Protein kinase inhibitors and uses thereof for treatment of diseases and conditions

Abstract

To provide compounds that demonstrate protein kinase inhibitory activity.SOLUTION: Compounds demonstrate protein kinase inhibitory activity and has the following structure, where A is N or CH, R1 and R2 are Me or the like, and R3 is a moiety shown below. The compounds are demonstrated to inhibit receptor interacting kinase 2 (RIPK2) and / or Activin-like kinase 2 (ALK2) and / or receptor interacting kinase 3 (RIPK3). Compounds that are either dual RIPK2 / ALK2 inhibitors or that preferentially inhibit RIPK2 or ALK2 or RIPK3 could provide therapeutic benefit.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 848,648, filed May 16, 2019, entitled "Pyrido[2,3-d]pyrimidin-7-ones and Related Compounds as Inhibitors of RIPK3 to Treat Inflammatory and Degenerative Conditions," and U.S. Provisional Patent Application No. 62 / 848,719, filed May 16, 2019, entitled "Protein Kinase Inhibitors and Uses Thereof for the Treatment of Diseases and Conditions," the contents of which are incorporated herein by reference.

[0002] This invention was made with Government support under Grants CA190542 and AG058642 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0003] The present disclosure relates to compounds that exhibit protein kinase inhibitory activity. Protein kinases are important enzymes in cell signal transduction. In many pathological conditions, abnormal signal transduction occurs. Therefore, protein kinase inhibitors can be used as therapeutic agents for the treatment of various diseases. [Background technology]

[0004] Human autoimmune, autoinflammatory and degenerative conditions involve a continuous loop of cell death and inflammation that leads to tissue damage and dysfunction. This is seen in progressive chronic diseases with an inflammatory etiology, including atherosclerosis and TNF-driven pathologies such as rheumatoid arthritis and ulcerative colitis. It also occurs during deleterious acute inflammatory responses, such as during infection with influenza viruses. Although treatments exist for some of these conditions, many inflammatory diseases remain poorly controlled and new therapeutic strategies are desperately needed.

[0005] Influenza A virus (IAV) infections are responsible for up to 700,000 hospitalizations and 50,000 deaths annually in the United States alone. Worryingly, highly pathogenic H5 and H7 strains of avian IAV have so far shown limited human-to-human spread, but only few mutations are required to become transmissible.

[0006] The pulmonary epithelium is the primary site of influenza A virus pathology in mammals. Influenza A viruses (IAV) are negative-sense RNA viruses of the Orthomyxoviridae family. In waterfowl, the main natural hosts of IAV, viral replication occurs in the gastrointestinal tract and is usually asymptomatic. In contrast, mammalian IAV strains replicate in the respiratory tract, resulting in symptoms ranging from a mild "flu" to severe, sometimes fatal, disease. IAV infects epithelial cells along the entire respiratory tract. Transmission is associated with infection of the upper respiratory tract, whereas severe disease is associated with infection in the lower lungs, and the degree of lung involvement correlates with disease outcome in humans and animal models.

[0007] As a lytic virus, IAV kills most lung cell types in which it replicates. However, not all cell death by IAV is pathogenic. While programmed cell death is essential for early control of IAV replication and prevention of viral spread throughout the lung, death of the lung epithelial layer is also one of the major drivers of morbidity and mortality associated with IAV infection. In particular, loss of type I airway epithelial cells (AECs, which are essential for gas exchange), above a threshold of approximately 10%, strongly correlates with mortality in IAV-infected mouse models. If cell death is well controlled and apoptotic, this represents a host defense mechanism that limits both viral spread and immunopathology. However, if cell death is uncontrolled or predominantly necrotic (i.e., highly proinflammatory), this can lead to a hyperinflammatory response, severe deterioration of the airway epithelium, and eventual host death, even if the virus is cleared. Such severe pathology is observed in mouse models, where destruction of the airway epithelium is a common feature of lethal IAV infection, and in humans, where distal lung epithelial death, indicated by areas of bronchoalveolar necrosis, is a typical feature of IAV-induced acute respiratory distress syndrome (ARDS). Thus, an ideal treatment for IAV disease should promote clearance-facilitating death while suppressing exacerbation-inducing inflammatory death and viral and secondary bacterial pneumonia.

[0008] Receptor-interacting protein kinase 3 (RIPK3) is a multifunctional protein involved in cell death pathways in various tissues. Previous studies have demonstrated that loss of RIPK3 kinase activity confers protection in various animal models of inflammatory and degenerative conditions, including sepsis, brain trauma, acute kidney injury, lung injury associated with influenza (IAV) infection, atherosclerosis, and many others. For example, it has recently emerged that RIPK3 is a targetable signaling pathway that accounts for nearly all of IAV-activated pathogenic death in infected cells. This pathway is initiated when the host sensor protein DAI detects IAV genomic RNA and activates RIPK3 kinase. RIPK3 then triggers a form of cell death called necroptosis (or programmed necrosis), which is responsible for much of the lung damage seen during IAV infection. Interestingly, eliminating necroptosis not only significantly reduces lung damage and improves animal survival, but does so without impeding viral clearance. This is because RIPK3 also activates a parallel pathway of non-pathogenic cell death (apoptosis) that mediates viral clearance. Because only necroptosis, but not apoptosis, is dependent on RIPK3 kinase activity, inhibitors of RIPK3 kinase function are expected to ameliorate necrotic lung injury without affecting viral clearance, which may represent an entirely new strategy for the treatment of IAV disease.Similarly, two separate studies revealed that animals lacking the Ripk3 gene were resistant to the development of atherosclerosis due to suppression of macrophage cell death and systemic inflammation.

[0009] Selective RIPK3 kinase inhibitors are not in clinical use and have not progressed to clinical trials. In 2014, a panel of RIPK3 selective inhibitors was reported, but these compounds showed only moderate activity in cells and unfortunately induced unexpected conformational changes in the structure of RIPK3, which resulted in toxicity. Because current vaccine and antiviral strategies are limited in their efficacy or susceptible to viral resistance and evasion mechanisms, it is urgent to identify novel therapeutic entry points for seasonal and virulent IAV pulmonary disease, preferably those targeting pathogenic host signaling pathways. There is a significant unmet need for clinically viable inhibitors of RIPK3 kinase activity. Summary of the Invention

[0010] overview The present disclosure relates generally to compounds that exhibit receptor interacting kinase 2 (RIPK2) inhibitory activity and / or activin-like kinase 2 (ALK2) and / or receptor interacting kinase 3 (RIPK3) inhibitory activity.

[0011] RIPK2 mediates proinflammatory signaling and has become a new therapeutic target in autoimmune and inflammatory diseases, such as inflammatory bowel disease (IBD) and multiple sclerosis. RIPK2 inhibitors may provide therapeutic benefits in the treatment of these and other conditions. Activin-like kinase 2 (ALK2) is involved in many diseases, such as bone disease (e.g., fibrodysplasia ossificans progressiva, ankylosing spondylitis), heart disease (e.g., atherosclerosis and vascular calcification), some cancers (e.g., diffuse intrinsic pontine glioma) and burns. Many of these diseases also have an inflammatory component that can exacerbate the condition and / or worsen clinical outcomes. Compounds that are RIPK2 / ALK2 dual inhibitors or preferentially inhibit RIPK2 or ALK2 may provide therapeutic benefits in the treatment of these and other conditions.

[0012] RIPK3 inhibitors potently and specifically inhibit RIPK3-driven necroptosis. These molecules are based on a pyrido[2,3-d]pyrimidine scaffold and target the ATP pocket and allosteric Glu-out pocket of RIPK3. This series is already approximately 10-fold more potent in inhibiting necroptosis in cells than previously reported RIPK3 inhibitor compounds. Importantly, compounds that block necroptosis without toxicity have been identified.

