Protein kinase inhibitors for promoting liver regeneration or inhibiting or preventing liver cell death - Patents.com

Abstract

The present invention relates to compounds that are inhibitors of MKK4 (mitogen-activated protein kinase 4) and their use for promoting liver regeneration or reducing or preventing liver cell death. The compounds of the invention selectively inhibit the protein kinase MKK4 over the protein kinases JNK and MKK7.

Description

[Technical field]

[0001] The present invention relates to protein kinase inhibitors that inhibit mitogen-activated protein kinase 4 (MKK4), and in particular to protein kinase inhibitors that selectively inhibit MKK4 over the protein kinases JNK1 and MKK7. [Background technology]

[0002] Liver disease can be caused by infection, injury, exposure to toxic compounds such as alcohol and drugs, autoimmune processes, genetic defects, and other factors. Although the liver has a remarkable regenerative capacity, in pathological conditions this may not function properly and therefore may be insufficient to compensate for the loss of liver cells and organ function.

[0003] US Patent No. 5,999,943 describes compounds that are protein kinase inhibitors useful for the treatment of diseases and conditions associated with abnormal activity of protein kinases. These compounds are inhibitors of Raf protein kinases, particularly B-Raf and c-Raf and their mutants, and are therefore useful for the treatment of cancer. They are also said to inhibit a wide variety of other protein kinases, particularly c-Jun N-terminal kinase (JNK), in particular JNK1.

[0004] Patent Document 2 has a similar disclosure, and Patent Documents 3 and 4 disclose modified compounds having Raf protein kinase inhibitory activity. H. Vin et al. refer to two compounds in Patent Document 5 as B-Raf inhibitors that suppress apoptosis via off-target inhibition of JNK signaling.

[0005] US Patent No. 6,399,663 describes pyrazolo[3,4-b]pyridine compounds that are protein kinase inhibitors useful in the treatment of diseases and conditions mediated by Raf protein kinase, such as cancer. In addition, they are said to inhibit a wide variety of other protein kinases, particularly c-Jun N-terminal kinase (JNK), especially JNK1. US Patent No. 6,399,663 discloses several compounds that are described as inhibitors of mitogen-activated protein kinase 4 (MKK4) and are useful in the treatment of liver failure, protection of liver cells from apoptosis, and regeneration of liver cells.

[0006] Non-Patent Document 1 describes a functional genetic approach for the identification of gene targets that can be used to enhance the regenerative capacity of hepatocytes. In particular, Non-Patent Document 1 identifies the protein kinase MKK4 as a key regulator of hepatocyte regeneration, and reports that inhibition of MKK4 increased hepatocyte regeneration through compensatory upregulation of MKK7 and JNK1-dependent activation of ATF2 and ELK1. Based on the findings of the prior art, it was concluded that MKK4 and JNK1 inhibitors may be useful for the treatment of JNK1-mediated diseases.

[0007] However, there is a recognized failure to treat liver disease with such compounds in clinical practice. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2007 / 002433 [Patent Document 2] International Publication No. 2010 / 002325 [Patent Document 3] International Publication No. 2012 / 109075 [Patent Document 4] International Publication No. 2014 / 194127 [Patent Document 5] International Publication No. 2007 / 002433 [Patent Document 6] International Publication No. 2010 / 111527 [Patent Document 7] International Publication No. 2012 / 136859 [Non-patent literature]

[0009] [Non-Patent Document 1] Wuestefeld et al.(Cell 153:389-401,2013) Summary of the Invention [Problem to be solved by the invention]

[0010] The problem underlying the present invention was to provide useful compounds which are MKK4 inhibitors, in particular MKK4 inhibitors which selectively inhibit MKK4 over MKK7 and JNK1. A further problem was to provide compounds which are MKK4 inhibitors which selectively inhibit MKK4 over MKK7 and JNK1 and are useful in the treatment of liver disease, in particular MKK4 inhibitors which are useful in promoting liver regeneration or inhibiting or preventing liver cell death. [Means for solving the problem]

[0011] This problem has been solved by providing compounds of formula (I): Thus, the present invention relates to the following embodiments:

[0012] 1. Compounds having formula (I) and pharma-ceutically acceptable salts, solvates and optical isomers thereof:

[0013] [ka]

[0014] R 1 is H, or alkyl; R 2 is H, or alkyl; R 4 is H, or alkyl; R6 is H, or alkyl; R W -NR 10 SO2R 12 ; R 10 is H, alkyl, or phenylalkyl; R 12 is H, alkyl, haloalkyl, or phenylalkyl (wherein the phenyl group is optionally substituted with 1 or 2 groups independently selected from alkyl and halogen);

[0015] R x , R y , R z , and R zz is selected from: a)R x and R y is F and R z and R zz is H; and b)R x , R y , and R zz are independently halogens, and R z is H;

[0016] R 5 is selected from: (a) phenyl substituted with 1, 2, or 3 groups independently selected from: -POdi(alkyl); and Hydroxyalkyl-ONH-CO-; (b) pyrimidinyl substituted by 1, 2 or 3 groups independently selected from the following: Alkyl; Cycloalkyl; Alkoxy; and Alkylsulfanyl.

[0017] 2.R 5 is phenyl substituted with 1 or 2 groups independently selected from -POdi(alkyl) and hydroxyalkyl-ONH-CO-, and pharma- ceutically acceptable salts, solvates, and optical isomers thereof.

[0018] 3.R 5 is pyrimidinyl substituted with 1 or 2 groups independently selected from alkyl, cycloalkyl, alkoxy, and alkylsulfanyl, and pharma- ceutically acceptable salts, solvates, and optical isomers thereof.

[0019] 4. The compound of embodiment 3, wherein the pyrimidinyl is attached to 1H-pyrrolo[2,3-b]pyridine at the 5-position, substituted at the 2-position with alkyl, cycloalkyl, or alkoxy, and optionally substituted at the 4-position with alkyl or alkylsulfanyl, as well as pharmaceutically acceptable salts, solvates, and optical isomers thereof.

[0020] 5. The compound of embodiment 4, wherein the pyrimidinyl is attached to 1H-pyrrolo[2,3-b]pyridine at the 5-position and substituted at the 2-position with cycloalkyl or alkyl, and pharma- ceutically acceptable salts, solvates and optical isomers thereof.

[0021] 6. The compound of embodiment 3, wherein the pyrimidinyl is attached to the 1H-pyrrolo[2,3-b]pyridine at the 5-position and substituted with alkyl at the 4-position or the 2-position and the 4-position, and the alkyl at the 4-position and the 6-position may be the same or different, as well as pharma-ceutically acceptable salts, solvates and optical isomers thereof.

