1]
Step 3: Synthesis of N-[2-(2-methylcyclohexyl)-3H-benzimidazol-5-yl] benzenesulfonamide
To a solution of 4 (0.12 g, 0.52 mmol) in dichloromethane (10 mL) at 0°C was added pyridine (0.04 mL, 0.52 mmol) followed by benzene sulfonyl chloride (0.06 mL, 0.47 mmol). The resulting reaction mixture was stirred at room temperature for 2 hours. After the completion of reaction, the reaction mixture was quenched with water (5 mL) and the product was extracted with ethyl acetate (2 X 15 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the crude product that was purified by column chromatography using 10-30% ethyl acetate in hexanes as eluent to afford the desired product Compound 23 (0.023 g, 13%) as a yellow solid; 1HNMR (400 MHz, DMSO-d6): d 0.65 (d, J = 6 Hz, 3H), 1.03-1.06 (m, 1 H), 1.29- 1.34 (m, 2H), 1.53-1.57 (m, 1 H), 1.71 -1.77 (br. m, 5H), 2.33-2.39 (m, 1 H), 6.82 (br. s, 1 H), 7.14-7.33 (m, 2H), 7.49- 7.59 (m, 3H), 7.71 (d, J = 7.6 Hz, 2H), 10.0 [d (Splitting due to solvent effect), 1 H], 12.05 [d (Splitting due to solvent effect), 1 H]; LCMS: m/z 368.1 [M++1]; HPLC purity: 95.15% [Retention time: 13.22]
Example 13 - Preparation of Compound 24: N-(2-(4-(4-methylpiperazin-1 -yl)cvclohexyl)-1 H-benzo[d1imidazol-6- vDbenzenesulfonamide
Figure imgf000049_0001
Step 1: Synthesis of tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3)
To a stirred solution of 4-nitrobenzene-1 , 2-diamine (1) (1 g, 6.53 mmol) in dry DMF (20 mL) was added 4-((tert- butoxycarbonyl)amino)cyclohexane-1 -carboxylic acid (2) (1.58 g, 6.53 mmol) at 0 °C. HATU (2.73 g, 7.18 mmol) was added followed by the addition of DIPEA (5.5 mL, 32.65 mmol). The reaction mixture was allowed to stir at RT for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was quenched with ice cold water (50 mL) to get precipitate which was filtered. The solid was washed with water (50 mL) and dried to get tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3) (1.3 g, yield = 53% ) which was taken to next step without further purification. LCMS: 379.4 (M+1 ).
Step 2: Synthesis of tert-butyl (4-(b-nitro-1H-benzo[d]\midazoi-2-yi)cyciohexyi)carbamate (4)
tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3) (1.3 g, 3.4 mmol) in glacial acetic acid (20 mL) was heated to 65 °C and stirred at the same temperature for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated to get a residue. The residue was diluted with ethyl acetate (250 mL) and washed with 10% aqueous sodium bicarbonate solution (200 mL). The ethyl acetate layer was separated, dried over sodium sulphate and concentrated. The crude was triturated with diethyl ether (100 mL) to obtain tert-butyl (4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamate (4) (0.8 g, yield = 65%) as a pale yellow solid which was dried well and taken to next step without further purification. LCMS: 361.2 (M+1 ).
Step 3: Synthesis of 4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexan-1 -amine (5)
To an ice-cooled solution of tert-butyl (4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamate (4) (0.8 g, 2.2 mmol) in dry DCM (15 mL) was added 4N HCI in dioxane (5 mL). The mixture was stirred at RT for 5 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to get a crude. The crude was triturated with diethyl ether (100 mL) to get the solid 4-(6-nitro-1 H- benzo[d]imidazol-2-yl)cyclohexan-1 -amine (5) (0.65 g, yield = 99%) as HCI salt. LCMS: 261.3 (M+1).
Step 4: Synthesis of2-(4-(4-methylpiperazin-1-yl)cyclohexyl)-6-nitro-1H-benzo[d]imidazole (7)
To a stirred solution of 4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexan-1-amine hydrochloride (5) (0.3 g, 1 .0 mmol) in dry acetonitrile (10 mL) was added 2-chloro-N-(2-chloroethyl)-N-methylethan-1-amine hydrochloride (6) (0.19 g, 1.0 mmol). Then DIPEA (0.85 mL, 5.0 mmol) was added. The reaction mixture was stirred at RT for 2 h and then heated to 90 °C for 16 h. The reaction mixture was concentrated under reduced pressure to obtain a residue of 2- (4-(4-methylpiperazin-1 -yl)cyclohexyl)-6-nitro-1 H-benzo[d]imidazole (7) (0.45 g crude) which was dried and taken to next step without further purification. LCMS: 344.4 (M+1 )
Step 5: Synthesis of tert-butyl 2-(4-(4-methyipiperaz\n-1-yi)cyciohexyi)-%-nitro-1H-benzo[d]\midazoie-1-carboxyiate
(8) To an ice-cooled solution of 2-(4-(4-methylpiperazin-1 -yl)cyclohexyl)-6-nitro-1 H-benzo[d]imidazole (7) (0.45 g, 1.3 mmol) in DCM (10 mL) was added di-tert-butyl dicarbonate (0.85 ml, 3.92 mmol) and triethylamine (0.9 ml, 6.5 mmol). The mixture was stirred at RT for 12 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was diluted with DCM (150 mL) and water (100 mL). The organic layer was separated, dried over sodium sulphate and the volatiles were removed under reduced pressure. The crude was purified using Isolera® silica column by eluting with 1 to 15 % MeOH in DCM. The pure fractions were collected and concentrated to get tert-butyl 2-(4-(4-methylpiperazin-1 -yl)cyclohexyl)-6-nitro-1 H-benzo[d]imidazole-1 -carboxylate
(8) (0.1 1 g, Yield = 25% for two steps) as a mixture of desired product (mixture of cisltrans and regio isomers). LCMS: 444.5 (M+1 )
Step 6: Synthesis of tert-butyl 6-amino-2-(4-(4-methylpiperazin-1-yl)cyclohexyl)-1H-benzo[d]imidazole-1 -carboxylate
(9)
To a solution of tert-butyl 2-(4-(4-methylpiperazin-1 -yl)cyclohexyl)-6-nitro-1 H-benzo[d]imidazole-1 -carboxylate (8) (0.1 1 g, 0.25 mmol) in dry MeOH/THF (15 mL, 7:3) was added 10% Pd/C (50 mg) at RT. The mixture was stirred under hydrogen (bladder) for 16 h. The reaction mixture was filtered through a celite bed and the filtrate was concentrated to get the crude tert-butyl 6-amino-2-(4-(4-methylpiperazin-1-yl)cyclohexyl)-1 H-benzo[d]imidazole-1 - carboxylate (9) (0.09 g, yield: 90%) which was taken to next step without purification. LCMS: 414.6 (M+1 )
Step 7: Synthesis of tert-butyl 2-(4-(4-methylpiperazin-1-yl)cyclohexyl)-6-(phenylsulfonamido)-1H- benzo[d]imidazole-1 -carboxylate (11)
To an ice-cooled solution of 9 (0.09 g, 0.22 mmol) in dry DCM (15 ml) was added triethylamine (0.15 mL, 0.66 mmol) and then benzenesulfonylchloride 10 (0.038 g, 0.22 mmol). The reaction mixture allowed to stir at RT for 16 h. The reaction mixture was diluted with DCM (75 mL) and water (50 mL). The DCM layer was separated, dried over sodium sulphate and concentrated to get the crude tert-butyl 2-(4-(4-methylpiperazin-1-yl)cyclohexyl)-6- (phenylsulfonamido)-1 H-benzo[d]imidazole-1-carboxylate (11) (0.15 g, crude) which was taken to next step without further purification. LCMS: 554.7 (M+1 )
Step 8: Synthesis ofN-(2-(4-(4-methy/piperazin-1-y/)cyc/ohexy/)-1H-benzo[d]imidazo/-6-y/)benzenesu/fonamide To a solution of tert-butyl 2-(4-(4-methylpiperazin-1 -yl)cyclohexyl)-6-(phenylsulfonamido)-1 H-benzo[d]imidazole-1- carboxylate (11) (0.15 g, crude) in dry DCM (10 mL) was added 4N HCL in dioxane (3 mL) at 0 °C. The reaction mixture was stirred at RT for 6 h. The reaction mixture was concentrated under reduced pressure to get the crude which was purified by preparative HPLC using 0.1 % HCOOH in acetonitrile. The product fractions were concentrated to get 20 mg of Compound 24. 1H-NMR (400 MHz, MeOH-d4, 25 °C): d [ppm] 1.34 (t, J 7.28 Hz, 1 H), 1.55-1.65 (m, 1 H), 1.83-1.95 (m, 5H), 2.18-2.20 (m, 1 H), 2.28-2.32 (m, 2H), 2.65-2.67 (m, 1 H), 2.80-2.83 (m, 4H), 3.06-3.09 (m, 1 H), 3.20-3.26 (m, 2H), 3.35 (s, 3H), 7.14 (d, 0 = 8.72 Hz, 1 H), 7.47-7.57 (m, 5H), 7.76-7.79 (m, 2H), 8.09 (s, 1 H). LCMS: 454 (M+1 ). HPLC Purity: 99.7% Example 14- Preparation of Compound 25: N-(4-(6-(phenylsulfonamido)-1 H-benzo[d1imidazol-2- yl)cvclohexyl)piperazine-1-carboxamide
Figure imgf000052_0001
Step 1: Synthesis of tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3)
To a stirred solution of 4-nitrobenzene-1 , 2-diamine (1) (1 g, 6.53 mmol) in dry DMF (20 mL) was added 4-((tert- butoxycarbonyl)amino)cyclohexane-1 -carboxylic acid (2) (1.58 g, 6.53 mmol) at 0 °C. HATU (2.73 g, 7.18 mmol) was added followed by the addition of DIPEA (5.5 mL, 32.65 mmol). The reaction mixture was allowed to stir at RT for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was quenched with ice cold water (50 mL) to get precipitate which was filtered. The solid was washed with water (50 mL) and dried to get tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3) (1.3 g, yield = 53% ) which was taken to next step without further purification. LCMS: 379.4 (M+1).
Step 2: Synthesis of tert-butyl (4-(b-nitro-1H-benzo[0]\m\0azoi-2-yi)cyciohexyi)carbamate (4)
tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3) (1.3 g, 3.4 mmol) in glacial acetic acid (20 mL) was heated to 65 °C and stirred at the same temperature for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated to get a residue. The residue was diluted with ethyl acetate (250 mL) and washed with 10% aqueous sodium bicarbonate solution (200 mL). The ethyl acetate layer was separated, dried over sodium sulphate and concentrated. The crude was triturated with diethyl ether (100 mL) to obtain tert-butyl (4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamate (4) (0.8 g, yield: 65%) as a pale yellow solid which was dried well and taken to next step without further purification. LCMS: 361.2 (M+1). Step 3: Synthesis of 4-(6-nitro-1H-benzo[d]imidazol-2-yl)cyclohexan-1 -amine (5)
To an ice-cooled solution of tert-butyl (4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamate (4) (0.8 g, 2.2 mmol) in dry DCM (15 mL) was added 4N HCI in dioxane (5 mL). The mixture was stirred at RT for 5 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to get a crude. The crude was triturated with diethyl ether (100 mL) to get the solid 4-(6-nitro-1 H- benzo[d]imidazol-2-yl)cyclohexan-1 -amine (5) (0.65 g, yield = 99%) as HCI salt. LCMS: 261.3 (M+1).
Step 4 and 5: Synthesis of tert-butyl 4-((4-(6-nitro-1H-benzo[d]imidazol-2-yl)cyclohexyl)carbamoyl)piperazine-1- carboxylate (7)
4-(6-Nitro-1 H-benzo[d]imidazol-2-yl)cyclohexan-1-amine hydrochloride (5) (0.25 g, 0.96 mmol) in dry DCM was taken in a RB flask under N2 and was cooled to 0 °C. Triethylamine (0.7 mL, 4.8 mmol) and phenylchloroformate (0.16 g, 1.05 mmol) were added to the mixture at 0 °C and stirred for 2 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was diluted with DCM (75 mL) and washed with water (50 mL). The DCM layer was separated, dried over sodium sulphate and concentrated under reduced pressure to get the crude phenyl (4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamate (0.32 g, crude). The crude was washed with hexane (50 mL) and dried.