[0013] This disclosure describes a targetable signaling pathway that accounts for nearly all of IAV-activated pathogenic death in infected cells. This pathway is initiated when the host sensor protein DAI detects IAV genomic RNA and activates RIPK3 kinase. RIPK3 then triggers a form of cell death called necroptosis (or programmed necrosis), which is responsible for much of the lung damage seen during IAV infection. Interestingly, eliminating necroptosis not only significantly reduces lung damage and improves animal survival, but does so without impeding viral clearance. This is because RIPK3 also activates a parallel pathway of non-pathogenic cell death (apoptosis) that mediates viral clearance. Because only necroptosis, but not apoptosis, is dependent on RIPK3 kinase activity, inhibitors of RIPK3 kinase function would ameliorate necrotic lung injury without affecting viral clearance, representing an entirely new strategy for the treatment of IAV disease.

[0014] RIPK3 is a central mediator of epithelial cell death or necroptosis triggered by IAV. Necroptosis is activated by viral and microbial infections (including IAV), several innate immune signaling pathways (TNF, interferon, TLRs, among others), certain proinflammatory stimuli (e.g., asbestos, oxidized LDL), and genotoxic stress. For decades, cell death associated with acute infection with IAV was thought to be a passive, unprogrammed consequence of infection by unspecified apoptosis, autophagy, or simply a "lytic" virus. However, these conclusions were mainly derived from cell culture studies on transformed cell lines that were grossly defective in cell death pathways (e.g., A549 cells) and / or unrelated to IAV biology (e.g., HeLa, MDCK cells). In 2016, a cell death pathway was reported that is active in lung epithelial cells and accounts for most cell death triggered by replicating IAV in these and other primary cell types. This pathway is initiated when the host protein DNA-dependent activator of interferon regulatory factor (DAI) senses IAV genomic RNA and recruits RIPK3 kinase. RIPK3 then activates two distinct parallel arms of cell death: necroptosis and apoptosis. The necroptosis arm requires RIPK3 kinase activity and targets the pseudokinase mixed lineage kinase domain-like (MLKL). The apoptosis arm requires FAS-associated protein with death domain (FADD) and caspase-8 and proceeds without the kinase activity of RIPK3. From within the necrosome, RIPK3 phosphorylates and activates MLKL, which then oligomerizes and translocates to the plasma membrane, where it punctures the membrane causing cell swelling and lysis. Such necroptotic lysis of cells is responsible for the release of "danger-associated molecular patterns" (DAMPs) into the extracellular space and is thought to be highly inflammatory. In certain cases, such as during IAV infection, RIPK3 can also activate a parallel pathway of apoptosis mediated by caspase-8, which promotes viral clearance.Importantly, this apoptotic pathway does not require the catalytic activity of RIPK3 and can indeed be driven by blockade of the RIPK3 kinase.

[0015] Figure 1 shows the RIPK3-mediated cell death or necroptosis pathways, including those activated by tumor necrosis factor (TNF) (left) and IAV (right). Collectively, this branched pathway is responsible for almost all cell death induced by replicating IAV. Most mammalian cell lines commonly used in IAV research (e.g., A549, HeLa, MDCK, etc.) do not express DAI, RIPK3 and / or MLKL, explaining why it remained undiscovered for so long.

[0016] Blockade of RIPK3 kinase can prevent inflammatory pathology without affecting viral clearance. Mice deficient in the DAI-RIPK3 cell death pathway cannot control IAV spread in the lung and succumb to the virus, highlighting the importance of DAI-RIPK3 signaling in anti-IAV host defense. However, the necroptosis branch downstream of RIPK3 is not only redundant for viral clearance but also actively promotes epithelial degradation and inflammation. Thus, selective blocking of necroptosis downstream of RIPK3 would prevent pathology without affecting the otherwise normally beneficial viral clearance by caspase-8-mediated apoptosis. Interestingly, only RIPK3-mediated necroptosis, but not apoptosis, requires the kinase activity of RIPK3 and is therefore easily targetable pharmacologically by the use of RIPK3 kinase inhibitors. Clinically viable RIPK3 small molecule inhibitors would represent a promising novel therapeutic avenue against IAV-induced necroptosis, epithelial degradation, inflammation, and resulting lung injury, as seen on the right side of Figure 1.

[0017] RIPK3-mediated necroptosis potently amplifies inflammation in various chronic conditions, including atherosclerosis and many TNF-associated pathologies, and underlies lung injury during acute IAV infection and ARDS. Moreover, RIPK3 can directly activate inflammatory gene expression independent of cell death, highlighting its central role for host inflammatory processes. There is consensus that genetic ablation of RIPK3 in mice ameliorated atherosclerotic plaque development, almost completely abolished TNF-induced inflammatory shock, significantly reduced injury resulting from chronically produced TNF, and eliminated IAV-induced necrotic lung injury without affecting viral clearance. In addition to the above examples, RIPK3 has been associated with lipid and lysosomal storage diseases, e.g., Niemann-Pick and Gaucher diseases, respectively. RIPK3 also has a pathogenic role in autoimmune diseases, including multiple sclerosis and lupus. RIPK3 activity has also been linked to neurogenesis in different tissues and disease paradigms, indicating that it is a target in a wide range of neurodegenerative diseases. RIPK3 has also been found as a target in a wide range of ischemia-reperfusion injuries, including stroke, myocardial infarction, kidney, retina and liver ischemia.

[0018] The present disclosure relates to a novel panel of RIPK3 inhibitors, sometimes referred to as the UH15 series. These compounds are based on a pyrido[2,3-d]pyrimidine scaffold that is structurally distinct from conventional RIPK3 inhibitor molecules. Members of this series are approximately 10-fold more potent at blocking necroptosis compared to other disclosed RIPK3 inhibitors. Interestingly, the present UH15 compounds block RIPK3 without triggering apoptotic activity and thus without the toxicity of other RIPK3 inhibitors. For most indications (e.g., TNF pathologies), blocking RIPK3 necroptosis without simultaneously activating apoptosis is ideal. However, for IAV, a RIPK3 inhibitor that can simultaneously enhance apoptosis of infected cells while efficiently blocking necroptosis would not only halt deleterious necrotic lung injury but also accelerate viral clearance. Of course, such induction of apoptosis must be restricted to infected cells (i.e., stimulus-specific) or toxicity would result (as seen with previous compounds).

[0019] In particular, the present RIPK3 inhibitor targets RIPK3 necroptosis as a novel therapeutic entry point for IAV disease. Seasonal and pandemic strains of IAV cause necrotic lung injury underlying both ARDS and viral / bacterial pneumonia, each of which remains a major cause of morbidity and mortality, with few effective treatment options. An ideal therapy for severe IAV would block RIPK3-dependent necroptosis but not impede (or may even simultaneously promote) non-inflammatory viral clearance via RIPK3-dependent apoptosis.