[0022] 7.R 12 The compound according to any one of embodiments 1 to 6, wherein is C1-C4-alkyl or phenylalkyl, wherein phenylalkyl is in particular benzyl, and the pharma- ceutically acceptable salts, solvates and optical isomers thereof.

[0023] 8.R 12 is C1-C4-alkyl, preferably methyl, ethyl, or propyl, and the pharma- ceutically acceptable salts, solvates, and optical isomers thereof.

[0024] 9. The compound of any one of embodiments 1 to 8 having formula (Ia), and pharma- ceutically acceptable salts, solvates and optical isomers thereof:

[0025] [ka]

[0026] In the formula, R 1 , R 2 , R 4 , R 5 , R 6 , R x , R w , and R y is as defined in any one of embodiments 1 to 8.

[0027] 10. The compound of any one of embodiments 1 to 8, having formula (Ib), and pharma- ceutically acceptable salts, solvates and optical isomers thereof:

[0028] [ka]

[0029] In the formula, R 1 , R 2 , R 4 , R 5 , R 6 , R x , R w , R y , and R zz is as defined in any one of embodiments 1 to 8.

[0030] 11.R 1 , R 2 , R 3 , R 4 , and R 6 The compound of any one of embodiments 1-10, wherein is H or alkyl, particularly H.

[0031] 12.R 10 The compound of any one of embodiments 1-11, wherein is H or alkyl, particularly H. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] [Detailed Description of the Invention] In one embodiment, the present invention relates to a compound of selective MKK4 inhibitors, and pharma- ceutically acceptable salts, solvates, and optical isomers thereof, of formula I, where R 1 ~R 6 , R 10 , A and Q are as defined in the above embodiments, in any combination.

[0033] In one embodiment, the compounds of the invention, and their pharma- ceutically acceptable salts, solvates, and optical isomers, selectively inhibit the protein kinase MKK4 over the protein kinases JNK1 and MKK7.

[0034] Furthermore, the present invention relates to said compounds for use in promoting liver regeneration or inhibiting or preventing liver cell death and at the same time increasing liver cell proliferation.

[0035] The term "compounds of the invention" or "compounds of formula (I)" includes pharma- ceutically acceptable salts, prodrugs, biologically active metabolites, solvates, and stereoisomers thereof.

[0036] The present invention includes pharma- ceutically acceptable salts of the above compounds. The pharma- ceutically acceptable salts are, in particular, acid or base addition salts using pharma- ceutical acceptable acids or bases. Examples of suitable pharma- ceutical acceptable organic and inorganic acids are hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, C1-C4-alkylsulfonic acids such as sulfamic acid and methanesulfonic acid, alicyclic sulfonic acids such as S-(+)-10-camphorsulfonic acid, aromatic sulfonic acids such as benzenesulfonic acid and toluenesulfonic acid, di- and tricarboxylic acids and hydroxycarboxylic acids having 2 to 10 carbon atoms, such as oxalic acid, malonic acid, maleic acid, fumaric acid, lactic acid, tartaric acid, citric acid, glycolic acid, adipic acid and benzoic acid.

[0037] Other usable acids are described, for example, in "Fortschritte der Arzneimittelforschung [Advances in drug research], Volume 10, pages 224 ff., Birkhaeuser Verlag, Basel and Stuttgart, 1966". Examples of suitable pharma-ceutically acceptable organic and inorganic bases are alkali metal hydroxides such as sodium hydroxide or potassium hydroxide, alkaline earth metal hydroxides such as calcium hydroxide or magnesium hydroxide, ammonium hydroxide, organic nitrogen bases such as dimethylamine, trimethylamine, ethanolamine, diethanolamine, triethanolamine, choline, 2-amino-2-hydroxymethyl-propane-1,3-diol, meglumine, procaine, etc., L-arginine, L-lysine, ethylenediamine, or hydroxyethylpyrrolidine.

[0038] The present invention also includes any tautomers, crystal forms and polymorphs of the compounds of the present invention and salts thereof, and mixtures thereof.

[0039] The present invention also includes solvates such as hydrates.

[0040] The compounds of the present invention may contain one or more chiral centers and therefore exist in different optically active forms, such as enantiomers and diastereomers.

[0041] As used herein, the term "prodrug" refers to an agent that is converted into the parent drug in vivo by some physiological chemical process. An example of a prodrug is, but is not limited to, an ester form of a compound of the present invention.

[0042] Prodrugs have many useful properties. For example, they may be more water soluble than the ultimate drug, facilitating intravenous administration of the drug. They may also have a higher level of oral bioavailability than the ultimate drug. After administration, the prodrug is enzymatically or chemically cleaved, and the ultimate drug is delivered to the blood or tissues.

[0043] Exemplary prodrugs include, but are not limited to, those in which the free hydrogen is (C1-C4) alkyl, (C1-C 12 )alkanoyloxy-methyl, (C4-C9)1-(alkanoyloxy)ethyl, 1-methyl-1-(alkanoyloxy)-ethyl having 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having 3 to 6 carbon atoms, 1-(alkoxycarbonyl-oxy)ethyl having 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)-ethyl having 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)aminomethyl having 4 to 10 carbon atoms Examples of suitable carboxylic acid substituents include N,N-(C1-C2)ethyl, 3-phthalidyl, 4-crotono-lactonyl, γ-butyrolactone-4-yl, di-N,N-(C1-C2)alkylamino(C2-C3)alkyl (e.g., β-dimethylaminoethyl), carbamoyl-(C1-C2)alkyl, N,N-di(C1-C2)-alkylcarbamoyl-(C1-C2)alkyl, and compounds having carboxylic acid substituents substituted with piperidino-, pyrrolidino-, or morpholino(C2-C3)alkyl.

[0044] Other exemplary prodrugs release alcohols of formula (I), where the free hydrogen of the hydroxyl substituent (e.g., an R group containing a hydroxyl) is (C1-C6)alkanoyloxy-methyl, 1-((C1-C6)alkanoyloxy)-ethyl, 1-methyl-1-((C1-C6)alkanoyloxy)ethyl, (C1-C 12)alkoxy-carbonyl-oxymethyl, N-(C1-C6)-alkoxy-carbonylaminomethyl, succinoyl, (C1-C6)alkanoyl, α-amino(C1-C4)alkanoyl, aryl, acyl, and α-aminoacyl or α-aminoacyl-α-aminoacyl, where the α-aminoacyl moieties are independently either naturally occurring L-amino acids found in proteins, P(O)(OH)2, -P(O)(O(C1-C6)alkyl)2, or glycosyl (the group resulting from removal of the hydroxyl of a carbohydrate hemiacetal).