The above crude phenyl (4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamate (0.32 g, crude) was taken in dry DMF (8 mL). tert-butyl piperazine-1 -carboxylate 6 (0.36 g, 1.92 mmol) was added followed by the addition of triethylamine (0.4 mL, 2.88 mmol) and stirred at RT for 15 min, then heated to 65 °C for 16 h. The mixture was diluted with DCM (150 mL) and water (100 mL). The organic layer was separated out and washed with water (50 mL), brine (50 mL) and then dried under sodium sulphate. The organic layer was concentrated to get tert-butyl 4-((4-(6-nitro- 1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamoyl)piperazine-1-carboxylate (7) (0.3 g, crude) which was taken to the next step. LCMS: 473.6 (M+1 )
Step 6: Synthesis of tert-butyl 2-(4-(4-(ted-butoxycarbonyi)piperaz\ne-1-carboxamido)cyciohexyi)-b-nitro-1H- benzo[d]imidazole- 1 -carboxylate
To a stirred solution of tert-butyl 4-((4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamoyl)piperazine-1 - carboxylate (7) (0.3 g, 0.63 mmol) in DCM (10 mL) was added di-tert-butyl dicarbonate (0.41 ml, 1.9 mmol) and triethylamine (0.5 ml, 3.15 mmol) at 0 °C. The reaction mixture was stirred at RT for 12 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was diluted with DCM (75 mL) and water (50 mL). The organic layer was separated, dried over sodium sulphate and the volatiles were removed under reduced pressure. The crude was purified using Isolera® silica column by eluting with 1 to 10% MeOH in DCM. The pure fractions were collected and concentrated to afford tert-butyl 2-(4-(4-(tert-butoxycarbonyl)piperazine-1 - carboxamido)cyclohexyl)-6-nitro-1 H-benzo[d]imidazole-1-carboxylate (0.15 g, yield = 44% for two steps), (mixture of cisltrans and regio isomers). LCMS: 573.4 (M+1 ) Step 7: Synthesis of tert-butyl 6-amino-2-(4-(4-(tert-butoxycarbonyl)piperazine-1-carboxamido)cyclohexyl)-1H- benzo[d]imidazole- 1 -carboxylate (8)
To a solution of tert-butyl 2-(4-(4-(tert-butoxycarbonyl)piperazine-1 -carboxamido)cyclohexyl)-6-nitro-1 H- benzo[d]imidazole-1 -carboxylate (0.15 g, 0.26 mmol) in dry MeOH/THF (15 mL, 7:3) was added 10% Pd/C (0.05 g) under nitrogen. The mixture was stirred under hydrogen atmosphere (bladder) at RT for 16 h. The reaction mixture was filtered through a celite bed and the filtrate was concentrated. The crude tert-butyl 6-amino-2-(4-(4-(tert- butoxycarbonyl)piperazine-1 -carboxamido)cyclohexyl)-1 H-benzo[d]imidazole-1 -carboxylate (8) (0.1 1 g, yield = 78%), (mixture of cisltrans and regio isomers) was taken to next step without purification. LCMS: 543.7 (M+1)
Step 8: Synthesis of tert-butyl 2-(4-(4-(ted-butoxycarbonyi)piperaz\ne-1-carboxamido)cyciohexyi)-8- (phenylsulfonamido)-l H-benzo[d]imidazole- 1 -carboxylate
To an ice-cooled solution of tert-butyl 4-((4-(6-amino-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamoyl)piperazine-1 - carboxylate (8) (0.1 1 g, 0.20 mmol) in dry DCM (10 ml) was added triethylamine (0.15 mL, 0.81 mmol) and benzenesulfonylchloride (0.040 g, 0.22 mmol). The reaction mixture was allowed to stir at RT for 16 h. The reaction mixture was diluted with DCM (75 mL) and water (50 mL). The DCM layer was separated, dried over sodium sulphate and concentrated. The crude tert-butyl 2-(4-(4-(tert-butoxycarbonyl)piperazine-1-carboxamido)cyclohexyl)-6- (phenylsulfonamido)-1 H-benzo[d]imidazole-1-carboxylate (0.15 g, crude) was taken to next step without further purification. LCMS: 583.3 (M+1 )
Step 9: Synthesis of N-(4-(6-(phenylsulfonamido)-1H-benzo[d]imidazol-2-yl)cyclohexyl)piperazine-1 -carboxamide To a stirred solution of tert-butyl 2-(4-(4-(tert-butoxycarbonyl)piperazine-1-carboxamido)cyclohexyl)-6- (phenylsulfonamido)-1 H-benzo[d]imidazole-1-carboxylate (0.15 g, 0.22 mmol) in dry DCM (5 mL) was added 4N HCL in dioxane (5 mL). The reaction mixture was stirred at RT for 6 h. The reaction mixture was concentrated under reduced pressure to get the crude which was purified by preparative HPLC using 0.1 % HCOOH in acetonitrile. The product fractions were concentrated to afford N-(4-(6-(phenylsulfonamido)-1 H-benzo[d]imidazol-2- yl)cyclohexyl)piperazine-1 -carboxamide (15 mg) of Compound 25 as a formic acid salt. 1H-NMR (400 MHz, MeOH- d4, 25 °C): d [ppm] 1.77-1.81 (m, 4H), 1.88-1.92 (m, 2H), 2.18-2.22 (m, 2H), 3.07-3.10 (m, 1 H), 3.20-3.24 (m, 4H), 3.65-3.68 (m, 4H), 3.83-3.86 (m, 1 H), 6.90 (d, 0 = 7.88 Hz, 1 H), 7.32-7.36 (m, 2H), 7.42-7.47 (m, 2H), 7.55 (t, J = 7.36 Hz, 1 H), 7.72 (d, 0 = 7.36 Hz, 2H), 8.34 (br s, 2H). LCMS: 483 (M+1 ). HPLC Purity: 97.2%
Example 15- Preparation of Compound 26: 3-methyl-N-(4-(6-(phenylsulfonamido)-1 H-benzo[dlimidazol-2- vDcyclohexyDbenzamide Step 1: Synthesis of tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3)
To a stirred solution of 4-nitrobenzene-1 , 2-diamine (1) (1 g, 6.53 mmol) in dry DMF (20 mL) was added 4-((tert- butoxycarbonyl)amino)cyclohexane-1 -carboxylic acid (2) (1.58 g, 6.53 mmol) at 0 °C. HATU (2.73 g, 7.18 mmol) was added followed by the addition of DIPEA (5.5 mL, 32.65 mmol). The reaction mixture was allowed to stir at RT for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was quenched with ice cold water (50 mL) to get precipitate which was filtered. The solid was washed with water (50 mL) and dried to get tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3) (1.3 g, yield = 53% ) which was taken to next step without further purification. LCMS: 379.4 (M+1 ).
Step 2: Synthesis of tert-butyl (4-(b-nitro-1H-benzo[d]\midazoi-2-yi)cyciohexyi)carbamate (4)
tert-butyl (4-((2-amino-4-nitrophenyl)carbamoyl)cyclohexyl)carbamate (3) (1.3 g, 3.4 mmol) in glacial acetic acid (20 mL) was heated to 65 °C and stirred at the same temperature for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated to get a residue. The residue was diluted with ethyl acetate (250 mL) and washed with 10% aqueous sodium bicarbonate solution (200 mL). The ethyl acetate layer was separated, dried over sodium sulphate and concentrated. The crude was triturated with diethyl ether (100 mL) to obtain tert-butyl (4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamate (4) (0.8 g, yield: 65%) as a pale yellow solid which was dried well and taken to next step without further purification. LCMS: 361.2 (M+1 ).
Step 3: Synthesis of 4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexan-1 -amine (5)
To an ice-cooled solution of tert-butyl (4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)carbamate (4) (0.8 g, 2.2 mmol) in dry DCM (15 mL) was added 4N HCI in dioxane (5 mL). The mixture was stirred at RT for 5 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to get a crude. The crude was triturated with diethyl ether (100 mL) to get the solid 4-(6-nitro-1 H- benzo[d]imidazol-2-yl)cyclohexan-1 -amine (5) (0.65 g, yield = 99%) as HCI salt. LCMS: 261.3 (M+1).
Step 4: Synthesis o†3-methyl-N-(4-(6-nitro-1H-benzo[d]imidazol-2-yl)cyclohexyl)benzamide (7)
To a stirred solution of 4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexan-1-amine hydrochloride salt (5) (0.5 g, 1.7 mmol) in dry DMF (10 mL) was added 3-methylbenzoic acid (6) (0.23 g, 1.7 mmol) at 0 °C. HATU (0.7 g, 1.9 mmol) was added to the mixture followed by DIPEA (1.5 mL, 8.5 mmol). The mixture was stirred at RT for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was quenched with water (50 mL) and ethyl acetate (150 mL). The ethyl acetate layer was separated, dried over sodium sulphate and concentrated to get crude. Purification of the crude by Isolera® silica column by eluting with 1 to 10% MeOH in DCM gave 3-methyl-N-(4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)benzamide (7) (0.25 g, yield = 34%) ( cisltrans mixture). LCMS: 379.5 (M+1 ).
Step 5: Synthesis of tert-butyl 2-(4-(3-methylbenzamido)cyclohexyl)-8-nitro-1H-benzo[d]imidazole-1-carboxylate (8) 3-Methyl-N-(4-(6-nitro-1 H-benzo[d]imidazol-2-yl)cyclohexyl)benzamide (7) (0.25 g, 0.66 mmol) in DCM (10 mL) was taken in a RB flask (50 mL) under N2 at 0 °C. Di-tert-butyl di carbon ate (0.43 ml, 1.98 mmol) and triethylamine (0.5 ml, 3.3 mmol) were added to the reaction mixture which was stirred at RT for 12 h. The reaction mixture was diluted with DCM (75 mL) and water (50 mL). The organic layer was separated, dried over sodium sulphate and concentrated under reduced pressure. The crude was purified using Isolera® silica column chromatography by eluting with 1 to 10% MeOH in DCM. The pure fractions were collected and concentrated to afford tert-butyl 2-(4-(3- methylbenzamido)cyclohexyl)-6-nitro-1 H-benzo[d]imidazole-1-carboxylate (8) (0.17 g, yield = 55%) compound (mixture of cisltrans and regio isomers). LCMS: 479.6 (M+1 )
Step 6: Synthesis of tert-butyl 8-amino-2-(4-(3-methyibenzamido)cyciohexyi)-1H-benzo[d]\midazoie-1-carboxyiate
(9)
To a stirred solution of tert-butyl 2-(4-(3-methylbenzamido)cyclohexyl)-6-nitro-1 H-benzo[d]imidazole-1 -carboxylate (8) (0.17 g 0.35 mmol) in dry MeOH/THF (15 mL, 7:3) was added 10% Pd/C (0.1 g). The reaction was stirred under hydrogen atmosphere (bladder) at RT for 16 h. The reaction mixture was filtered through a celite bed and the filtrate was concentrated. The crude tert-butyl 6-amino-2-(4-(3-methylbenzamido)cyclohexyl)-1 H-benzo[d]imidazole-1 - carboxylate (9) (0.15 g, crude) was taken to next step without purification. LCMS: 449.5 (M+1)
Step 7 and Step 8: Synthesis of 3-methyl-N-(4-(6-(phenylsulfonamido)-1H-benzo[d]imidazol-2- yl)cyclohexyl)benzamide
tert-butyl-6-amino-2-(4-(3-methylbenzamido)cyclohexyl)-1 H-benzo[d]imidazole-1 -carboxylate (9) (0.15 g, 0.31 mmol) in dry DCM (15 mL) was taken in a RB flask (50 mL) and cooled to 0 °C. Triethylamine (0.25 mL, 1.65 mmol) and benzene sulfonyl chloride (0.058 g, 0.33 mmol) were added to the mixture. The reaction mixture was allowed to stir at RT for 16 h. The reaction mixture diluted with DCM (75 mL) and water (75 mL). The DCM layer was separated, dried over sodium sulphate and concentrated to get the crude which was taken to next step.
To the above crude was dissolved in dry DCM (10 mL) and cooled to 0 °C. 4N HCL in dioxane (2 mL) was added and the reaction mixture was stirred at RT for 5 h. The reaction mixture was concentrated under reduced pressure to get the crude which was purified using Isolera® silica column by eluting with 1 to 10% MeOH in DCM to afford 3- methyl-N-(4-(6-(phenylsulfonamido)-1 H-benzo[d]imidazol-2-yl)cyclohexyl)benzamide Compound 26 (15 mg). 1H- NMR (400 MHz, DMSO-d6, 25 °C): d [ppm] 1.61 -1.67 (m, 4H), 1.77-1.80 (m, 2H), 2.26-2.30 (m, 2H), 2.31 (s, 3H), 3.02-3.06 (m, 1 H), 3.91 -3.95 (m, 1 H), 6.82-6.89 (m, 1 H), 7.17-7.24 (m, 1 H), 7.26-7.28 (m, 2H), 7.37 (d, 0 = 8.80 Hz, 1 H), 7.49-7.53 (m, 2H), 7.56-7.63 (m, 3H), 7.71 (d, 0 = 7.60 Hz, 2H), 8.01-8.03 (m, 1 H), 9.97-10.06 (m, 1 H), 12.02- 12.09 (m, 1 H). LCMS: 489 (M+1 ). HPLC Purity: 97.3%
Example 16- Preparation of Compound 27 and Compound 28: N-(2-cvclopentyl-1 H-benzo[dlimidazol-6-yl)-1 - phenylmethanesulfonamide and N-benzhydryl-2-cyclopentyl-1 H-benzo[dlimidazol-6-amine, respectively
Figure imgf000057_0001
Step 1: Synthesis ofN-(2-amino-4-nitrophenyl)cyclopentanecarboxamide (3)
To a stirred solution of 4-nitrobenzene-1 , 2-diamine (1) (0.25 g, 1.6 mmol) in dry DMF (10 mL) was added cyclopentyl carboxylic acid (2) (0.19 g, 1.6 mmol) at 0 °C under N2. HATU (0.730 g, 1.19 mmol) and DIPEA (0.8 mL, 4.8 mmol) were added to the mixture at the same temperature. The reaction mixture was allowed to stir at room temperature for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was quenched with ice-cold water (10 mL) to get the solid. The solid was filtered and washed with water (25 mL) and dried to get N-(2-amino-4-nitrophenyl)cyclopentanecarboxamide (3) (0.25 g, yield = 62.5%) which was taken to next step without further purification. LCMS: 250.3 (M+1 ).
Step 2: Synthesis of 2-cyclopentyl-6-nitro-1 H-benzo[d]imidazole (4)
N-(2-Amino-4-nitrophenyl)cyclopentanecarboxamide (3) (0.25 g, 0.1 mmol) in glacial acetic acid (10 mL) was heated at 65 °C for 16 h. The conversion was monitored by TLC/LCMS. After the reaction was completed, the reaction mixture was concentrated to get a residue which was diluted with ethyl acetate (150 mL) and washed with 10% aqueous sodium bicarbonate solution (75 mL). The ethyl acetate layer was separated, dried over sodium sulphate and concentrated. The crude was triturated with diethyl ether (25 mL) to get (4) (0.125 g, yield: 54%) as a pale yellow solid. This solid was taken to the next step without further purification. LCMS: 232.5 (M+1 ).