[0020] In addition, the present disclosure supports RIPK3 blockade as a fundamental and novel approach to acute and chronic inflammation. Evidence from genetic models demonstrates that RIPK3 has a central role in multiple inflammatory diseases in addition to those seen during IAV infection. These include both acute and chronic inflammatory conditions known to be driven by TNF, such as colitis and rheumatoid arthritis. Furthermore, RIPK3 is essential for all known pathways of necroptosis, while the similar kinase RIPK1 (currently the subject of research by others) is only involved in a subset of necroptosis pathways, such as those activated by TNF (see the right side of Figure 1). Thus, RIPK3 inhibitors would have benefits in a broader range of acute and chronic inflammatory conditions than would function as anti-TNF approaches or RIPK1 inhibitors. [The present invention 1001] A compound exhibiting protein kinase inhibitory activity and having the following structure: TIFF2024170457000002.tif23128 formula, A is N or CH; R1 is Me, TIFF2024170457000003.tif24128, where X is F or Me, where Me is methyl; R2 is Me, Et, Et-O-Me, or isobutyl, where Et is ethyl; and R3 is The file is TIFF2024170457000004.tif13128. [The present invention 1002] A pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention 1001 and a pharma- ceutically acceptable excipient, adjuvant, carrier, buffer, stabilizer, or mixtures thereof. [The present invention 1003] Administering the pharmaceutical composition of the present invention 1002 4. A method for treating a protein kinase-associated disease or condition, comprising: [The present invention 1004] Administering the pharmaceutical composition of the present invention 1002 23. A method for treating a disease or condition in which activity of receptor interacting kinase 2 (RIPK2), activin-like kinase 2 (ALK2), or receptor interacting kinase 3 (RIPK3) is implicated, comprising: [The present invention 1005] Administering the pharmaceutical composition of the present invention 1002 2. A method for treating an inflammatory or degenerative disease or condition in which activity of receptor interacting kinase 3 (RIPK3) is implicated, comprising: [The present invention 1006] The compound of the present invention has the following structure: TIFF2024170457000005.tif123128, wherein R2 is Et, Et-O-Me, or isobutyl, and wherein X is F or Me. [The present invention 1007] A pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention 1006 and a pharma- ceutically acceptable excipient, adjuvant, carrier, buffer, stabilizer, or mixtures thereof. [The present invention 1008] Administering the pharmaceutical composition of the present invention 1007 4. A method for treating a protein kinase-associated disease or condition, comprising: [The present invention 1009] Administering the pharmaceutical composition of the present invention 1007 23. A method for treating a disease or condition in which activity of receptor interacting kinase 2 (RIPK2), activin-like kinase 2 (ALK2), or receptor interacting kinase 3 (RIPK3) is implicated, comprising: [The present invention 1010] Administering the pharmaceutical composition of the present invention 1007 2. A method for treating an inflammatory or degenerative disease or condition in which activity of receptor interacting kinase 3 (RIPK3) is implicated, comprising: [The present invention 1011] 1. A method for treating an inflammatory or degenerative disease or condition in which receptor interacting kinase 3 (RIPK3) inhibitory activity is implicated, comprising the steps of: The following structure: TIFF2024170457000006.tif36128[In the formula, At one available ring position, R is H or R is TIFF2024170457000007.tif24128, where Me is methyl and Et is ethyl; A and D are independently N or CH; E is N, CH, or CR; B and C are independently N, CH, or C-Cl; At one available ring position, R 1 is H or R 1 C-Cl, CF, C-OCH 3 , CC(CH 3 ) 3 or C—OH, and XY is C=C or TIFF2024170457000008.tif10128, where R 2 is H, alkyl, alkylhydroxyl, alkylalkoxyl, or alkylaryl. and a pharma- ceutical composition comprising a therapeutically effective amount of a compound having the formula: [The present invention 1012] The compound has the following structure: The method of the present invention 1011 having TIFF2024170457000009.tif174128. [The present invention 1013] The method of the present invention, wherein the inflammatory or degenerative disease or condition in which RIPK3 activity is implicated is influenza A virus (IAV). [Brief description of the drawings]

[0021] [Figure 1] A diagram of the RIPK3-mediated cell death pathway activated by IAV (left) and TNF (right) is shown. [Diagram 2]1 shows structures of exemplary protein kinase inhibitors identified as UH15 analogs. [Diagram 3] 1 shows the structures of exemplary RIPK3 inhibitors. [Figure 4] 1 shows an overall schematic synthesis scheme for exemplary preferred compounds disclosed herein as inhibitors of protein kinase activity, generally referred to as UH15 analogs or UH15. [Diagram 5] 1 shows steps in the synthesis of an exemplary RIPK3 inhibitor. [Figure 6] 1 shows steps in the synthesis of an exemplary RIPK3 inhibitor. [Figure 7] 1 shows steps in the synthesis of an exemplary RIPK3 inhibitor. [Figure 8] 1 shows a synthetic scheme of intermediate compounds used in the synthesis of exemplary inhibitors of RIPK3. [Figure 9] 1 shows a synthetic scheme of intermediate compounds used in the synthesis of exemplary inhibitors of RIPK3. [Figure 10] 1 shows structures of intermediate compounds used in the synthesis of exemplary inhibitors of RIPK3. [Figure 11] 1 shows a synthetic scheme of intermediate compounds used in the synthesis of exemplary inhibitors of RIPK3. [Figure 12] 1 shows structures of intermediate compounds used in the synthesis of exemplary inhibitors of RIPK3. [Figure 13] 1 shows structures of intermediate compounds used in the synthesis of exemplary inhibitors of RIPK3. [Figure 14] 1 shows a synthetic scheme of intermediate compounds used in the synthesis of exemplary inhibitors of RIPK3. [Figure 15] 1 shows a synthetic scheme of intermediate compounds used in the synthesis of exemplary inhibitors of RIPK3. [Figure 16] FIG. 16 shows structures of intermediate compounds used in the synthesis of exemplary inhibitors of RIPK3. [Figure 17] 1 shows a synthesis scheme of an exemplary inhibitor of RIPK3 according to a preferred embodiment. [Figure 18] 1 shows the structures of exemplary inhibitors of RIPK3 according to preferred embodiments. [Figure 19] 1 shows the structures of exemplary inhibitors of RIPK3 according to preferred embodiments. [Figure 20] 1 shows the structures of exemplary inhibitors of RIPK3 according to preferred embodiments. [Figure 21] 1 shows the structures of exemplary inhibitors of RIPK3 according to preferred embodiments. [Figure 22] 1 shows a synthesis scheme of an exemplary inhibitor of RIPK3 according to a preferred embodiment. [Figure 23] 1 shows a synthesis scheme of an exemplary inhibitor of RIPK3 according to a preferred embodiment. [Figure 24] Figure 24A shows a synthesis scheme of an exemplary inhibitor of RIPK3 according to a preferred embodiment. Figure 24B shows the structure of an intermediate compound used in the synthesis of an exemplary inhibitor of RIPK3 according to a preferred embodiment. [Diagram 25] 1 shows the structures of exemplary inhibitors of RIPK3 according to preferred embodiments. [Figure 26] 1 shows a synthesis scheme of an exemplary inhibitor of RIPK3 according to a preferred embodiment. [Figure 27] 1 shows a synthesis scheme of an exemplary inhibitor of RIPK3 according to a preferred embodiment. [Figure 28] 1 shows a synthesis scheme of an exemplary inhibitor of RIPK3 according to a preferred embodiment. [Figure 29] 1 shows a synthesis scheme of an exemplary inhibitor of RIPK3 according to a preferred embodiment. [Diagram 30] 1 shows a synthesis scheme of an exemplary inhibitor of RIPK3 according to a preferred embodiment. [Diagram 31] 1 shows the activity of selected UH15 compounds and GSK'872 in blocking phosphorylation of RIPK3 and MLKL in RAW264.7 macrophages. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present disclosure relates to protein kinase inhibitors and their uses.

[0023] The diagram below shows the schematic structures of preferred embodiments of compounds that inhibit protein kinases, including receptor interacting kinase 2 (RIPK2), activin-like kinase 2 (ALK2), and receptor interacting kinase 3 (RIPK3). TIFF2024170457000010.tif23128

[0024] In the above structure, A can be N or CH. R1 is Me, TIFF2024170457000011.tif23128, where X is F or Me, where Me is methyl. R2 may be Me, Et, Et-O-Me, or isobutyl. R3 may be TIFF2024170457000012.tif13128, where Et is ethyl.