[0045] The phrase "MKK4 inhibitor" means one which upon administration inhibits the kinase activity of MKK4 with an IC50 of <10 μmol / l, preferably <1 μmol / l, in particular <0.5 μmol / l. As used herein, the expression "selectively inhibits the protein kinase MKK4 over the protein kinases JNK1 and MKK7" means that the ratio of MKK7 inhibitory activity to MKK4 inhibitory activity, or the ratio of JNK1 inhibitory activity to MKK4 inhibitory activity, expressed either as a percentage of the control or as a Kd, is ≧10, as measured using KINOMEscan™.

[0046] As used herein, the expression "promoting liver regeneration or inhibiting or preventing liver cell death" means that the relative number of proliferating liver cells is increased by at least 30%, preferably at least 50%, compared to the number of proliferating cells at the start of treatment. In particular, the expression means an increase of ≧100% compared to the number of proliferating cells at the start of treatment.

[0047] In this context, experimental measurements and quantification are performed using standard methods, such as the quantification of the protein Ki67, which is precisely related to cell proliferation. For the quantification of proliferating hepatocytes in tissue slides, several immunohistochemical standard methods are available, which use a primary anti-Ki67 antibody, followed by visualization of anti-Ki67 binding, for example, using a horseradish peroxidase-conjugated secondary antibody. The amount of peroxidase activity, visualized by enzymatic conversion of a chromogenic substrate, correlates with the amount of Ki67 protein and the number of proliferating cells.

[0048] In the experiments described below, hepatocyte proliferation was quantified by Ki67 staining using the fluorophore tetramethylrhodamine containing primary polyclonal rabbit anti-Ki67 antibody from Abcam (cat. no. ab 15580, Abcam, Cambridge, USA) and secondary goat polyclonal antibody from Invitrogen (cat. no. 16101, Invitrogen / ThermoFisher).

[0049] Based on data from several preclinical mouse models, it was found that shRNA-mediated inhibition of MKK4 in a mouse model of chronic CCl4-induced liver injury increased hepatocyte proliferation from 13% to 27% (compared to control shRNA) and was associated with reduced liver damage (transaminases) and decreased liver fibrosis.

[0050] According to the definition in the previous chapter, the relative increase in proliferating cells was 108%. In a model of alcohol-induced steatohepatitis (ASH), shRNA-mediated silencing of MKK4 resulted in a 4% hepatocyte proliferation rate compared to 2% with control shRNA (relative increase: 100%). Repeated hepatocyte proliferation was associated with reduced steatosis (fatty deposits) and reduced liver damage measured by transaminases. Similarly, in a partial hepatectomy model (48 hours after surgical resection of two-thirds of the liver), shRNA-mediated MKK4 silencing increased hepatocyte proliferation from 16% (control shRNA) to 33% (relative increase: 106%). Again, increased hepatocyte proliferation was associated with improved liver regeneration and faster recovery of liver mass.

[0051] The organic moieties mentioned in the definitions of the variables above are generic to the individual lists of members of the individual groups, as is the term halogen. n -C m indicates in each case the possible number of carbon atoms in the group.

[0052] The term halogen stands in each case for fluorine, bromine, chlorine or iodine, in particular for fluorine or chlorine.

[0053] Alkyl is a linear or branched alkyl group, preferably a C1-C6-alkyl group (i.e. an alkyl group having 1 to 6 carbon atoms), more preferably a C1-C4-alkyl group. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl, tert-butyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, and 1-ethyl-2-methylpropyl.

[0054] The definition of alkyl is equally applicable to any group that contains an alkyl group.

[0055] Cycloalkyl is an alicyclic group, preferably C3-C8-cycloalkyl, i.e. a cycloalkyl group having 3 to 8 carbon atoms. In particular, 3 to 6 carbon atoms form a ring structure, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The ring structure may be unsubstituted or may carry 1, 2, 3 or 4 C1-C4 alkyl groups, preferably one or more methyl groups.

[0056] The compounds of the present invention (including pharma- ceutically acceptable salts, prodrugs, biologically active metabolites, solvates and stereoisomers thereof) can be prepared as disclosed in WO2007 / 002433, the entirety of which is incorporated herein by reference, or by similar procedures. Acid or base addition salts are prepared in a conventional manner by mixing a free base with the corresponding acid, or by mixing a free acid with the desired base. Optionally, the reaction is carried out in a solution of an organic solvent, for example a lower alcohol such as methanol, ethanol or propanol, an ether such as methyl tert-butyl ether or diisopropyl ether, a ketone such as acetone or methyl ethyl ketone, or an ester such as EtOAc.

[0057] The compounds of the present invention are useful for promoting liver regeneration or inhibiting or preventing liver cell death while simultaneously increasing liver cell proliferation. Thus, the compounds of the present invention are useful for treating, regulating, ameliorating, or preventing diseases involving acute or chronic damage to the liver, which may be caused by infection, injury, exposure to toxic compounds, abnormal accumulation of normal substances in the blood, autoimmune processes, genetic defects, or unknown causes.

[0058] These liver diseases include all diseases in which it is believed to be useful to promote liver regeneration and inhibit or prevent liver cell death in order to achieve a potential therapeutic effect (i.e. partial or complete restoration of liver function). These diseases include: Acute and chronic liver disease or acute exacerbation of liver disease, for example acute and chronic viral hepatitis such as hepatitis B, hepatitis C, hepatitis E, Epstein-Barr virus, cytomegalovirus, herpes simplex virus and other viral hepatitis, all types of autoimmune hepatitis, primary sclerosing hepatitis, alcoholic hepatitis;

[0059] Metabolic liver diseases, such as metabolic syndrome, fatty liver such as nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), Wilson's disease (Morbus Wilson), hemochromatosis, alpha1-antitrypsin deficiency, glycogen storage disease;

[0060] All types of cirrhosis, e.g. primary biliary cirrhosis, ethyl toxic cirrhosis, cryptogenic cirrhosis;

[0061] Acute (fulminant) or chronic liver failure, e.g. acetaminophen (paracetamol) induced liver failure, alpha-amanitin induced liver failure, drug induced hepatotoxicity e.g. liver failure caused by antibiotics, nonsteroidal anti-inflammatory drugs and anticonvulsants, toxic liver failure such as acute liver failure caused by herbal supplements (e.g. kava, ephedra, skullcap, pennyroyal), liver disease and liver failure due to vascular diseases such as Budd-Chiari syndrome, acute liver failure of unknown cause, chronic liver disease due to right heart failure;

[0062] Galactosemia, cystic fibrosis, porphyria, hepatic ischemia / perfusion injury, small-for-size graft syndrome after liver transplantation, primary sclerosing cholangitis or hepatic encephalopathy.

