Heteroaryl-substituted pyrazolopyridine protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death

Abstract

The present invention relates to pyrazolopyridine compounds which inhibit mitogen-activated protein kinase kinase 4 (MKK4), and in particular selectively inhibit MKK4 relative to protein kinases JNK1 and MKK7. The compounds can be used to promote liver regeneration or reduce or prevent liver cell death. They can also be used to treat osteoarthritis or rheumatoid arthritis or CNS related diseases.

Description

Technical Field

[0001] The present invention relates to heteroaryl-substituted pyrazolo pyridine protein kinase inhibitors which inhibit mitogen-activated protein kinase kinase 4 (MKK4), in particular selectively inhibit MKK4 relative to protein kinases JNK1 and MKK7. Background Art

[0002] Liver disease can be caused by infection, injury, exposure to toxic compounds such as alcohol or drugs, autoimmune processes, genetic defects, and other factors. Although the liver has a remarkable regenerative capacity, this can be impaired in disease states and may therefore be insufficient to compensate for the loss of liver cells and organ function.

[0003] WO 2007 / 002433 describes compounds as protein kinase inhibitors that can be used to treat 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 mutations thereof, and are therefore useful in cancer treatment. In addition, they are said to inhibit a variety of other protein kinases, including c-Jun N-terminal kinase (JNK), particularly JNK1. WO 2007 / 002325 has similar disclosures, and WO 2012 / 109075 and WO 2014 / 194127 disclose modified compounds with Raf protein kinase inhibitory activity. H. Vin et al. refer to two compounds of WO2007 / 002433 as B-Raf inhibitors that inhibit apoptosis by off-target inhibition of JNK signaling. WO 2010 / 111527 describes pyrazolo[3,4-b]pyridine compounds that are protein kinase inhibitors for treating diseases or conditions mediated by Raf protein kinases, such as cancer. In addition, it is said that they inhibit a variety of other protein kinases, including c-Jun N-terminal kinase (JNK), particularly JNK1. WO 2012 / 136859 discloses some compounds, which are described as inhibitors of mitogen-activated protein kinase 4 (MKK4) and can be used to treat liver failure, for protecting liver cells from apoptosis and for liver cell regeneration. Wuestefeld et al. (Cell 153:389-401, 2013) and Willebring et al. (Cell 153:283-284) describe functional genetic methods for identifying gene targets, which can be used to increase the regenerative capacity of liver cells. In particular, Wuetefeld et al. identified protein kinase MKK4 as a key regulator of liver regeneration, and reported that MKK4 inhibits the activation of ATF2 and ELK1 by compensatory upregulation of MKK7 and JNK1 dependence and increased liver cell regeneration.

[0004] Based on the findings of the prior art, it has been concluded that MKK4 and JNK1 inhibitors can be used to treat JNK1-mediated diseases. However, despite the recognition that inhibition of JNK1 can be beneficial in treating liver diseases, clinical studies have not yet been conducted. WO 2018 / 134254 discloses pyrrolopyridine compounds, which are protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death. Summary of the invention

[0005] The problem underlying the present invention is to provide compounds that are useful MKK4 inhibitors, in particular MKK4 inhibitors that selectively inhibit MKK4 relative to MKK7 and JNK1. Another problem is to provide compounds that are MKK4 inhibitors that selectively inhibit MKK4 relative to MKK7 and JNK1, which can be used to treat liver diseases, in particular for promoting liver regeneration or reducing or preventing liver cell death.

[0006] This problem was solved by providing compounds of the formula (I).

[0007] Therefore, the present invention relates to the following embodiments:

[0008] 1. A compound of formula (I)

[0009]

[0010] and pharmaceutically acceptable salts, prodrugs, biologically active metabolites, solvates and stereoisomers thereof,

[0011] The variables in formula (I) have the following meanings:

[0012] R 1 H or alkyl

[0013] R 4 is H or alkyl;

[0014] R 5 Selected from

[0015] a) pyrimidinyl, which is substituted by 1 or 2 independently selected from cycloalkyl, alkyl, -COOR 10 , -OH, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, tetrazolyl, CN, halogen, alkoxy, -(NR 10 =) S(=O)-alkyl [S-alkylsulfonimidoyl] and Substituents are substituted, and

[0016] b1) pyridinyl, which is substituted by 1 or 2 substituents independently selected from alkyl and halogen and which is optionally further selected from -OH, alkoxy, CN, -COOR10 ,CF3,-(NR 10 =)S(=O)-alkyl and The group substitution, and

[0017] b2) pyridyl, which is -COOR 10 substituted and further substituted by -OH, CN or CF3;

[0018] R 6 is H or alkyl;

[0019] R w For-NR 10 S02R 12 ;

[0020] R x is H, halogen or alkyl;

[0021] R y is H, halogen or alkyl;

[0022] R z is H, halogen or alkyl;

[0023] Where R x , R y or R z One, two or three of them are halogen, and R x , R y or R z The rest are H or alkyl;

[0024] R 10 is independently at each occurrence H or alkyl;

[0025] R 12 is H, alkyl or phenylalkyl; and

[0026] n is 1 or 2.

[0027] 2. The compound of embodiment 1 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 1 For H.

[0028] 3. The compound of embodiment 1 or 2 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R x , R y or R z 2 or 3 of them are halogen.

[0029] 4. The compound of any one of embodiments 1 to 3 and its pharmaceutically acceptable salts, prodrugs, solvates and optical isomers, wherein R x , R y or R zThe one or more halogen atoms are independently F or Cl, in particular F.

[0030] 5. The compound of any one of the preceding embodiments and pharmaceutically acceptable salts, prodrugs, solvates and optical isomers thereof, wherein R 4 and R 6 For H.

[0031] 6. The compound of embodiment 6 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 5 is pyrimidinyl, which is substituted by 1 or 2 independently selected from cycloalkyl, alkyl, -COOR 10 , alkoxy, OH, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, halogen, CN and tetrazolyl substituents.

[0032] 7. The compound of embodiment 6 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 5 is pyrimidinyl substituted by 1 or 2 substituents independently selected from cycloalkyl, alkyl, alkoxy, OH, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, halogen, CN and tetrazolyl.

[0033] 8. The compound of embodiment 7 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 5 is pyrimidinyl substituted by 1 or 2 substituents independently selected from cycloalkyl, alkoxy, OH, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, halogen, CN and tetrazolyl.

[0034] 9. The compound of embodiment 8 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 5 is pyrimidinyl substituted by 1 or 2 substituents independently selected from cycloalkyl, alkoxy, OH, alkylsulfanyl, halogen, CN and tetrazolyl.

[0035] 10. The compound of embodiment 9 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 5 is pyrimidinyl substituted by 1 or 2 substituents independently selected from cycloalkyl, alkoxy, -OH, halogen and alkylsulfanyl.

[0036] 11. The compound of any one of embodiments 1 to 5 and its pharmaceutically acceptable salts, prodrugs, solvates and optical isomers, wherein R 5 is a pyrimidinyl group selected from cycloalkyl, alkyl, -COOR 10 , alkoxy, OH, alkylsulfanyl, alkylsulfinyl and alkylsulfonyl, and further substituted by a group selected from halogen, CN, tetrazolyl, -(NR10 =)S(=O)-alkyl and The group is substituted.

[0037] 12. The compound of embodiment 7 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 5 is a pyrimidinyl group substituted with a group selected from the group consisting of a cycloalkyl group, an alkyl group, an alkoxy group, -OH and an alkylsulfanyl group, and further substituted with a group selected from the group consisting of an alkyl group, an alkoxy group and an alkylsulfanyl group.

[0038] 13. The compound of embodiment 12 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 5 is a pyrimidinyl group substituted with a group selected from a cycloalkyl group, an alkoxy group, -OH and an alkylsulfanyl group, and further substituted with a group selected from an alkyl group, an alkoxy group and an alkylsulfanyl group.

[0039] 14. The compound of any one of embodiments 1 to 5 and its pharmaceutically acceptable salts, prodrugs, solvates and optical isomers, wherein R 5 is pyrimidinyl, which is substituted by cycloalkyl, and which is optionally further selected from cycloalkyl, alkyl, -COOR 10 , -OH, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, tetrazolyl, CN, halogen, alkoxy, -(NR 10 =)S(=O)-alkyl[S-alkylsulfimidoyl] and The group is substituted.

[0040] 15. The compound of embodiment 14 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 5 is a pyrimidinyl group, which is substituted by a cycloalkyl group, and which is optionally further selected from a cycloalkyl group, an alkyl group, -COOR 10 , -OH, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, tetrazolyl, CN, halogen and alkoxy groups.

[0041] 16. The compound of embodiment 15 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 5 is a pyrimidinyl group, which is substituted by a cycloalkyl group, and which is optionally further selected from cycloalkyl, alkyl, -COOR 10 , -OH, alkylsulfanyl, tetrazolyl, CN, halogen and alkoxy groups.

[0042] 17. The compound of embodiment 16 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 5is a pyrimidinyl group, which is substituted by a cycloalkyl group, and which is optionally further selected from alkyl, -COOR 10 , -OH, alkylsulfanyl, CN, halogen and alkoxy groups.

[0043] 18. The compound of any one of the preceding embodiments and pharmaceutically acceptable salts, prodrugs, solvates and optical isomers thereof, wherein R 5 is a pyrimidinyl group substituted at the 2-position.

[0044] 19. The compound of any one of embodiments 1 to 5 and its pharmaceutically acceptable salts, prodrugs, solvates and optical isomers, wherein R 5 For -COOR in position 2 10 Pyridin-3-yl substituted and further substituted by -OH, CN or CF3 (the pyridinyl group is bonded to the pyrazolopyridinyl group in the 3-position).

[0045] 20. The compound of any one of embodiments 1 to 5 and its pharmaceutically acceptable salts, prodrugs, solvates and optical isomers, wherein R 5 It is a pyridin-4-yl group substituted at the 3-position by an alkyl group or a halogen group (the pyridinyl group is bonded to the pyrazolopyridinyl group at the 4-position).

[0046] 21. The compound of any one of the preceding embodiments and pharmaceutically acceptable salts, prodrugs, solvates and optical isomers thereof, wherein R 10 is H or alkyl, especially H.

[0047] 22. The compound of any one of the preceding embodiments and pharmaceutically acceptable salts, prodrugs, solvates and optical isomers thereof, wherein R 12 It is an alkyl group or a phenylalkyl group.

[0048] 23. The compound of embodiment 22 and its pharmaceutically acceptable salt, prodrug, solvate and optical isomer, wherein R 12 is C1-C3-alkyl or benzyl.

[0049] 24. The compound of any one of the preceding embodiments and its pharmaceutically acceptable salts, prodrugs, solvates and optical isomers, which has formula (Ia):

[0050]

[0051] in:

[0052] R x is a halogen;

[0053] R y is a halogen; and

[0054] R1 , R 4 , R 5 , R 6 and R w As defined in any of the preceding embodiments.

[0055] 25. The compound of any one of embodiments 1 to 23 and its pharmaceutically acceptable salts, prodrugs, solvates and optical isomers, which has formula (Ib)

[0056]

[0057] in:

[0058] R x is a halogen;

[0059] R y is a halogen; and

[0060] R 1 , R 4 , R 5 , R 6 and R w As defined in any one of embodiments 1 to 15.

[0061] 26. The compound of any one of embodiments 1 to 23 and its pharmaceutically acceptable salts, prodrugs, solvates and optical isomers, which has the formula (Ic)

[0062]

[0063] in:

[0064] R x is a halogen;

[0065] R y is a halogen;

[0066] R z is a halogen; and

[0067] R 1 , R 4 , R 5 , R 6 and R w As defined in any one of embodiments 1 to 15.

[0068] 27. The compound of any one of embodiments 24 to 26 and its pharmaceutically acceptable salts, prodrugs, solvates and optical isomers, wherein R x , R y and R z (if present) is F or Cl, especially F. DETAILED DESCRIPTION

[0069] In one embodiment, the present invention relates to compounds of formula I and pharmaceutically acceptable salts, prodrugs, esters, solvates and optical isomers thereof, wherein R 1 , R 4 To R 6 , R w , R x , R y and R z As defined above in any combination.

[0070] In another embodiment, the present invention is directed to compounds of formula (Ia), (Ib) and (Ic) and pharmaceutically acceptable salts, prodrugs, esters, solvates and optical isomers thereof, wherein each variable is as defined in the above embodiments.

[0071] In another embodiment, R x , R y or R z At least two of them are halogens, and R x , R y and R z The other of is H, halogen or alkyl, in particular alkyl or halogen. Halogen is preferably F or Cl, in particular F.

[0072] In another embodiment, R 1 , R 4 and R 6 For H.

[0073] In another embodiment, R 12 It is methyl, ethyl or propyl.

[0074] In one embodiment, the present invention relates to MKK4 inhibitors of formula (I) and (Ia) to (Ic) and pharmaceutically acceptable salts, prodrugs, solvates and optical isomers thereof, and in particular to MKK4 inhibitors that selectively inhibit protein kinase MKK4 relative to protein kinases JNK1 and MKK7.

[0075] Furthermore, the present invention also relates to compounds according to the invention for use in inhibiting the protein kinase MKK4 and in particular for selectively inhibiting the protein kinase MKK4 relative to the protein kinases JNK1 and MKK7.

[0076] Furthermore, the present invention also relates to said compounds for promoting liver regeneration or reducing or preventing liver cell death and simultaneously increasing liver cell proliferation.

[0077] The present invention also includes pharmaceutically acceptable salts of the above compounds. Pharmaceutically acceptable salts are in particular acid or base addition salts formed with pharmaceutically acceptable acids or bases. Examples of suitable pharmaceutically acceptable organic and inorganic acids are hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, aminosulfonic acid, C1-C4-alkylsulfonic acids such as methanesulfonic acid, alicyclic sulfonic acids such as S-(+)-10-camphorsulfonic acid, aromatic sulfonic acids such as benzenesulfonic acid and toluenesulfonic acid, dicarboxylic acids and tricarboxylic acids having 2 to 10 carbon atoms and hydroxycarboxylic acids such as oxalic acid, malonic acid, maleic acid, fumaric acid, lactic acid, tartaric acid, citric acid, glycolic acid, adipic acid and benzoic acid. Other available acids are described, for example, in Fortschritte der Arzneimittelforschung [Advances in Drug Research], Vol. 10, p. 224 ( Verlag, Basel and Stuttgart, 1966). Examples of suitable pharmaceutically 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-1,3-propanediol, meglumine, procaine, etc., L-arginine, L-lysine, ethylenediamine or hydroxyethylpyrrolidine.

[0078] The present invention also includes any tautomers, crystals and polymorphs of the compounds and salts of the present invention, and mixtures thereof.

[0079] The present invention also includes solvates such as hydrates.

[0080] The compounds of the present invention may contain one or more chiral centers and exist in different optically active forms such as enantiomers and diastereomers.

[0081] As used herein, the term "pro-drug" refers to an agent that is converted into the parent drug in vivo by some physiological chemical process. A non-limiting example of a prodrug is an ester form of a compound of the invention.