[0025] Further preferred embodiments of compounds that inhibit RIPK2, ALK2, and RIPK3 are shown in FIG.

[0026] Further preferred embodiments of compounds that inhibit RIPK2, ALK2, and RIPK3 are provided below. TIFF2024170457000013.tif129128 where R2 is Et (UH15-4), Et-O-Me (UH15-6), or isobutyl (UH15-10), and where X is F (UH15-18) or Me (UH15-20).

[0027] The following diagram shows the schematic structures of further preferred embodiments of compounds that inhibit RIPK3. TIFF2024170457000014.tif36128

[0028] In the above structure, R can be H or R can be substituted on any one available position of the phenyl ring. TIFF2024170457000015.tif25128, where Me is methyl and Et is ethyl. A and D may independently be N or CH. E may be N, CH, or CR, and R is as defined above. B and C may independently be N, CH, or C-Cl. R 1 may be H, or R 1 is C-Cl, CF, C-OCH at any one available position on the ring. 3 , CC(CH 3 ) 3 XY may be C=C or they may be TIFF2024170457000016.tif10128, where R 2 is H; alkyl, including but not limited to methyl, ethyl, or isobutyl; alkylhydroxyl, including but not limited to 2-hydroxyethyl; alkylalkoxyl, including but not limited to 2-methoxyethyl; or alkylaryl, including but not limited to benzyl or phenethyl.

[0029] The following diagram shows the schematic structures of further preferred embodiments of compounds that inhibit RIPK3. TIFF2024170457000017.tif37128

[0030] In the above structure, A and D may independently be N or CH. E may be N, CH, or CR. B and C may independently be N, CH, or C-Cl. XY may be C=C or they may be TIFF2024170457000018.tif10128, where R 2is H; alkyl, including but not limited to, methyl, ethyl, or isobutyl; alkylhydroxyl, including but not limited to, 2-hydroxyethyl; alkylalkoxyl, including but not limited to, 2-methoxyethyl; or alkylaryl, including but not limited to, benzyl or phenethyl. R is H, TIFF2024170457000019.tif12128, where Me is methyl and Et is ethyl. 1 may be any alkyl group, including but not limited to methyl, ethyl, or propyl, or R 1 R may be any aryl group, including, but not limited to, naphthyl, thienyl, indoyl, and the like. 3 may be H, or R 3 is C-Cl, CF, C-OCH at any one available position on the ring. 3 , CC(CH 3 ) 3 , or C—OH.

[0031] Further preferred embodiments of compounds that inhibit RIPK3 are shown in FIG.

[0032] The exemplary compounds described herein that inhibit protein kinases, including receptor interacting kinase 2 (RIPK2), activin-like kinase 2 (ALK2), and receptor interacting kinase 3 (RIPK3), may exist in different geometric and enantiomeric forms, and both the pure form and mixtures of these individual isomers, as well as any physiologically functional or pharmacologically acceptable salt derivatives or prodrugs thereof, are included within the scope of the present invention.The preparation of these alternative forms is well within the capabilities of those skilled in the art.

[0033] The present invention also relates to a method of preventing or treating inflammatory and degenerative conditions, including diseases in which protein kinase activity is implicated, such as influenza A virus (IAV) infection and TNF-driven inflammatory conditions, comprising administering a compound that inhibits protein kinase activity according to the preferred embodiments disclosed herein. In a preferred embodiment, the method of preventing or treating an inflammatory or degenerative disease in which protein kinase activity is implicated comprises administering a compound that is a compound depicted in FIG. 2 or FIG. 3.

[0034] In another aspect of the present invention, a pharmaceutical composition is provided that includes a therapeutically effective amount of a compound that inhibits protein kinase activity as defined above, and a pharma- ceutically acceptable excipient, adjuvant, carrier, buffer, or stabilizer. A "therapeutically effective amount" is to be understood as the amount of an exemplary protein kinase inhibitor compound sufficient to exhibit an inhibitory effect on protein kinase activity. The actual amount, rate, and time-course of administration will vary according to the nature and severity of the disease being treated. Prescription of treatment is within the responsibility of general practitioners and other physicians. The pharma-ceutically acceptable excipient, adjuvant, carrier, buffer, or stabilizer should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material will vary according to the route of administration, whether oral, or by injection, such as cutaneous, subcutaneous, or intravenous injection, or by dry powder inhaler.

[0035] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders or liquids. Tablets may contain solid carriers or adjuvants. Liquid pharmaceutical compositions generally contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oils. Physiological saline, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Capsules may contain solid carriers such as gelatin. For intravenous, cutaneous or subcutaneous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as sodium chloride solution, Ringer's solution, lactated Ringer's solution, etc. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as necessary.

[0036] In another aspect there is provided the use of a therapeutically effective amount of a protein kinase inhibitor compound as defined above in the manufacture of a medicament for administration to a subject.

[0037] The term "pharmacologically acceptable salts" as used throughout the specification should be taken to mean any acid or base derived salt formed from hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, methanesulfonic acid, isoethonic acids, and the like, as well as potassium carbonate, sodium or potassium hydroxide, ammonia, triethylamine, triethanolamine, and the like.

[0038] The term "prodrug" refers to a pharmacological agent that is administered in an inactive or significantly less active form. Upon administration, the prodrug is metabolized in vivo to an active metabolite.

[0039] The term "therapeutically effective amount" refers to a drug amount that is sufficient to provide the desired therapeutic effect without toxicity. The "effective" amount will vary from subject to subject, depending on the age and general condition of the individual, the specific concentration and composition administered, etc. Thus, it is not always possible to specify an exact effective amount. However, the appropriate effective amount in any individual case can be determined by those skilled in the art using routine experimentation. Moreover, an effective amount is a concentration that is within a range sufficient to allow immediate application of the formulation to deliver an amount of drug within the therapeutically effective range.

[0040] Further aspects of the present invention will become apparent from the following description, which is given by way of example only. EXAMPLES

[0041] Example 1. Synthesis Figure 4 shows the overall schematic synthesis scheme of the exemplary preferred compound disclosed herein as an inhibitor of protein kinase activity, generally referred to as UH15 analog or UH15. In particular, in the following discussion, the compound may be referred to in the form of, for example, UH15-1 or UH15_1. These compounds have different structures. For example, UH15-15 is a different compound from UH15_15.

[0042] All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions unless otherwise stated. All commercially available chemicals and reagent grade solvents were used directly without further purification unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) on Baker-flex® silica gel plates (IB2-F) using UV light (254 and 365 nm) as visualization agent and either an ethanolic solution of phosphomolybdic acid or ninhydrin solution as developer and heat. Flash chromatography was performed on silica gel (230-400 mesh) using a Teledyne ISCO CombiFlash® Rf. NMR spectra were recorded at room temperature using a JEOL ECA-500 (1H NMR and 13C NMR at 400, 500, 600 MHz) with tetramethylsilane (TMS) as internal standard. Chemical shifts (δ) are given in parts per million (ppm) with reference to the solvent signal [1H-NMR: CDCl 3 (7.26ppm), CD 3 OD(3.30ppm), DMSO-d6(2.49ppm);13C-NMR:CDCl 3 (77.0ppm), CD 3 OD (49.0 ppm), DMSO-d6 (39.5 ppm)]. Signal patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), td (triplet of doublets), m (multiplet) and brs (broad singlet). Coupling constants (J) are given in Hz. High-resolution mass spectra (HRMS) were performed by the Mass Spectrometry Facility of the Department of Chemistry at The University of Texas at Austin using an Agilent 6530 Q-TOF instrument. Electrospray ionization (ESI) was used as the ionization source and spectra were analyzed for the molecules [M] or [M+H]. +The m / z (relative intensity) of the ionic species was reported. Purity of the compounds was determined by high performance liquid chromatography (HPLC) analysis using a binary hplc pump (Waters) and a Kinetex 5 μm C18 100A column (250 × 4.6 mm). UV absorption was monitored at λ = 254 nm. Injection volume was 15 μL. The HPLC gradient was 2% acetonitrile / 98% water to 90% acetonitrile / 10% water (both solvents containing 0.1% trifluoroacetic acid), with a total run time of 30 min and a flow rate of 1 mL / min.