[0063] To promote liver regeneration or inhibit or prevent liver cell death, the compounds of the present invention are administered to patients in need thereof in therapeutically effective amounts. To detect the presence of liver disease, various diagnostic methods are available. Blood levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) that are above the clinically accepted normal range are known to indicate ongoing liver damage. Blood bilirubin levels or other liver enzymes are used as detection or diagnostic criteria.

[0064] Routine monitoring of liver disease patients for blood levels of ALT and AST is used to measure the progression of liver disease during treatment. The reduction of elevated ALT and AST levels to acceptable limits is considered clinical evidence reflecting a decrease in the severity of the patient's liver damage. Commercial tests such as FibroTest / FibroSURE, HepaScore™, FibroMeter or Cirrhometer evaluate the combined results of five or more biochemical parameters for the detection of fatty liver, fibrosis and cirrhosis. In addition, non-invasive and innovative physical imaging methods such as magnetic resonance imaging, sonography and especially elastography are available to detect and monitor the status and progression of liver disease.

[0065] Furthermore, shRNA-mediated inhibition of MKK4 has been shown to attenuate TNF-α-driven cartilage matrix degradation in osteoarthritis (Cell Death and Disease (2017)8,e3140). Thus, inhibition of MKK4 activity using the compounds of the present invention is further useful for the treatment of osteoarthritis and rheumatoid arthritis.

[0066] The compounds of the present invention are conventionally administered in the form of a pharmaceutical composition containing at least one compound according to the invention, optionally together with an inert carrier (e.g., a pharma- ceutically acceptable excipient), and, where appropriate, other drugs. These compositions can be administered, for example, orally, rectally, transdermally, subcutaneously, intraperitoneally, intravenously, intramuscularly, or intranasally.

[0067] Examples of suitable pharmaceutical compositions are solid pharmaceutical forms such as powders, granules, tablets, in particular film tablets, troches, sachets, cachets, dragees, semi-solid pharmaceutical forms such as capsules, such as hard and soft gelatin capsules or suppositories, ointments, creams, hydrogels, pastes or salves, and liquid pharmaceutical forms such as solutions, emulsions, in particular oil-in-water emulsions, suspensions (for example lotions), injection and infusion preparations. It is also possible to use liposomes or microspheres.

[0068] When preparing compositions, the compounds according to the invention are optionally mixed or diluted with one or more carriers (excipients). A carrier (excipient) can be a solid, semi-solid or liquid material that acts as a vehicle, carrier or medium for the active compound.

[0069] Suitable carriers (excipients) are described in specialized pharmaceutical monographs.Furthermore, the formulation can contain pharma-ceutically acceptable auxiliary substances.For example, wetting agents; emulsifiers and suspending agents; preservatives; antioxidants; anti-irritants; chelating agents; coating aids; emulsion stabilizers; film formers; gel formers; odor masking agents; flavoring agents; resins; hydrocolloids; solvents; solubilizers; neutralizing agents; diffusion promoters; pigments; quaternary ammonium compounds; refatting and superfatting agents; raw materials for ointments, creams or oils; silicone derivatives; diffusion aids; stabilizers; sterilizing agents; suppository bases; tableting aids such as binders, fillers, glidants, disintegrants or coatings; spraying agents; drying agents; opacifiers; thickeners; waxes; plasticizers and white mineral oil. Formulations in this respect are based on the expert knowledge described, for example, in "Fiedler, HP, Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete [Encyclopedia of auxiliary substances in pharmacy, cosmetics and related fields], 4th edition, Aulendorf: ECV-Editio-Cantor-Verlag, 1996".

[0070] The compounds of the present invention may also be suitable for combination with other therapeutic agents.Therefore, the present invention further relates to a combination comprising the compounds of the present invention and one or more additional therapeutic agents, particularly for use in promoting liver regeneration or suppressing or preventing liver cell death.The combination therapy of the present invention can be administered adjunctively.Adjunctive administration refers to the simultaneous or overlapping administration of each component in the form of separate pharmaceutical compositions or devices.

[0071] This regime of therapeutic administration of two or more therapeutic agents is generally referred to by those skilled in the art and herein as adjunctive therapeutic administration; also known as additive therapeutic administration. Any and all treatment regimens in which a patient receives separate but simultaneous or overlapping therapeutic administration of a compound of the present invention and at least one additional therapeutic agent are within the scope of the present invention. In one embodiment of adjunctive therapeutic administration described herein, a patient is typically stabilized for a period of time on a therapeutic administration of one or more components, and then receives administration of another component.

[0072] The combination therapy of the present invention can also be administered simultaneously. By simultaneous administration, it is meant that each component is administered together in the form of a single pharmaceutical composition or device that has or contains both components, or as separate compositions or devices, each of which contains one of the components that is administered simultaneously. This combination of each separate component for simultaneous combination can be provided in the form of a kit of parts.

[0073] Suitable agents for use in combination with the compounds of the present invention include, for example:

[0074] ACC inhibitors such as TOFA (5-(tetradecyloxy)-2-furoic acid), filsocostat (formerly known as GS 0976), PF-05221304, and the ACC inhibitors disclosed in WO2016 / 112305; Angiotensin II receptor antagonists; angiotensin-converting enzyme (ACE) inhibitors such as enalapril; ASK1 (apoptosis signal-regulating kinase 1; MAP3K5) inhibitors, such as selonsertib (formerly known as GS-4997) or SRT-015; caspase inhibitors such as emricasan; Cathepsin B inhibitors such as mixed cathepsin B / Hepatitis C virus NS3 protease inhibitors (e.g., VBY-376); CCR2 chemokine antagonists, such as mixed CCR2 / CCR5 chemokine antagonists (e.g., cenicriviroc); CCR5 chemokine antagonists; Chloride channel stimulants such as cobiprostone; Cholesterol solubilizers;

[0075] copper amine oxidase 3 (AOC3) inhibitors, such as BI 1467335 (formerly known as PXS-4728A); Diacylglycerol O-acyltransferase 1 (DGAT1) inhibitors such as LCQ908 or GSK-3008356; diacylglycerol O-acyltransferase 2 (DGAT2) inhibitors such as PF-06865571; Dipeptidyl peptidase IV (DPPIV) inhibitors such as linagliptin; Farnesoid X receptor (FXR) agonists such as INT-747 (obeticholic acid), Cryofexor (formerly known as GS-9674 or PX-102), Tropifexor (formerly known as LJN452), EDP-305, or LMB-763;