[0082] Prodrugs have many useful properties. For example, a prodrug may be more soluble in water than the ultimate drug, thereby facilitating intravenous administration. A prodrug may also have a higher level of oral bioavailability than the ultimate drug. After administration, the prodrug is enzymatically or chemically cleaved to deliver the ultimate drug in the blood or tissue. Exemplary prodrugs include, but are not limited to, compounds having carboxylic acid substituents in which the free hydrogen is replaced by (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-(alkoxycarbonyloxy)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)amino)methyl having 4 to 10 carbon atoms (C1-C6)alkanoyloxy-methyl, 1-((C1-C6)alkanoyloxy)-ethyl, 1-methyl-1-((C1-C6)alkanoyloxy)ethyl, 1-methyl-1-((C1-C6)alkanoyloxy)- ... 12 )alkoxy-carbonyloxy-methyl, N-(C1-C6)-alkoxy-carbonylaminomethyl, succinyl, (C1-C6)alkanoyl, α-amino(C1-C4)alkanoyl, arylactyl and α-aminoacyl, or α-aminoacyl-α-aminoacyl, wherein the α-aminoacyl moiety is independently any naturally occurring L-amino acid found in proteins, P(O)(OH)2, -P(O)(O(C1-C6)alkyl)2, or a glycosyl (a radical derived from the removal of a hydroxyl group from a carbohydrate hemiacetal)

[0083] The expression of MKK4 inhibitor means that the kinase activity of MKK4 is inhibited, where IC 50 <10 μmol / l, preferably <1 μmol / l, in particular <0.5 μmol / l. As used herein, the expression "selectively inhibit protein kinase MKK4 over 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 as a percentage of control or Kd) is ≥10 as measured using KINOMEscanTM.

[0084] The expression "promoting liver regeneration or reducing or preventing hepatocyte death" as used herein means that the relative number of proliferating hepatocytes increases 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. In this context, experimental determination and quantification will be performed using standard methods, such as quantitative and Cell proliferation Strictly related protein Ki67. To quantify proliferating hepatocytes in tissue slides, several standard immunohistochemical methods can be used, which use a primary anti-Ki67 antibody followed by visualization of anti-Ki67 binding by using, for example, 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.

[0085] In the experiments described below, hepatocyte proliferation was quantified by Ki67 staining using a primary polyclonal rabbit anti-Ki67 antibody from Abcam (Cat. No. ab15580, Abcam, Cambridge, USA) and a secondary goat polyclonal antibody from Invitrogen containing the fluorophore tetramethylrhodamine (Cat. No. 16101, Invitrogen / ThermoFisher).

[0086] Based on data obtained from several preclinical mouse models, it was found that in a chronic CCl4 (carbon tetrachloride)-mediated liver injury mouse model, shRNA (small hairpin RNA)-mediated inhibition of MKK4 increased hepatocyte proliferation from 13% to 27% (compared to control shRNA) and was associated with reduced liver damage (transaminases) and reduced liver fibrosis. The relative increase in proliferating cells was 108% as defined in the previous chapter. In the alcohol-induced steatohepatitis (ASH) model, shRNA-mediated silencing of MKK4 resulted in a hepatocyte proliferation rate of 4%, compared to 2% when control shRNA was used (relative increase: 100%). The duplication of hepatocyte proliferation was associated with reduced steatosis (fat deposition) and reduced liver damage measured by transaminases. Similarly, in a partial hepatectomy model (48 hours after surgical removal of two-thirds of the liver), shRNA-mediated silencing of MKK4 increased hepatocyte proliferation from 16% (control shRNA) to 33% (relative increase: 106%). Likewise, increased hepatocyte proliferation was associated with improved liver regeneration and faster recovery of liver mass. In summary, these studies validate MKK4 as a therapeutic target for the treatment of acute and chronic liver diseases. In addition, WO 2018 / 134254 discloses novel compounds that selectively inhibit MKK4 relative to MKK7 and JNK1. In in vitro and in vivo experimental models of liver regeneration, these compounds effectively prevented acute liver failure induced by administration of Jo2 antibodies and induced proliferation of isolated primary mouse hepatocytes.

[0087] The novel compounds disclosed in the present application are potent MKK4 inhibitors that are selective for MKK7 and JNK1 and therefore, similar to the compounds disclosed in WO2018 / 134254, can be used to treat liver diseases and to promote liver regeneration or reduce or prevent hepatocyte death.

[0088] The organic moieties mentioned in the definitions of the above variables - such as the term halogen - are collective terms for the individual group members listed individually. n -C m In each case the possible number of carbon atoms in the group is indicated.

[0089] The term halogen denotes in each case fluorine, bromine, chlorine or iodine, in particular fluorine or chlorine and preferably fluorine.

[0090] Alkyl is a straight-chain or branched alkyl group, preferably a C1-C6-alkyl group, ie an alkyl group having 1 to 6 carbon atoms, more preferably a C1-C4-alkyl group, in particular a C1-C3-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.

[0091] The definition of alkyl applies equally to any group including an alkyl group, for example, alkoxy, alkylsulfinyl, phenylalkyl, and the like.

[0092] Haloalkyl is a halogenated alkyl group as defined above, wherein at least one, for example 1, 2, 3, 4 or all hydrogen atoms are replaced by 1, 2, 3, 4 or a corresponding number of identical or different halogen atoms, such as trifluoromethyl, chloromethyl, bromomethyl, difluoromethyl, fluoromethyl, difluoroethyl, etc. Specific examples include fluorinated C1-C4 alkyl as defined above, for example trifluoromethyl, difluoromethyl, fluoromethyl or difluoroethyl.

[0093] Cycloalkyl is an alicyclic radical, preferably a 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 radicals, preferably one or more methyl radicals.

[0094] The compounds of the present invention can be prepared as disclosed in WO 2010 / 111527, which is incorporated herein by reference in its entirety, or according to similar methods. Acid or base addition salts are prepared in a conventional manner by mixing a free base with a corresponding acid or by mixing a free acid with a desired base. Optionally, the reaction is carried out in a solution of an organic solvent, such as 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 ethyl acetate.

[0095] The compounds of the present invention can be used to promote liver regeneration or reduce or prevent liver cell death while increasing liver cell proliferation. Therefore, the compounds can be used to treat, regulate, improve or prevent diseases involving acute or chronic damage to the liver that 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.

[0096] These liver diseases include all diseases in which increasing liver regeneration and reducing or preventing hepatocyte death may help achieve a potential therapeutic effect (i.e., partial or complete restoration of liver function). These diseases include:

[0097] Acute and chronic or acute on chronic liver diseases, such as: acute and chronic viral hepatitis such as hepatitis B, hepatitis C, hepatitis E, hepatitis caused by Epstein-Barr virus, cytomegalovirus, herpes simplex virus and other viruses, all types of autoimmune hepatitis, primary sclerosing hepatitis, alcoholic hepatitis;

[0098] Metabolic liver diseases such as metabolic syndrome, fatty liver such as non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), Wilson's disease (Morbus Wilson), hemochromatosis, alpha-1 antitrypsin deficiency, glycogen storage disease;

[0099] All types of cirrhosis, such as primary biliary cirrhosis, ethyl toxic liver cirrhosis, cryptogenic cirrhosis;

[0100] Acute (fulminant) or chronic liver failure, such as toxic liver failure such as acetaminophen (paracetamol)-induced liver failure, alpha-amanitin-induced liver failure, drug-induced hepatotoxicity and liver failure such as caused by antibiotics, nonsteroidal anti-inflammatory drugs, anticonvulsants, acute liver failure induced by herbal supplements (kava, ephedra, skullcap, peppermint, etc.), liver disease and liver failure caused by vascular disease such as Budd-Chiari syndrom, acute liver failure of unknown cause, chronic liver disease caused by right heart failure;

[0101] Galactosemia, cystic fibrosis, porphyria, ischemic perfusion injury of the liver, small liver syndrome after liver transplantation, primary sclerosing cholangitis, or hepatic encephalopathy.

[0102] In order to promote liver regeneration or reduce or prevent liver cell death, the compounds of the present invention are administered to patients in need in a therapeutically effective amount. Various diagnostic methods can be used to detect the presence of liver disease. Blood levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) known to be higher than the clinically acceptable normal range indicate on-going liver damage. Blood bilirubin levels or other liver enzymes can be used as detection or diagnostic criteria. Routine monitoring of ALT and AST blood levels in patients with liver disease is used to measure the progression of liver disease during medical treatment. Reducing elevated ALT and AST levels to within an acceptable normal range is considered clinical evidence reflecting a reduction in the severity of liver damage in the patient. Commercial tests such as FibroTest / FibroSURE, The FibroMeter or Cirrhometer evaluates the combined results of five or more biochemical parameters for the detection of liver steatosis, fibrosis and cirrhosis. In addition, non-invasive innovative physical imaging techniques such as magnetic resonance imaging, ultrasonography and especially elastography can be used to detect and monitor the status and progression of liver diseases.

[0103] It was also found that shRNA-mediated inhibition of MKK4 attenuated TNF-α-driven cartilage matrix degradation in osteoarthritis (Cell Death and Disease (2017) 8, e3140). Therefore, using the compounds of the present invention to inhibit the activity of MKK4 can also be used to treat osteoarthritis and rheumatoid arthritis.

[0104] In addition, MKK4 inhibitors can also be used to treat neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Grueninger et al. found that in human neuroblastoma cells, MKK4 plays a key role in the phosphorylation of Tau protein at serine 422, which promotes Tau aggregation (Mol Cell Biochem (2011) 357: 199-207). Tau phosphorylation inhibitors that prevent Tau aggregation are considered to be useful in preventing or treating Alzheimer's disease.

[0105] Recently, MKK4 inhibitors with potent neuroprotective effects in vitro and in vivo have been described. In hippocampal cultures, incubation with MKK4 inhibitors prevented glutamate-induced cell death and caspase-3 activation, and also inhibited cell death induced by N-methyl-4-phenylpyridinium iodide and amyloid beta 1-42 in SH-SY5Y cells. The same compound also alleviated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced degeneration of nigrostriatal dopaminergic neurons in mice (Biochemical Pharmacology (2018), Vol. 162, April 2019, 109-122; doi: https: / / doi.org / 10.1016 / j.bcp.2018.10.008 ).

[0106] The compounds of the present invention are usually administered in the form of a pharmaceutical composition comprising at least one compound according to the present invention, optionally together with an inert carrier (e.g., a pharmaceutically acceptable excipient) and, if necessary, other drugs. These compositions can be administered, for example, orally, rectally, transdermally, subcutaneously, intraperitoneally, intravenously, intramuscularly or intranasally.

[0107] Examples of suitable pharmaceutical compositions are solid pharmaceutical forms such as powders, granules, tablets, in particular film tablets, lozenges, sachets, cachets, sugar-coated tablets; capsules such as hard gelatin capsules and soft gelatin capsules; or suppositories; semisolid pharmaceutical forms such as ointments, creams, hydrogels, pastes or plasters; and liquid pharmaceutical forms such as solutions, emulsions, in particular oil-in-water emulsions; suspensions such as lotions, injection preparations and infusion preparations. In addition, liposomes or microspheres can also be used.

[0108] When preparing the composition, the compound of the present invention is optionally mixed with or diluted by one or more carriers (excipients). A carrier (excipient) can be a solid, semi-solid or liquid material that serves as a vehicle, carrier or medium for the active compound.

[0109] Suitable carriers (excipients) are listed in the specialist medical monographs. In addition, the preparations may contain pharmaceutically acceptable auxiliary substances, such as wetting agents; emulsifiers and suspending agents; preservatives; antioxidants; antiirritants; chelating agents; coating aids; emulsion stabilizers; film formers; gel formers; odor masking agents; taste correctors; resins; hydrophilic colloids; solvents; solubilizers; neutralizers; diffusion promoters; pigments; quaternary ammonium compounds; refatting agents and overfatting agents; raw materials for ointments, creams or oils; silicone derivatives; dispersing aids; stabilizers; sterilizers; suppository bases; tablet aids such as binders, fillers, glidants, disintegrants or coatings; propellants; desiccants; opacifiers; thickeners; waxes; plasticizers and white mineral oil. The formulations in this regard are based on specialist knowledge, as described, for example, in Fiedler, HP, Lexikon der Hilfsstoffe für Pharmazie, Kosmetik undangrenzende Gebiete [Encyclopedia of auxiliary substances for pharmacy, cosmetics and related fields], 4th edition, Aulendorf: ECV-Editio-Cantor-Verlag, 1996.

[0110] The compounds of the present invention may also be suitable for combination with other therapeutic agents. Therefore, the present invention also relates to a combination comprising the compounds of the present invention and one or more other therapeutic agents, particularly for promoting liver regeneration or reducing or preventing liver cell death. The combined therapy of the present invention can be administered adjunctively. Adjunctive administration refers to the continuous or superimposed administration of each component in the form of a separate pharmaceutical composition or device. Such therapeutic administration schemes of two or more therapeutic agents are generally referred to as adjunctive therapeutic administration by those skilled in the art herein; it is also referred to as add-on therapeutic administration. Any and all therapeutic schemes in which the patient receives a separate but continuous or superimposed therapeutic administration of the compounds of the present invention and at least one other therapeutic agent are within the scope of the present invention. In one embodiment of adjunctive therapeutic administration as described herein, the patient is generally stabilized after a period of therapeutic administration of one or more components, and then receives administration of another component.

[0111] The combination therapy of the present invention can also be administered simultaneously. Simultaneous administration refers to a treatment regimen in which the individual components are administered together, either in the form of a single pharmaceutical composition or device comprising or containing both components, or as separate compositions or devices, each comprising one of the components, administered simultaneously. This combination of the individual components for simultaneous combination can be provided in the form of a kit.