[0043] Compounds UH15-4, UH15-6, UH15-10, UH15-18 and UH15-20 were prepared by adapting the method described in Cuny et al., WO2018 / 213219.

[0044] 2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-ethyl-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (UH15-4): TIFF2024170457000020.tif28128

[0045] Yellow solid (54% yield): TIFF2024170457000021.tif34150 Purity 95.8%(t R 20.50 minutes).MP133~135℃.

[0046] 6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-(2-methoxyethyl)pyrido[2,3-d]pyrimidin-7(8H)-one (UH15-6): TIFF2024170457000022.tif33128

[0047] Yellow solid (61% yield): TIFF2024170457000023.tif48150 Purity 97.4%(t R 20.76 minutes).MP133~135℃.

[0048] 6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-isobutylpyrido[2,3-d]pyrimidin-7(8H)-one (UH15-10): TIFF2024170457000024.tif30128

[0049] Yellow solid (36% yield): TIFF2024170457000025.tif54151 Purity 97.7%(t R 22.55 minutes).MP196~198℃.

[0050] 6-(2-chloro-4-fluorophenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (UH15-18): TIFF2024170457000026.tif28128

[0051] White solid (60% yield): TIFF2024170457000027.tif48150 Purity 97.6%(t R 22.72 minutes).MP248~250℃.

[0052] Synthesis of UH15-20: TIFF2024170457000028.tif26128

[0053] The synthesis of UH15-20 proceeded as shown in Figure 5. Reagents and conditions: (a) NaH, THF, reflux, 2.5 h, 42%; (b) Pd(PPh 3 ) 4 , Na 2 CO 3 , DMF:AcN, 90°C, 5 hours, 57%; (c) mCPBA, DCM, room temperature, 5 hours, 68%; (d) NaH, THF:DMF, 0°C to room temperature, 2.5 hours, 75%.

[0054] 6-Chloro-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (A19):

[0055] A mixture of A7 (20 mg, 0.11 mmol) and 60% NaH (8 mg, 0.33 mmol) in THF (1.5 mL) was stirred at room temperature for 10 min under argon. A solution of ethyl 2-chloro-2-(diethoxyphosphoryl)acetate A18 (42 mg, 0.16 mmol) in THF (0.5 mL) was added dropwise to the above mixture and refluxed for 2.5 h. The reaction mixture was cooled to room temperature, concentrated, and extracted with EtOAc and water. The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated to give a residue which was purified by column chromatography on silica gel (30% EtOAc / Hexanes) to give A19 as a white solid (42% yield). TIFF2024170457000029.tif20150

[0056] 6-(2-chloro-4-methylphenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (A20):

[0057] A19 (24 mg, 0.11 mmol), (2-chloro-4-methylphenyl)boronic acid (28 mg, 0.16 mmol) and Pd(PPh 3 ) 4 (13 mg, 0.011 mmol) was placed in a round-bottom flask and purged with argon for 10 min. DMF (1 mL) and CH 3 CN (2 mL) was added to the above mixture and purged for 10 min. 2 CO 3 A solution of (23 mg, 0.21 mmol) (220 μL) was added dropwise and the reaction mixture was heated at 90° C. for 5 h. The reaction mixture was cooled to room temperature, the solvent was evaporated and the residue was partitioned between water and EtOAc. The organic layer was washed with anhydrous Na 2 SO 4The mixture was dried at 40° C., filtered and concentrated to give a residue which was purified by column chromatography on silica gel (10% EtOAc / DCM) to give A20 as a white solid (57% yield). TIFF2024170457000030.tif27151

[0058] 6-(2-chloro-4-methylphenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (A21):

[0059] A21 was synthesized by adapting the method described by Cuny et al. in WO2018 / 213219. White solid (68% yield): TIFF2024170457000031.tif27150

[0060] Synthesis of A16c:

[0061] To 3-(methylsulfonyl)aniline in a round-bottom flask containing molecular sieves (4 Å, 8–12 mesh) was added formic acid (1 mL). The reaction mixture was heated at 60 °C for 6 h and then cooled to 5° C. with NaHCO 3 The organic layer was then washed with brine solution and then concentrated to give A16c, which was used without purification.

[0062] N-(3-(methylsulfonyl)phenyl)formamide (A16c) TIFF2024170457000032.tif27128

[0063] White solid (80% yield): TIFF2024170457000033.tif20150

[0064] 6-(2-chloro-4-methylphenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (UH15-20): TIFF2024170457000034.tif27128

[0065] UH15-20 was prepared from A21 and A16c by adapting the method described in Cuny et al., WO2018 / 213219. White solid (75% yield): TIFF2024170457000035.tif34151 Purity 99.6%(t R 23.51 min).MP182~184℃.

[0066] Synthesis of UH15-28: TIFF2024170457000036.tif29128

[0067] The synthesis of UH15-28 proceeded as shown in Figure 6. Reagents and conditions: (a) CH 3 NH 2 (aq), THF, 0°C to room temperature, 2 hours, 67%; (b) LAH, THF, 0°C to room temperature, 1.5 hours, 96%; (c) MnO 2 , DCM, room temperature, overnight, 93%; (d) KF / Al 2 O 3 , DMA, room temperature, 2 hours, 75%; (e) Pd 2 (dba) 3 , Xantphos, K 2 CO 3 , DMF, reflux, 24 h, 37%; (f) Pd / C, H 2 , MeOH, room temperature, overnight, 94%; (g) Pd 2 (dba) 3 , Xantphos, Cs 2 CO 3 , dioxane, 80°C, overnight, 36%.

[0068] Ethyl 6-chloro-4-(methylamino)nicotinate (D2):

[0069] To a solution of D1 (100 mg, 0.45 mmol) in THF (2 mL) was added aqueous methylamine solution (0.4 mL) at 0° C. The mixture was stirred for 30 min at the same temperature and then at room temperature for 2 h. After evaporation in vacuum to remove THF, the crude mixture was then 2 The organic layer was washed with brine and anhydrous Na2 SO 4 The residue was purified by silica gel column chromatography (10% EtOAc / Hexanes) to give D2 (65 mg, 67%) as a white solid. TIFF2024170457000037.tif21150

[0070] (6-chloro-4-(methylamino)pyridin-3-yl)methanol (D3):

[0071] A suspension of LAH (27 mg, 0.69 mmol) in THF (2 mL) was cooled at 0° C. to which a solution of D2 (100 mg, 0.46 mmol) in THF (2 mL) was added dropwise under argon. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was then cooled to 0° C. and 15% NaOH (0.5 mL) and water (1 mL) were added dropwise. The reaction mixture was stirred for 1 h, filtered and washed with EtOAc. Evaporation of EtOAc in vacuum gave a residue which was purified by silica gel column chromatography (5% MeOH / DCM) to give D3 (77 mg, 96%) as a white solid. TIFF2024170457000038.tif20150