Step 3: Synthesis of 2-cyclopentyl-1 H-benzo[d]imidazol-6-amine (5)
2-Cyclopentyl-6-nitro-1 H-benzo[d]imidazole (4) (0.125 g, 0.54 mmol) in dry MeOH/THF (15 mL, 7:3) was taken in a RB flask (50 mL). 10% Pd/C (75 mg) was added and the mixture was stirred under hydrogen atmosphere (bladder) at RT for 16 h. The reaction mixture was filtered through a celite bed and the filtrate was concentrated. The crude 2- cyclopentyl-1 H-benzo[d]imidazol-6-amine (5) (0.09 g, crude) was taken to next step without purification. LCMS: 202.6 (M+1 ).
Step 4a: Synthesis ofN-(2-cyclopentyl-1H-benzo[d]imidazol-6-yl)-1-phenylmethanesulfonamide
To an ice-cooled solution of 2-cyclopentyl-1 H-benzo[d]imidazol-6-amine (5) (0.04 g, 0.2 mmol) in dry DCM (15 ml) was added DIPEA (0.12 mL, 1.0 mmol) and then phenylmethanesulfonyl chloride (0.038 g, 0.2 mmol). The reaction mixture allowed to stir at RT for 16 h. The reaction mixture was diluted with DCM (75 mL) and water (50 mL). The DCM layer was separated, dried over sodium sulphate and concentrated. The crude was purified by Isolera® silica column eluting with 1 to 10% MeOH in DCM. The pure fractions were collected and concentrated to afford 22 mg of N-(2-cyclopentyl-1 H-benzo[d]imidazol-6-yl)-1-phenylmethanesulfonamide Compound 27. 1H-NMR (400 MHz, DMSO-de, 25 °C): d [ppm] 1.64-1.68 (m, 2H), 1.71-1.77 (m, 2H), 1.86-1.91 (m, 2H), 2.04-2.09 (m, 2H), 3.26 (quin, J = 8.00 Hz, 1 H), 4.34-4.36 (m, 2H), 6.98-7.06 (m, 1 H), 7.26-7.27 (m, 2H), 7.35 (s, 4H), 7.44-7.48 (m, 1 H), 9.56-9.66 (m, 1 H), 12.09-12.15 (m, 1 H). LCMS: 356 (M+1 ). HPLC Purity: 99.6%
Step 4b: Synthesis of N-benzhydryl-2-cyclopentyl-1 H-benzo[d]imidazol-6-amine
To a dry THF solution (10 mL) of 2-Cyclopentyl-1 H-benzo[d]imidazol-6-amine (5) (0.04 g, 0.2 mmol) and benzophenone 7 (0.073 g, 0.4 mmol) was cooled to 0 °C. Ti(OiPr)4 (0.142 g, 0.5 mmol) was added to the mixture. The resultant mixture was allowed to stir at RT for 2 h and then heated to 65 °C for 14 h. The reaction mixture was quenched with 10% aq. NaHC03 solution (10 mL) and extracted with DCM (2 x 30 mL). The DCM layer was separated, dried over sodium sulphate and concentrated. The crude was dissolved with dry MeOH/THF (1 : 1 , 10 mL) and cooled to 0 °C. NaCNBH3 (0.05 g) was added and stirred at RT for 5 h. The reaction mixture diluted with DCM (50 mL) and water (40 mL). The organic layer was separated, dried over sodium sulphate and concentrated. The crude was purified using Isolera® silica column by eluting with 1 to 10% MeOH in DCM. The pure fractions were collected and concentrated to afford 24 mg of N-benzhydryl-2-cyclopentyl-1 H-benzo[d]imidazol-6-amine Compound 28. 1H-NMR (400 MHz, DMSO-de, 25 °C): d [ppm] 1.59-1.62 (m, 2H), 1.69-1.72 (m, 2H), 1.77-1.84 (m, 2H), 1.96-
I .99 (m, 2H), 3.12 (quin, 0 = 8.00 Hz, 1 H), 5.59 (d, 0 = 6.40 Hz, 1 H), 6.13 (d, 0 = 6.40 Hz, 1 H), 6.51 (s, 1 H), 6.69 (d, 0 = 8.80 Hz, 1 H), 7.14 (d, 0 = 8.40 Hz, 1 H), 7.19-7.23 (m, 2H), 7.31 (t, 0 = 7.60 Hz, 4H), 7.44 (d, 0 = 7.60 Hz, 4H),
I I .70 (br s, 1 H). LCMS: 368 (M+1 ). HPLC Purity: 97.3% Example 17- Preparation of compound 29: N-(2-cvclopentyl-1 H-pyrrolo[3,2-b1pyridin-6-yl)benzenesulfonamide
Figure imgf000059_0001
Step f: Synthesis of 5-bromo-2-iodo-3-nitropyridine (2)
5-Bromo-3-nitropyridin-2-amine (1) (5 g, 22.9 mmol) was dissolved in dry acetonitrile (100 mL) then cooled to 0 °C under nitrogen atmosphere. Iodine (1 1.6 g, 45.8 mmol) was added to the reaction mixture followed by the slow addition of tert-butylnitrite (3.5 mL, 34.3 mmol). The mixture was stirred at 0 °C for 5 min then allowed to stir at RT for 2 h. The resultant mixture was heated at 70 °C for 14 h. The reaction was diluted with ethyl acetate (300 mL) and water (150 mL). The ethyl acetate layer was separated out and then dried over sodium sulphate and concentrated. The obtained crude was purified using Isolera® silica column by eluting with 1 to 20 % EtOAc in hexane. The pure fractions were collected and concentrated to afford 5-bromo-2-iodo-3-nitropyridine (2) (1 g, 13% yield) as a yellow solid. 1H-NMR (400 MHz, CDCI3): d 8.21 (d, J 2.40 Hz, 1 H), 8.66 (d, 0 = 2.00 Hz, 1 H).
Step 2: Synthesis of 5-bromo-2-(cyclopentylethynyl)-3-nitropyridine (4)
5-Bromo-2-iodo-3-nitropyridine (2) (0.5 g, 1.5 mmol), triethylamine (1 ml, 7.6 mmol), Cul (0.057 g, 0.3 mmol) in acetonitrile (20 mL) were taken in a seal tube (25 mL) which was bubbled with nitrogen for 10 min. Then Pd(PPh3)4 (0.35 g, 0.3 mmol) was added to the mixture followed by ethynylcyclopentane (3) (0.14 g, 1.5 mmol). The mixture was bubbled with nitrogen for another 5 min. The seal tube was closed tightly and heated to 55 °C for 5 h. The mixture was filtered through a celite bed. The filtrate was evaporated under reduced pressure to get the crude which was purified by Isolera® silica column using 1 to 10% EtOAc in hexane as eluent to afford 5-bromo-2- (cyclopentylethynyl)-3-nitropyridine (4) (0.3 g, yield = 66%) as a yellow gummy solid. LCMS: 344.4 (M+1 ). 1H-NMR (400 MHz, CDCI3): d 1.66-1.68 (m, 2H), 1.81-1.91 (m, 4H), 2.03-2.05 (m, 2H), 2.94-2.95 (m, 1 H), 8.46 (d, 0 = 1.96 Hz, 1 H), 8.83 (d, 0 = 1.96 Hz, 1 H).
Step 3: Synthesis of 5-bromo-2-(cyclopentylethynyl)pyridin-3-amine (5)
5-Bromo-2-(cyclopentylethynyl)-3-nitropyridine (4) (0.3 g, 1.0 mmol) in MeOH/water (30 mL, 7:3) was taken in a RB flask (50 mL). Iron (0.28 g, 5 mmol) and NH4CI (0.55 g, 10.0 mmol) were added to the mixture. The reaction mass was heated at 60 °C for 5 h. The reaction mixture was filtered through a celite bed and the filtrate was concentrated to get a residue. The residue was diluted with ethyl acetate (75 mL) with water (50 mL). The ethyl acetate layer was separated then dried over sodium sulphate and evaporated under reduced pressure. The crude was purified by Isolera® silica column using 1 to 10% EtOAc in hexane as eluent to obtain 5-bromo-2-(cyclopentylethynyl)pyridin-3- amine (5) (0.21 g, yield = 77%). LCMS: 265.5 (M+1 ), 267.5 (M+3).
Step 4: Synthesis of 6-bromo-2-cyclopentyl-1 H-pyrrolo[3,2-b]pyridine (6)
To a stirred solution of 5-bromo-2-(cyclopentylethynyl)pyridin-3-amine (5) (0.2 g, 0.75 mmol) in dry DMF (5 mL) was added potassium tert-butoxide (0.17 g, 1.51 mmol) at 0 °C. The reaction mixture was allowed to stir at RT for 5 h. The reaction was again cooled to 0 °C and then quenched by the slow addition of ice-cooled water (15 mL). The mixture was diluted with DCM (75 mL) and water (50 mL). The organic layer was separated, dried over sodium sulphate and concentrated to get the 0.45 g of the crude material. Purification of the crude using Isolera® silica column chromatography by eluting with 1 to 20% EtOAc in hexane gave 6-bromo-2-cyclopentyl-1 H-pyrrolo[3,2- b] pyridine (6) (0.1 1 g, yield = 65%). 1H-NMR (400 MHz, CDCI3): d 1.74-1.84 (m, 6H), 2.15-2.18 (m, 2H), 3.19-3.23 (m, 1 H), 6.44 (s, 1 H), 7.74 (s, 1 H), 8.43 (s, 1 H).
Step 5: Synthesis of tert-butyl %-bromo-2-cyciopenfyi-1H-pyrroio[3,2-b]pyridine-1-ca\boxyiate (7)
To a stirred solution of 6-bromo-2-cyclopentyl-1 H-pyrrolo[3,2-b]pyridine (6) (0.1 1 g, 0.41 mmol) in dry DCM (10 mL) was taken in R. B flash (100 mL) which was cooled to 0 °C. DIPEA (0.35 mL, 2.0 mmol) was added followed by the addition of cat. DMAP. Then di-tert-butyl dicarbonate (0.18 mL, 0.82 mmol) was added and the reaction mixture was allowed to stir at RT for 5 h. The reaction mixture was diluted with DCM (75 mL) and water (50 mL). The organic layer was separated, dried over sodium sulphate and concentrated. The crude was purified by Isolera® silica column using 1 to 10% EtOAc in hexane to obtain tert-butyl 6-bromo-2-cyclopentyl-1 H-pyrrolo[3,2-b]pyridine-1 -carboxylate (7) (0.10 g, yield = 66%). LCMS: 365.7 (M+1), 367.6 (M+3). 1H-NMR (400 MHz, CDCI3): d 1.65 (s, 9H), 1.66-1.76 (m, 6H), 2.1 1-2.42 (m, 2H), 3.69-3.71 (m, 1 H), 6.72 (s, 1 H), 8.42 (d, J = 2.00 Hz, 1 H), 8.52 (d, J = 2.00 Hz, 1 H).
Step 6 and Step 7: Synthesis of N-(2-cyclopentyl-1H-pyrrolo[3,2-b]pyridin-6-yl)benzenesulfonamide
To a stirred solution of tert-butyl 6-bromo-2-cyclopentyl-1 H-pyrrolo[3,2-b]pyridine-1-carboxylate (7) (0.1 g, 0.27 mmol) in 1 ,4-dioxane (3 mL) was added benzene sulfonamide (0.13 g, 0.82 mmol) at RT. The mixture was purged with N2 for 10 min. Cul (5 mg, 0.027 mmol) and N,N'-dimethylethylenediamine (5 mg, 0.05 mmol) were added to the reaction which was bubbled with N2 for another 10 min. K2C03 (0.1 1 g, 0.81 mmol) was added and the reaction mixture was heated at 1 10 °C for 16 h. The reaction mixture was diluted with DCM (60 mL) and water (50 mL). The organic layer was separated, dried over sodium sulphate and concentrated. The crude was purified using Isolera® silica column by eluting with 30 to 50 % of EtOAc in hexane to obtain (0.045 g, yield = 51 %) an off-white solid. LCMS: 442.6 (M+1).
The above solid was dissolved in dry DCM (5 mL) and 4N HCI in dioxane (2 mL) was added. The reaction mixture was stirred at RT for 14 h. The reaction mixture was concentrated. The crude was neutralised with triethylamine and then purified using Isolera® silica column by eluting with 1 to 5 % MeOH in DCM to obtain (30 mg, yield = 85%) Compound 29 as an off-white solid. 1H-NMR (400 MHz, DMSO-d6, 25 °C): d [ppm] 1.63-1.73 (m, 6H), 2.02-2.04 (m, 2H), 3.13-3.18 (m, 1 H), 6.19 (s, 1 H), 7.34 (s, 1 H), 7.51 -7.54 (m, 2H), 7.58-7.62 (m, 1 H), 7.68 (d, 0 = 8.00 Hz, 2H), 7.87 (s, 1 H), 10.07 (s, 1 H), 1 1.06 (s, 1 H). LCMS: 342 (M+1 ). HPLC Purity: 96.8%
Example 18: Preparation of Compound 30: N-(2-(3-methoxy-4-(1 H-pyrazol-4-yl)phenyl)-1 H-benzo[d]imidazol-6- yhbenzene sulfonamide
Figure imgf000061_0001
Step 1: N-(4-amino-3-nitrophenyl)benzenesulfonamide (2)
To an ice-cooled solution of 2-nitrobenzene-1 , 4-diamine 1 (5 g, 32.67 mmol) in dry DCM (100 mL) was added triethylamine (5.4 mL, 39.2 mmol) and benzenesulfonylchloride (5.7 g, 32.67 mmol). The reaction mixture was stirred at RT for 16 h. The reaction mixture was diluted with water (200 mL) to obtain a precipitate which was filtered. The solid was washed with DCM (100 mL) and then dried to get the title compound 2 which was used for next step without further purification. (Yield = 6 g, 62%). LCMS: 294 (M+1 ).