[0112] Suitable pharmaceutical agents for use in combination with the compounds of the present invention include, for example:

[0113] ACC inhibitors, such as TOFA (5-(tetradecyloxy)-2-furoic acid), firsocostat (formerly GS 0976), PF-05221304, and ACC inhibitors as disclosed in WO 2016 / 112305;

[0114] Angiotensin II receptor antagonists;

[0115] angiotensin-converting enzyme (ACE) inhibitors, such as enalapril;

[0116] ASK1 (apoptosis signal-regulating kinase 1, MAP3K5) inhibitors, such as selonsertib (formerly known as GS-4997) or SRT-015;

[0117] Caspase inhibitors, such as emricasan;

[0118] Cathepsin B inhibitors, such as mixed cathepsin B / HCV NS3 protease inhibitors such as VBY-376;

[0119] CCR2 chemokine antagonists, such as mixed CCR2 / CCR5 chemokine antagonists such as cenicriviroc;

[0120] CCR5 chemokine antagonists;

[0121] Chloride channel stimulators, such as cobiprostone;

[0122] Cholesterol solubilizer;

[0123] Copperamine oxidase 3 (AOC3) inhibitors, such as BI 1467335 (formerly known as PXS-4728A);

[0124] Diacylglycerol O-acyltransferase 1 (DGAT1) inhibitors, such as LCQ908 or GSK-3008356;

[0125] Diacylglycerol O-acyltransferase 2 (DGAT2) inhibitors, such as PF-06865571;

[0126] Dipeptidyl peptidase IV (DPPIV) inhibitors, such as linagliptin;

[0127] Farnesoid X receptor (FXR) agonists, such as INT-747 (obeticholic acid), cliofexor (formerly GS-9674 or PX-102), tropifexor (formerly LJN452), EDP-305, or LMB-763;

[0128] Fibroblast growth factor (FGF) and its analogs, such as long-acting analogs of FGF19 (e.g., aldafermin, formerly known as NGM-282) or long-acting analogs of FGF21 (e.g., TEV-47948, also named Bio89-100 or ARK01 or PF-05231023);

[0129] FXR / TGR5 dual agonists, such as INT-767;

[0130] Galectin-3 inhibitors, such as GR-MD-02;

[0131] Glucagon-like peptide 1 (GLP1) agonists, such as liraglutide or exenatide;

[0132] Glucagon-like peptide 1 (GLP1) / glucagon dual agonists, such as cotadutide;

[0133] Dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonists, such as tirzepatide (formerly known as LY3298176);

[0134] Glutathione precursor;

[0135] Hepatitis C virus NS3 protease inhibitors, such as mixed cathepsin B / hepatitis C virus NS3 protease inhibitors such as VBY-376;

[0136] HMG CoA reductase inhibitors, such as statins like atorvastatin;

[0137] 11β-hydroxysteroid dehydrogenase (11β-HSD1) inhibitors, such as R05093151;

[0138] IL-1β antagonists;

[0139] IL-6 antagonists, such as mixed IL-6 / IL-1β / TNFα ligand inhibitors such as BLX-1002;

[0140] IL-10 agonists, such as peg-ilodecakin;

[0141] Anti-IL-11 antibody or IL-11 antagonist;

[0142] IL-17 antagonists, such as KD-025;

[0143] ileal sodium bile acid cotransporter inhibitors, such as volixibat (formerly SHP-626);

[0144] Integrin inhibitors, such as selective αvβ1-inhibitors (e.g. PLN-1474 or those reviewed by Wilkinson et al., Eur. J. Pharmacol., 842, 239-247 (2019));

[0145] Hexokinases inhibitors, such as PF-06835919;

[0146] Leptin analogs, such as metreleptin;

[0147] 5-lipoxygenase inhibitors, such as mixed 5-lipoxygenase / PDE3 / PDE4 / PLC inhibitors such as tipelukast;

[0148] LPL gene stimulators, such as alipogene tiparvovec;

[0149] Lysyl oxidase homolog 2 (LOXL2) inhibitors, such as anti-LOXL2 antibodies such as simtuzumab (formerly known as GS-6624), or small molecule inhibitors such as those disclosed in WO2017 / 136870;

[0150] Small molecule inhibitors of NOD-like receptor family pyocyanin domain-containing 3 (NLRP3) inflammasome, such as MCC950;

[0151] Omega-3 polyunsaturated fatty acids and their derivatives, such as icosabutate and the examples disclosed in US 8,735,436 B2;

[0152] Oxysterol sulfates, such as 25-hydroxycholesterol 3-sulfate and 25-hydroxy-cholesterol 3,25-hydrogen sulfate;

[0153] PDE4 inhibitors, such as ASP-9831;

[0154] PPARα agonists, such as the mixed PPARαPA agonist elafibranor (formerly known as GFT-505), the mixed PPARα / γ / δ agonist lanibibranor, or the mixed PPARα / γ agonist saroglitazar);

[0155] PPARγ agonists, such as pioglitazone;

[0156] PPARδ agonists, such as seladelpar;

[0157] Rho-associated protein kinase 2 (ROCK2) inhibitors, such as KD-025;

[0158] Sodium glucose transporter-2 (SGLT2) inhibitors, such as repagliflozin ectopic acid;

[0159] Sodium glucose transporter-1 / 2 (SGLT1 / 2) inhibitors, such as linagliptin;

[0160] Stearoyl CoA desaturase-1 inhibitors, such as aramchol or CVT-12805;

[0161] Thyroid hormone receptor beta agonists, such as MGL-3196 or VK2809;

[0162] Tumor necrosis factor alpha (TNFα) ligand inhibitors;

[0163] Transglutaminase inhibitors and transglutaminase inhibitor precursors, such as mercaptoethylamine;

[0164] 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

[0165] Namacizumab, an antibody that stabilizes the cannabinoid 1 receptor (CB1) in an inactive conformation.

[0166] In some embodiments, the one or more additional therapeutic agents are selected from acetylsalicylic acid, alipogenetiparvovec, aramchol, atorvastatin, BLX-1002, cenicriviroc, cobiprostone, colesevelam, emncasan, enalapril, GFT-505, GR-MD-02, hydrochlorothiazide, icosapentaenoic acid ethyl (ethyl eicosapentaenoic acid), IMM-124E, KD-025, linagliptin, liraglutide, mercaptoethylamine, MGL-3196, obeticholic acid, olesoxime, peg-ilodecakin, pioglitazone, GS-9674, remogliflozin ectoate, etabonate), SHP-626, solithromycin, tipelukast, TRX-318, ursodeoxycholic acid, and VBY-376.

[0167] In some embodiments, one of the one or more additional therapeutic agents is selected from acetylsalicylic acid, alipogene tiparvovec, aramchol, atorvastatin, BLX-1002, and cenicriviroc.

[0168] In one embodiment, the invention relates to a method, which:

[0169] Inhibits protein kinase MKK4;

[0170] Selectively inhibit protein kinase MKK4 relative to protein kinases JNK1 and MKK7, promote liver regeneration or prevent hepatocyte death;

[0171] Treatment of acute, acute-on-chronic or chronic liver disease;

[0172] Treatment of acute and chronic or acute-on-chronic liver diseases, such as: acute and chronic viral hepatitis such as hepatitis B, hepatitis C, hepatitis E, hepatitis caused by Epstein-Barr virus, cytomegalovirus, herpes simplex virus and other viruses, all types of autoimmune hepatitis, primary sclerosing hepatitis, alcoholic hepatitis;

[0173] Treatment of metabolic liver diseases such as metabolic syndrome, fatty liver such as non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), Wilson's disease, hemochromatosis, alpha 1-antitrypsin deficiency, glycogen storage disease;

[0174] Treatment of all types of liver cirrhosis, such as primary biliary cirrhosis, alcoholic cirrhosis, cryptogenic cirrhosis;

[0175] Treatment of acute (fulminant) or chronic liver failure, such as toxic liver failure such as acetaminophen (paracetamol)-induced liver failure, alpha-amanitin-induced liver failure, drug-induced hepatotoxicity and liver failure such as caused by antibiotics, nonsteroidal anti-inflammatory drugs, anticonvulsants, acute liver failure induced by herbal supplements (kava, ephedra, skullcap, peppermint, etc.), liver disease and liver failure caused by vascular disease such as Budd-Chiari syndrome, acute liver failure of unknown cause, chronic liver disease caused by right heart failure;

[0176] Treatment of galactosemia, cystic fibrosis, porphyria, liver ischemia-perfusion injury, small liver syndrome after liver transplantation, primary sclerosing cholangitis, or hepatic encephalopathy;

[0177] Treat osteoarthritis, rheumatoid arthritis or CNS-related diseases such as Alzheimer's disease and Parkinson's disease;

[0178] It comprises administering to a subject in need thereof an effective amount of a compound or composition as defined above.

[0179] In one embodiment, the compound of the invention is administered at a dose of 0.2-15 mg / kg or 0.5-12 mg / kg of the subject being treated. The compound may be administered once or several times a day. The compound is administered for 4-12 weeks.

[0180] The following examples illustrate the invention without limiting it.

[0181] Example

[0182] abbreviation:

[0183] Boc2O Di-tert-butoxycarbonate

[0184] CPME Cyclopentyl Methyl Ether

[0185] DCM Dichloromethane

[0186] 4-DMAP 4-Dimethylaminopyridine

[0187] DME Dimethyl ether

[0188] DMF Dimethylformamide

[0189] DMSO Dimethyl sulfoxide

[0190] EtOAc Ethyl acetate

[0191] HPLC High Performance Liquid Chromatography

[0192] KOH Potassium Hydroxide

[0193] LDA Lithium diisopropylamide

[0194] MeCN Acetonitrile

[0195] MeOH Methanol

[0196] NaHCO3 Sodium bicarbonate

[0197] NH4Cl Ammonium chloride

[0198] Na2SO4 Sodium sulfate

[0199] O / N Overnight

[0200] PdCl2(PPh3)2 Bis(triphenylphosphine)palladium(II) dichloride

[0201] Pd(dppf)Cl2 DCM 1,1'-Bis(diphenylphosphino)ferrocenepalladium(II) chloride complex with DCM

[0202] Pd2(dba3) Tris(dibenzylideneacetone)dipalladium(0)

[0203] pTSA p-Toluenesulfonic acid

[0204] PE Petroleum Ether

[0205] RT Room temperature

[0206] TEA Triethylamine

[0207] THF Tetrahydrofuran

[0208] TLC Thin Layer Chromatography

[0209] Xantphos 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene

[0210] Example 1: Synthesis of N-(3-(5-(2-cyclopropylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4-difluorophenyl)-1-phenylmethanesulfonamide

[0211]

[0212] Step 1-1: 5-Bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine (II)

[0213] To a stirred mixture of 5-bromo-1H-pyrazolo[3,4-b]pyridine ((I), 6.81 g, 34.4 mmol) and potassium hydroxide (KOH, 6.75 g, 120.4 mmol) in DMF (45 mL) was added iodine (9.60 g, 37.8 mmol) at room temperature. After a short induction period, an exothermic reaction began. After 1 h, another 1 g of iodine was added and the mixture was stirred at 45 °C for 1 h. The mixture was poured into 300 mL of dilute Na2SO3 solution and acidified with 2N HCl. The solid was collected by suction filtration, washed with water and dried in an oven at 110 °C. Yield: 10.92 g,

[0214] Analyze the data:

[0215] HPLC purity: 95%,

[0216] 1 H NMR (200MHz, DMSO) δ14.29(s,1H),8.62(s,1H),8.17(s,1H); 13 C NMR (50MHz, DMSO) δ150.53,150.17,131.86,120.58,112.43,91.95;

[0217] MS (ESI - ):m / z 322.0 / 324.0[MH] - .

[0218] Step 1-2: 5-Bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid (III)

[0219] 5-Bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine ((II), 10.44 g, 32.2 mmol) was combined with DMF, MeOH and triethylamine (TEA, 75 mL each). The vessel was evacuated and flushed with argon (4×). XantPhos (1.12 g, 1.93 mmol) and Pd(OAc)2 (217 mg, 0.97 mmol) were added, and carbon monoxide (generated from formic acid and sulfuric acid) was bubbled through the solution while heating to 60°C. The mixture was stirred under a carbon monoxide (balloon) atmosphere for 8 h. Carbon monoxide was bubbled through the solution for 5 min every 1.5 h. The mixture was concentrated under reduced pressure and the residue was triturated with 2N HCl. The solid was heated at 95°C overnight (O / N) in about 100 mL of 1N NaOH. After cooling to room temperature, the mixture was acidified with concentrated HCl, the precipitate was collected by suction filtration and washed with water. The solid was dried in an oven at 110°C to constant mass. The solid was sonicated in 100 mL of toluene for 5 min and stirred for 30 min. The product was filtered, washed with another 20 mL of toluene and dried at 110°C. Yield: 7.92 g.

[0220] Analyze the data:

[0221] HPLC purity: >99%,

[0222] 1 H NMR (200MHz, DMSO) δ8.64 (d, J = 7.9Hz, 2H), 5.69 (bs, 1H); 13 C NMR (50MHz, DMSO) δ163.27,150.97,149.67,136.69,132.65,115.73,113.6;

[0223] MS (ESI - ):m / z 239.9 / 241.9[MH] - .

[0224] Step 1-3: 5-Bromo-N-methoxy-N-methyl-1H-pyrazolo[3,4-b]pyridine-3-carboxamide (IV)

[0225] 5-Bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid ((III), 7.91 g, 32.7 mmol) and 1,1'-carbonyldiimidazole (5.83 g, 35.9 mmol) were stirred in 200 mL DMF at 60 °C for 45 min. N,O-dimethylhydroxylamine hydrochloride (3.51 g, 35.9 mmol) was added to the resulting suspension and the mixture was stirred at 65 °C for 4 h. Most of the solvent was removed under vacuum and the residue was half saturated. NaHCO3 solution was added. The solid was collected by suction filtration, washed with water and dried at 110 °C. Yield: 7.94 g,

[0226] Analyze the data:

[0227] HPLC purity: 96%,

[0228] 1 H NMR (200MHz, DMSO) δ14.46 (s, 1H), 8.62 (d, J = 20.4Hz, 2H), 3.76 (s, 3H), 3.44 (s, 3H),

[0229] MS (ESI - ):m / z 283.0 / 285.0[MH] -

[0230] Step 1-4: Synthesis of (3-amino-2,6-difluorophenyl)(5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)methanone (V)

[0231] 2,4-Difluoroaniline (6.25 g, 48.4 mmol) was dissolved in 50 mL of anhydrous THF and cooled to -78°C under argon. A 2.5 M solution of n-butyl lithium in hexane (19.4 mL, 48.4 mmol) was added dropwise. After 15 min, 15 mL of 1,2-bis(chlorodimethylsilyl)ethane (10.9 g, 49.5 mmol) in anhydrous THF was added dropwise and the mixture was stirred for 30 min. A 2.5 M solution of n-butyl lithium in hexane (19.4 mL, 48.4 mmol) was added dropwise and the mixture was allowed to reach room temperature within 1 h. After cooling to -78°C, a 2.5 M solution of n-butyl lithium in hexane (19.4 mL, 48.4 mmol) was added dropwise and stirred at -78°C for 1 h. (=Solution A).

[0232] 5-Bromo-N-methoxy-N-methyl-1H-pyrazolo[3,4-b]pyridine-3-carboxamide ((IV), 6.00 g, 21.1 mmol) was suspended in 50 mL of anhydrous THF and cooled to 0° C. under an argon atmosphere. NaH (60% solution in mineral oil, 0.88 g, 22.1 mmol) was added portionwise and the solution was stirred at room temperature for 1 h. (=solution B).

[0233] Solution B was added dropwise to solution A at -78°C. After the addition was complete, the mixture was allowed to warm to room temperature over 30 min. 12 ml of concentrated HCl was carefully added and the mixture was stirred for 30 min. Solid NaHCO3 was added to neutralize the solution, the solid was filtered off and washed with THF. The filtrate was evaporated and the residue was triturated with MeOH and water and dried at 110°C. Yield: 4.03 g;

[0234] Analyze the data:

[0235] HPLC purity: 97%,

[0236] 1 H NMR (200MHz, DMSO-d6) δ14.91 (s, 1H), 8.77 (dd, J = 5.4, 2.1Hz, 2H), 7.18–6.59 (m, 2H), 5.25 (s, 2H); 13 C NMR (50MHz, DMSO) δ183.95,151.04,150.79,150.27(dd,J=161.0,6.8Hz),145.50(dd,J=167.3,6.8Hz),141.34,133.35(dd,J=12 .8, 2.6Hz), 132.28, 117.45 (dd, J = 8.4, 6.5Hz), 116.24 (dd, J = 22.7, 19.1Hz), 115.55, 114.81, 111.26 (dd, J = 21.7, 3.5Hz); MS (ESI - ):m / z 351.1 / 353.1[MH] - .