[0072] 6-Chloro-4-(methylamino)nicotinaldehyde (D4):

[0073] A solution of D3 (495 mg, 2.86 mmol) in DCM (10 mL) was diluted with MnO 2 (1496 mg, 17.20 mmol) was added and the mixture was stirred overnight at room temperature under argon. The reaction mixture was then filtered, concentrated to remove DCM, and purified by silica gel column chromatography (5% MeOH / DCM) to give D4 (453 mg, 93%) as a white solid. TIFF2024170457000039.tif20150

[0074] 7-Chloro-3-(2-chlorophenyl)-1-methyl-1,6-naphthyridin-2(1H)-one (D6):

[0075] A stirred solution of D4 (300 mg, 1.75 mmol) and D5 (325 mg, 1.75 mmol) in dry DMA (5 mL) was diluted with KF / Al 2 O 3 (1800 mg, 40 wt%) was added and the reaction mixture was stirred at room temperature for 2 h under argon. Upon completion, the reaction mixture was filtered through Celite and the remaining solid was washed with DCM. The filtrate was concentrated and the residue was purified by silica gel column chromatography using 25% EtOAc / Hexane to give D6 (400 mg, 75%) as a white solid. TIFF2024170457000040.tif13128

[0076] 4-(3-nitrophenyl)thiomorpholine 1,1-dioxide (D14):

[0077] Add Pd to a preheated round-bottom flask. 2 (dba) 3 (68 mg, 0.073 mmol), Xantphos (85 mg, 0.147 mmol) and potassium carbonate (408 mg, 2.95 mmol) were added and flushed with argon for 10 min. DMF (4 mL) was added to the mixture and flushed for another 5 min, followed by addition of D12 (200 mg, 1.47 mmol) and D13 (356 mg, 1.77 mmol) and then refluxed for 24 h. The reaction mixture was then partitioned between ethyl acetate and water and anhydrous Na 2 SO 4 The mixture was dried at rt, filtered, concentrated and purified by column chromatography on silica gel (1.5% MeOH / DCM) to give D14 (140 mg, 37%) as a pale yellow solid. TIFF2024170457000041.tif13128

[0078] 4-(3-aminophenyl)thiomorpholine 1,1-dioxide (D15):

[0079] CH 3To a solution of D14 (140 mg, 0.54 mmol) in OH (10 ml) was added 10% Pd / C (50 mg) and the reaction was incubated at room temperature with H 2 (g) (1 atm) for 4 h. The reaction mixture was then filtered through Celite and concentrated to give D15 (115 mg, 94%) as a brown solid, which was used in the next step without purification.

[0080] 3-(2-chlorophenyl)-7-((3-(1,1-dioxidothiomorpholino)phenyl)amino)-1-methyl-1,6-naphthyridin-2(1H)-one (UH15-28):

[0081] In a preheated round-bottom flask, add D6 (40 mg, 0.13 mmol), D15 (30 mg, 0.13 mmol), and Pd 2 (dba) 3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol) and cesium carbonate (86 mg, 0.26 mmol) were added and flushed with argon for 10 minutes. Dioxane (3 ml) was added to the mixture and flushed again for 5 minutes and then heated at 80° C. overnight. The reaction mixture was then partitioned between ethyl acetate and water and anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by column chromatography on silica gel (20% EtOAc / DCM) to give Nap-5 (23 mg, 36%) as a pale yellow solid. TIFF2024170457000042.tif47150

[0082] Synthesis of UH15-37: TIFF2024170457000043.tif30128

[0083] The synthesis of UH15-37 proceeded as shown in Figure 7. Reagents and conditions: (a) Pd 2 (dba) 3 , Xantphos, Cs 2 CO 3 , dioxane, 80°C, overnight, 47%.

[0084] 3-(2-chlorophenyl)-1-methyl-7-((3-(4-methylpiperazin-1-yl)phenyl)amino)-1,6-naphthyridin-2(1H)-one (UH15-37):

[0085] In a preheated, dry round-bottom flask, add D6 (30 mg, 0.098 mmol), 3-(4-methylpiperazin-1-yl)aniline (19 mg, 0.098 mmol), Pd 2 (dba) 3 (9 mg, 0.009 mmol), Xantphos (12 mg, 0.019 mmol) and cesium carbonate (64 mg, 0.19 mmol) were added and flushed with argon for 10 min. Dry dioxane (1.5 mL) was added to the mixture and after flushing for 10 min, the reaction mixture was heated at 80° C. overnight. The reaction mixture was cooled to room temperature, the solvent was evaporated and the residue was partitioned between water and EtOAc. The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated to give a residue which was purified by column chromatography on silica gel (5% MeOH / DCM) to give UH15-37 (21 mg, 47%) as a yellow solid. TIFF2024170457000044.tif47150

[0086] Figures 8-9, 11, and 14-15 show synthetic schemes of intermediate compounds used in the synthesis of exemplary inhibitors of protein kinases according to preferred embodiments. Figures 10, 12-13, and 16 show structures of intermediate compounds used in the synthesis of exemplary inhibitors of protein kinases according to preferred embodiments.

[0087] Ethyl 4-amino-2-(methylthio)pyrimidine-5-carboxylate (2):

[0088] To a solution of ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate (1) (100 mg, 0.43 mmol) in dry THF (2 mL) was added triethylamine (0.2 mL, 1.29 mmol) and ammonium hydroxide (0.5 mL). The resulting mixture was stirred at room temperature for 2 h until completion. After evaporation in vacuo to remove THF, the crude mixture was purified by H 2 The mixture was partitioned between 200 and EtOAc. The organic layer was washed with brine and then with anhydrous Na 2 SO 4 The residue was purified by silica gel column chromatography (15% EtOAc / Hexanes) to give 2 (90 mg, 98%) as a white solid. TIFF2024170457000045.tif26150

[0089] Ethyl 4-(methylamino)-2-(methylthio)pyrimidine-5-carboxylate (3):

[0090] To a solution of ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate (1) (3000 mg, 12.93 mmol) in dry THF (20 mL) was added aqueous methylamine (aq) (6 mL). The resulting mixture was stirred at room temperature for 2 h until completion. After evaporation in vacuo to remove THF, the crude mixture was then purified by H 2 The mixture was partitioned between 200 and EtOAc. The organic layer was washed with brine and then with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated. The residue was purified by silica gel column chromatography (10% EtOAc / Hexanes) to give 3 (2500 mg, 85%) as a white solid. TIFF2024170457000046.tif20150

[0091] (4-amino-2-(methylthio)pyrimidin-5-yl)methanol (4):

[0092] LiAlH in THF (4 mL) 4(120 mg, 3.16 mmol) was cooled at 0° C. to which a solution of 2 (450 mg, 2.11 mmol) in THF (2 mL) was added dropwise under argon and stirred at room temperature for 30 min. The reaction mixture was then cooled at 0° C. and 15% NaOH (0.5 mL) and water (1 mL) were added dropwise. The reaction mixture was stirred for 1 h, filtered and washed with EtOAc. Evaporation in vacuum to remove EtOAc gave 4 (220 mg, 61%) as a pale yellow solid, which was used in the next step without purification. TIFF2024170457000047.tif13130

[0093] S(4-(methylamino)-2-(methylthio)pyrimidin-5-yl)methanol (5):

[0094] LiAlH in THF (10 mL) 4 (626 mg, 16.50 mmol) suspension was cooled at 0° C. to which a solution of 3 (2500 mg, 11.00 mmol) in THF (5 mL) was added dropwise under argon and stirred at room temperature for 30 min. The reaction mixture was then cooled at 0° C. and 15% NaOH (2 mL) and water (4 mL) were added dropwise. The reaction mixture was stirred for 1 h, filtered and washed with EtOAc. Evaporation in vacuum to remove EtOAc gave 5 (1790 mg, 95%) as a light yellow solid, which was used in the next step without purification. TIFF2024170457000048.tif13129