[0076] Fibroblast growth factors (FGFs) and their analogues, such as long-acting analogues of FGF19 (e.g., aldafermin; formerly known as NGM-282) or long-acting analogues of FGF21 (e.g., TEV-47948; also referred to as Bio89-100 or ARK01 or PF-05231023); FXR / TGR5 dual agonists such as INT-767; Galectin-3 inhibitors such as GR-MD-02; Glucagon-like peptide 1 (GLP1) agonists, such as liraglutide or exenatide; Glucagon-like peptide 1 (GLP1) / glucagon dual agonists (e.g., coreceptor agonists); dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonists (e.g., tirzepatide (formerly known as LY3298176)); Glutathione precursor;

[0077] Hepatitis C virus NS3 protease inhibitors, such as mixed cathepsin B / Hepatitis C virus NS3 protease inhibitors (e.g., VBY-376); HMG CoA reductase inhibitors such as statins (e.g., atorvastatin); 11β-hydroxysteroid dehydrogenase (11β-HSD1) inhibitors such as R05093151; IL-1β antagonists; IL-6 antagonists such as mixed IL-6 / IL-1β / TNFα ligand inhibitors (e.g., BLX-1002); IL-10 agonists such as pegylodecaquine;

[0078] Anti-IL-11 antibodies or IL-11 antagonists; IL-17 antagonists such as KD-025; ileal sodium bile acid cotransporter inhibitors, such as vorixibat (formerly known as SHP-626); Integrin inhibitors such as selective αvβ1-inhibitors (e.g., PLN-1474, or those reported in Wilkinson et al., Eur. J. Pharmacol., 842, 239-247 (2019)); ketohexokinase inhibitors such as PF-06835919; leptin analogues such as metreleptin; 5-lipoxygenase inhibitors, such as mixed 5-lipoxygenase / PDE3 / PDE4 / PLC inhibitors (e.g., tipelukast);

[0079] LPL gene stimulators such as Alipogene tiparvovec; lysyl oxidase homolog 2 (LOXL2) inhibitors, such as anti-LOXL2 antibodies like simtuzumab (formerly known as GS-6624), or small molecule inhibitors such as those disclosed in WO2017 / 136870; NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome small molecule inhibitors such as MCC950; Omega-3 polyunsaturated fatty acids and their derivatives (e.g., icosabtate and examples disclosed in US 8,735,436 B2); Oxysterol sulfates such as 25-hydroxycholesterol 3-sulfate and 25-hydroxycholesterol 3,25-disulfate;

[0080] PDE4 inhibitors such as ASP-9831; PPARα agonists (e.g., the mixed PPARα / δ agonist elafibranor (formerly known as GFT-505), the mixed PPARα / γ / δ agonist lanifibranor, or the mixed PPARα / γ agonist saroglitazar); PPARγ agonists such as pioglitazone; PPARδ agonists such as Seradelpar; Rho-associated protein kinase 2 (ROCK2) inhibitors, such as KD-025; Sodium glucose transporter-2 (SGLT2) inhibitors such as remogliflozin etabonate; sodium glucose transporter-1 / 2 (SGLT1 / 2) inhibitors, such as licogliflozin; Stearoyl-CoA desaturase-1 inhibitors such as aramchol or CVT-12805; Thyroid hormone receptor beta agonists, such as MGL-3196 or VK2809;

[0081] Tumor necrosis factor alpha (TNFα) ligand inhibitors; Transglutaminase inhibitors and transglutaminase inhibitor precursors such as mercaptamines; PTPlb inhibitors such as A119505, A220435, A321842, CPT633, ISIS-404173, JTT-551, MX-7014, MX-7091, MX-7102, NNC-521246, OTX-001, OTX-002, or TTP814; and Namacizumab; an antibody that stabilizes the cannabinoid 1 receptor (CB1) in an inactive form.

[0082] In some embodiments, the one or more additional therapeutic agents are selected from acetylsalicylic acid, alipogen tiparvovec, aramchol, atorvastatin, BLX-1002, cenicriviroc, cobiprostone, colesevelam, emricasan, enalapril, GFT-505, GR-MD-002, hydrochlorothiazide, icosapent ethyl ester (eicosapentaenoic acid ethyl), IMM-124E, KD-025, linagliptin, liraglutide, mercaptoamine, MGL-3196, obeticholic acid, olesoxime, pegylodecaquin, pioglitazone, GS-9674, remogliflozin etabonate, SHP-626, solithromycin, tipelukast, TRX-318, ursodeoxycholic acid, and VBY-376.

[0083] In some embodiments, one of the one or more additional therapeutic agents is selected from acetylsalicylic acid, alipogen tiparvovec, aramchol, atorvastatin, BLX-1002, and cenicriviroc.

[0084] In one embodiment, the present invention relates to a method of:

[0085] Inhibition of protein kinase MKK4; Selectively inhibiting MKK4 over the protein kinases JNK1 and MKK7 to promote liver regeneration or prevent liver cell death; Treatment of acute, acutely exacerbated, or chronic liver disease;

[0086] Treatment of acute and chronic or acutely exacerbated liver diseases, such as acute and chronic viral hepatitis, including hepatitis B, C, E, Epstein-Barr virus, cytomegalovirus, herpes simplex virus and other viral hepatitis, all types of autoimmune hepatitis, primary sclerosing hepatitis, alcoholic hepatitis;

[0087] Treatment of metabolic liver diseases, e.g. metabolic syndrome, fatty liver such as nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), Wilson's disease (Morbus Wilson), hemochromatosis, alpha-1-antitrypsin deficiency, glycogen storage disease;

[0088] Treatment of all types of cirrhosis, e.g. primary biliary cirrhosis, ethyl toxic cirrhosis, cryptogenic cirrhosis;

[0089] Treatment of acute (fulminant) or chronic liver failure, e.g. acetaminophen (paracetamol) induced liver failure, alpha-amanitin induced liver failure, drug induced hepatotoxicity e.g. liver failure caused by antibiotics, nonsteroidal anti-inflammatory drugs and anticonvulsants, toxic liver failure such as acute liver failure caused by herbal supplements (e.g. kava, ephedra, skullcap, pennyroyal), liver disease and liver failure due to vascular diseases such as Budd-Chiari syndrome, acute liver failure of unknown cause, chronic liver disease due to right heart failure;

[0090] Treatment of galactosemia, cystic fibrosis, porphyria, hepatic ischemia / perfusion injury, small-for-size graft syndrome after liver transplantation, primary sclerosing cholangitis, or hepatic encephalopathy; or

[0091] Treating osteoarthritis or rheumatoid arthritis;

[0092] The method comprises administering to a subject in need thereof an effective amount of a compound or composition as defined above.