[0237] Step 1-5: N-(3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide (VI)

[0238] (3-amino-2,6-difluorophenyl)-(5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)methanone (579 mg, 1.64 mmol) and 4-dimethylaminopyridine (4-DMAP, 0.401 g, 3.28 mmol) were dissolved in pyridine (3.31 mL, 41.0 mmol) under heating. After cooling to -10 ° C, phenylmethanesulfonyl chloride (406 mg, 2.13 mmol) was added to the formed suspension. The mixture was stirred at -10 ° C for 10 min, stirred at room temperature for another 10 min, then warmed to 50 ° C and stirred for 30 min. The mixture was concentrated under reduced pressure, reconstituted in 2N NaOH (2.46 mL, 4.92 mmol) and stirred at room temperature for 10 min. The mixture was diluted with water and slowly added to 25 mL 2N HCl with stirring. After 10 min, the solid formed was collected by suction filtration, washed with water and dried at 75°C (0.590 g, 1.16 mmol, yield 71%).

[0239] Analyze the data:

[0240] 1 H NMR (200MHz, CDCl3) δ14.22(s,1H),8.96(s,1H),8.79(d,J=2.2Hz,1H),8.59( d,J=2.1Hz,1H),7.52–7.25(m,6H),6.87(td,J=9.1,1.6Hz,1H),4.32(s,2H);

[0241] MS (ESI - ):m / z 504.7[M-1] - .

[0242] Step 1-6: N-[3-[5-Bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-1-phenylmethanesulfonamide (VII)

[0243] N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4-difluorophenyl]-1-phenylmethanesulfonamide (0.419 g, 0.826 mmol), p-toluenesulfonic acid (p-TSA) monohydrate (15.7 mg, 0.0826 mmol) and dihydropyran (0.0904 mL, 0.991 mmol) were dissolved in 4.13 ml of DCM and refluxed for 1.5 h. After cooling, the mixture was diluted with DCM, washed with saturated NaHCO3 solution, dried and filtered. n-heptane was added and DCM was removed. After cooling in an ice bath, the product was collected by suction filtration. (0.393 g, 0.6650 mmol, yield 80%).

[0244] Analyze the data:

[0245] 1 H NMR (200MHz, CDCl3) δ8.88(d,J=1.8Hz,1H),8.68(d,J=1.8Hz,1H),7.62(td,J=9.0,5.6Hz,1H),7.34(s,5H),6.96(t,J=8.1Hz,1H),6. 48(s,1H),6.14(dd,J=9.8,2.0Hz,1H),4.39(s,2H),4.03(d,J=11.1Hz,1H),3.90–3.56(m,1H),2.56–2.36(m,1H),2.09–1.46(m,5H);

[0246] MS (ESI-): m / z 591.3 / 589.4 [MH] - .

[0247] Step 1-7: N-[2,4-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]-1-phenylmethanesulfonamide (VIII)

[0248] To a vessel was added N-[3-[5-bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-1-phenylmethanesulfonamide (0.365 g, 0.617 mmol), bis(pinacolato)diboron (0.313 g, 1.23 mmol), potassium acetate (0.182 g, 1.85 mmol) and DMF (3.09 mL). The mixture was heated to 90 °C and the vessel was evacuated and filled with argon (3×). Bis(triphenylphosphine)palladium(II) dichloride (PdCl2(PPh)3)2, 4.33 mg, 0.00617 mmol) was added and the reaction was stirred at 90 °C for 4 h. After cooling, the reaction was concentrated, taken up in EtOAc, washed with water, half-saturated brine and brine, dried and filtered. Activated carbon was added to the filtrate and the mixture was refluxed for 15 min. After cooling, the mixture was filtered through celite and the solvent was removed. The residue was dissolved in a small amount of DCM, n-heptane was added, and DCM was removed under reduced pressure. The solid was collected by suction filtration, washed with n-hexane, and dried to give a pale solid product (0.298 g, 0.4670 mmol, 76% yield), which was used without further purification.

[0249] Analyze the data:

[0250] 1H NMR (200MHz, CDCl3) δ9.16(s,1H),8.97(s,1H),7.63(td,J=9.1,5.7Hz,1H),7.34(s,5H),6.96(t,J=8.6Hz,1H),6.45(s,1H),6. 23(d,J=8.8Hz,1H),4.39(s,2H),4.12–3.94(m,1H),3.80(t,J=9.9Hz,1H),2.60–2.31(m,1H),2.09–1.55(m,5H),1.39(s,12H).

[0251] Step 1-8: N-(3-(5-(2-cyclopropylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4-difluorophenyl)-1-phenylmethanesulfonamide (IX)

[0252] N-[2,4-difluoro-3-[1-(oxane-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]-1-phenylmethanesulfonamide (0.275 g, 0.431 mmol), 5-bromo-2-cyclopropylpyrimidine (0.111 g, 0.560 mmol), PdCl2(PPh)3)2 (3.02 mg, 0.00431 mmol) and 1,4-dioxane (1.44 mL) were added to the vessel. The vessel was evacuated and filled with argon (3×). Degassed 3M K2CO3 aqueous solution (0.431 mL, 1.29 mmol) was added and the mixture was stirred at 60°C for 1 h. After cooling, the reactant was diluted with EtOAc and neutralized with NH4Cl solution. The organic phase was dried, evaporated and the main product was isolated by flash chromatography (DCM / EtOAc, 10 to 50%). To remove the THP protecting group, the isolated product was refluxed in 2.5 N HCl (3 mL) for 1 h.

[0253] Analyze the data:

[0254] 1 H NMR (200MHz, DMSO-d6) δ15.01 (s, 1H), 9.86 (s, 1H), 9.12 (s, 2H), 9.08 (d, J = 2.0Hz, 1H), 8. 90(d,J=2.0Hz,1H),7.60–7.16(m,7H),4.54(s,2H),2.39–2.19(m,1H),1.27–1.04(m,4H).

[0255] MS (ESI - ):m / z 545.5[MH] -.

[0256] Example 2: Synthesis of N-(3-(5-(4-chlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)methanesulfonamide

[0257]

[0258] Step 2-1: N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl]methane-sulfonamide

[0259] (3-Amino-2,4-difluorophenyl)(5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)methanone (1.29 g, 3.65 mmol) was dissolved in 18 ml THF and 5.09 ml TEA (36.5 mmol, 10 eq). The mixture was heated to achieve complete dissolution. The solution was then cooled to 0 °C, followed by the addition of 0.99 ml mesylchloride (12.8 mmol, 3.5 eq). After 10 min, the mixture was warmed to room temperature, 12 ml 2N KOH was added and the mixture was stirred for 10 min. After acidification with 3N HCl, THF was removed under reduced pressure, the solid was collected by suction filtration, washed with 3N HCl / MeOH (1+1) and dried (1.46 g, 93%).

[0260] Analyze the data:

[0261] 1 H NMR (400MHz, DMSO-d6) δ14.87(s,1H),9.75(s,1H),8.71(d,J=13.9Hz,2H),7.85(d,J=6.6Hz,1H),7.39(t,J=8.5Hz,1H),3.12(s,3H).

[0262] MS (ESI - ):m / z 431.1 / 429.1[MH] - ,411.1 / 409.1[MH-HF] - .

[0263] Step 2-2: N-[3-[5-bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]methanesulfonamide

[0264] To a suspension of dihydropyran (0.614 mL, 6.73 mmol) and N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl]methanesulfonamide (1.45 g, 3.36 mmol) in 16.8 mL DCM was added p-TSA monohydrate (64.0 mg, 0.336 mmol) and the mixture was refluxed for 1 h. The mixture was diluted with DCM, washed with saturated NaHCO3 solution, dried over Na2SO4 and concentrated. The residue was dissolved in a small amount of acetone and transferred to diethyl ether under stirring. The product was collected by suction filtration and washed with diethyl ether to give the product as a white powder (1.07 g, 2.08 mmol, 62% yield).

[0265] Analyze the data:

[0266] 1 H NMR(200MHz,DMSO-d6)δ9.78(s,1H),8.80(dd,J=13.4,1.6Hz,2H),7.88(dd,J=14.6,7.8Hz,1H),7.43(t,J=9.0Hz,1H),6.1 4(d,J=8.6Hz,1H),3.94(d,J=11.6Hz,1H),3.72(dd,J=14.6,9.1Hz,1H),3.13(s,3H),2.46-2.19(m,1H),2.00-1.18(m,5H);

[0267] MS (ESI - ):m / z 515.2 / 513.2[MH] - .

[0268] Step 2-3: N-[3-[5-(4-chlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]methanesulfonamide

[0269] N-[3-[5-bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]methanesulfonamide (119 mg, 0.231 mmol), (4-chlorophenyl)boronic acid (36.1 mg, 0.231 mmol), PdCl2(PPh)3)2 (3.24 mg, 0.00462 mmol) were added to the vessel and purged with argon. Degassed 1,4-dioxane (0.770 mL) and degassed potassium fluoride (95.7 mg, 0.693 mmol) were added and the reaction was stirred at 60°C for 15 min. After cooling, EtOAc and NH4Cl solution were added and the aqueous phase was discarded. The organic phase was dried over Na2SO4, concentrated and the residue was taken up in ethanol containing 2.5N HCl (3 mL) and stirred at 60°C overnight. 30 ml of isopropanol was added, the solid was collected by centrifugation and the solvent was discarded. The solid was taken up in THF / NaHCO3 solution and shaken. The organic phase was dried, the solvent was evaporated and the residue was triturated with DCM (yield: 44.0 mg, 0.0941 mmol, 41%).

[0270] Analyze the data:

[0271] 1 H NMR (400MHz, DMSO-d6) δ14.82(s,1H),9.75(s,1H),9.00(d,J=2.2Hz,1H),8.76(d,J= 2.2Hz,1H),7.90–7.81(m,3H),7.61–7.57(m,2H),7.40(t,J=8.4Hz,1H),3.11(s,3H);

[0272] Calculated exact mass: 462.04, MS (ESI - ):m / z:461.0[M-1]-.

[0273] Example 3: Synthesis of N-(3-(5-(4-chlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)ethanesulfonamide

[0274] In analogy to Example 2, N-(3-(5-(4-chlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)ethanesulfonamide was prepared.

[0275] Analyze the data:

[0276] 1H NMR (400MHz, DMSO-d6) δ9.00(d,J=2.2Hz,1H),8.76(d,J=2.2Hz,1H),7.84(dd,J=13.4,8.4Hz, 3H),7.60(d,J=8.6Hz,2H),7.38(t,J=8.6Hz,1H),3.17(q,J=7.3Hz,2H),1.32(t,J=7.3Hz,3H)

[0277] Calculated exact mass: 476.05, MS (ESI - ):m / z:474.9[M-1]-.

[0278] Example 4: Synthesis of ethyl 4-(3-(2,4-difluoro-3-(methylsulfonylamino)benzoyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate

[0279]

[0280] Step 4-1: 4-[3-[2,4-difluoro-3-(methylsulfonylamino)benzoyl]-1-(oxan-2-yl)pyrazolo[3,4-b]pyridin-5-yl]benzoic acid

[0281] N-[3-[5-bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]methanesulfonamide (( I ), 355 mg, 0.689 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (188 mg, 0.758 mmol), XPhos Pd G3 ((2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]methanesulfonate palladium(II), commercially available from Aldrich; 17.5 mg, 0.0207 mmol) were combined in degassed 1,4-dioxane (2.30 mL) and 1.5 M potassium carbonate (2.07 mL, 3.10 mmol). The reaction was evacuated and flushed with argon (3x). XPhos Pd G3 (17.5 mg, 0.0207 mmol) was added and the reaction was stirred at 60 ° C (oil bath temperature) for 3 h. After cooling, the mixture was acidified with 2N HCl and extracted with EtOAc. The extract was washed with brine, dried over sodium sulfate (Na2SO4) and concentrated. The residue was purified by flash chromatography (DCM + MeOH 3%-25%) and triturated with n-hexane (276 mg, 0.4960 mmol, 72% yield).

[0282] Step 4-2: Ethyl 5-(3-(2,4-difluoro-3-(methylsulfonylamino)benzoyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate

[0283] 50mg( II ) (0.09 mmol) was dissolved in a mixture of 300 μl H2SO4 and 800 μl ethanol and stirred at 75°C for 16 h. After the mixture was cooled to room temperature, it was poured into a NaHCO3 aqueous solution, and the precipitate was collected by centrifugation and washed with water and diethyl ether (22 mg, 46%, chemical purity (HPLC / UV): 95%).

[0284] Analyze the data:

[0285] 1 H NMR (400MHz, DMSO) δ14.86(s,1H),9.76(s,1H),9.06(s,1H),8.83(s,1H),8.10(d,J=7.8Hz,2H),8.04–7.94(m,2H ),7.87(dd,J=14.5,7.6Hz,1H),7.40(t,J=8.8Hz,1H),4.35(q,J=7.0Hz,2H),3.11(s,3H),1.35(t,J=7.1Hz,3H);

[0286] Calculated exact mass: 500.10, MS (ESI - ):m / z:499.4[M-1]-.

[0287] Example 5: Synthesis of 5-(3-(2,4-difluoro-3-(methylsulfonylamino)benzoyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)picolinic acid methyl ester

[0288]

[0289] Step 5-1: N-[2,6-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]methanesulfonamide

[0290] Add N-[3-[5-bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]methanesulfonamide (( I), 0.630 g, 1.22 mmol), bis(pinacolato)diboron (341 mg, 1.34 mmol), anhydrous potassium acetate (360 mg, 3.67 mmol) and anhydrous 1,4-dioxane (4.08 mL). The vessel was evacuated and filled with argon (3×). 1,1′-bis(diphenylphosphino)-ferrocene-dichloropalladium (1:1 complex with DCM) (Pd(dppf)Cl2, 17.9 mg, 0.0245 mmol) was added and the reaction was stirred at 80°C overnight. After cooling, EtOAc was added and the suspension was stirred for 30 min and filtered through Celite. The solvent was concentrated, n-heptane was added and the solid was collected by suction filtration, washed with hexane and dried (0.690 g, 1.23 mmol, 100% yield).

[0291] Step 5-2: Methyl 5-(3-(2,4-difluoro-3-(methylsulfonylamino)benzoyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)picolinate

[0292] Add N-[2,6-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]methanesulfonamide (( II ), 116 mg, 0.206 mmol), 5-bromopyridine-2-carboxylic acid methyl ester (49.0 mg, 0.227 mmol), potassium fluoride (36.0 mg, 0.619 mmol), Pd(dppf)Cl2DCM (8.42 mg, 0.0103 mmol) and degassed 1,4-dioxane / water (4+1) (0.6 mL), the vessel was evacuated and filled with argon (3×). The mixture was stirred at 50°C overnight, then diluted with EtOAc, washed with brine and the solvent removed. The product was isolated by flash chromatography (DCM+EtOAc 0%-20%); 81.0 mg was a white solid (0.1420 mmol, yield 69%).

[0293] Step 5-3: Methyl 5-(3-(2,4-difluoro-3-(methylsulfonylamino)benzoyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)picolinate

[0294] To 5-[3-[2,4-difluoro-3-(methylsulfonylamino)benzoyl]-1-(oxan-2-yl)pyrazolo[3,4-b]pyridin-5-yl]pyridine-2-carboxylic acid methyl ester (( III), 60.0 mg, 0.105 mmol) in methanol (0.525 mL) was added with methanesulfonic acid (0.0273 mL, 0.420 mmol), and the mixture was stirred at 65 ° C for 1.5 h. The mixture was cooled to room temperature and slowly added to diethyl ether (15 mL). The solid was collected by suction filtration, washed with diethyl ether and dried in vacuo; 36.0 mg was a white solid (0.0739 mmol, yield 70%).