[0095] 4-Amino-2-(methylthio)pyrimidine-5-carbaldehyde (6):

[0096] A solution of 4 (220 mg, 1.28 mmol) in DCM (5 mL) was diluted with MnO 2 (670 mg, 7.71 mmol) was added and the mixture was stirred overnight at room temperature under argon. The reaction mixture was then filtered, concentrated to remove DCM and purified by silica gel column chromatography (30% EtOAc / Hexanes) to give 6 (180 mg, 83%) as a light yellow solid. TIFF2024170457000049.tif19150

[0097] 4-(Methylamino)-2-(methylthio)pyrimidine-5-carbaldehyde (7):

[0098] A solution of 5 (1795 mg, 10.489 mmol) in DCM (15 mL) was diluted with MnO 2 (5471 mg, 62.93 mmol) was added and the mixture was stirred overnight at room temperature under argon. The reaction mixture was then filtered, concentrated to remove DCM and purified by silica gel column chromatography (20% EtOAc / Hexanes) to give 7 (1470 mg, 77%) as a light yellow solid. TIFF2024170457000050.tif20150

[0099] General procedure for preparation of 9a-c:

[0100] 4-(Methylamino)-2-(methylthio)pyrimidine-5-carbaldehyde (7) (250 mg, 1.37 mmol), 2-(2,4-dichlorophenyl)acetonitrile (8a) (381 mg, 2.05 mmol), and K 2 CO 3 (944 mg, 6.82 mmol) was added DMF (4 mL) under argon and the solution was refluxed at 105° C. for 18 h. After completion, the reaction mixture was partitioned between water and EtOAc, and the organic layer was washed with brine solution. Evaporation in vacuum to remove EtOAc gave a residue, which was purified by silica gel column chromatography (5% MeOH / DCM) to give 9a.

[0101] 6-(2,4-dichlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-imine (9a):

[0102] Yield 63%, pale red solid; TIFF2024170457000051.tif20150

[0103] 6-(2-chlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-imine (9b):

[0104] 7 (500 mg, 2.73 mmol) and 8b (621 mg, 4.10 mmol) were used to make 9b, which was used in the next step without purification.

[0105] 8-Methyl-2-(methylthio)-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-imine (9c):

[0106] 7 (600 mg, 3.28 mmol) and 8c (576 mg, 4.91 mmol) were used to make 9c.

[0107] Yield 54%, light red solid; TIFF2024170457000052.tif13132

[0108] General procedure for preparation of 13a-c:

[0109] A suspension of 9a (200 mg, 0.57 mmol) in acetic anhydride (3 mL) was refluxed at 139° C. for 30 min. Evaporation of the solvent in vacuo gave a residue which was treated with concentrated HCl (2 mL) and refluxed at 100° C. for 5 min. The reaction mixture was then diluted with NaHCO 3 The mixture was neutralized with a saturated solution of 1,2-dichloromethane and partitioned between water and EtOAc. The organic layer was then washed with brine solution and after concentration, the residue was purified by silica gel column chromatography (30% EtOAc / Hexane) to give 13a.

[0110] 6-(2,4-dichlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13a):

[0111] Yield 65%, pale yellow solid; TIFF2024170457000053.tif13132

[0112] 6-(2-chlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13b):

[0113] 9b (350 mg, 1.10 mmol) was used to prepare 13b.

[0114] Yield 61%, pale yellow solid; TIFF2024170457000054.tif27150

[0115] 8-Methyl-2-(methylthio)-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (13c):

[0116] 9c (200 mg, 0.71 mmol) was used to prepare 13c.

[0117] Yield 55%, yellow solid; TIFF2024170457000055.tif27150

[0118] General procedure for preparation of 13d-p:

[0119] A stirred solution of 7 (15 mg, 0.08 mmol) and 11d (23 mg, 0.12 mmol) in dry DMA (1.5 mL) was diluted with KF / Al 2 O 3 (76 mg, 40 wt%) was added and the reaction mixture was stirred at room temperature for 24 h under argon. Upon completion, the reaction mixture was filtered through Celite, the remaining solid was washed with DCM and the filtrate was concentrated. The residue was purified by silica gel column chromatography using 15% EtOAc / Hexane to give 13d as a light yellow solid.

[0120] 6-(4-chlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13d):

[0121] Yield 58%, pale yellow solid; TIFF2024170457000056.tif27150

[0122] 6-(4-(tert-butyl)phenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13e):

[0123] Yield 41%, yellow solid; TIFF2024170457000057.tif27150

[0124] 6-(2,6-dichlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13f):

[0125] Yield 30%, white solid; TIFF2024170457000058.tif27150

[0126] 6-(2,3-dichlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13g):

[0127] Yield 38%, white solid; TIFF2024170457000059.tif13139

[0128] 6-(2-chloro-4-fluorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13h):

[0129] Yield 35%, white solid; TIFF2024170457000060.tif34150

[0130] Preparation of (13o):

[0131] 2-(2,5-Dichlorophenyl)methyl acetate (11o):

[0132] 11o in methanol (3 mL) *To a stirred solution of (100 mg, 0.537 mmol) was added thionyl chloride (0.5 mL) dropwise at 0° C. The reaction mixture was maintained at the same temperature for 5 h and then stirred at room temperature overnight. After completion, the reaction mixture was concentrated and diluted with EtOAc. The organic layer was then washed with sodium bicarbonate and brine solution and after concentration, the residue was purified by silica gel column chromatography (20% EtOAc / Hexane) to give 11o. Yield 59%, colorless liquid: TIFF2024170457000061.tif20150

[0133] 6-(2,5-dichlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13o)

[0134] Yield 23%, white solid; TIFF2024170457000062.tif13128

[0135] 6-(4-hydroxyphenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13p)

[0136] Yield 55%, pale yellow solid; TIFF2024170457000063.tif20150

[0137] General procedure for preparation of 12i, 12k and 12n:

[0138] A stirred solution of 6 (37 mg, 0.22 mmol) and methyl 2-phenylacetate (11i) (30 mg, 0.20 mmol) in dry DMA (2.0 mL) was diluted with KF / Al 2 O 3 (187 mg, 40 wt%) was added and the reaction mixture was stirred at room temperature for 24 h under argon. Upon completion, the reaction mixture was filtered through Celite, the remaining solid was washed with DCM and the filtrate was concentrated. The residue was purified by silica gel column chromatography using 30% EtOAc / Hexane to give 12i as a white solid.

[0139] 2-(Methylthio)-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (12i):

[0140] Yield 51%, white solid; TIFF2024170457000064.tif27150

[0141] 6-(2,6-dichlorophenyl)-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (12k):

[0142] Yield 28%, pale yellow solid; TIFF2024170457000065.tif27150

[0143] 6-(4-chlorophenyl)-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (12n):

[0144] Yield 40%, white solid; TIFF2024170457000066.tif27150

[0145] General procedure for preparation of 13i-r:

[0146] To a mixture of 12i (20 mg, 0.07 mmol) and NaH (3 mg, 0.11 mmol) under argon was added DMF (1.5 mL), followed by dropwise addition of iodoethane (10 μl, 0.11 mmol). The reaction mixture was heated at 50° C. for 1 h and upon completion was partitioned between water and EtOAc. The organic layer was then washed with brine solution and diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated, and the residue was purified by silica gel column chromatography (10% EtOAc / Hexanes) to give 13i as a colorless liquid.