[0093] In one embodiment, the compounds of the present invention are administered at a dosage of 0.2-15 mg / kg or 0.5-12 mg / kg of the subject being treated. The compounds can be administered once or several times daily. The compounds are administered for 4-12 weeks.

[0094] The following examples illustrate the invention without limiting it. EXAMPLES

[0095] Abbreviation: ATP = adenosine triphosphate DCM = dichloromethane 4-DMAP = (4-) dimethylaminopyridine DMA = Dimethylacetamide DMF = dimethylformamide DMSO = dimethyl sulfoxide DPPA = diphenylphosphoryl azide DTT = dithiothreitol EtOH = ethanol EtOAc = ethyl acetate

[0096] FCC = flash column chromatography HEPES = 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid HOBt = hydroxybenzotriazole HPLC = High Performance Liquid Chromatography IPA = Isopropyl Alcohol (or iPrOH) LCMS = Liquid Chromatography Mass Spectrometry MeCN = acetonitrile MeOH = methanol NaHCO3 = sodium bicarbonate NBS = N-bromosuccinimide

[0097] NMP = N-methylpyrrolidone NMR = nuclear magnetic resonance Pd(dppf)Cl2 = [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride PE = Petroleum Ether rt or RT = RT TEA = triethylamine TFA = trifluoroacetic acid THF = Tetrahydrofuran TLC = thin layer chromatography

[0098] [Example] Example 01: Synthesis of 2,4-difluoro-3-nitrobenzoic acid Chemical formula: C7H3F2NO4(MW:203.10)

[0099] [ka]

[0100] Step 1: Synthesis of 1-bromo-2,4-difluoro-3-nitrobenzene To a stirred solution of 1,3-difluoro-2-nitrobenzene (1 eq., 50.0 g, 314.29 mmol) in H2SO4 (400 mL) was added NBS (1.20 eq., 67.12 g, 377.14 mmol) and the reaction mixture was heated to 80 °C for 16 h. The reaction was monitored by TLC (100% hexane) and after completion, the reaction mixture was poured into ice-cold water and extracted with EtOAc (2 L). The organic layer was separated and washed with saturated NaHCO3 solution and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by FCC (gradient 1-5% EtOAc in n-hexane) to give 1-bromo-2,4-difluoro-3-nitrobenzene (50 g, 67%) as a yellow liquid.

[0101] Analytical data: 1 H NMR (400MHz, CDCl3) δ7.78-7.71(m,1H),7.09-7.03(m,1H).

[0102] Step 2: Synthesis of methyl 3-amino-2,4-difluorobenzoate A stirred solution of 1-bromo-2,4-difluoro-3-nitrobenzene (1 eq., 60.0 g, 252.21 mmol) in MeOH (1000 mL) was degassed with nitrogen for 15 min. TEA (5 eq., 127.49 g, 1261.03 mmol) was added and the reaction mixture was degassed for another 5 min. Pd(dppf)Cl2x DCM (0.10 eq., 20.58 g, 25.22 mmol) was added and the reaction mixture was flushed twice with carbon monoxide. The reaction mixture was stirred at room temperature for 30 min and further heated to 100° C. under carbon monoxide (200 psi) for 12 h. The reaction was monitored by TLC (100% DCM). After completion, the reaction mixture was filtered through a pad of Celite and the volatiles were removed under reduced pressure. Crude diethyl ether was added, stirred at room temperature for 30 min, filtered through a pad of Celite and the volatiles removed under reduced pressure to give methyl 3-amino-2,4-difluorobenzoate (38.0 g, crude) as a yellow syrup.

[0103] Analytical data: 1 H NMR(400MHz,DMSO-d6)δ7.08-6.96(m,2H),5.46(s,2H),3.81(s,3H)

[0104] Step 3: Synthesis of methyl 2,4-difluoro-3-nitrobenzoate 30% H2O2 (68.40 mL) was stirred and a solution of methyl 3-amino-2,4-difluorobenzoate (19.0 g, 101.52 mmol) in TFA (205 mL) was added dropwise at room temperature over 60 min. The reaction mixture was stirred at 80 °C for 1 h. The reaction was monitored by TLC (30% EtOAc in hexanes). After completion, the reaction mixture was poured onto crushed ice and the resulting precipitate was filtered and washed with water. The crude compound was dried under vacuum to give methyl 2,4-difluoro-3-nitrobenzoate (19.0 g, crude) as a light brown solid, which was used in the next reaction without further purification.

[0105] Analytical data: 1H NMR (400MHz, DMSO-d6) δ8.30-8.21 (m, 1H), 7.62 (t, J = 9.29Hz, 1H), 3.90 (d, J = 0.98Hz, 3H).

[0106] Step 4: Synthesis of 2,4-difluoro-3-nitrobenzoic acid To a stirred solution of methyl 2,4-difluoro-3-nitrobenzoate (19.0 g, 87.51 mmol) in THF (200 mL) was added LiOHxH2O (1.20 eq., 4.40 g, 105.01 mmol) dissolved in HO (100 mL) in portions over 30 min at room temperature. The reaction mixture was further stirred at room temperature for 3 h. The reaction was monitored by TLC (10% MeOH in DCM). After completion, the reaction mixture was neutralized with 1N HCl solution and concentrated under reduced pressure. The resulting crude product was acidified with 1N HCl solution and extracted with EtOAc. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 2,4-difluoro-3-nitrobenzoic acid (15.50 g, crude) as an off-white solid.

[0107] Analytical data: 1 H NMR (400MHz, DMSO-d6) δ13.85 (br s, 1H), 8.27-8.16 (m, 1H), 7.62-7.53 (m, 1H).

[0108] Example 02: Synthesis of N-(3-(1-(2,6-dichlorobenzoyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide Chemical formula:C 30 H 28 BCl2F2N3O6S(MW:678.34)

[0109] [ka]

[0110] Step 1: Synthesis of (5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)(2,4-difluoro-3-nitrophenyl)methanone Step 1a: 2,4-Difluoro-3-nitrobenzoic acid (12.4 g, 60.9 mmol) was suspended in nitromethane (50 ml, 20% of the total amount) and oxalyl chloride (5.44 mL, 63.4 mmol) and DMF (0.197 mL, 2.54 mmol) were added dropwise.