[0295] Analyze the data:

[0296] 1H NMR (200MHz, CDCl3) δ14.90(s,1H),9.74(s,1H),9.16(dd,J=12.4,1.9Hz,2H),8.93(d,J=2.0Hz,1H),8.47(dd,J=8. 2,2.3Hz,1H),8.19(d,J=8.3Hz,1H),7.88(dd,J=14.8,7.7Hz,1H),7.41(t,J=8.7Hz,1H),3.93(s,3H),3.12(s,3H);

[0297] Calculated exact mass: 487.08; MS (ESI + ):m / z:510.4[M+Na + ]+.

[0298] Example 6: Synthesis of N-(2,6-difluoro-3-(5-(pyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide

[0299]

[0300] Step 6-1: N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl]propane-1-sulfonamide

[0301] At -10 ° C, (3-amino-2,4-difluorophenyl)-(5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)methanone (( I), 65.0 g, 184 mmol) and TEA (282 mL, 2020 mmol) in THF (614 mL) suspension was added dropwise a 1:1 (v / v) mixture of 1-propylsulfonyl chloride (68.4 mL, 607 mmol) and DCM, keeping the temperature below -5 °C. After the addition was complete, the mixture was stirred at 0 °C for 1 h. 2N NaOH (736 mL, 1470 mmol) was added and the mixture was stirred at room temperature for another 30 min. THF and TEA were removed from the mixture by evaporation under reduced pressure at 45 °C. The solution was cooled to 20 °C and carefully washed with EtOAc (4×300 mL) under vortex (without shaking). The residual EtOAc was removed under reduced pressure, and the solution was slowly added to 3N HCl (736 mL, 2210 mmol) under effective stirring. The solid was collected by suction filtration, washed with a large amount of water and dried at 100°C to give N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl]propane-1-sulfonamide (73.9 g, 161 mmol, yield 87%) as an off-white solid.

[0302] Step 6-2: N-[3-[5-bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide

[0303] Towards( II To a suspension of 4-(4-(4-(4-(4-piperidin-2-yl)-1-yl)-2-nitropropene) (70.7 g, 154 mmol) in DCM (440 mL) were added p-TSA monohydrate (2.93 g, 15.4 mmol) and dihydropyran (15.5 mL, 169 mmol) and the mixture was refluxed for 1 h. After cooling, the mixture was diluted with 200 mL DCM, washed with saturated NaHCO3 solution and brine, dried over Na2SO4 and evaporated. The oily residue was dissolved in warm (45°C) MeOH (150 mL), seeds from the previous synthesis were added and the mixture was stirred at room temperature. After precipitation was observed, the mixture was diluted with cold MeOH, the suspension was cooled to -20°C for 2 h, the solid was collected by suction filtration and washed with 50 mL MeOH at -20°C. The product was dried in a vacuum oven at 50 °C to give N-[3-[5-bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide (73.4 g, 135 mmol, 88% yield).

[0304] Step 6-3: N-[2,6-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide

[0305] Add N-[3-[5-bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide (( III ), 4.74 g, 8.72 mmol), bis(pinacolato)diboron (4.65 g, 18.3 mmol) and anhydrous potassium acetate (2.57 g, 26.2 mmol). The container was evacuated and filled with argon (3×). Anhydrous DMF (29.1 mL) was added and the mixture was heated to 90°C. The container was evacuated and filled with argon (3×) again. PdCl2(PPh3)2 (30.6 mg, 0.0436 mmol) was added under an argon stream, the container was sealed and the reactants were stirred at 90°C for 4 h. After cooling, the reactants were concentrated under reduced pressure, the residue was dissolved in about 50 ml of EtOAc, washed with water, half-saturated NaCl solution and brine. After drying, the extract was filtered, activated carbon was added and the mixture was heated to reflux temperature for 15 min. After cooling, the mixture was filtered through diatomaceous earth, n-heptane (50 mL) was added to the filtrate and the solvent was removed. The solid was stirred in n-hexane (100 mL) for 30 min, and the product was collected by suction filtration. After drying, N-[2,6-difluoro-3-[1-(oxane-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide (4.37 g, 7.4 mmol, yield 85%) was obtained as a colorless solid.

[0306] Step 6-4: N-[2,6-difluoro-3-(5-pyrimidin-5-yl-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl]propane-1-sulfonamide

[0307] Add N-[2,6-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide (( IV), 120 mg, 0.203 mmol), 5-bromopyrimidine (35.5 mg, 0.224 mmol), potassium fluoride (35.4 mg, 0.610 mmol), PdCl2(PPh3)2 (3.32 mg, 0.00406 mmol) and degassed 1,4-dioxane / water (0.5 mL, (4+1)). The vessel was evacuated and filled with argon (3×) and heated to 60°C for 2 h. The reaction was acidified with concentrated HCl (0.3 mL), diluted with MeOH (0.2 mL) and heated to 60°C overnight. An additional 0.3 mL of concentrated HCl was added and stirring was continued for 4 h. After cooling, the mixture was diluted with water, neutralized with NaHCO3 solution and extracted with THF. The organic phase was washed with brine, dried over Na2SO4 and evaporated. The residue was purified by flash chromatography (DCM / methanol, 2%-8%) and triturated with acetone to give N-[2,6-difluoro-3-(5-pyrimidin-5-yl-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl]propane-1-sulfonamide (35.0 mg, 0.0741 mmol, 36% yield).

[0308] Analyze the data:

[0309] 1H NMR (400MHz, DMSO-d6) δ14.87(s,1H),9.71(s,1H),9.28(d,J=10.4Hz,3H),9.10(d,J=2.2Hz,1H),8.94(d,J=2.2Hz,1 H),7.87(dd,J=14.7,7.6Hz,1H),7.40(t,J=8.6Hz,1H),3.20–3.07(m,2H),1.89–1.71(m,2H),0.99(t,J=7.4Hz,3H);

[0310] C 20 H 16 The calculated exact mass of F2N6O3S (molecular weight: 458.44) is 458.10;

[0311] MS (ESI - ):m / z:456.9[M-1]-.

[0312] Example 7: Synthesis of N-(2,6-difluoro-3-(5-(2-methylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide

[0313]

[0314] To a vessel was added N-[2,6-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide (120 mg, 0.203 mmol), 5-bromo-2-methylpyrimidine (38.7 mg, 0.224 mmol), potassium fluoride (35.4 mg, 0.610 mmol), Pd(dppf)Cl2.DCM (3.32 mg, 0.00406 mmol) and degassed 1,4-dioxane / water (0.5 mL, (4+1)). The vessel was evacuated and filled with argon (3×) and heated to 60° C. for 2 h. The mixture was acidified with concentrated HCl (0.3 mL), diluted with MeOH (0.2 mL) and heated to 60 ° C overnight. Another 0.3 mL of concentrated HCl was added and stirring was continued for 3 h. After cooling, the mixture was diluted with EtOAc and water. The organic phase was evaporated and the product was purified by flash chromatography.

[0315] Analyze the data:

[0316] 1H NMR (400MHz, DMSO-d6) δ14.82(s,1H),9.66(s,1H),9.16(s,2H),9.06(d,J=2.1Hz,1H),8.89(d,J=2.2Hz,1H),7.86(dd,J=14.6, 7.6Hz,1H),7.40(t,J=8.7Hz,1H),3.15(dd,J=8.7,6.5Hz,2H),2.70(s,3H),1.81(dq,J=14.9,7.4Hz,2H),0.99(t,J=7.5Hz,3H);

[0317] C 21 H 18 The calculated exact mass of F2N6O3S (molecular weight: 472.47) is 472.11;

[0318] MS (ESI - ):m / z:471.0[M-1]-

[0319] Example 8: Synthesis of 5-(3-(2,4-difluoro-3-(propylsulfonylamino)benzoyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidine-2-carboxylic acid

[0320]

[0321] Step 8-1: 5-(3-(2,4-difluoro-3-(propylsulfonylamino)benzoyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidine-2-carboxylic acid methyl ester

[0322] Add N-[2,6-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide (( I ), 134 mg, 0.227 mmol), methyl 5-bromopyrimidine-2-carboxylate (54.2 mg, 0.250 mmol), potassium fluoride (39.6 mg, 0.681 mmol), Pd(dppf)Cl2 DCM (3.71 mg, 0.00454 mmol) and degassed 1,4-dioxane / water (0.5 mL, (4+1)). The vessel was evacuated and filled with argon (3×) and heated to 60°C for 2 h. NH4Cl solution and EtOAc were added and the mixture was evaporated over celite. The product was isolated by flash chromatography (DCM / EtOAc, 30%-100%); 121 mg solid (0.2010 mmol, yield 89%).

[0323] Step 8-2: 5-(3-(2,4-difluoro-3-(propylsulfonylamino)benzoyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidine-2-carboxylic acid

[0324] 5-[3-[2,4-difluoro-3-(propylsulfonylamino)benzoyl]-1H-pyrazolo[3,4-b]pyridin-5-yl]pyrimidine-2-carboxylic acid methyl ester (( II ), 87.0 mg, 0.1640 mmol, 82% yield) was dissolved in 3 mL 2.5 M HCl in ether and refluxed for 1 h. The solvent was removed in vacuo and the residue was dissolved in 2 mL MeOH and 2 mL 2N NaOH and refluxed for 1 h. After cooling, the mixture was acidified to pH 3 with 1 N HCl, brine was added, and the mixture was extracted with THF and THF / EtOAc (1:1). The extract was washed with brine (acidified with some 1 N HCl), dried over Na2SO4 and filtered. The solvent was removed to give 5-[3-[2,4-difluoro-3-(propylsulfonylamino)benzoyl]-1H-pyrazolo[3,4-b]pyridin-5-yl]pyrimidine-2-carboxylic acid (87.0 mg, 0.1640 mmol, 82% yield).

[0325] Analyze the data:

[0326] 1H NMR (400MHz, DMSO-d6) δ14.94(s,1H),9.68(d,J=4.6Hz,1H),9.45(s,2H),9.16(d,J=1.7Hz,1H),9.04(s,1H),7.88( dd,J=14.5,7.9Hz,1H),7.41(t,J=8.9Hz,1H),3.15(dd,J=8.7,6.6Hz,3H),1.87–1.77(m,2H),1.00(t,J=7.5Hz,3H);

[0327] C 21 H 16 The calculated exact mass of F2N6O5S (molecular weight: 502.45) is 502.09;

[0328] MS (ESI - ):m / z:501.0[M-1]-

[0329] Example 9: Synthesis of N-(3-(5-(2-cyanopyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0330]

[0331] Step 9-1: N-[3-[5-(2-cyanopyrimidin-5-yl)-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide

[0332] Add N-[2,6-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide (( I), 398 mg, 0.674 mmol), 5-bromopyrimidine-2-carbonitrile (136 mg, 0.741 mmol), potassium fluoride (117 mg, 2.02 mmol), PdCl2(PPh3)2 (11.8 mg, 0.0169 mmol) and degassed dioxane / water (4+1, 2.5 mL). The vessel was evacuated and filled with argon (3×) and heated to 60° C. for 2 h. After cooling, the reaction mixture was diluted with NH4Cl solution and extracted with EtOAc. The extract was washed with brine, dried over Na2SO4 and evaporated. The residue was purified by flash chromatography (DCM / EtOAc, 5%-30%) to give N-[3-[5-(2-cyanopyrimidin-5-yl)-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide (270 mg, 0.4760 mmol, 71% yield).

[0333] Step 9-2: N-[3-[5-(2-cyanopyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide

[0334] N-[3-[5-(2-cyanopyrimidin-5-yl)-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide (( II ), 50.0 mg, 0.0881 mmol) was stirred in 1 mL TFA at room temperature for 5 h. The mixture was concentrated, absorbed in water and the pH was adjusted to 4 using 1 N NaOH. The aqueous phase was extracted with EtOAc, the extract was washed with brine, dried over Na2SO4 and evaporated. The residue was triturated with ether to give N-[3-[5-(2-cyanopyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide (27.0 mg, 0.0542 mmol, 61% yield).

[0335] Analyze the data:

[0336] 1 HNMR (400MHz, DMSO-d6) δ14.98(s,1H),9.68(s,1H),9.55(s,2H),9.31(s,1H),9.17(d,J=2.2Hz,1H),9.08(d,J=2.1Hz ,1H),7.88(dd,J=14.5,7.8Hz,1H),7.41(t,J=8.8Hz,1H),3.19–3.10(m,2H),1.86–1.74(m,2H),1.00(t,J=7.5Hz,3H);

[0337] C 21 H 15 The calculated exact mass of F2N7O3S (molecular weight: 483.45) is 483.09;

[0338] MS (ESI - ):m / z:482.0[M-1]-.

[0339] Example 10: Synthesis of N-(3-(5-(2-(1H-tetrazolyl-5-yl)pyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0340]

[0341] Modified from Vorona, S. et al. (Synthesis-Stuttgart 46(6):781-786 (2014)), N-[3-[5-(2-cyanopyrimidin-5-yl)-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide (75.0 mg, 0.132 mmol), zinc bromide (29.8 mg, 0.132 mmol) and sodium azide (9.45 mg, 0.145 mmol) were heated to 95° C. in 1-propanol (0.661 mL) for 2 h. The reaction was cooled, 0.25 N NaOH (2.64 mL, 0.661 mmol) was added, the n-propanol was removed under reduced pressure and the suspension was stirred at room temperature for 15 min. The mixture was filtered and the filter was washed with 0.25N NaOH (2.64mL, 0.661mmol). The filtrate was acidified with 2N HCl and extracted with EtOAc. The extract was washed with brine, dried over Na2SO4 and evaporated. The residue was dissolved in 2.5N HCl in EtOH (2mL) and heated to 70°C for 2h. The reactant was concentrated, diluted with ether, and the solid was collected by suction filtration to obtain N-[2,6-difluoro-3-[5-[2-(1H-tetrazol-5-yl)pyrimidin-5-yl]-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide hydrochloride (44.0mg, 0.0782mmol, 59% yield).

[0342] Analyze the data:

[0343] 1H NMR (400MHz, DMSO-d6) δ14.96(s,1H),9.68(s,1H),9.57(s,2H),9.22(d,J=2.1Hz,1H),9.08(d,J=2.1Hz,1H),7 .88(dd,J=14.8,7.6Hz,1H),7.41(t,J=8.5Hz,1H),3.21–3.09(m,2H),1.89–1.72(m,2H),1.00(t,J=7.4Hz,3H);

[0344] C 21 H 16 F2N 10 The calculated exact mass of O3S (molecular weight: 526.48) is 526.11;

[0345] MS (ESI-): m / z: 525.0 [M-1] -.

[0346] Example 11: Synthesis of N-(3-(5-(2-cyclopropylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0347]

[0348] To a vessel was added N-[2,6-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide (176 mg, 0.298 mmol), 5-bromo-2-cyclopropylpyrimidine (65.3 mg, 0.328 mmol), potassium fluoride (52.0 mg, 0.894 mmol), PdCl2(PPh3)2 (4.18 mg, 0.00596 mmol) and degassed 1,4-dioxane / water (1 mL, (4+1)). The vessel was evacuated and filled with argon (3×) and heated to 60° C. for 2 h. After cooling, the mixture was distributed between saturated NH4Cl solution and EtOAc, and the organic phase was dried and evaporated with Na2SO4. The main product was separated by flash chromatography (DCM / EtOAc, 20%-80%) and dissolved in 2mL of 2.5N HCl in ethanol and refluxed for 2h. The reaction mixture was concentrated and the solid was ground with EtOAc to give N-[3-[5-(2-cyclopropylpyrimidine-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide hydrochloride (55.0mg, 0.1030mmol, 34% yield).