Step 2: N-(3, 4-diaminophenyl)benzenesulfonamide (3)
To a solution of N-(4-amino-3-nitrophenyl)benzenesulfonamide (3 g, 10.23 mmol) in dry ethyl acetate (60 mL) was added 10% Pd/C (300 mg) at RT. The mixture was stirred under hydrogen atmosphere (bladder) for 16 h. The reaction mixture was filtered through a celite bed and the filtrate was concentrated to get the crude which was purified by flash silica (230-400) column using Isolera to get 3. Yield (1.5 g, 57%). 1H-NMR (400 MHz, DMSO-d6): d [ppm] 9.36 (s, 1 H), 7.65-7.67 (m, 2H), 7.55-7.57 (m, 1 H), 7.49-7.53 (m, 2H), 6.28-6.32 (m, 2H), 6.03 (dd, 0 = 2.40, 8.00 Hz, 1 H), 4.42 (br s, 4H). LCMS: 264 (M+1 ).
Step 3: N-(2-amino-4-(phenylsulfonamido) phenyl)-3-methoxy-4-(1H-pyrazol-4-yl)benzamide (5) To a stirred solution of N-(3, 4-diaminophenyl)benzenesulfonamide 3 (200 mg, 0.760 mmol) in dry DMF (4 mL) was added 3-methoxy-4-(1 H-pyrazol-4-yl)benzoic acid 4 (132 mg, 0.60 mmol) at 0 °C. Then HATU (346 mg, 0.912 mmol) and DIPEA (0.24 mL, 1.52 mmol) were added. The mixture was allowed to stir at RT for 16 h. The conversion was monitored by LCMS. After completion of the reaction, the reaction mixture was quenched with ice-cooled water (20 mL) and extracted with ethyl acetate (2 x 25 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The crude was purified by flash column chromatography (isolera) using silica gel (230-400 mesh) to get the title compound 5 (Yield = 100 mg, 28%). LCMS: 464 (M+1 ).
Step 2: N-(2-(3-methoxy-4-(1H-pyrazol-4-yl)phenyl)-1H-benzo[d]imidazol-6-yl)benzene sulfonamide
N-(2-amino-4-(phenylsulfonamido)phenyl)-3-methoxy-4-(1 H-pyrazol-4-yl)benzamide 2 (100 mg, 0.215 mmol) in glacial acetic acid (4 mL) was heated at 60°C for 16 h. The conversion was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated and purified by prep HPLC to get N-(2-(3-methoxy-4-(1 H-pyrazol- 4-yl)phenyl)-1 H-benzo[d]imidazol-6-yl)benzene sulfonamide (Yield = 30 mg, 31 %). 1H-NMR (400 MHz, DMSO-d6): d [ppm] 10.52 (s, 1 H), 8.23 (s, 2H), 7.93 (d, J = 8.00 Hz, 1 H), 7.82-7.73 (m, 4H), 7.60-7.53 (m, 4H), 7.45 (s, 1 H), 7.17 (d, 0 = 8.80 Hz, 1 H), 4.02 (s, 3H), LCMS: 446 (M+1 ).
Example 19: Preparation of Compound 31 : N-(2-cvclopentyl-1 H-benzo[dlimidazol-6-yl)-4-hvdroxybenzene- sulfonamide
Figure imgf000062_0001
Step-1: N-(2-amino-4-nitrophenyl) cyclopentane carboxamide (2)
To a stirred solution of 4-nitrobenzene-1 , 2-diamine (1) (1 g, 6.53 mmol) in dry DMF (20 mL) was added cyclopentane carboxylic acid (0.75 g, 6.53 mmol). HATU (2.48 g, 6.53 mmol) was added followed by the addition of DIPEA (3.35 mL, 19.59 mmol). The reaction was allowed to stir at RT for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was quenched with ice-cooled water (50 mL), extracted with EtOAc (75 mL x 2). The combined organic layer was washed with brine (50 mL) then dried over anhydrous sodium sulphate, filtered and concentrated. The crude was purified by silica column chromatography using Isolera by eluting with 35% to 40% ethyl acetate in pet. ether to afford N-(2-amino-4-nitrophenyl) cyclopentane carboxamide (2) (1.3 g, yield: 80%). 1H- NMR (400 MHz, CDCI3): d 8.06-8.01 (m, 2H), 7.1 1 (brs, 1 H), 6.80 (d, 0 = 8.80 Hz, 1 H), 4.63 (brs, 2H), 2.82-2.80 (m, 1 H), 2.06-2.03 (m, 2H), 2.00-1.93 (m, 2H), 1.84-1.81 (m, 2H), 1.71-1.69 (m, 2H). LCMS: 250 (M+1 ). Step-2: 2-cyclopentyl-6-nitro-1H-benzo[d]imidazole (3)
N-(2-amino-4-nitrophenyl) cyclopentane carboxamide (2) (1.3 g, 5.21 mmol) in glacial acetic acid (40 mL) was heated to 60 °C for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated to get a residue. The residue was diluted with ethyl acetate (100 mL) and washed with 10% aqueous sodium bicarbonate solution (50 mL). The ethyl acetate layer was separated, dried over anhydrous sodium sulphate, filtered and concentrated to afford 2-cyclopentyl-6-nitro-1 H-benzo[d]imidazole (3) (1.15 g, yield: 96%). 1H-NMR (400 MHz, CDCI3): d 9.84-9.64 (m, 1 H), 8.64-8.36 (m, 1 H), 8.20 (m, 1 H), 7.76-7.47 (m, 1 H), 3.42-3.37 (m, 1 H), 2.30-2.21 (m, 2H), 2.06-1.95 (m, 2H), 1.93-1.87 (m, 2H), 1.83-1.73 (m, 2H). (Note: 1H NMR shows mixture of two regio-isomers). LCMS: 232 (M+1 ).
Step-3: tert-butyl 2-cyclopent/l-5-nitro-1H-benzo[d]\midazole-1-carboxylate (4)
To a stirred solution of 2-cyclopentyl-6-nitro-1 H-benzo[d]imidazole (3) (0.55 g, 2.37 mmol) in DCM (20 mL) was added di-tert-butyl dicarbonate (0.62 mL, 2.85 mmol) at 0 °C. Triethylamine (0.99 ml, 7.1 1 mmol) and catalytic DMAP were added and the mixture was stirred at RT for 12 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was diluted with DCM (75 mL), washed with water (30 mL). The organic layer was separated, dried over anhydrous sodium sulphate, filtered and concentrated. The crude was purified by silica column chromatography using Isolera by eluting with 7% EtOAc in pet. ether to afford tert-butyl 2-cyclopentyl-6-nitro-1 H- benzo[d]imidazole-1 -carboxylate (4) (0.7 g, yield = 89%). 1H-NMR (400 MHz, CDCI3): d 8.87-8.59 (m, 2H), 8.27-8.22 (m, 2H), 8.03-7.76 (m, 2H), 4.00-3.92 (m, 2H), 2.23-2.22 (m, 4H), 2.08-2.04 (m, 4H), 1.92-1.88 (m, 4H), 1.78-1.73 (m, 22H). (Note: 1H NMR shows mixture of two regio-isomers). LCMS: 232 (M+1 ).
Step-4: tert-butyl 5-amino-2-cyclopent/l-1H-benzo[d]\midazole-1-carboxylate (5)
To a solution of tert-butyl 2-cyclopentyl-6-nitro-1 H-benzo[d]imidazole-1 -carboxylate (4) (0.7 g, 2.1 1 mmol) in EtOAc (20 mL) was added 10% Pd/C (0.3 g) under nitrogen. The mixture was stirred under hydrogen atmosphere (bladder) at RT for 16 h. The reaction was monitored by TLC. The mixture was filtered through a celite bed and the filtrate was concentrated. The crude was purified by silica column chromatography using Isolera by eluting with 30-40% EtOAc in pet. ether to afford tert-butyl 6-amino-2-cyclopentyl-1 H-benzo[d]imidazole-1 -carboxylate (5) (0.59 g, yield = 93%). 1H-NMR (400 MHz, CDCI3): d 7.67 (d, J 8.40 Hz, 1 H), 7.01 (d, J 2.00 Hz, 1 H), 6.69 (dd, J 2.40, 8.80 Hz, 1 H), 3.92-3.84 (m, 1 H), 3.69 (br s, 2H), 2.18-2.15 (m, 2H), 2.07-2.01 (m, 2H), 2.00-1.85 (m, 2H), 1.71-1.69 (m, 1 1 H). LCMS: 302 (M+1 ).
Step-5: tert-butyl 2-cyclopentyl-6-((4-hydroxyphenyl) sulfonamido)-1H-benzo[d]imidazole-1-carboxylate (6)
To an ice-cooled solution of tert-butyl 6-amino-2-cyclopentyl-1 H-benzo[d]imidazole-1-carboxylate (5) (0.05 g, 0.166 mmol) in pyridine (1 mL) was added 4-hydroxy benzene sulfonylchloride (0.035 g, 0.182 mmol). The reaction was allowed to stir at RT for 2 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated as such and the residue was dissolved in EtOAc (10 mL), washed with water (10 mL), brine (10 mL) then dried over anhydrous sodium sulphate, filtered and concentrated to get 60 mg crude tert-butyl 2-cyclopentyl-6- ((4-hydroxyphenyl) sulfonamido)-1 H-benzo[d]imidazole-1 -carboxylate (6). LCMS: 458 (M+1 ).
Step-6: N-(2-cyclopentyl-1H-benzo[d]imidazol-6-yl)-4-hydroxybenzenesulfonamide
To a stirred solution of tert-butyl 2-cyclopentyl-6-((4-hydroxyphenyl) sulfonamido)-1 H-benzo[d]imidazole-1 - carboxylate (6) (0.06 g, 0.131 mmol) in dry DCM (5 mL) was added TFA (0.05 mL, 0.655 mmol). The reaction was stirred at RT for 3 h. The mixture was concentrated under reduced pressure to get the crude which was purified by preparative HPLC using 0.1 % TFA in acetonitrile. The product fractions were lyophilized to afford N-(2-cyclopentyl- 1 H-benzo[d]imidazol-6-yl)-4-hydroxybenzenesulfonamide as a TFA salt (35 mg, yield: 59%). 1H-NMR (400 MHz, DMSO-de): 6 10.51 (br s, 1 H), 10.33 (s, 1 H), 7.60-7.57 (m, 3H), 7.39 (d, 0 = 1.60 Hz, 1 H), 7.18 (dd, 0 = 2.00, 8.80 Hz, 1 H), 6.83 (d, 0 = 7.00 Hz, 2H), 3.47-3.41 (m, 1 H), 2.34-2.33 (m, 2H), 1.87-1.77 (m, 4H), 1.71-1.67 (m, 2H). LCMS: 358 (M+1 ).
Example 20: Preparation of Compound 32: N-(2-(((1 r,3r)-3-aminocyclobutyl)methyl)-1 H-benzo[dlimidazol-6-yl) benzenesulfonamide
Figure imgf000064_0001
Step 1: N-(4-amino-3-nitrophenyl)benzenesulfonamide (2)
To an ice-cooled solution of 2-nitrobenzene-1 , 4-diamine 1 (1 g, 6.53 mmol) in dry DCM (20 mL) was added triethylamine (1.1 mL, 7.84 mmol) and benzenesulfonylchloride (1.15 g, 6.53 mmol). The reaction mixture was stirred at RT for 16 h. The reaction mixture was diluted with water (50 mL) to obtain a precipitate which was filtered to get crude which was purified by reverse phase column to get the title compound (2) (1 g, yield = 52%). LCMS: 294 (M+1 ).
Step 2: N-(3, 4-diaminophenyl)benzenesulfonamide (3)
To a solution of N-(4-amino-3-nitrophenyl)benzenesulfonamide (1 g, 3.41 mmol) in dry ethyl acetate (20 mL) was added 10% Pd/C (100 mg) at RT. The mixture was stirred under hydrogen atmosphere (bladder) for 16 h. The reaction mixture was filtered through a celite bed and the filtrate was concentrated to get the crude which was purified by flash silica (230-400) column using Isolera to get 0.85 g of (3). LCMS: 264 (M+1 ). Step 3: tert-butyl((1r,3r)-3-(2-((2-amino-4-(phenylsulfonamido) phenyl) amino)-2-oxoethyl) cyclobutyl) carbamate (4) To a stirred solution of N-(3, 4-diaminophenyl) benzenesulfonamide (3) (0.1 g, 0.379 mmol) in dry DMF (3 mL) was added 2-(trans-3-((tert-Butoxycarbonyl) amino) cyclobutyl) acetic acid (96 mg, 0.418 mmol) at O °C. HATU (216 mg, 0.569 mmol) and DIPEA (0.13 mL, 0.759 mmol) were added and the mixture was stirred at RT for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was diluted with ice cooled water (10 mL) and extracted with ethyl acetate (2 x 10 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to get 0.05 g of crude tert-butyl ((1 r,3r)-3-(2-((2-amino-4- (phenylsulfonamido) phenyl) amino)-2-oxoethyl) cyclobutyl) carbamate (4). The crude was use as such for next step. LCMS: 475 (M+1 ).
Step 4: tert-butyl ((1r,3r)-3-((6-(phenylsulfonamido)-1H-benzo[cl]imiclazol-2-yl) methyl) cyclobutyl) carbamate (5) tert-butyl ((1 r,3r)-3-(2-((2-amino-4-(phenylsulfonamido) phenyl) amino)-2-oxoethyl) cyclobutyl) carbamate (4) (50 mg, 0.105 mmol) in glacial acetic acid (2 mL) was heated at 60 °C for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated under reduced pressure to get tert-butyl ((1 r,3r)-3- ((6-(phenylsulfonamido)-1 H-benzo[d]imidazol-2-yl) methyl) cyclobutyl) carbamate (5). (20 mg, yield = 42%). The crude product was taken to next step without any purification. LCMS: 457 (M+1 ).