[0111] Step 1b: In a second flask, 5-bromo-1H-pyrrolo[2,3-b]pyridine (10 g, 50.8 mmol) and aluminum chloride (33.8 g, 254 mmol) were dissolved in nitromethane (204 ml, 80% of the total amount) and stirred for about 1 h. 2,4-difluoro-3-nitrobenzoyl chloride generated in situ along with the solution was added to the second flask over 10 min. The solution was stirred overnight at 55° C. TLC analysis showed the reaction was complete. The mixture was cooled to 0° C. and after dropwise addition of MeOH (102 ml), the product started to precipitate. Water (169 ml) was added and the precipitate was filtered off and dried in vacuum. The product was obtained as a beige solid (15.2 g; 75% yield; 95% purity).

[0112] Analytical data: 1 H NMR(400MHz,DMSO-d6)δ13.12(br s,1H),8.64(d,J=2.45Hz,1H),8.52-8.48(m,1H),8.38-8.34(m,1H),8.07-7.99(m,1H),7.64(t,J=9.29Hz,1H).

[0113] Step 2: Synthesis of (3-amino-2,4-difluorophenyl)(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)methanone (5-Bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-(2,4-difluoro-3-nitrophenyl)methanone (15.1 g, 37.5 mmol) was suspended in EtOAc (659 mL) and THF (659 mL). The mixture was heated to 60° C. and tin(II) chloride dihydrate (29.6 g, 131 mmol) was added portionwise. After stirring for 2 days, TLC analysis showed the reaction was complete. The mixture was cooled to room temperature and half-saturated NaHCO3 solution was added. The precipitate was filtered off through a pad of sea sand and Celite. The filtrate was washed with brine, dried over sodium sulfate and the solvent was evaporated under reduced pressure. The solid was suspended in MeCN (50 ml), filtered off and dried in vacuum. (3-Amino-2,4-difluorophenyl)-(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)methanone (11.9 g, 31.8 mmol, 85% yield) was obtained as a pale orange solid.

[0114] Step 3: Synthesis of (3-(3-amino-2,4-difluorobenzoyl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-1-yl)(2,6-dichlorophenyl)methanone To an ice-bath cooled solution of (3-amino-2,4-difluorophenyl)-(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)methanone (11.9 g, 31.7 mmol) in THF (338 mL) was added TEA (4.65 mL, 33.3 mmol). 2,6-Dichlorobenzoyl chloride (4.60 mL, 32.1 mmol) was added dropwise followed by 4-dimethylaminopyridine (0.194 g, 1.59 mmol). The ice-bath was removed and the mixture was stirred at room temperature until the starting material was completely consumed. The amount of solvent was reduced to approximately 1 / 10th the volume and the residue was diluted with EtOAc (500 ml). The organic phase was washed twice with 1M HCl (100 ml each), once with brine (100 ml) and dried over sodium sulfate. The solvent was removed under reduced pressure and the residue was dissolved in THF (66 ml). n-Heptane (330 ml) was added portionwise with vigorous stirring and upon completion stirring was continued for 1 h at room temperature. The precipitate was filtered off and dried in vacuum. (3-amino-2,4-difluorophenyl)-[5-bromo-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridin-3-yl]methanone was obtained as a beige solid (14.7 g, 26.5 mmol, 84% yield).

[0115] Step 4: Synthesis of N-(3-(5-bromo-1-(2,6-dichlorobenzoyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide Step 4a: To a cooled solution (-10°C, MeOH / ice) of (3-amino-2,4-difluorophenyl)-[5-bromo-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridin-3-yl]methanone (8.00 g, 14.5 mmol) in THF (76.2 mL) was added TEA (15.5 mL, 111 mmol). A solution of 1-propanesulfonyl chloride (3.58 mL, 31.8 mmol) in THF (3.6 mL) was added dropwise and the reaction mixture was stirred at -10°C until TLC showed completion.

[0116] Step 4b: To the cold solution was added 2N NaOH (8 eq., 61 mL) and the solution was allowed to warm to room temperature. After TLC showed complete hydrolysis of the disulfonamide, the mixture was acidified with 1M HCl (pH=2) and extracted twice with EtOAc. The combined organic phases were dried over sodium sulfate and the solvent was removed in vacuum. The residue was dissolved in EtOAc (50 mL) and treated with n-pentane until no further precipitate formed. The solid was filtered off and dried in vacuum (major fraction of product). The filtrate was concentrated under reduced pressure and purified by flash chromatography using PE / EE 30% as eluent (second fraction of product).

[0117] Step 5: Synthesis of (3-(3-amino-2,4-difluorobenzoyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)(2,6-dichlorophenyl)methanone To a stirred solution of (3-(3-amino-2,4-difluorobenzoyl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-1-yl)(2,6-dichlorophenyl)methanone (1 eq., 7.0 g, 13.33 mmol) in dry 1,4-dioxane (100 mL) was added bis(pinacolato)diboron (1.20 eq., 4.06 g, 16.00 mmol) followed by potassium acetate (molten, 2.50 eq., 3.28 g, 33.33 mmol). Pd(dppf)Cl2×DCM (0.05 eq., 0.54 g, 0.67 mmol) was added to the mixture and stirring was continued at 80° C. for 3 h. The progress of the reaction was monitored by TLC (20% EtOAc in hexanes) and LC-MS. After completion, the reaction mixture was cooled to room temperature and filtered through a pad of Celite. The organic layer was concentrated under reduced pressure. The crude product was dissolved in diethyl ether (200 mL), filtered, and the organic layer was concentrated under reduced pressure. The crude product was triturated with acetonitrile, followed by filtration to give (3-(3-amino-2,4-difluorobenzoyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridin-1-yl)(2,6-dichlorophenyl)methanone 5 (7.0 g,) as a light brown solid.

[0118] Example 03: Synthesis of 4-(3-(2,4-difluoro-3-(propylsulfonamido)benzoyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-N-(2-hydroxyethoxy)benzamide Step 1: Synthesis of 4-bromo-N-(2-hydroxyethoxy)benzamide

[0119] [ka]

[0120] 4-Bromobenzoyl chloride (1 eq., 0.200 mg, 0.91 mmol) was dissolved in dichloromethane (5 ml) and triethylamine (4 eq., 0.262 mg, 3.64 mmol) was added followed by 2-(aminooxy)ethan-1-ol (1 eq., 0.070 mg, 0.91 mmol). The reaction was monitored by TLC. After stirring at room temperature for 16 h, the reaction mixture was poured into ice-cold water and extracted with DCM (10 ml). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to give the crude compound. The crude compound was purified by column chromatography to give 4-bromo-N-(2-hydroxyethoxy)benzamide (0.120 g, 50%).