[0349] Analyze the data:

[0350] 1H NMR (400MHz, DMSO) δ14.90(s,1H),9.68(s,1H),9.11(s,2H),9.04(d,J=2.2Hz,1H),8.87(d,J=2.2Hz,1H),7.86(dd,J=14.9,7.6H z,1H),7.40(t,J=8.5Hz,1H),3.20–3.10(m,2H),2.35–2.26(m,1H),1.86–1.74(m,2H),1.16–1.04(m,4H),0.99(t,J=7.5Hz,3H);

[0351] C 23 H 20 The calculated exact mass of F2N6O3S (molecular weight: 498.51) is 498.13;

[0352] MS (ESI - ):m / z:497.0[M-1]-.

[0353] Example 12: Synthesis of N-(3-(5-(2-chloropyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0354]

[0355] To a vessel was added N-[2,6-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide (120 mg, 0.203 mmol), 5-bromo-2-chloropyrimidine (43.2 mg, 0.224 mmol), potassium fluoride (35.4 mg, 0.610 mmol), Pd(dppf)Cl2 DCM (3.32 mg, 0.00406 mmol) and degassed 1,4-dioxane / water (0.5 mL, (4+1)). The vessel was evacuated and filled with argon (3×) and heated to 60° C. for 2 h. After cooling, the mixture is distributed between saturated NH4Cl solution and EtOAc, and the organic phase is dried and evaporated with Na2SO4. The residue is dissolved in 1mL DCM and 1mL TFA and stirred overnight. The mixture is poured into water, extracted with EtOAc, and the extract is washed with NH4Cl solution, dried and evaporated with Na2SO4. The product is passed through flash chromatography (solvent: MeOH; DCM / EtOAc of 20%-80% is used as solvent system, and the product is not eluted from chromatographic column). The fraction containing the product is evaporated to dryness and the residue is ground with toluene and MeOH. HPLC shows that the purity is only 87%. The solid is washed and dried with DCM, MeCN and ether (33.0mg, 0.0596mmol, yield 29%).

[0356] Analyze the data:

[0357] 1H NMR (400MHz, DMSO-d6) δ14.92(s,1H),9.67(s,1H),9.28(s,2H),9.09(d,J=2.1Hz,1H),8.98(d,J=2.2Hz,1H),7 .87(dd,J=14.9,7.6Hz,1H),7.40(t,J=8.7Hz,1H),3.22–3.10(m,2H),1.86–1.74(m,2H),1.00(t,J=7.5Hz,3H);

[0358] C 20 H 15 Calculated exact mass of ClF2N6O3S (molecular weight: 492.89): 492.06;

[0359] MS (ESI - ):m / z:490.9[M-1]-.

[0360] Example 13: Synthesis of N-(3-(5-(4-chlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4,6-trifluorophenyl)propane-1-sulfonamide

[0361]

[0362] Step 13-1: (3-amino-2,4,6-trifluorophenyl)-(5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)methanone

[0363] A solution of 2,4,6-trifluoroaniline (941 mg, 6.40 mmol) and chlorotrimethylsilane (1.62 mL, 12.8 mmol) in THF (5.82 mL) was cooled to -78 °C and a 2M solution of lithium diisopropylamide (6.40 mL, 12.8 mmol) in THF / heptane / ethylbenzene was added dropwise. The mixture was warmed to room temperature and stirred for 30 min. 5-bromo-N-methoxy-N-methyl-1H-pyrazolo[3,4-b]pyridine-3-carboxamide (829 mg, 2.91 mmol) was added and the mixture was cooled to -30 °C. A solution of lithium diisopropylamide (4.65 mL, 9.30 mmol) in THF / heptane / ethylbenzene was added dropwise and the mixture was stirred at -15 °C for 20 min. Concentrated HCl (3 mL) was added and the mixture was stirred at room temperature for 10 min. The reaction was neutralized with 2N NaOH, the aqueous phase was separated and extracted with THF. The combined organic phases were dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography (DCM / EtOAc, 0%-25%) and triturated with a small amount of DCM to give (3-amino-2,4,6-trifluorophenyl)-(5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)methanone (0.423 g, 1.14 mmol, 39% yield) as a bright yellow solid.

[0364] Analyze the data:

[0365] 1 H-NMR (200MHz, CDCl3) δ14.96 (s, 1H), 8.77 (d, J = 2.8Hz, 3H), 7.21 (t, J = 10Hz, 2H), 5.33 (s, 4H).

[0366] MS (ESI + ):m / z=370.8 / 368.8[MH] - ,350.8 / 348.8[MH-HF] - .

[0367] Step 13-2: N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4,6-trifluorophenyl]propane-1-sulfonamide

[0368] To a solution of (3-amino-2,4,6-trifluorophenyl)-(5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)methanone (0.378 g, 1.02 mmol) and TEA (1.56 mL, 11.2 mmol) in THF (5.09 mL) was slowly added 1-propylsulfonyl chloride (0.378 mL, 3.36 mmol) in 1 mL THF at -10 °C. The reactant was stirred at 0 °C for 30 min and 2N NaOH (4.07 mL, 8.15 mmol) was added. After stirring at room temperature for 10 min, THF was evaporated from the mixture and the aqueous solution was washed with EtOAc. The solution was acidified with 2N HCl and extracted with EtOAc. The extract was dried over Na2SO4, evaporated and the residue was triturated with n-hexane to give N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4,6-trifluorophenyl]propane-1-sulfonamide (0.344 g, 0.7210 mmol, 71% yield).

[0369] Analyze the data:

[0370] 1 H NMR (200MHz, DMSO-d6) δ8.78(dd,J=5.7,2.2Hz,2H),7.57(td,J=9.8,2.0Hz,1H),3.18-3.05(m,3H),1.90–1.68(m,2H),0.98(t,J=7.4Hz,3H);

[0371] MS (ESI - ):476.8 / 474.8[MH] - ,456.9 / 454.9[MH-HF] - .

[0372] Step 13-3: N-[3-[5-(4-chlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]-2,4,6-trifluorophenyl]propane-1-sulfonamide

[0373] A microwave vessel was charged with N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4,6-trifluorophenyl]propane-1-sulfonamide (78.0 mg, 0.163 mmol), (4-chlorophenyl)boronic acid (26.8 mg, 0.172 mmol) and purged with argon. Degassed 1,4-dioxane (0.545 mL) and degassed 1.5 M aqueous potassium carbonate solution (0.327 mL, 0.490 mmol) were added and the reaction was heated to 110 °C under microwave irradiation for 1 h. The reaction was partitioned between NH4Cl solution and EtOAc and the organic phase was evaporated. The residue was purified by flash chromatography (DCM / EtOAc 5%-35%) and triturated with a mixture of DCM and n-hexane to give N-[3-[5-(4-chlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]-2,4,6-trifluorophenyl]propane-1-sulfonamide (44.0 mg, 0.0865 mmol, 53% yield).

[0374] Analyze the data:

[0375] 1H NMR (400MHz, DMSO-d6) δ14.97(s,1H),9.66(s,1H),9.04(d,J=2.2Hz,1H),8.77(d,J=2.2Hz,1H),8 .05–7.69(m,2H),7.71–7.44(m,3H),3.21–3.02(m,2H),1.92–1.62(m,2H),0.99(t,J=7.4Hz,3H);

[0376] C 22 H 16 Calculated exact mass of ClF3N4O3S (molecular weight: 508.90): 508.06;

[0377] MS (ESI - ):m / z:507.0[M-1]-.

[0378] Example 14: Synthesis of N-(3-(5-(2-cyclopropylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)-1-phenylmethanesulfonamide

[0379] Example 14 was synthesized similarly to Example 11.

[0380]

[0381] Analyze the data:

[0382] 1H NMR(400MHz,dmso)δ15.02–14.46(m,1H),9.77(s,1H),9.10(s,2H),9.05(d,J=2.1Hz,1H),8.88(d,J=2.1H z,1H),7.86(dd,J=14.3,7.5Hz,1H),7.46–7.34(m,6H),4.51(s,2H),2.35–2.24(m,1H),1.15–1.06(m,4H).

[0383] C 27 H 20 Calculated exact mass of F2N6O3S (molecular weight: 546.55): 546.13

[0384] MS (ESI + ):m / z 546.0[M+H] + .

[0385] Example 15: Synthesis of N-(2,6-difluoro-3-(5-(2-methoxypyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide

[0386] Example 15 was synthesized similarly to Example 7.

[0387]

[0388] Analyze the data:

[0389] 1 H NMR (400MHz, DMSO-d6) δ14.87(s,1H),9.67(s,1H),9.09(s,2H),9.04(d,J=1.7Hz,1H),8.86(d,J=1.6Hz,1H),7.86(dd,J=1 4.3,7.6Hz,1H),7.40(t,J=8.8Hz,1H),4.00(s,3H),3.19–3.09(m,2H),1.81(td,J=14.8,7.3Hz,2H),0.99(t,J=7.3Hz,3H).

[0390] C 21 H 18 Calculated exact mass of F2N6O4S (molecular weight: 488.47): 488.11

[0391] MS (ESI + ):m / z 489.05[M+H] + .

[0392] Example 16: Synthesis of N-(2,6-difluoro-3-(5-(2-hydroxypyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide

[0393] Example 16 was synthesized similarly to Example 7.

[0394]

[0395] Analyze the data:

[0396] 1 H NMR(400MHz,DMSO-d6)δ14.78(s,1H),12.38(s,1H),9.66(s,1H),8.95(s,1H),8.77(s,1H),8.49(s,2H),7.85(dd, J=14.7,7.7Hz,1H),7.39(t,J=9.0Hz,1H),3.18–3.11(m,2H),1.81(td,J=14.8,7.3Hz,2H),1.00(t,J=7.4Hz,3H).

[0397] C 20 H 16 Calculated exact mass of F2N6O4S (molecular weight: 474.44): 474.09

[0398] MS (ESI + ):m / z 475.00[M+H] + .

[0399] Example 17: Synthesis of N-(3-(5-(3-chloropyridin-4-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0400] Example 17 was synthesized similarly to Example 7.

[0401]

[0402] Analyze the data:

[0403] 1H NMR (400MHz, DMSO-d6) δ15.05–14.58(m,1H),9.66(s,1H),8.83(s,2H),8.76(d,J=1.6Hz,1H),8.68(d,J=4.8Hz,1H),7.87(dd,J=14 .3,8.0Hz,1H),7.70(d,J=4.9Hz,1H),7.40(t,J=8.9Hz,1H),3.19–3.10(m,2H),1.81(dq,J=14.8,7.3Hz,2H),1.00(t,J=7.4Hz,3H).

[0404] C 21 H 16 Calculated exact mass of ClF2N5O3S (molecular weight: 491.90): 491.06

[0405] MS (ESI + ):m / z 491.95[M+H] + .

[0406] Example 18: Synthesis of N-(2,6-difluoro-3-(5-(3-methylpyridin-4-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide

[0407] Example 18 was synthesized similarly to Example 7.

[0408]

[0409] Analyze the data:

[0410] 1 H NMR (400MHz, DMSO-d6) δ14.82(s,1H),9.88–9.37(m,1H),8.75(d,J=1.9Hz,1H),8.56(d,J=1.6Hz,1H),8.55(d,J=0.9Hz,1H),7.85(dd,J=14.7,7. 5Hz,1H),7.78(d,J=7.6Hz,1H),7.39(dd,J=10.8,5.9Hz,2H),3.17–3.11 (m,2H),2.48(s,3H),1.81(dq,J=14.9,7.4Hz,2H),1.00(t,J=7.4Hz,3H).

[0411] C 22 H 19 Calculated exact mass of F2N5O3S (molecular weight: 471.48): 471.12

[0412] MS (ESI+ ):m / z 471.9[M+H] + .

[0413] Example 19: Synthesis of N-(3-(5-(4-(tert-butyl)phenyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0414] Example 19 was synthesized similarly to Example 7.

[0415]

[0416] Analyze the data:

[0417] 1 H NMR (400MHz, DMSO-d6) δ14.78(s,1H),9.67(s,1H),9.00(d,J=2.1Hz,1H),8.73(d,J=2.1Hz,1H),7.86(dd,J=14.7,7.6Hz,1H),7.75(d,J=8. 3Hz,2H),7.57(d,J=8.4Hz,2H),7.40(t,J=8.8Hz,1H),3.18–3.12(m,2H),1.81(td,J=15.0,7.4Hz,2H),1.34(s,9H),1.00(t,J=7.4Hz,3H).

[0418] C 26 H 26 Calculated exact mass of F2N4O3S (molecular weight: 512.56): 512.17

[0419] MS (ESI + ):m / z 513.0[M+H] + .

[0420] Example 20: Synthesis of N-(3-(5-(2-chloro-4-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0421] Example 20 was synthesized similarly to Example 7.

[0422]

[0423] Analyze the data:

[0424] 1H NMR(400MHz,DMSO-d6)δ14.82(s,1H),9.66(s,1H),8.72(d,J=1.8Hz,1H),8.5 7(d,J=1.8Hz,1H),7.86(dd,J=15.0,7.4Hz,1H),7.53(d,J=8.6Hz,1H),7.39( t,J=8.7Hz,1H),7.25(d,J=2.3Hz,1H),7.10(dd,J=8.6,2.4Hz,1H),3.86(s,3 H), 3.15 (t, J = 7.6Hz, 2H), 1.81 (dq, J = 15.0, 7.5Hz, 2H), 1.00 (t, J = 7.4Hz, 3H).

[0425] C 23 H 19 Calculated exact mass of ClF2N4O4S (molecular weight: 520.94): 520.08

[0426] MS (ESI + ):m / z 521.0[M+H] + .

[0427] Example 21: Synthesis of N-(3-(5-(2-(tert-butyl)pyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0428] Example 21 was synthesized similarly to Example 7.

[0429]

[0430] Analyze the data:

[0431] 1 H NMR (400MHz, DMSO-d6) δ14.88(s,1H),9.66(s,1H),9.22(s,2H),9.08(s,1H),8.91(s,1H),7.86(dd,J=14.7, 7.3Hz,1H),7.40(t,J=8.7Hz,1H),3.18–3.11(m,2H),1.87–1.74(m,2H),1.43(s,9H),1.00(t,J=7.2Hz,3H).

[0432] C 24 H 24 Calculated exact mass of F2N6O3S (molecular weight: 514.55): 514.16

[0433] MS (ESI +):m / z 515.15[M+H] + .

[0434] Example 22: Synthesis of N-(2,6-difluoro-3-(5-(2-(methylthio)pyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide

[0435] Example 22 was synthesized similarly to Example 7.

[0436]

[0437] Analyze the data:

[0438] 1 H NMR (400MHz, DMSO-d6) δ14.87(s,1H),9.67(s,1H),9.13(s,2H),9.06(d,J=1.9Hz,1H),8.90(d,J=1.9Hz,1H),7.86(dd ,J=14.3,7.5Hz,1H),7.40(t,J=8.8Hz,1H),3.19–3.12(m,2H),2.60(s,3H),1.87–1.75(m,2H),1.00(t,J=7.4Hz,3H).