Step 5: N-(2-(((1r,3r)-3-aminocyclobutyl) methyl)-1H-benzo[d]imidazol-6-yl) benzenesulfonamide
To a stirred solution of tert-butyl ((1 r,3r)-3-((6-(phenylsulfonamido)-1 H-benzo[d]imidazol-2-yl) methyl) cyclobutyl) carbamate (5) (0.02 g, 0.043 mmol) in dry DCM (5 mL) was added TFA (0.02 mL, 0.219 mmol). The reaction was stirred at RT for 3 h. The mixture was concentrated under reduced pressure to get the crude which was purified by preparative HPLC using 0.1 % TFA in acetonitrile. The product fraction was lyophilized to afford N-(2-cyclopentyl-1 H- benzo[d]imidazol-6-yl)-4-hydroxybenzenesulfonamide as a TFA salt (16 mg, yield = 80%). 1H-NMR (400 MHz, DMSO-de): d 10.37 (br s, 1 H), 7.96 (br s, 3H), 7.76-7.73 (m, 1 H), 7.62-7.49 (m, 4H), 7.34 (s, 1 H), 7.06-7.04 (m, 1 H), 3.79-3.77 (m, 3H), 3.12-3.10 (m, 3H), 2.88-2.79 (m, 1 H), 2.26-2.21 (m, 2H), 2.16-2.12 (m, 2H), 1.28-1.25 (m, 2H). LCMS: 357 (M+1 ).
Example 21 : Preparation of Compound 33: N-((1 r,3r)-3-((6-(phenylsulfonamido)-1 FI-benzo[d1imidazol-2-yl)methyl) cyclobutyl) acetamide Step 1: Synthesis of2-((1r,3r)-3-acetamidocyclobutyl)acetic acid (2):
To an ice-cooled solution of trans-(3-aminocyclobutyl)acetic acid hydrochloride (1) (0.05 g, 0.302 mmol) in dioxane/water (3 mL, 2: 1 ) was added NaHC03 (126 mg, 1.51 mmol). Acetic anhydride (0.06 mL, 0.604 mmol) was added and the mixture was stirred at RT for 16 h. The mixture was diluted with water (5 mL) and washed with EtOAc (2 x 5 mL). The aqueous layer was acidified using 1.5N HCI and extracted with 10% MeOH in DCM mixture (2 x 10 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to get 0.038 g of 2-((1 r,3r)-3-acetamidocyclobutyl)acetic acid (2). The crude was taken as such to the next step. 1H-NMR (400 MHz, DMSO-d6): d 8.12 (d, 0 = 7.60 Hz, 1 H), 4.26-4.20 (m, 1 H), 2.41 -2.38 (m, 2H), 2.03- 1.91 (m, 5H), 1.77 (s, 3H). LCMS: 172 (M+1 ).
Step 2: 2-((1r,3r)-3-acetamidocyclobutyl)-N-(2-amino-4-(phenylsulfonamido)phenyl) acetamide (4)
To a stirred solution of N-(3, 4-diaminophenyl) benzenesulfonamide (3) (0.058 g, 0.22 mmol) in dry DMF (3 mL) was added 2-((1 r,3r)-3-acetamidocyclobutyl)acetic acid (2) (38 mg, 0.22 mmol) at 0 °C. HATU (92 mg, 0.242 mmol) and DIPEA (0.08 mL, 0.44 mmol) were added and the mixture was stirred at RT for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was diluted with ice-cooled water (10 mL) and extracted with ethyl acetate (2 x 10 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to get 0.06 g of crude 2-((1 r,3r)-3-acetamidocyclobutyl)-N-(2-amino-4- (phenylsulfonamido)phenyl) acetamide (4). The crude was taken as such to the next step. LCMS: 417 (M+1 ).
Step 3: N-((1r,3r)-3-((6-(phenylsulfonamido)-1H-benzo[d]imidazol-2-yl)methyl)cyclobutyl) acetamide
2-((1 r,3r)-3-acetamidocyclobutyl)-N-(2-amino-4-(phenylsulfonamido)phenyl) acetamide (4) (60 mg, 0.144 mmol) in glacial acetic acid (2 mL) was heated at 60 °C for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated under reduced pressure. The crude was purified by prep. HPLC to get N- ((1 r,3r)-3-((6-(phenylsulfonamido)-1 H-benzo[d]imidazol-2-yl)methyl) cyclobutyl) acetamide as a TFA salt (24 mg, yield = 33%). 1H-NMR (400 MHz, DMSO-d6): d 10.55 (s, 1 H), 8.15 (d, 0 = 7.20 Hz, 1 H), 7.77-7.75 (m, 2H), 7.63-7.53 (m, 4H), 7.41 (s, 1 H), 7.18-7.12 (m, 1 H), 4.35-4.31 (m, 1 H), 3.18 (d, J = 8.00 Hz, 2H), 2.68-2.61 (m, 1 H), 2.08-2.05 (m, 4H), 1.77 (s, 3H). LCMS: 399 (M+1 ).
Example 22: Preparation of Compound 34 and 35: 4: 4-(6-(phenylsulfonamido)-1 H-indole-2-carbonyl) piperazin-1 - ium and N-(3-aminopropyl)-N-methyl-6-(phenylsulfonamido)-1 H-indole-2-carboxamide
Figure imgf000067_0001
Step 1: 1 -(tert-butyl) 2-methyl 6-(phenylsulfonamido)-1 H-indole-1 ,2-dicarboxylate (2)
To a stirred solution of 1 -(tert-butyl) 2-methyl 6-amino-1 H-indole-1 ,2-dicarboxylate (300 mg, 1.03 mmol) in dry DCM (10 mL) was added triethyl amine (0.27 mL, 1.06 mmol) at 0 °C. Benzene sulfonyl chloride (0.15 mL, 1.14 mmol) was added and the mixture was slowly warm to RT and stirred for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was quenched with ice-cooled water (10 mL) and extracted with MTBE (50 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The crude was purified by silica column chromatography (Isolera) to get 1 -(tert-butyl) 2-methyl 6- (phenylsulfonamido)-l H-indole-1 ,2-dicarboxylate (2) (400 mg crude, 60% purity). LCMS: 469 (M-1 ).
Step 2: 1-(tert-butoxycarbonyl)-6-(phenylsulfonamido)-1H-indole-2-carboxylic acid (3)
To the solution of l-(tert-butyl) 2-methyl 6-(phenylsulfonamido)-1 H-indole-1 ,2-dicarboxylate (2) (400 mg, 0.929 mmol) in THF / MeOH / water (10 mL, 4: 3: 3 ratio) was added LiOH. H2O (234 mg, 5.57 mmol) at RT. The reaction was stirred at RT for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was diluted with water (20 mL) then washed with EtOAc (2 x 10 mL). The aqueous layer was neutralized with citric acid and extracted with mixture of 10% MeOH in DCM (2 x 40 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated to get 1-(tert-butoxycarbonyl)-6-(phenylsulfonamido)-1 H-indole-2- carboxylic acid (3) (230 mg crude). LCMS: 317 (M+1 - Boo group).
Step 3: tert-butyl 4-(6-(phenylsulfonamido)-1 H-indole-2-carbonyl) piperazine-1 -carboxylate (4) To a stirred solution of 1 -(tert-butoxycarbonyl)-6-(phenylsulfonamido)-1 H-indole-2-carboxylic acid (3) (75 mg, 0.072 mmol) in dry DMF (3 mL) was added 1 -Boc piperazine (37 mg, 0.079 mmol) at 0 °C. HATU (103 mg, 0.108 mmol) and DIPEA (0.06 mL, 0.144 mmol) were added to the mixture and then stirred at RT for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was diluted with EtOAc (30 mL) and washed with water (10 mL), brine (10 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The crude was purified by prep HPLC to get tert-butyl 4-(6-(phenylsulfonamido)-1 H-indole- 2-carbonyl) piperazine-1-carboxylate (4). (40 mg, yield = 46%). 1H-NMR (400 MHz, CDCI3): d 9.15 (br s, 1 H), 7.76 (dd, J = 1.20, 8.40 Hz, 2H), 7.56-7.53 (m, 2H), 7.52-7.41 (m, 2H), 7.31-7.27 (m, 1 H), 6.79-6.72 (m, 1 H), 6.63 (s, 1 H), 3.90 (m, 4H), 3.58-3.52 (m, 4H), 1.50 (s, 9H). LCMS: 483 (M-1 ).
Step 4: 4-(6-(phenylsulfonamido)-1H-indole-2-carbonyl) piperazin-1-ium
To a stirred solution of tert-butyl 4-(6-(phenylsulfonamido)-1 H-indole-2-carbonyl) piperazine-1 -carboxylate (4) (0.04 g, 0.082 mmol) in dry DCM (2 mL) was added TFA (0.02 mL, 0.247 mmol). The reaction was stirred at RT for 3 h. The mixture was concentrated under reduced pressure to get residue which was lyophilized to afford 4-(6- (phenylsulfonamido)-1 H-indole-2-carbonyl) piperazin-1-ium as a TFA salt (20 mg, yield = 49%). 1H-NMR (400 MHz, DMSO-de): d 1 1.51 (s, 1 H), 10.19 (s, 1 H), 8.84 (br s, 2H), 7.75-7.73 (m, 2H), 7.58-7.44 (m, 3H), 7.45 (d, 0 = 8.40 Hz, 1 H), 7.25 (s, 1 H), 6.85-6.81 (m, 2H), 3.91 (br s, 4H), 3.20 (br s, 4H). LCMS: 385 (M+1 ).
Step 5: tert-butyl (3-(N-methyl-6-(phenylsulfonamido)-1H-indole-2-carboxamido) propyl) carbamate (4)
To a stirred solution of 1 -(tert-butoxycarbonyl)-6-(phenylsulfonamido)-1 H-indole-2-carboxylic acid (3) (75 mg, 0.072 mmol) in dry DMF (3 mL) was added tert-butyl (3-(methylamino) propyl) carbamate (45 mg, 0.079 mmol) at 0 °C. HATU (103 mg, 0.108 mmol) and DIPEA (0.06 mL, 0.144 mmol) were added and the mixture was stirred at RT for 16 h. The mixture was diluted with EtOAc (20 mL), washed with water (10 mL), brine (10 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The crude was purified by prep HPLC to get tert-butyl (3-(N-methyl-6-(phenylsulfonamido)-1 H-indole-2-carboxamido) propyl) carbamate (4). (20 mg, yield = 23%). 1H-NMR (400 MHz, CDCI3): d 9.51 (br s, 1 H), 7.77-7.75 (m, 2H), 7.54-7.51 (m, 2H), 7.44-7.40 (m, 2H), 7.24 (s, 1 H), 6.83-6.82 (m, 3H), 5.34 (br s, 1 H), 3.71 (m, 2H), 3.41 (m, 3H), 3.23-3.17 (m, 2H), 1.85 (m, 2H), 1.45 (s, 9 H). LCMS: 485 (M-1 ).
Step 6: N-( 3-aminopropyl) -N-methyl-6-(phenylsulfonamido) - 1 H-indole-2-carboxamide
To a stirred solution of tert-butyl (3-(N-methyl-6-(phenylsulfonamido)-1 H-indole-2-carboxamido) propyl) carbamate (4) (0.03 g, 0.062 mmol) in dry DCM (2 mL) was added TFA (0.02 mL, 0.184 mmol). The reaction was stirred at RT for 3 h. The mixture was concentrated under reduced pressure to get the residue which was lyophilized to afford N- (3-aminopropyl)-N-methyl-6-(phenylsulfonamido)-1 H-indole-2-carboxamide as a TFA salt (1 1 mg, yield = 52%). 1H- NMR (400 MHz, DMSO-de): d 1 1.43 (s, 1 H), 10.17 (s, 1 H), 7.74-7.72 (m, 5H), 7.60-7.44 (m, 4H), 7.26 (s, 1 H), 6.82 (dd, 0 = 2.00, 8.80 Hz, 1 H), 3.50 (s, 3H), 3.25 (br s, 2H), 2.84-2.80 (m, 2H), 1.87 (m, 2H). LCMS: 385 (M-1 ). Example 23: Preparation of Compound 36: N-((1 -carbamoylcvclopropyl)methyl)-N-methyl-6-(phenylsulfonamido)-
1 H-indole-2-carboxamide
Figure imgf000069_0001
Step 1: 1-(((tert-butoxycarbonyl)amino)methyl)cyclopropane-1 -carboxylic acid (2)
To a stirred solution of 1 -(aminomethyl)cyclopropane-1-carboxylic acid hydrochloride (1) (0.25 g, 1.649 mmol) in dioxane/water (5 mL, 4: 1 ) was added NaHC03 (0.866 g, 8.245 mmol). Boo anhydride (0.72 mL, 3.298 mmol) was added and the mixture was stirred at RT for 16 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was diluted with water (10 mL), washed with EtOAc (2 x 10 mL). The aqueous layer was neutralised with citric acid and extracted with 10% MeOH in DCM (2 x 25 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to get 1 -(((tert- butoxycarbonyl)amino)methyl)cyclopropane-1 -carboxylic acid (2) (0.4 g, yield = 99%). 1H-NMR (400 MHz, CDCI3): d 5.23 (br s, 1 H), 3.30 (d, 0 = 6.40 Hz, 2H), 1.47 (s, 9H), 1.35-1.28 (m, 2H), 1.07-1.06 (m, 2H). LCMS: 214 (M-1).
Step 2: methyl 1-(((tert-butoxycarbonyl)(methyl)amino)methyl)cyclopropane-1-carboxylate (3)
To the solution of 1-(((tert-butoxycarbonyl)amino)methyl)cyclopropane-1 -carboxylic acid (2) (0.21 g, 0.975 mmol) in DMF (5 mL) was added NaH (60% in mineral oil, 0.1 17 g, 2.92 mmol) at 0 °C and stirred for 20 min. Methyl iodide (0.15 mL, 2.43 mmol) was added and the mixture was slowly warm to RT and stirred for 3 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was quenched with ice-cooled water (10 mL) and extracted with EtOAc (2 x 10 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated to get methyl 1 -(((tert-butoxycarbonyl) (methyl)amino) methyl)cyclopropane-1-carboxylate (3) (0.14 g, yield = 59%).