[0121] 1 H NMR(400MHz,DMSO-d6)δ11.85(s,1H),7.72(m,4H),4.76(s,1H),3.92(m,2H),3.60(m,2H) LCMS: 260.80 [M+H] + ,261.80[M+H] + , 261.8[M+Na] + , 260.8[M+Na] +

[0122] Step 2: 4-(3-(2,4-difluoro-3-(propylsulfonamido)benzoyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-N-(2-hydroxyethoxy)benzamide

[0123] [ka]

[0124] N-(3-(1-(2,6-dichlorobenzoyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide (180 mg, 0.265 mmol), 4-bromo-N-(2-hydroxyethoxy)benzamide (75 mg, 290 mmol) and potassium fluoride (46.2 mg, 0.795 mmol) were suspended in 1,4-dioxane (0.9 mL) and water (0.45 mL) and degassed with argon for 5 min. Pd(dppf)Cl2 (8.09 mg, 0.0111 mmol) was added and the mixture was heated to 90 °C for 6 h. The solvent was evaporated and the residue was dissolved in 10 mL of MeOH. Potassium carbonate (1 g) was added and the mixture was stirred at room temperature. After 6 h, water was added and the pH was adjusted to about 7 with aqueous HCl (1N). The aqueous phase was extracted three times with EtOAc, the combined organics were dried over sodium sulfate and the solvent was removed in vacuo. The product was purified by preparative RP-HPLC.

[0125] Analytical data: 1H NMR(400MHz,DMSO-d6)δ12.94(s,1H),11.85(s,1H),9.66(s,1H),8.74(d, J=8.7Hz,2H),8.10(s,1H),7.89(dd,J=19.2,8.2Hz,4H),7.70-7.58(m,1H) ,7.35(t,J=8.7Hz,2H),4.79(t,J=5.6Hz,1H),3.95(t,J=4.6Hz,2H),3.69 -3.61(m,2H),3.18-3.10(m,2H),1.88-1.77(m,2H),1.00(t,J=7.4Hz,3H). LC-MS: 559.20 [M+H] + .

[0126] Analogously to Examples 2 and 3, the following products were prepared:

[0127] [Table 1]

[0128] [Table 2]

[0129] [Table 3]

[0130] Example 15: Biological activity The kinase activity of the compounds of the present invention was measured using the 33PanQinase™ assay service provided by ProQinase GmbH (Freiburg, Germany). Detailed assay conditions are disclosed on the ProQinase website (https: / / www.proqinase.com / products-services-biochemical-assay-services / kinase-assays). Briefly, all kinase assays were performed in 50 μl reaction volumes using 96-well FlashPlates™ from PerkinElmer (Boston, MA, USA). The reaction cocktail was pipetted in four steps in the following order: 20 μl Assay Buffer (standard buffer) 5 μl of ATP solution (in H2O) 5 μl of test compound (10% DMSO solution) 20 μl enzyme / substrate mix

[0131] All protein kinase assays contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 50 μg / ml PEG20000, ATP (variable concentrations, corresponding to the apparent ATP-Km of each kinase), [γ-33P]-ATP (approximately 8 × 1005 cpm per well), protein kinase and substrate. The reaction cocktail was incubated for 60 min at 30° C. The reaction was stopped with 50 μl of 2% (v / v) H3PO4, and the plates were aspirated and washed twice with 200 μl of 0.9% (w / v) NaCl. 33 Pi incorporation was determined using a microplate scintillation counter (Microbeta, Wallac). All assays were performed on a BeckmanCoulter / SAGIAN™ core system.

[0132] For single concentration assays, efficacy of test compounds is expressed as % remaining activity. To determine IC50 values, serial dilutions were tested with a final concentration range of 100 μM to 3 nM (10 concentrations). The fitting model for the determination of IC50 values ​​was "sigmoidal response (variable slope)" with parameters "top" fixed at 100% and "bottom" fixed at 0%. The fitting method used was least squares.

[0133] MKK4 Efficacy:

[0134] [Table 4]

[0135] The selectivity of test compounds against BRaf, JNK1, and MKK7, all referred to as off-targets, was calculated by the ratio IC50(off-target) / IC50(MKK4) and classified as follows:

[0136] [Table 5]

[0137] The results are shown in Table 1 below.

[0138] [Table 6]

Claims

1. A compound having the formula (I) or a pharma- ceutically acceptable salt, solvate or optical isomer thereof: 【Chemistry 1】 During the ceremony, R １ is H, or alkyl; R ２ is H, or alkyl; R ４ is H, or alkyl; R ６ is H, or alkyl; R Ｗ is -NR １０ SO ２ R １２ ; R １０ is H, alkyl, or phenylalkyl; R １２ is H, alkyl, or phenylalkyl (wherein the phenyl group is optionally substituted with 1 or 2 groups independently selected from alkyl and halogen); R ｘ , R ｙ , R ｚ , and R ｚｚ is selected from the following: a) R ｘ and R ｙ is F and R ｚ and R ｚｚ is H; and b) R ｘ , R ｙ , and R ｚｚ is F and R ｚ is H; R ５ is phenyl substituted with 1, 2 or 3 groups independently selected from: -POdi(alkyl); and Hydroxyalkyl -ONH-CO-.

2. 2. The compound of claim 1 having formula (Ia) or formula (Ib), or a pharma- ceutically acceptable salt, solvate or optical isomer thereof. 【Chemistry 2】 【Chemistry 3】

3. R １ , R ２ , R ４ , and R ６ is H, or a pharma- ceutically acceptable salt, solvate or optical isomer thereof.

4. 1. A compound selected from the following: or a pharma- ceutically acceptable salt, solvate, or optical isomer thereof: 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】

5. A pharmaceutical composition comprising the compound according to any one of claims 1 to 4 or a pharma- ceutically acceptable salt, solvate or optical isomer thereof.

6. A compound according to any one of claims 1 to 4 or a pharma- ceutically acceptable salt, solvate or optical isomer thereof, or a pharmaceutical composition according to claim 5, for use in selectively inhibiting the protein kinase MKK4 over the protein kinases JNK1 and MKK7.

7. A compound according to any one of claims 1 to 4, or a pharma- ceutically acceptable salt, solvate or optical isomer thereof, or a pharmaceutical composition according to claim 5, for use in promoting liver regeneration or preventing liver cell death.

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