[0439] C 21 H 18 Calculated exact mass of F2N6O3S2 (molecular weight: 504.53): 504.08

[0440] MS (ESI + ):m / z 505.05[M+H] + .

[0441] Example 23: Synthesis of N-(2,6-difluoro-3-(5-(2-isopropylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide

[0442] Example 23 was synthesized similarly to Example 7.

[0443]

[0444] Analyze the data:

[0445] 1H NMR (400MHz, DMSO-d6) δ14.87(s,1H),9.67(s,1H),9.13(s,2H),9.06(d,J=1.9Hz,1H),8.90(d,J=1.9Hz,1H),7.86(dd ,J=14.3,7.5Hz,1H),7.40(t,J=8.8Hz,1H),3.19–3.12(m,2H),2.60(s,3H),1.87–1.75(m,2H),1.00(t,J=7.4Hz,3H).

[0446] C 23 H 22 Calculated exact mass of F2N6O3S (molecular weight: 500.52): 500.14

[0447] MS (ESI + ):m / z 501.05[M+H] + .

[0448] Example 24: Synthesis of N-(2,6-difluoro-3-(5-(4-fluoro-2-methylphenyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide

[0449] Example 24 was synthesized similarly to Example 7.

[0450]

[0451] Analyze the data:

[0452] 1 H NMR (400MHz, DMSO-d6) δ14.82(s,1H),9.67(s,1H),8.68(d,J=2.1Hz,1H),8.48(d,J=2.1Hz,1H),7.86(dd,J=14.8,7.6Hz,1H),7.42–7.36(m,2H) ,7.26(dd,J=10.1,2.5Hz,1H),7.17(td,J=8.5,2.6Hz,1H),3.18–3.11( m,2H),2.28(s,3H),1.81(dq,J=15.0,7.4Hz,2H),1.00(t,J=7.4Hz,3H).

[0453] C 23 H 19 Calculated exact mass of F3N4O3S (molecular weight: 488.49): 488.11

[0454] MS (ESI + ):m / z 489.05[M+H]+ .

[0455] Examples 25 and 26: Synthesis of N-(2,6-difluoro-3-(5-(4-methoxy-2-methylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (Example 25) and N-(2,6-difluoro-3-(5-(4-hydroxy-2-methylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (Example 26)

[0456] Step 1: Similarly to Example 9, Step 9-1, conversion of N-(2,6-difluoro-3-(1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide with 5-bromo-4-chloro-2-methylpyrimidine was carried out.

[0457] Two different deprotection methods afforded Example 25 or Example 26.

[0458]

[0459] Analytical data of N-(2,6-difluoro-3-(5-(4-methoxy-2-methylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide:

[0460] 1 H NMR (400MHz, DMSO-d6) δ14.84(s,1H),9.66(s,1H),8.86(d,J=2.0Hz,1H),8.75(d,J=2.0Hz,1H),8.64(s,1H),7.85(dd,J=15.4,7. 4Hz,1H),7.39(t,J=8.7Hz,1H),3.99(s,3H),3.18–3.10(m,2H),2.62(s,3H),1.81(td,J=15.3,7.7Hz,2H),1.00(t,J=7.5Hz,3H).

[0461] C 22 H 20 Calculated exact mass of F2N6O4S (molecular weight: 502.50): 502.12

[0462] MS (ESI + ):m / z 502.95[M+H] + .

[0463] Analytical data of N-(2,6-difluoro-3-(5-(4-hydroxy-2-methylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide:

[0464] 1 H NMR (400MHz, DMSO-d6) δ14.84(s,1H),9.66(s,1H),8.86(d,J=2.0Hz,1H),8.75(d,J=2.0Hz,1H),8.64(s,1H),7.85(dd,J=15.4,7. 4Hz,1H),7.39(t,J=8.7Hz,1H),3.99(s,3H),3.18–3.10(m,2H),2.62(s,3H),1.81(td,J=15.3,7.7Hz,2H),1.00(t,J=7.5Hz,3H).

[0465] C 21 H 18 Calculated exact mass of F2N6O4S (molecular weight: 488.47): 488.11

[0466] MS (ESI + ):m / z 489.15[M+H] + .

[0467] Example 27: Synthesis of N-(2,6-difluoro-3-(5-(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide

[0468] Example 27 was synthesized similarly to Example 11.

[0469]

[0470] Analyze the data:

[0471] 1 H NMR (400MHz, DMSO-d6) δ14.86(s,1H),9.67(s,1H),9.05(dd,J=10.7,2.0Hz,2H),8.83(d,J=2.2Hz,1H),8.66(dd,J=4.8,1.5Hz,1H),8.30–8.23(m, 1H),7.87(dd,J=14.9,7.5Hz,1H),7.57(dd,J=7.5,4.8Hz,1H),7.40(t,J =8.5Hz,1H),3.17–3.12(m,2H),1.86–1.75(m,2H),1.00(t,J=7.5Hz,3H);

[0472] C 21 H 17 Calculated exact mass of F2N5O3S (molecular weight: 457.46): 457.10

[0473] MS (ESI - ):m / z:455.9[M-1]-.

[0474] Example 28: Synthesis of 5-(3-(2,4-difluoro-3-(propylsulfonylamino)benzoyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)picolinic acid

[0475] Example 28 was synthesized similarly to Example 11.

[0476]

[0477] Analyze the data:

[0478] 1 H NMR (400MHz, DMSO-d6) δ14.92(s,1H),9.68(s,1H),9.15(dd,J=16.8,2.0Hz,2H),8.93(d,J=2.0Hz,1H),8.45(dd,J=8.1,2.3Hz,1H),8 .18(d,J=8.1Hz,1H),7.88(dd,J=14.5,7.7Hz,1H),7.41(t,J=8.7Hz,1H),3.19–3.10(m,2H),1.86–1.75(m,2H),1.00(t,J=7.5Hz,3H);

[0479] C 22 H 17 The calculated exact mass of F2N5O5S (molecular weight: 501.46) is 501.09;

[0480] MS (ESI - ):m / z:500.1[M-1]-

[0481] Example 29: Synthesis of N-[2,6-difluoro-3-[5-(4-methylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide

[0482] Example 29 was synthesized similarly to Example 7.

[0483]

[0484] Analyze the data:

[0485] 1H NMR (400MHz, DMSO-d6) δ14.90(s,1H),9.66(s,1H),9.12(s,1H),8.80(d,J=2.1Hz,1H),8.76(s,1H),8.68(d,J=2.1Hz, 1H),7.90–7.82(m,1H),7.43–7.36(m,1H),3.30(s,3H),3.19–3.11(m,3H),1.88–1.73(m,3H),1.00(t,J=7.4Hz,3H).;

[0486] C 21 H 18 The calculated exact mass of F2N6O3S (molecular weight: 472.47) is 472.11;

[0487] MS (ESI + ):m / z:473.55[M+H] + .

[0488] Example 30: Synthesis of N-[3-[5-(2,4-dimethylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide

[0489] Example 30 was synthesized similarly to Example 7.

[0490]

[0491] Analyze the data:

[0492] 1 H NMR (400MHz, DMSO-d6) δ14.88(s,1H),9.67(s,1H),8.77(d,J=1.6Hz,1H),8.67–8.59(m,2H),7.86(dd,J=14.6,7.5H z,1H),7.40(t,J=8.8Hz,1H),3.20–3.10(m,2H),2.66(s,3H),2.45(s,3H),1.87–1.74(m,2H),0.99(t,J=7.4Hz,3H).

[0493] C 22 H 20 The calculated exact mass of F2N6O3S (molecular weight: 486.49) is 486.13;

[0494] MS (ESI + ):m / z:486.9[M+H] + .

[0495] Example 31: Synthesis of N-(3-(5-(2-cyclopropyl-4-methylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0496]

[0497] Step 1: Synthesis of N-(3-(5-(2-cyclopropyl-4-methylpyrimidin-5-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide (C)

[0498] A solution of N-(2,6-difluoro-3-(1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (300 mg, 0.51 mmol), compound B (119 mg, 0.56 mmol) and KF (144 mg, 1.53 mmol) in 1,4-dioxane / H2O (10 mL) was stirred at room temperature and then purged with argon for 5 min. PdCl2(dppf).CH2Cl2 (42 mg, 0.051 mmol) was added and the mixture was purged with argon again for 5 min. The reactants were then heated to reflux for 8 h. The progress of the reaction was monitored by TLC using (20% EtOAc in hexanes v / v). After completion, the reaction mixture was gradually cooled to room temperature and diluted with ethyl acetate (20 mL), filtered through a celite pad and concentrated under reduced pressure to obtain the crude compound. The crude product was finally purified by FCC to obtain 135 mg (45%) of the desired compound C.

[0499] 1 H NMR(400MHz, DMSO-d6)δ:9.69(s,1H),8.81(d,1H,J=2.0Hz),8.64(d,1H,J=2.0Hz),8.56(s,1H),7.93–7.85(m,1H) ,7.43(t,1H,J=8.8Hz),6.21(d,1H,J=9.6Hz),3.93–3.97(m,1H),3.71–3.80(m,1H),3.14(t,2H,J=7.6Hz),2.43(br s,3H),2.21–2.27(m,1H),1.95–2.01(m,2H),1.67–1.75(m,3H),1.55–1.62(m,3H),1.10–1.20(m,4H),0.99(t,3H,J=7.2Hz).

[0500] MS (ESI + ):m / z 597.1(M+H + )

[0501] Step-2: Synthesis of N-(3-(5-(2-cyclopropyl-4-methylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0502] p-TSA (28 mg, 0.24 mmol) was added to a solution of compound C (135 mg, 0.22 mmol) in MeOH (10 mL) at room temperature, and the reaction mixture was heated to 50 ° C for 6 h. The reaction progress was monitored by TLC using (5% MeOH in DCM v / v). After the reaction was complete, the solvent was evaporated in vacuo, water (5 mL) was added, the mixture was neutralized with a saturated NaHCO3 aqueous solution, and extracted with EtOAc (3 × 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude compound, which was ground to obtain 55 mg (48%) of the desired compound as an off-white solid.

[0503] 1 H NMR(400MHz,DMSO-d6)δ:9.67(br s,1H),8.76(d,1H,J=2.0Hz),8.61(d,1H,J=2.0Hz),8.55(s,1H),7.82–7.88(m,1H),7.39(t,1H,J=8.80Hz),3.14(t ,2H,J=7.60Hz),2.42(s,3H),2.20–2.28(m,1H),1.80(q,2H,J=7.60Hz),1.05–1.11(m,4H),0.99(t,3H,J=7.20Hz).

[0504] C 24 H 22 The calculated exact mass of F2N6O3S (molecular weight: 512.53) is 512.14;

[0505] MS (ESI + ):m / z 513.60[M+H] + .

[0506] Example 32: Synthesis of N-(3-(5-(2-cyclopropyl-4-(methylthio)pyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0507] Part A. Synthesis of 5-bromo-2-cyclopropyl-4-(methylthio)pyrimidine (D)

[0508]

[0509] Step-1: 5-Bromo-4-chloro-2-(methylsulfonyl)pyrimidine (B)

[0510] To a stirred solution of 5-bromo-4-chloro-2-(methylthio)pyrimidine (3g, 12.6mol) in THF (25mL) at room temperature was added potassium monopersulfate (oxone) (1.16g, 37.8mmol) dissolved in water (15mL). The mixture was subsequently stirred for 4h. As observed by TLC, after the reaction was completed using EtOAc / n-hexane (30 / 70% v / v) as a solvent, the reactant was diluted with water (50mL) and extracted with EtOAc (3×25mL). The combined separated organic layer was washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure to obtain 3.2g (94%) of compound B as an off-white solid.

[0511] 1 H NMR(400MHz,DMSO-d6)δ:9.38(s,1H),3.41(s,3H)

[0512] (ESI + ):m / z 272.80[M+H] +

[0513] Step-2: 5-Bromo-4-chloro-2-cyclopropylpyrimidine (C)

[0514] 1M cyclopropylmagnesium bromide (6.66mL, 6.66mmol) was slowly added to a stirred solution of anhydrous THF (20mL) of compound B (1.5g, 5.55mmol) at 0°C. The mixture was then gradually warmed to room temperature and stirred for 2h. As observed by TLC, after the reaction was completed using EtOAc / n-hexane (20 / 80% v / v) as a solvent, NH4Cl aqueous solution was added to quench the reaction, and extracted with EtOAc (2×25mL). The separated combined organic layer was washed with brine (15mL), dried over anhydrous Na2SO4 and evaporated under reduced pressure to obtain a crude compound. The crude product was finally purified by FCC to obtain 600mg (52%) of compound C required as an off-white solid.

[0515] 1 H NMR(400MHz,DMSO-d6)δ:8.59(s,1H),2.05–2.25(m,1H),1.05–1.10(m,4H)

[0516] MS (ESI + ):m / z 234.8[M+H] + .

[0517] Step-3: 5-Bromo-2-cyclopropyl-4-(methylthio)pyrimidine (D)

[0518] At room temperature, NaSMe (544 mg, 7.75 mol) was added to a stirred solution of compound C (1.2 g, 5.17 mmol) in DMF (10 mL), and the mixture was stirred at room temperature for 16 h. As observed by TLC, after the reaction was completed using EtOAc / n-hexane (10 / 90% v / v) as a solvent, water (30 mL) was added and extracted with EtOAc (2×25 mL), and the separated organic layer was washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure to obtain 800 mg (80%) of compound D as a yellow liquid.

[0519] 1 H NMR(400MHz, CDCl3)δ:8.25(s,1H),2.52(s,3H),4.10–4.23(m,1H),1.10–1.20(m,4H)

[0520] MS (ESI + ):m / z 246.8[M+H] + .

[0521] Part B: Synthesis of N-(3-(5-(2-cyclopropyl-4-(methylthio)pyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0522]

[0523] Step-1: N-(3-(5-(2-cyclopropyl-4-(methylthio)pyrimidin-5-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide (C)

[0524] To a stirred solution of N-(2,6-difluoro-3-(1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (400 mg, 0.67 mmol) and compound D (182 mg, 0.74 mmol) in 5 ml of 1,4-dioxane / H2O (75 / 25% v / v) was added 118 mg (2.03 mmol) of KF dissolved in 1 mL of water. The resulting mixture was purged with argon for 5 min, then Pd(dppf)Cl2 DCM (0.1 eq, 55 mg, 0.067 mmol) was added and the mixture was purged with argon again for 5 min. The reaction was then heated at 90°C for 6 h. After the reaction was completed, water (10 mL) was added and the pH was adjusted to 6 with 1N HCl. The mixture was extracted with EtOAc (3×15 mL), and the organic layer was separated, dried and concentrated to give a crude residue. The crude material was purified by reverse phase preparative HPLC to give 60 mg (yield 14%) of compound C.

[0525] MS (ESI + ):m / z 629.20[M+H] + .