Step 3: 1-(((tert-butoxycarbonyl)(methyl)amino)methyl)cyclopropane-1-carboxylic acid (4)
To the solution of 1 -(((tert-butoxycarbonyl) (methyl)amino) methyl)cyclopropane-1 -carboxylate (3) (0.14 g, 0.573 mmol) in THF/MeOH/water (5 mL, 3: 1 : 1 ) was added LiOH. H2O (0.055 g, 1.15 mmol) and stirred at RT for 4 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was diluted with water (10 mL) then washed with EtOAc (2 x 10 mL). The aqueous layer was neutralized with citric acid and extracted with EtOAc (2 x 10 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated to get 1 - (((tert-butoxycarbonyl)(methyl)amino)methyl)cyclopropane-1 -carboxylic acid (4) (0.05 g yield = 38%). 1H-NMR (400 MHz, CDCI3): d 3.60 (s, 2H), 2.94 (s, 3H), 1.48 (s, 9H), 1.40-1.37 (m, 2H), 1.05-1.02 (m, 2H). LCMS: 228 (M-1 ).
Step 4: tert-butyl ((1-carbamoylcyclopropyl)methyl)(methyl)carbamate (5)
To an ice-cooled solution of 1-(((tert-butoxycarbonyl)(methyl)amino)methyl)cyclopropane-1 -carboxylic acid (4) (0.05 g, 0.082 mmol) in dry DMF (2 mL) was added EDC.HCI (0.063 g, 0.322 mmol). Then HOBT (0.044 g, 0.322 mmol), NH4CI (0.058 g, 1.09 mmol) and triethylamine (0.45 mL, 3.27 mmol) were added and the mixture was stirred at RT for 16 h. The mixture was diluted with water (10 mL) and extracted with EtOAc (2 x 10 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated to get tert-butyl ((1- carbamoylcyclopropyl)methyl)(methyl)carbamate (5) (0.05 g yield = 99%). LCMS: 129 (M+1 , -Boc group cleaved).
Step 5: 1-((methylamino)methyl)cyclopropane-1 -carboxamide (6)
To a stirred solution of tert-butyl ((1 -carbamoylcyclopropyl)methyl)(methyl)carbamate (5) (0.05 g, 0.219 mmol) in dry DCM (2 mL) was added TFA (0.05 mL, 0.657 mmol) and stirred at RT for 16 h. The mixture was concentrated under reduced pressure to get 1-((methylamino)methyl)cyclopropane-1 -carboxamide (6). (0.03 g, crude). LCMS: 129 (M+1 ).
Step 6: tert-butyl 2-(((1-carbamoylcyclopropyl)methyl)(methyl)carbamoyl)-8-(phenylsulfonamido)-1H-indole-1- carboxylate (8)
To a stirred solution of 1 -((methylamino)methyl)cyclopropane-1 -carboxamide (6) (0.025 g, 0.195 mmol) in dry DMF (3 mL) was added 1 -(tert-butoxycarbonyl)-6-(phenylsulfonamido)-1 H-indole-2-carboxylic acid (7) (0.097 g, 0.234 mmol). HATU (0.13 g, 0.292 mmol) and DIPEA (0.08 mL, 0.390 mmol) were added and the mixture was stirred at RT for 16 h. The mixture was diluted with EtOAc (20 mL), washed with water (10 mL) and brine (10 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to get tert-butyl 2-(((1-carbamoylcyclopropyl)methyl)(methyl)carbamoyl)-6-(phenylsulfonamido)-1 H-indole-1-carboxylate (8) (0.03 mg, crude). Step 7: N-((1-carbamoylcyclopropyl)methyl)-N-methyl-6-(phenylsulfonamido)-1H-indole-2-carboxamide
To a stirred solution of tert-butyl 2-(((1 -carbamoylcyclopropyl)methyl)(methyl)carbamoyl)-6-(phenylsulfonamido)-
1 H-indole-1 -carboxylate (8) (0.03 g, 0.057 mmol) in dry DCM (2 mL) was added TFA (0.02 mL, 0.285 mmol) and stirred at RT for 3 h. The mixture was concentrated under reduced pressure to get the residue which was purified by prep. HPLC to get N-((1 -carbamoylcyclopropyl)methyl)-N-methyl-6-(phenylsulfonamido)-1 H-indole-2-carboxamide as a TFA salt (6 mg, yield = 25%). 1H-NMR (400 MHz, DMSO-d6): d 1 1.49 (s, 1 H), 10.14 (br s, 1 H), 7.74-7.72 (m, 2H), 7.62-7.41 (m, 4H), 7.24 (s, 1 H), 7.00 (br s, 1 H), 6.83-6.80 (m, 2H), 3.84 (s, 2H), 3.17 (s, 3H), 1.07-1.04 (m, 2H), 0.81 (m, 2H). LCMS: 427 (M+1 ).
Example 24 - Purification of recombinant GST-p38a protein
Human p38a with a N-terminal GST tag (64 kDa) was produced in BL21 (BL21-DE3 or BL21 -DE3-pLysS) E. Coli. Bacteria was grown in 250-1000 ml of LB medium containing ampicillin (50 pg/ml) at 37°C (25 °C with BL21-DE3- pLysS) until an O.D. of 0.5-0.8 and induced for 3 h at 18°C with IPTG (0.05 mM). After induction, cells were harvested by centrifugation (4000 rpm for 10 min at 4 °C), resuspended in 13.5 ml of cold PBS containing 1 mg/ml lysozyme, 5 mM EDTA, 1 complete EDTA-free protease inhibitor cocktail tablet (Roche, 1 1873580001 ), and sonicated. Then, Triton X-100 was added to a final concentration of 1 % (v/v) and cell debris was cleared by centrifugation (10000 rpm for 20 min at 4 °C). Glutathione-Sepharose™ 4 Fast Flow beads (GE Healthcare, GE17-5132-01) were washed in cold PBS buffer, resuspended in PBS (50:50), and 70 mI of the bead slurry were added per 1 ml of supernatant. After
2 h rotating at 4°C, samples were centrifuged (1000 rpm for 2 min at 4 °C) and the supernatant was removed. Beads were washed three times with 10 ml of cold PBS and once with 10 ml of cold 50 mM Tris-HCI pH 8.0, and then centrifuged to remove residual buffer. Beads were incubated with 1 ml of 10 mM glutathione (Sigma-Aldrich®, G4251) in 50 mM Tris-HCI pH 8.0 to elute the GST-p38a protein. After 2-3 min at RT, the beads were centrifuged (1000 rpm for 1 min at 4 °C), and the supernatant was collected. Protein elution was repeated three times. To remove glutathione, eluted proteins were put into a dialysis bag (12-14 MWCO, SpectralPor) and dialyzed against 2 1 of buffer containing 20 mM Tris pH 8.0, 50 mM NaCI, 0, 1 mM EDTA, 0.5 mM DTT and 5% glycerol, overnight at 4°C. Following dialysis, samples were collected, aliquoted and stored at -80°C. An aliquot was analyzed in 10% SDS-PAGE gel and stained with Coomassie blue to quantify the GST-p38a using BSA as a reference.
Example 25 - Dephosphorylation of p38a
Purified and dialyzed recombinant GST-p38a protein (5 g) was dephosphorylated with 1 pi of Lambda Phosphatase (BioLabs, P0753S) in 20 mI of PMP Buffer (50 mM HEPES 100 mM NaCI, 2 mM DTT and 0.01 % Brij 35) containing 1 mM MnCI2. After 2 h at 37°C, 2 mI of 100 mM Na3V04 were added to stop the reaction. Dephosphorylated GST- p38a protein was stored in aliquots at -80 °C. Purified GST-p38a protein from BL21-DE3-pLysS was not treated with Lambda Phosphatase. Example 26 - Phosphorylation of p38a by MKK6
Purified and dialyzed recombinant GST-p38a protein (4 pg) was incubated with purified MBP-MKK6 (1 pg) in 20 mI of buffer containing 50 mM Tris-HCI pH 7.5, 2 mM DTT, 10 mM MgCh, 100 pM NaaVC , 1 mM PMSF, 10 pg/ml aprotinin, 10 pg/ml leupeptin and 200 pM ATP for 30 min at 37 °C. Reactions were stopped by the addition of 5x sample loading buffer and boiling at 95 °C for 5 min. Phosphorylation of GST-p38a was detected by immunoblotting using phospho antibodies against the activation loop residues Thr180 and Tyr182 of p38a (BD Biosciences, 612288). Particularly preferred are compounds that show no inhibition of MKK6-induced p38a phosphorylation at 10 mM, more preferably 30 mM.
Example 27 - p38a kinase activity assay
Active GST-p38a (200 ng) was prepared by incubation with MKK6 as describe above, and then was incubated with purified GST-MK2 protein (500 ng) in 20 mI of buffer containing 50 mM Tris-HCI pH 7.5, 2 mM DTT, 10 mM MgCh, 100 pM Na3V04, 1 mM PMSF, 10 pg/ml aprotinin, 10 pg/ml leupeptin and 200 pM ATP for 30 min at 30 °C. Reactions were stopped by the addition of 5x sample loading buffer and boiling at 95 °C for 5 min. Phosphorylation of MK2 on Thr334 was detected by immunoblotting using phospho-specific antibodies (Cell Signalling, 3007S).
Particularly preferred are compounds that show no inhibition of p38a kinase activity on MK2 at 2 mM, more preferred are those that show no inhibition on MK2 at 10 mM or even 30 mM.
Example 28 - p38a autophosphorylation assay
Many protein kinases, including p38a, have the capacity to autophosphorylate spontaneously, as part of the protein dynamics. In some cases, the autophosphorylation can be stimulated allosterically by the binding to certain proteins (such as TAB1 for p38a). It seems very likely that the underlying molecular mechanism is the same in both cases. Dephosphorylated GST-p38a protein (2 pg) was incubated with 600 pM ATP in 20 mI of buffer containing 100 mM NaCI, 20 mM T ris-HCI pH 7.5, 2 mM DTT, 2 mM MgCh for 2 h at 37°C. Reactions were stopped by the addition of 5x sample loading buffer and boiling at 95 °C for 5 min. Autophosphorylation was detected by immunoblotting using an antibody that recognizes phosphorylated Thr180 and Tyr182 in the activation loop of p38a (BD Biosciences, 612288).
Particularly preferred are compounds that show inhibition of p38a autophosphorylation of at least 40% at 5 mM and/or at least 60 % at 10 mM and/or at least 90 % at 30 mM.
Example 29 - TAB1-induced autophosphorylation of p38a
A peptide corresponding to amino acids 386-414 of TAB1 [RVYPVSVPYSSAQSTSKTSVTLSLVMPSQ] (GenScript) was resuspended in 50 mM Tris-base adjusted to pH 7.5, at a concentration of 100-300 mM and stored in aliquots at -80°C. Dephosphorylated GST-p38a protein (2 pg) was incubated with 15 pM TAB1 peptide in 20 mI of buffer containing 100 mM NaCI, 20 mM Tris-HCI pH 7.5, 2 mM DTT, 2 mM MgCh and 600 pM ATP for 2 h at 37°C. Reactions were stopped by the addition of 5x sample loading buffer and boiling at 95 °C for 5 min. Autophosphorylation was detected by immunoblotting using an antibody that recognizes phosphorylated Thr180 and Tyr182 in the activation loop of p38a (BD Biosciences, 612288).
Particularly preferred are compounds that show inhibition of TAB1 -induced p38a phosphorylation of at least 20% at 5 mM and/or at least 60 % at 10 mM and/or at least 90 % at 30 mM.
Example 30 - Analysis of the effect of chemical compounds on p38a autophosphorylation
Compounds were resuspended in DMSO to prepare 10 mM stocks and stored at -80°C. As controls, reactions were performed in the presence of the ATP-competitor PH797804 (2 mM) or DMSO. The results with the assays described in Examples 26 to 29 were as follows:
Figure imgf000073_0001
Figure imgf000074_0001
* These compounds were analysed using GST-p38a not treated with Lambda Phosphatase. Example 31 - Permeability in Caco-2 cells in vitro
Caco-2 cells were grown as monolayers for 21 days in 96-well plates and used as a model to study transport across the intestinal barrier. Compounds were assayed at 10 mM (in triplicates) in HBSS-Hepes buffer, pH 7.4 and added to the donor side, while the receiver side was filled only with HBSS-Hepes buffer, pH 7.4. Teer and Lucifer yellow Assay were used as markers of Integrity of the cell monolayer. After incubation at 37 °C for 2 h, media was removed from the receiver side and samples were analyzed by HPLC-MS to calculate, the Papp values. Metoprolol was used as reference compound. In the table below, the apparent permeability (Papp) values for the indicated compounds in Caco-2 cells is shown.
Figure imgf000075_0001
According to the results, the compounds of the invention showed high permeability in Caco-2 cells.
Example 32 - Stability in human liver microsomes in vitro
Compounds at 10 mM in pH 7.4 buffer were incubated with human liver microsomes (0.4 mg protein/mL) for 0 and 1 h at 37 °C in triplicates. The degree of hepatic metabolism was measured by LC-MS/MS as the decrease in the peak area of the parent compound. Terfenadine and Imipramine were assayed as high and low metabolized reference compounds. In the table below, the percentage of the indicated compounds remaining after incubation with human microsomes is shown.
Figure imgf000075_0002
According to the results, the compounds of the invention 10, 11 and 15 showed high stability in human liver microsomes in vitro.
Example 33 - Human hERG inhibition in vitro
CHO-hERG DUO cells were cultured in Ham's Nutrient Mixture F-12 (Lonza, BE12-615F), supplemented with 10% Fetal Bovine Serum (Sigma, F7524), 5% Penicillin/Streptomycin (Lonza, DE17-602E), G418 (Sigma, G8168; 1 mL of 50mg/mL stock solution) and Hygromycine B (Invivogen, ant-hm-5; 0.5 mL of 100 mg/mL stock solution). 2x106 CHO-hERG DUO cells were seeded 48 h before the experiment onto a T225 flask. Just before the experiment, cells were washed twice with DPBS (Euroclone, ECB4004L) and detached from the flask with trypsin-EDTA (Sigma, T4174; diluted 1/10). Cells were then re-suspended in 25 mL EX-CELL ACF CHO medium (Sigma,
C5467); 0.25 mL of 100x Penicillin/Streptomycin (Euroclone, ECB3001 D), 0.1 mL of Soybean Trypsin Inhibitor 10 mg/mL (Sigma, T6522) and placed on the GPatch 16X. Compounds were prepared at 10 mM in 100% DMSO, and were tested at five concentrations: 10 mM, 2 pM, 10 pM, 0.4 pM and 0.08 pM. Dilutions were prepared from stocks just before the experiments in the extracellular solution (0.1 % final DMSO concentration), and the successive dilutions were performed directly in the extracellular solution. Verapamil (Sigma, V4629; stock 100 mM in DMSO) was used as a reference agonist.
The following solutions were used for Patch clamp. Intracellular solution: 120 mM KCI, 5.37 mM CaCI2, 1.75 mM MgCI2, 10 mM EGTA, 10 mM HEPES, 4 mM Na2- ATP (pH 7.2 with KOH). Extracellular solution: 145 mM NaCI, 4 mM KCI, 1 mM MgCI2, 2 mM CaCI2, 10 mM HEPES, 10 mM Glucose (pH 7.4 with NaOH).
For the voltage clamp experiments on hERG, data were sampled at 5 KHz. After establishment of the seal and the passage in the whole cell configuration, the cells were held at -90 mV and the hERG current was performed by applying 200 ms step at -50 mV; 2000 ms step at +20 mV; 2000 ms step at -50 mV; and back to -90 mV; every 15 in the absence (vehicle period, i.e. 0.1 % DMSO) and in the presence of increasing concentrations of the compounds. The table below shows the percentage of hERG inhibition by the indicated compounds in duplicates at 10 pM (left), and IC50 values higher than 50% inhibition (right).
Figure imgf000076_0001
*N.D. Not determined as the % of the highest concentration tested was less than 50% hERG inhibition
According to the results, the compounds of the invention 1 , 10, 11 , 12, 15 and 23 showed no inhibition of human hERG potassium channel. This means that the compounds of the invention are less likely to be cardiotoxic.
Example 34 - NanoBRET target engagement assay
HEK293 cells were grown in DMEM (Life Technologies, 11995) supplemented with 10 % Fetal Bovine Serum (HyClone, SH30070.03) (growth medium) and detached from the plate with 0.05 % Trypsin/EDTA (Life
Technologies, 25300). The cell suspension was diluted in growth media and centrifuged at 200xg for 5 min. Cell pellet was resuspended, counted and adjusted to 2x105 cells/ml in assay medium [Opti-MEM (Life Technologies 11058) supplemented with 1 % FBS (HyClone, SH30070.03)]. In parallel, cell transfection reagents were prepared as follows:
1. 10 pg/mL solution of DNA in Opti-MEM without serum, containing 9.0 pg/mL of Transfection Carrier DNA,
1.0 pg/mL of NanoLuc® fusion p38a DNA 1 mL of Opti-MEM without phenol red.
2. 30 pi of FuGENE FID (Promega, E2311) added into each mL of DNA mixture to form lipid:DNA complex, mixed by inversion 5-10 times and incubated at room temperature for 20 min to allow the formation of lipid:DNA complexes.
To transfect cells, 1 ml lipid:DNA complex was added to 20 ml HEK293T cells at 2x105 cells/ml and mixed by inversion. 100 pl/well was seeded into 96-well plates (Corning, 3917) and cultured for 20 h to allow NanoLuc® fusion p38a expression.
Then, 1X Tracer for BRET assay was prepared as follows:
1. First, 100X NanoBRET Tracer (in 100% DMSO) added to Tracer Dilution Buffer (ratio 1 :4) to generate Complete 20X NanoBRET Tracer Dilution Buffer.
2. 5 pL of Complete 20x NanoBRET Tracer Dilution Buffer added per well to cells in suspension.
3. Dilutions mixed on an orbital shaker for 15 sec at 700 rpm.
compounds were prepared by serially dilution inhibitor at 1000X final concentration in 100% DMSO and then diluted to 10X final concentration in Opti-MEM. 10pL per well of 10X compounds were added to the cells with 1X tracer. Plates were mixed on orbital shaker for 15 sec at 700 rpm and further incubated at 37°C 5% C02 for 2 h. Finally, plates were incubated for 15 min at room temperature to cool down before performing BRET
measurements.
Immediately prior to BRET measurements, 3X Complete NanoBRET™ Nano-Glo® Substrate was prepared in OptiMEM without serum or phenol red (1 : 166 dilution of NanoBRET™ Nano-Glo® Substrate plus a 1 :500 dilution of Extracellular NanoLuc Inhibitor in OptiMEM without serum or phenol red) and mixed gently by inversion 5-10 times in a conical tube. 50pL were added per well of 3X Complete NanoBRET™ Nano-Glo® Substrate for a 96-well plate and incubated 2-3 min at room temperature. Measurements were done within 10 min of substrate activation. Following addition of NanoBRET™ Nano-Glo® Substrate, donor emission (e.g. 450 nm) and acceptor emission (e.g. 610 nm or 630 nm) were measured using a NanoBRET™-compatible luminometer. To generate raw BRET ratio values, acceptor emission values (e.g. 610 nm) were divided by the donor emission value (e.g. 450 nm) for each sample. Then, raw BRET units were converted to % inhibition for better interpretation of the results.
FIG. 1 shows IC50 curves for VX-702 (ATP-competitive inhibitor) and the compounds 1 and 10. The lower table shows the NanoBRET inhibition (%) for the indicated compounds at 50 mM, 15 pM and 5 pM (in duplicates).
Figure imgf000077_0001
According to the results, the compounds of the invention 3, 5, 10 and 15 were able to interact with p38a in cells. Example 35 - Inhibition of ischemia-reperfusion induced cell death
H9c2 cells were cultured in high-glucose DMEM medium (Sigma, D5796), supplemented with 10% Fetal Bovine Serum (LifeTechnologies, 10500064), 1 % Penicillin/Streptomycin (LabClinics, L0022-100, 5 mL of 100x Solution) and 1 % L-glutamine (LabClinics, X0550-100)] in 10 cm plates at 37°C with 5% C02 until they reach 80-90% confluence. Then, cells were washed with 1X DPBS (Sigma, D1408), detached from the plates with trypsin-EDTA (Sigma, T3924) and counted with an automated cell counter (Bio-Rad T20). 250,000 cells in 3 ml of medium were seeded in 60 mm plates and grown for 24 h.
Compounds were prepared as stocks at 10 mM in 100% DMSO (Sigma, D5796), and were added to cells at 30 mM 24h before the experiment. SB203580 (ATP-competitor) at 10 pM was used as a control.
For simulated ischemia, the medium was changed to 3 ml of Ischemic Buffer (137 mM NaCI, 12 mM KCI, 0.5 mM MgCh, 0.9 mM CaCh, 4 mM HEPES, 10 mM 2-deoxy-glucose, and 20 mM sodium lactate (pH 6.2) containing either DMSO, SB203580 or the compounds. Cells were kept in H35-hypoxistation chamber flushed with 0.1 % O2 5% CO2 and 95 % N2 (65% HR) for 2 h. For simulated reperfusion, cells were switched back to the original medium and kept for 4 h in a normal incubator with 5%C02. Non-treated cells were maintained in a normal incubator with 5%C02 (normoxia).
After the treatment, media was collected and the cells were washed with 1X DPBS and the supernatant was also collected with the media. Cells were detached by treating with Trypsin-EDTA (Sigma, T3924) for 5 min at 37°C.
The cell suspension was centrifuged 5 min at 1500 rpm and the cell pellet was lysed in RIPA buffer (150 mM NaCI, 50 mM Tris-HCI pH 7.3, 1 % NP-40, 5mM EDTA, 20 mM NaF, 1 mM Na3V04, 1 mM PMSF, 1 mM DTT, 2.5 mM benzamide, 10 pg/ml pepstatin, 10 pg/ml aprotinin, 10 pg/ml leupeptin and 1 pM microcystin) for 30 min on ice. Then, samples were centrifuged 15 min at 13200 rpm at 4°C, and protein concentration in the supernatant was quantified with BioRad DC™ Protein Assay. Protein lysates were analyzed by immunoblotting using caspase-3 antibody (Cell signaling, 9662) 1 :1000 in TBS-Tween 0.1 % with 5% BSA. The band corresponding to cleaved caspase-3 (17-19 kDa) was quantified using the LI-COR system.
FIG 2 shows cleaved caspase-3 levels as a measure of cell death in H9c2 cardiomyocytes pre-treated with the indicated compounds and subjected to simulated ischemia-reperfusion. Values were normalized to DMSO treated cells. Each dot represents an independent experiment. According to these results the compounds of the invention 10, 11 , 12 and 15 were able to reduce simulated ischemia-reperfusion induced cell death in H9c2 cells.
Example 36 - Toxicity in cardiomyocytes and fibroblasts
Neonatal rat ventricular myocytes (NRVMs) were isolated from newborn (1-2 days) Wistar rats. Hearts (11-15) were collected in ice-cold CBFHH buffer (137 mM NaCI, 20 mM Hepes, 0.81 mM Mg2S04, 0.44 mM K2PO4, 0.33 mM Na2HP04, 5.6 mM glucose, 5.4 mM KCI, pH 7.3) and atria, blood vessels and tissues from other organs were existed. Minced ventricles were put into a 100 ml Erlenmeyer flask and NRVMs were purified by serial enzymatic digestions of tissue with 0.6 mg/ml of collagenase Type II (Worthington, LS004174) and 0.4 mg/ml pancreatin (Sigma, P3292) in CBFHH buffer shaking at 37°C. First digestion (20 min) was discarded. After the second (20 min), third (25 min), fourth (25 min) and fifth (15 min) digestions, the cell suspension was placed into 4 ml FBS (Pan Biotech, P30-3302) at 37°C. After all digestions, cells were centrifuged at 900 rpm for 5 min and the pellet was resuspended in 10 ml of high-glucose DMEM (Sigma, D5796): M199 (Gibco, 31150022) (4:1) supplemented with 10% Horse Serum (Gibco, 26050088), 5% FBS (Pan Biotech, P30-3302) and 1 pg/ml AraC (Sigma, C1768). The suspension was passed through a 40 pm nylon filter and pre-plated for 90 min on a 10 cm plate at 37°C, to allowing the attachment of fibroblasts.
The remaining cell suspension was collected and cardiomyocytes were counted with an automated cell counter (Bio-Rad T20), and cell survival being determined by Trypan Blue (Gibco, 15250061) exclusion. 25.000
NRVMs/well in a final volume of 100 pi were seeded onto 1 % gelatin-coated 96-well plates. After 24 h, media was changed and cells were kept at 37°C and 5% CO2 for 3 days, when a confluent monolayer of spontaneously beating myocytes was observed. At this point, compounds were added to the cells.
After pre-plating, fibroblasts were maintained with high-glucose DMEM medium (Sigma, D5796), supplemented with 10% Fetal Bovine Serum (LifeT echnologies, 10500064), 1 % Penicillin/Streptomycin (LabClinics, L0022-100, 5 mL of 100x Solution) and 1 % L-glutamine (LabClinics, X0550-100). After 48 h, cells were detached with Trypsin- EDTA (Sigma, T3924) and split 1 :2 for 3 days allowing them to proliferate. Then, 7.500 cells/well in a final volume of 100 pi were seeded onto 96-well plates and the next day compounds were added.
Stocks of the compounds 1 , 10 and 15 were prepared at 10 mM in 100% DMSO. Compounds were diluted serially in 100% DMSO to obtain solutions at 100X of the final concentrations used for cells. These solutions were diluted 1 :100 in 100 pi of media to obtain final concentrations of 100 pM, 30 pM, 10 pM, 5 pM and 1 pM, which all contain 1 % final DMSO concentration, and 100 pi were added per well.
Cell viability was determined after 48 h and 72 h using an MTT kit (Roche, 11465007001), by adding first 10 pi of reagent A to every well and 4 h later 100 pi of reagent B. Next day, absorbance was read at 570 nm using an Epoch 2 plate reader. Absorbance was proportional to the viable number of cells and values were normalized to the DMSO control.
According to the results, the compounds of the invention 1 , 10 and 15 showed no toxicity on cardiomyocytes or fibroblasts, neither at 48 h nor at 72 h.
References
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For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:
Clause 1. A compound of formula (I) for use in the prophylaxis or treatment of a disease associated with autophosphorylation of p38a kinase:
Figure imgf000081_0001
wherein
R1 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms selected from the group consisting of C, N, O, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C^alkyl, C^alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and— [NHC(0)]zR9, wherein z is 0 or 1 , and wherein R9 is aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2;
or R1 is an aliphatic acyclic group with 4 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl,
Figure imgf000082_0001
R3 is -R7-R8, wherein R7 is -(CH2)X-, with x being 0 to 3, and R8 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH2;
R6 is hydrogen, an alkyl group with 1 to 3 carbon atoms, or an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH2;
wherein R4, R5, and R have one of the following meanings:
(i) R4 is N; R5 is CH; and R is CH;
(ii) R4 is N; R5 is N; and R is CH;
(iii) R4 is N; R5 is N; and R is N;
(iv) R4 is CH; R5 is N; and R is N;
(v) R4 is CH; R5 is CH; and R is N;
(vi) R4 is CH; R5 is N; and R is CH; or
(vii) R4 is CH; R5 is CH; and R is CH;
a tautomer thereof, or a pharmaceutically acceptable salt thereof.
Clause 2. The compound according to clause 1 , wherein R4, R5, and R have one of the following meanings:
(i) R4 is N; R5 is CH; and R is CH; or
(ii) R4 is N; R5 is N; and R is CH.
Clause 3. The compound according to clause 1 or clause 2, wherein R1 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 4 to 6 ring atoms selected from the group consisting of C and N, wherein the ring has no or one N as ring atom, and which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)]zR9, wherein z is 0 or 1 , and wherein R9 is aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2. O
Figure imgf000083_0001
Clause 4. The compound according to one or more of the preceding clauses, wherein R2 is °
Clause 5. The compound according to one or more of the preceding clauses, wherein R3 is -R7-R8, wherein R7 is -(CH2)X-, with x being 0 or 1 , and R8 is an aromatic or heteroaromatic ring or ring system having 5 to 6 ring atoms selected from the group consisting of C, N, and 0, wherein the ring or ring system has no or one atom other than C as ring atom, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, and -NHC alkyl.