[0526] Step 2: N-(3-(5-(2-cyclopropyl-4-(methylthio)pyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3carbonyl)-2,6-difluorophenyl)propane-1-sulfonamide

[0527] pTSA (74 mg, 0.42 mmol) was added to a stirred solution of MeOH (2 mL) of compound C (90 mg, 0.143 mmol) at room temperature, and the reaction mixture was heated to 65 ° C for 16 h. EtOAc / n-hexane (70 / 30% v / v) was used to monitor the reaction progress by TLC. After the reaction was completed, the solvent was evaporated in vacuo, water (5 mL) was added, then neutralized (pH=7) with NaHCO aqueous solution, and extracted with EtOAc (3×5 mL). The separated organic layer was dried over anhydrous Na SO and concentrated under reduced pressure to obtain a crude product, which was purified by SFC to obtain 25 mg (32%) of the desired compound.

[0528] 1H NMR(400MHz, DMSO-d6)δ:9.66(s,1H),8.72(s,1H),8.69(s,1H),8.37(s,1H),7.86(q,1H,J=6.8Hz) ,7.39(t,1H,J=8.8Hz),3.14(t,2H,J=8.0Hz),2.20–2.30(m,1H),1.70–1.86(m,2H),1.10–1.15(br s,4H),0.99(t,3H,J=7.2Hz).

[0529] C 24 H 22 The calculated exact mass of F2N6O3S (molecular weight: 512.53) is 512.14;

[0530] MS (ESI + ):m / z 545.10[M+H] + .

[0531] Example 33: Synthesis of N-[2,6-difluoro-3-[5-(4-methyl-2-methanesulfonyl-pyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]phenyl]propane-1-sulfonamide

[0532] Example 33 was synthesized similarly to Example 7.

[0533]

[0534] Analyze the data:

[0535] 1 H NMR (400MHz, DMSO-d6) δ14.79(s,1H),9.73(s,1H),8.77(d,J=2.0Hz,1H),8.65(d,J=2.0Hz,1H),8.58(s,1H),7.86(dd,J=14 .6,7.8Hz,1H),7.39(t,J=9.0Hz,1H),3.21–3.05(m,2H),2.57(s,3H),2.44(s,3H),1.91–1.68(m,2H),0.99(t,J=7.4Hz,3H).

[0536] C 22 H 20 The calculated exact mass of F2N6O3S2 (molecular weight: 518.56) is 518.10;

[0537] MS (ESI - ):m / z:517.5[M-1] - .

[0538] Example 34: Synthesis of N-[3-[5-(2,4-dimethoxypyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide

[0539] Example 34 was synthesized similarly to Example 7.

[0540]

[0541] Analyze the data:

[0542] 1 H NMR (400MHz, DMSO-d6) δ14.80(s,1H),9.66(s,1H),8.81(d,J=2.1Hz,1H),8.69(d,J=2.1Hz,1H),8.54(s,1H),7.84(dd,J =14.5,7.7Hz,1H),7.38(t,J=8.8Hz,1H),4.05–3.89(m,6H),3.18–3.09(m,2H),1.86–1.72(m,2H),0.98(t,J=7.4Hz,3H).

[0543] C 22 H 20 The calculated exact mass of F2N6O5S (molecular weight: 518.49) is 518.12;

[0544] (ESI + ):m / z 519.15[M+H] +

[0545] Example 35: Synthesis of N-(3-(5-(2-cyclopropylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4,6-trifluorophenyl)propane-1-sulfonamide

[0546]

[0547] Step-1: Synthesis of (3-amino-2,4,6-trifluorophenyl)(5-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-3-yl)methanone (B)

[0548] Solution A: At -10 ° C, a solution of 2M isopropylmagnesium chloride (9.33 mL, 1 equivalent, 18.68 mmol) in anhydrous THF (24 mL) of compound A (4.22 g, 18.68 mmol) was added dropwise in THF, keeping the temperature below 10 ° C. After stirring at 25 ° C for 20 min, the reactant was cooled to 0 ° C and trimethylsilyl chloride (2.02 g, 1 equivalent, 18.68 mmol) was slowly added. The mixture was warmed to 25 ° C and stirred for 30 min. The solution was cooled to -10 ° C again and a THF solution of 2M isopropylmagnesium chloride (9.33 mL, 1 equivalent, 18.68 mmol) was added dropwise, keeping the temperature below 10 ° C. After stirring at 25 ° C for 20 min, the reactant was cooled to 0 ° C and trimethylsilyl chloride (2.02 g, 1 equivalent, 18.68 mmol) was slowly added. The mixture was warmed to 25 ° C and stirred for 30 min. The reaction was cooled to -10°C and 2M isopropylmagnesium chloride in THF (9.33 mL, 1 eq., 18.68 mmol) was added dropwise. After the addition was complete, the mixture was stirred at 0°C for 20 min.

[0549] Solution B: In a second flask, 2M isopropylmagnesium chloride in THF (9.33 mL, 1 eq., 18.68 mmol) was added dropwise to a suspension of 5-bromo-N-methoxy-N-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboxamide (3 g, 8.12 mmol) in THF (30 mL) at -10°C, keeping the temperature below 0°C. After the addition was complete, the mixture was stirred at 0°C for 10 min.

[0550] Solution B was added to solution A and the reaction was stirred at 25°C for 2 h. 4N aqueous HCl was then carefully added until all the magnesium salts were dissolved. 30% NaOH was used to aq Adjust the pH of the aqueous solution to 5, saturate with NaCl and separate the layers. Extract the aqueous layer with a mixture of THF and EtOAc (addition of EtOAc is necessary to separate the layers). Evaporate the extract, redissolve in THF and combine with the organic phase. Add concentrated HCl aq In 4%MeOH (5mL), add 4-nitropropene (2-nitropropene) (5mL), and at room temperature the mixture is stirred for 10min.By the mixture solid NaHCO3 neutralization, discard lower aqueous layer, organic layer is through Na2SO4 dry.After evaporating solvent, residue is suspended in ether (15mL), grinds with DCM (20mL) and collects solid by suction filtration.Evaporation filtrate, residue is dissolved in ether (20mL), and collects solid by suction filtration.Use the DCM solution of 0-5%MeOH to merge solid by FCC purification, obtain 900mg (38%) expected off-white solid.

[0551] 1 H NMR (400MHz, DMSO-d6) δ: 8.86 (d, 1H, J = 2.0Hz), 8.80 (d, 1H, J = 2.0Hz), 7.25–7.35 (m, 1H), 6.14 (d, 1H, J = 10Hz), 5.36 (br s,2H),3.90–3.95(m,1H),3.70–3.85(m,1H),2.25–2.33(m,1H),1.65–1.85(m,1H),1.50–1.62(m,2H),1.10–1.15(m,2H).

[0552] (ESI + ):m / z 455.18[M+H] +

[0553] Step 2: Synthesis of N-(3-(5-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4,6-trifluorophenyl)propane-1-sulfonamide (C)

[0554] At -10 ° C, an equal volume of THF solution of 1-propylsulfonyl chloride (0.8 mL, 7 mmol) was slowly added to a THF (20 mL) solution of compound B (900 mg, 1.98 mmol) and triethylamine (13.7 mL, 10 mmol), and the reactant was stirred at -10 ° C for 30 min. 2N NaOH (3.75 mL, 12 mmol) was added and THF was evaporated under reduced pressure at 45 ° C. The aqueous solution was washed with EtOAc (2 × 20 mL) and ether. The residual organic matter was evaporated, and the solution was acidified with 2N HCl and stirred vigorously for 30 min to break up larger lumps. The solid was collected by suction filtration, washed with water and dried at 100 ° C to obtain compound C (960 mg, yield 87%) as an off-white solid. 1 H NMR showed a mixture of mono- and di-sulfonamides which were used as such in the next step.

[0555] Step 3: Synthesis of N-(2,4,6-trifluoro-3-(1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (D)

[0556] To compound C (900mg, 1.60mmol) 1,4-dioxane (15mL) stirring solution, add bis (pinacol) diboron (612mg, 2.41mmol) and KF (278mg, 4.8mmol), and the resulting mixture is degassed with argon for 10min. Add Pd (dppf) Cl2 DCM (65mg, 0.08mmol), the mixture is further degassed with argon for 5min and stirred at 85 ° C for 8h. Using EtOAc / normal hexane (60 / 40% v / v) as solvent, the reaction process is monitored by TLC. After completion, the reaction mixture is cooled to room temperature, diluted with EtOAc (20mL), filtered through a diatomaceous earth pad and the organic layer is concentrated under reduced pressure to obtain a crude compound, which is purified by FCC to obtain compound D (960mg, 87%).

[0557] MS (ESI + ):m / z 609.0[M+H] + .

[0558] Step 4: Synthesis of N-(3-(5-(2-cyclopropylpyrimidin-5-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4,6-trifluorophenyl)propane-1-sulfonamide (F)

[0559] KF (271 mg, 4.68 mmol) was added to a stirred solution of 1,4-dioxane / H2O (10 mL) of compound D (950 mg, 1.56 mmol) and compound E (342 mg, 1.71 mmol), and the resulting mixture was degassed with argon for 10 min. Pd (dppf) Cl2 DCM (64 mg, 0.078 mmol) was added, and the reaction mixture was further degassed with argon for 5 min and stirred at 90 ° C for 8 h. EtOAc / normal hexane (40 / 60% v / v) was used as solvent, and the reaction progress was monitored by TLC. After completion, the reaction mixture was cooled to room temperature and diluted with EtOAc (20 mL), filtered through a diatomaceous earth pad and the organic layer was concentrated under reduced pressure to obtain a crude compound, which was ground with ether to obtain compound F (650 mg, 61%).

[0560] 1H NMR(400MHz, DMSO-d6)δ:9.10–9.15(m,3H),8.89(d,1H,J=2.4Hz),7.80(t,1H,J=8.8Hz),6.66(dd,1H,J=2.4&10.4Hz),3.60– 3.80(m,4H),2.22–2.38(m,3H),1.95–2.00(m,2H),1.72–1.93(m,3H),1.55–1.60(m,2H),1.06-1.09(m,2H),0.95–1.05(m,5H)

[0561] Step 5: Synthesis of N-(3-(5-(2-cyclopropylpyrimidin-5-yl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4,6-trifluorophenyl)propane-1-sulfonamide

[0562] In compound F (650mg, 1.08mmol), TFA (10mL) is added in room temperature and the mixture is stirred overnight.As observed by TLC, after using (MeOH / DCM 5 / 95%v / v) to complete the reaction, N2 stream is used at room temperature to evaporate the reaction mixture to obtain crude compound.Use saturated NaHCO3 aqueous solution to neutralize the pH of crude material to 8, then extract with DCM (2 × 20mL), separate the organic layer and through anhydrous Na2SO4 dry, evaporate the solvent to obtain crude material.Finally use the DCM solution (v / v) of 0-5%MeOH to purify the crude product by FCC, obtain 175mg (32%) as the required compound of off-white solid.

[0563] 1 H NMR(400MHz,DMSO-d6):13.05(br s,1H),9.66(br s,1H),9.11(s,2H),9.07(s,1H),8.88(s,1H),7.59(t,1H,J=8.0Hz),3.13(t,1H,J=8. 0Hz),2.25–3.35(m,1H),1.70–1.85(m,2H),1.05–1.15(m,4H),0.98(t,3H,J=7.60Hz)

[0564] MS (ESI + ):m / z 517.06[M+H] + .

[0565] Example 36: Synthesis of N-[3-[5-(3-ethyl-4-pyridinyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide

[0566] Example 36 was synthesized similarly to Example 7.

[0567]

[0568] Analyze the data:

[0569] 1 H NMR (400MHz, DMSO-d6) δ14.90(s,1H),9.67(s,1H),8.72(d,J=1.9Hz,1H),8.64(s,1H),8.59–8.50(m,2H),7.87(dd,J=14.7,7.6Hz,1H), 7.39(dd,J=14.1,6.6Hz,2H),3.21–3.06(m,2H),2.66(q,J=7.5Hz,2H),1.88–1.73(m,2H),1.06(t,J=7.5Hz,3H),1.00(t,J=7.4Hz,3H).

[0570] C 23 H 21 The calculated exact mass of F2N5O3S (molecular weight: 485.51) is 485.13;

[0571] MS (ESI - ):m / z:484.2[M+H] - .

[0572] Example 37: Synthesis of N-[3-[5-(3-cyano-4-pyridinyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonyl]-2,6-difluorophenyl]propane-1-sulfonamide

[0573] Example 37 was synthesized similarly to Example 7.

[0574]

[0575] Analyze the data:

[0576] 1 H NMR(400MHz,DMSO-d6)δ9.19(s,1H),9.04–8.91(m,3H),7.95–7.84(m,2H),7.41(t,J =8.8Hz,1H),3.19–3.10(m,2H),1.81(dq,J=15.0,7.5Hz,2H),1.00(t,J=7.4Hz,3H).

[0577] C 22 H 16 The calculated exact mass of F2N6O3S (molecular weight: 482.47) is 482.10;

[0578] MS (ESI - ):m / z:481.3[M+H] - .

[0579] Example 38: Biological Activity - Inhibitory Potency and Selectivity in Functional Enzyme Assays

[0580] The PCR products were provided by ProQinase GmbH (Freiburg, Germany). Assay Service to measure the kinase activity of the compounds of the present invention. https: / / www.proqinase.com / products-services-biochemical-assay-services / kinase-assays ) are published in the literature. Briefly, all kinase assays were performed in 96-well FlashPlates from PerkinElmer (Boston, MA, USA). TM The reaction mixture was pipetted in four steps in the following order:

[0581] 20 μl assay buffer (standard buffer)

[0582] 5 μl ATP solution (H2O solution)

[0583] 5 μl test compound (10% DMSO solution)

[0584] 20 μl enzyme / substrate mix

[0585] All protein kinase assays contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM sodium orthovanadate, 1.2 mM DTT, 50 μg / ml PEG20000, ATP (variable concentration, corresponding to the apparent ATP-Km of each kinase), [γ-33P]-ATP (approximately 8×1005 cpm / well), protein kinase and substrate.

[0586] The reaction mixture was incubated at 30°C for 60 h. The reaction was terminated with 50 μl of 2% (v / v) H3PO4, the plate was aspirated and washed twice with 200 μl of 0.9% (w / v) NaCl. The concentrations of the samples were determined using a microplate scintillation counter (Microbeta, Wallac). 33 Incorporation of Pi.

[0587] All assays were performed using the Beckman Coulter / SAGIANTM core system.

[0588] In case of single concentration assays, the potency of the test compound was expressed as % residual activity. To determine the IC 50IC values ​​were obtained by testing a series of dilutions with a final concentration range of 100 μM to 3 nM (10 concentrations). 50 The fitted model for the assay was "Sigmoidal response (variable slope)" with the parameter "top" fixed at 100% and "bottom" fixed at 0%. The fitting method used was a least-squares fit.

[0589] MKK4 Effect:

[0590] The inhibitory potency against MKK4 was categorized as follows:

[0591] Determination of IC 50 Test value:

[0592]

[0593] By IC 50 (Off-target) / IC 50 The selectivity of the test compounds against BRaf, JNK1, and MKK7 (collectively referred to as off-targets) was calculated based on the ratio of (to MKK4) and was classified as follows:

[0594]

[0595] Table 1: Representative examples based on IC 50 The values ​​show biochemical potency against MKK4 and selectivity against BRaf, MKK7 and JNK1.

[0596]

[0597]

Claims

1. A compound selected from or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 2 in the preparation of an MKK4 inhibitor for selectively inhibiting protein kinase MKK4 relative to protein kinases JNK1 and MKK7.

4. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 2 in the preparation of a medicament for promoting liver regeneration or reducing or preventing hepatocyte death.

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