Clause 6. The compound according to one or more of the preceding clauses, wherein R3 is phenyl, optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
Clause 7. The compound according to one or more of the preceding clauses, wherein said compound is selected from the group consisting of compounds 1 to 29, a tautomer thereof, or a pharmaceutically acceptable salt thereof.
Clause 8. The compound according to any one of clauses 1 to 7 in combination with one or more pharmaceutically acceptable carriers or excipients.
Clause 9. The compound according to any one of clauses 1 to 8, wherein said disease associated with autophosphorylation of p38a kinase is selected from the group consisting of cardiovascular diseases such as myocardial ischemia-reperfusion injury, cardiac amyloidosis, or atherosclerosis/hypercoagulability; inflammatory diseases such as skin inflammation or toxoplasmosis; t-cell senescence; cancer such as pancreatic cancer; and inflammatory autoimmune diseases.
Clause 10. A compound of formula (la):
Figure imgf000083_0002
wherein
R1 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, and -C(0)NH2; or R1 is an aliphatic acyclic group with 4 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)]zR9, wherein z is 0 or 1 , and wherein R9 is aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2;
or R1 is an aliphatic acyclic group with 4 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3I and -C(0)NH2;
Figure imgf000084_0001
R3 is -R7-R8, wherein R7 is -(CH2)X-, with x being 0 to 3, and R8 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, and 0, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, and -NHC alkyl;
R6 is hydrogen, an alkyl group with 1 to 3 carbon atoms, or an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH2;
wherein R4, R5, and R have one of the following meanings:
(i) R4 is N; R5 is CH; and R is CH;
(ii) R4 is N; R5 is N; and R is CH;
(iii) R4 is N; R5 is N; and R is N;
(iv) R4 is CH; R5 is N; and R is N;
(v) R4 is CH; R5 is CH; and R is N;
(vi) R4 is CH; R5 is N; and R is CH; or
(vii) R4 is CH; R5 is CH; and R is CH; a tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (la) is not:
Figure imgf000084_0002
Clause 1 1. The compound according to clause 10, wherein R4, R5, and R have one of the following meanings: (i) R4 is N; R5 is CH; and R is CH; or
(ii) R4 is N; R5 is N; and R is CH.
Clause 12. The compound according to clause 10 or clause 1 1 , wherein R1 is an aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C and N, wherein the ring or ring system has no or one N as ring atom, and which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, Ci_ alkoxy, amino, halogen, oxo, -NHC alkyl, -C(0)CH3, -C(0)NH2, and -[NHC(0)]zR9, wherein z is 0 or 1 , and wherein R9 is aliphatic cyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2.
O
Figure imgf000085_0001
Clause 13. The compound according to one or more of clauses 10 to 12, wherein R2 is ° , and
R3 is -R7-R8, wherein R7 is -(CH2)X-, with x being 0 or 1 , and R8 is an aromatic or heteroaromatic ring or ring system having 5 to 6 ring atoms selected from the group consisting of C, N, O, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH2.
Clause 14. The compound according to one or more of clauses 10 to 13, wherein R3 is phenyl, optionally substituted with one or more substituents selected from the group consisting of halogen and methyl.
Clause 15. The compound according to one or more of clauses 10 to 14, wherein said compound is selected from the group consisting of compounds 1 to 8, and 19 to 29, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

Claims

Claims
1. A compound A for use in the prophylaxis or treatment of a disease selected from the group consisting of cardiovascular diseases, autoimmune diseases, inflammatory diseases, neurodegenerative diseases and cancer, wherein the compound A is either a compound of formula (I):
Figure imgf000086_0001
wherein
R1 is an aliphatic carbocyclic ring or ring system having 4 to 10 ring atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and - [NHC(0)]zR9; or alternatively
R1 is an aliphatic heterocyclic or heteroaromatic monocyclic ring having 6 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C^alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is -CONR10R11, wherein
R10 is H or C alkyl; and R11 is Ci-6alkyl optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, Ci-4alkoxy, Ci-4alkylthio, amino, halogen, oxo, -NHCi-4alkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; and optionally two hydrogen atoms attached to one carbon atom of the Ci-6alkyl are replaced by two CH2 moieties which form together a carbocyclic ring having 3 to 6 ring atoms; or alternatively
R11 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC^alkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R10 and R11 form together a heterocyclic ring or ring system having 4 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCi-4alkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; or alternatively
R1 is -Y-R12, wherein
Y is selected from the group consisting of NR13, 0, S, -C(O)-;
R12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH¾ -C(0)NH¾ -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; and
R13 is H or Ci alkyl; or alternatively
R1 is -CH2-R14, wherein
R14 is an aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms , wherein the atoms of the heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; or alternatively
R14 is an aliphatic heterocyclic ring having 5 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, - C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; R3 is -R7-R8;
R7 is -(CH2)X-, with x being 0 to 3;
R8 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, -NHC alkyl and -C(0)NH2; z is O or 1;
R9 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2;
R15 is H or C alkyl;
R16 is R9 or alternatively is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2; wherein R4, R5, and R have one of the following meanings:
(i) R4 is N; R5 is CH; and R is CH;
(ii) R4 is N; R5 is N; and R is CH;
(iii) R4 is N; R5 is N; and R is N;
(iv) R4 is CH; R5 is N; and R is N;
(v) R4 is CH; R5 is CH; and R is N;
(vi) R4 is CH; R5 is N; and R is CH; or
(vii) R4 is CH; R5 is CH; and R is CH; an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof; or alternatively the compound A is one of the following:
Figure imgf000089_0001
or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof.
2. The compound A for use according to claim 1 , wherein the disease is associated with autophosphorylation of p38a kinase.
3. The compound A for use according to any of claims 1-2, wherein in the compound of formula (I), R4, R5, and R have one of the following meanings:
(i) R4 is N; R5 is CH; and R is CH;
(ii) R4 is N; R5 is N; and R is CH; or
(vii) R4 is CH; R5 is CH; and R is CH.
4. The compound A for use according to any of claims 1-3, wherein in the compound of formula (I), R1 is selected from the group consisting of an optionally substituted aliphatic carbocyclic ring or ring system, an optionally substituted aliphatic heterocyclic monocyclic ring, and an optionally substituted heteroaromatic monocyclic ring.
5. The compound A for use according to claim 4, wherein R1 is an aliphatic carbocyclic ring having 4 to 6 ring atoms.
6. The compound A for use according to any of claims 1 -3, wherein in the compound of formula (I), R1 is -CONR10R11; wherein R10 is H or C alkyl and and R11 is an optionally substituted Ci-6alkyl, wherein optionally two hydrogen atoms attached to one carbon atom of the Ci-6alkyl are replaced by two Chh moieties which form together a carbocyclic ring having 3 to 6 ring atoms, or alternatively R10 and R11 form together an optionally substituted heterocyclic ring or ring system.
7. The compound A for use according to claim 6, wherein in the compound of formula (I), R4, R5, and R have the following meaning:
(vii) R4 is CH; R5 is CH; and R is CH.
8. The compound A for use according to any of claims 1-3, wherein in the compound of formula (I), R1 is -Y-R12, and Y is selected from the group consisting of NR13, 0, and -C(O)-.
9. The compound A for use according to claim 6, wherein R12 is an optionally substituted aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 6 ring atoms.
10. The compound A for use according to any of claims 1 -3, wherein in the compound of formula (I), R1 is -CH2-R14, wherein R14 is an optionally substituted aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having
4 to 6 ring atoms, or alternatively R14 is an optionally substituted aliphatic heterocyclic ring.
11. The compound A for use according to any of claims 1-10, wherein in the compound of formula (I), R3 is— R7-R8, wherein R7 is -(CH2)X-, with x being 0 or 1 , and R8 is an aromatic or heteroaromatic ring or ring system having 5 to 6 ring atoms.
12. The compound A for use according to claim 1, wherein said compound is selected from the group consisting of compounds 1 to 38:
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof.
13. The compound A for use according to any of claims 1-12, wherein said disease is selected from the group consisting of myocardial ischemia-reperfusion injury, cardiac amyloidosis, or atherosclerosis/hypercoagulability; skin inflammation or toxoplasmosis; t-cell senescence; pancreatic cancer; and inflammatory autoimmune diseases.
14. A compound B which is either a compound of formula (la): wherein
R1 is an aliphatic carbocyclic ring or ring system having 5 to 10 ring atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is an aliphatic heterocyclic or heteroaromatic monocyclic ring having 6 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH¾ -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R1 is -CONR10R11, wherein
R10 is H or C alkyl; and R11 is C^alkyl optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, Ci-4alkoxy, Ci-4alkylthio, amino, halogen, oxo, -NHCi-4alkyl, -NR15R16, -C(0)CH3, -C(0)NH¾ -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; and optionally two hydrogen atoms attached to one carbon atom of the C^alkyl are replaced by two CH2 moieties which form together a carbocyclic ring having 3 to 6 ring atoms; or alternatively
R11 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH¾ -C(0)NR15R16, -NR15C(0)R16, and -[NHC(0)]zR9; or alternatively
R10 and R11 form together a heterocyclic ring or ring system having 4 to 10 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCi-4alkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; or alternatively
R1 is -Y-R12, wherein
Y is selected from the group consisting of NR13, 0, S, -C(O)-;
R12 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms, wherein the atoms of the aliphatic heterocyclic or heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHCualkyl, -NR15R16, -C(0)CH3, -C(0)NH2, -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; and
R13 is H or Ci alkyl; or alternatively
R1 is -CH2-R14, wherein
R14 is an aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 10 ring atoms , wherein the atoms of the heteroaromatic ring or ring system are selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, -C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9; or alternatively
R14 is an aliphatic heterocyclic ring having 5 ring atoms selected from the group consisting of C, N, 0, and S, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C haloalkyl, C alkoxy, C alkylthio, amino, halogen, oxo, -NHC alkyl, -NR15R16, - C(0)CH3I -C(0)NH2I -C(0)NR15R16, -NR15C(0)R16 and -[NHC(0)]zR9;
Figure imgf000094_0001
R3 is -R7-R8;
R7 is -(CH2)X-, with x being 0 to 3; R8 is an aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 5 to 10 ring atoms selected from the group consisting of C, N, and 0, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, halogen, oxo, and -NHC alkyl; z is 0 or 1;
R9 is aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring having 6 ring atoms selected from the group consisting of C and N, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2;
R15 is H or C alkyl;
R16 is R9 or alternatively is an aliphatic acyclic group with 5 to 10 carbon atoms, which is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, C alkyl, C alkoxy, amino, halogen, oxo, -NHC alkyl, and -C(0)NH2; wherein R4, R5, and R have one of the following meanings:
(i) R4 is N; R5 is CH; and R is CH;
(ii) R4 is N; R5 is N; and R is CH;
(iii) R4 is N; R5 is N; and R is N;
(iv) R4 is CH; R5 is N; and R is N;
(v) R4 is CH; R5 is CH; and R is N;
(vi) R4 is CH; R5 is N; and R is CH; or
(vii) R4 is CH; R5 is CH; and R is CH; an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof, wherein the compound of formula (la) is other than:
Figure imgf000095_0001
Figure imgf000096_0002
or alternatively compound B is a compound which is one of the following:
Figure imgf000096_0001
or an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof.
15. The compound B according to claim 14, wherein in the compound of formula (la), R4, R5, and R have one of the following meanings:
(i) R4 is N; R5 is CH; and R is CH;
(ii) R4 is N; R5 is N; and R is CH; or (vii) R4 is CH; R5 is CH; and R is CH.
16. The compound B according to any of claims 14-15, wherein in the compound of formula (la), R1 is is selected from the group consisting of an optionally substituted aliphatic carbocyclic ring or ring system, an optionally substituted aliphatic heterocyclic monocyclic ring, and an optionally substituted heteroaromatic monocyclic ring.
17. The compound B according to claim 16, wherein R1 is an aliphatic carbocyclic ring having 5 to 6 ring atoms.
18. The compound B according to any of claims 14-15, wherein in the compound of formula (la), R1 is -CONR10R11; wherein R10 is H or C alkyl and and R11 is an optionally substituted Ci-6alkyl, wherein optionally two hydrogen atoms attached to one carbon atom of the Ci-6alkyl are replaced by two Chh moieties which form together a carbocyclic ring having 3 to 6 ring atoms, or alternatively R10 and R11 form together an optionally substituted heterocyclic ring or ring system.
19. The compound B according to claim 18, wherein in the compound of formula (la), R4, R5, and R have the following meaning:
(vii) R4 is CH; R5 is CH; and R is CH.
20. The compound B according to any of claims 14-15, wherein in the compound of formula (la), R1 is -Y-R12, and Y is selected from the group consisting of NR13, 0, and -C(O)-.
21. The compound B according to claim 20, wherein R12 is an optionally substituted aliphatic carbocyclic, aliphatic heterocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 6 ring atoms.
22. The compound B according to any of claims 14-15, wherein in the compound of formula (la), R1 is -CH2-R14, wherein R14 is an optionally substituted aliphatic carbocyclic, aromatic, or heteroaromatic ring or ring system having 4 to 6 ring atoms, or alternatively R14 is an optionally substituted aliphatic heterocyclic ring.
23. The compound B according to any of claims 14-22, wherein in the compound of formula (la), R3 is— R7-R8, wherein R7 is— (CH2)X-, with x being 0 or 1 , and R8 is an optionally substituted aromatic or heteroaromatic ring or ring system having 5 to 6 ring atoms.
24. The compound B according to claim 14, wherein said compound is selected from the group consisting of:
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
an isomer thereof, or a pharmaceutically or veterinary acceptable salt thereof.
25. A pharmaceutical or veterinary composition comprising a therapeutically effective amount of a compound A or a compound B and one or more pharmaceutically or veterinary acceptable carriers or excipients.
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