[0147] 8-Ethyl-2-(methylthio)-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (13i):

[0148] 74% yield, colorless liquid; TIFF2024170457000067.tif27150

[0149] 8-(2-hydroxyethyl)-2-(methylthio)-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (13j):

[0150] Purification by silica gel column chromatography (40% EtOAc / hexanes) afforded 13j (48% yield) as a light yellow viscous liquid. TIFF2024170457000068.tif27150

[0151] 6-(2,6-dichlorophenyl)-8-(2-methoxyethyl)-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13k):

[0152] Yield 44%, colorless liquid; TIFF2024170457000069.tif33150

[0153] 8-Benzyl-6-(2,6-dichlorophenyl)-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13l):

[0154] Yield 29%, white semi-solid; TIFF2024170457000070.tif33150

[0155] 6-(2,6-dichlorophenyl)-8-isobutyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13m):

[0156] Yield 28%, white solid; TIFF2024170457000071.tif34150

[0157] 6-(4-chlorophenyl)-8-isobutyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13n):

[0158] Yield 73%, yellow solid; TIFF2024170457000072.tif20150

[0159] 6-(2,6-dichlorophenyl)-8-(3-(methylsulfonyl)benzyl)-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13q):

[0160] Yield 36%, pale yellow solid; TIFF2024170457000073.tif20150

[0161] 6-(2,6-dichlorophenyl)-8-(4-(methylsulfonyl)benzyl)-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13r):

[0162] Yield 53%, pale yellow solid; TIFF2024170457000074.tif20150

[0163] 6-(4-Methoxyphenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13s):

[0164] To a solution of 13p (20 mg, 0.06 mmol) in acetone (2 mL) was added anhydrous potassium carbonate (14 mg, 0.10 mmol), iodomethane (10 μl, 0.10 mmol) and the mixture was refluxed for 8 h. After completion, the crude mixture was extracted with ethyl acetate and water, concentrated and purified by column chromatography on silica gel (20% EtOAc / Hexane) to give 13s (20 mg, 95%) as a pale yellow solid. TIFF2024170457000075.tif27150

[0165] General procedure for preparation of 14a-s:

[0166] To a solution of 6-(2,4-dichlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13a) (38 mg, 0.108 mmol) in DCM (2 mL) was added mCPBA (85 mg, 55%) and stirred for 3 h. The reaction mixture was then partitioned between water and DCM and the organic layer was washed with brine. After concentration, the residue was purified by column chromatography on silica gel (40% EtOAc / Hexane) to give 14a as a white powder.

[0167] 6-(2,4-dichlorophenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14a):

[0168] Yield 63%, white solid; TIFF2024170457000076.tif26150

[0169] 6-(2-chlorophenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14b):

[0170] Yield 45%, white solid; TIFF2024170457000077.tif27150

[0171] 8-Methyl-2-(methylsulfonyl)-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (14c):

[0172] Yield 54%, pale yellow solid; TIFF2024170457000078.tif27150

[0173] 6-(4-chlorophenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14d):

[0174] Yield 72%, white solid; TIFF2024170457000079.tif27151

[0175] 6-(4-(tert-butyl)phenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14e):

[0176] Yield 81%, white solid; TIFF2024170457000080.tif27151

[0177] 6-(2,6-dichlorophenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14f):

[0178] Yield 93%, white solid; TIFF2024170457000081.tif13145

[0179] 6-(2,3-dichlorophenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14g):

[0180] Yield 65%, pale yellow solid; TIFF2024170457000082.tif27150

[0181] 6-(2-chloro-4-fluorophenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14h):

[0182] Yield 40%, white solid; TIFF2024170457000083.tif41150

[0183] 8-Ethyl-2-(methylsulfonyl)-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (14i):

[0184] Yield 55%, white solid; TIFF2024170457000084.tif20150

[0185] 8-(2-hydroxyethyl)-2-(methylsulfonyl)-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (14j):

[0186] Yield 81%, pale yellow solid, which was used in the next step without purification.

[0187] 6-(2,6-dichlorophenyl)-8-(2-methoxyethyl)-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14k):

[0188] Yield 58%, white solid; TIFF2024170457000085.tif33150

[0189] 8-Benzyl-6-(2,6-dichlorophenyl)-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14l):

[0190] Yield 61%, white solid; TIFF2024170457000086.tif27150

[0191] 6-(2,6-dichlorophenyl)-8-isobutyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14m):

[0192] Yield 87%, white solid; TIFF2024170457000087.tif34150

[0193] 6-(4-chlorophenyl)-8-isobutyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14n):

[0194] Yield 66%, light brown solid, which was used in the next step without purification.

[0195] 6-(2,5-dichlorophenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14o):

[0196] Yield 75%, white solid; TIFF2024170457000088.tif27150

[0197] 6-(4-((tert-butyldimethylsilyl)oxy)phenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (13p * ):

[0198] To a solution of 13p (160 mg, 0.53 mmol) in DMF (2 mL) was added imidazole (109 mg, 1.60 mmol) and DMAP (catalytic amount 3 mg) and the temperature was lowered to 0 °C. TBDMSCl (121 mg, 0.80 mmol) in DMF (1 mL) was added to the above solution and the resulting mixture was stirred at room temperature overnight. Upon completion, the reaction was partitioned between EtOAc and water, the organic layer was filtered, concentrated and purified by column chromatography on silica gel (5% EtOAc / DCM) to give 13p. * (194 mg, 88%) was obtained as a pale yellow solid. TIFF2024170457000089.tif21150

[0199] 6-(4-((tert-butyldimethylsilyl)oxy)phenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14p):

[0200] Yield 64%, pale yellow solid; TIFF2024170457000090.tif27150

[0201] 6-(2,6-dichlorophenyl)-2-(methylsulfonyl)-8-(3-(methylsulfonyl)benzyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14q):

[0202] Yield 79%, white powder; TIFF2024170457000091.tif20150

[0203] 6-(2,6-dichlorophenyl)-2-(methylsulfonyl)-8-(4-(methylsulfonyl)benzyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14r):

[0204] Yield 72%, white powder; TIFF2024170457000092.tif14148

[0205] 6-(4-Methoxyphenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (14s):

[0206] Yield 97%, yellow solid; TIFF2024170457000093.tif20150

[0207] 4-(2-(diethylamino)ethoxy)aniline (15a):

[0208] CH 3 To a solution of N,N-diethyl-2-(4-nitrophenoxy)ethanamine (490 mg, 2.06 mmol) in OH (10 mL) was added excess 10% Pd / C (125 mg) and stirred for 30 min at 37°C. 2 (g) was passed through a latex bulb bladder for 4 h. The reaction mixture was then filtered through Celite and concentrated to give 15a (412 mg, 96%) as a brown viscous liquid. TIFF2024170457000094.tif27150

[0209] N-(4-(2-(diethylamino)ethoxy)phenyl)formamide (16a):

[0210] Method A: 15a (100 mg, 0.48 mmol) was added with formic acid (1 mL) in a round-bottom flask containing molecular sieves. The reaction mixture was heated at 60 °C for 6 h, then NaHCO 3 The mixture was partitioned between a saturated solution of 16a and EtOAc, and the organic layer was then washed with brine solution and then concentrated to give 16a (90 mg, 79%) as a brown viscous liquid, which was used directly in the next step without purification.

[0211] N-(4-(methylsulfonyl)phenyl)formamide (16b):

[0212] 16b was prepared by method A by stirring the reaction mixture at room temperature overnight, 80% yield, white solid, which was used directly in the next step without purification.

[0213] N-(3-(methylsulfonyl)phenyl)formamide (16c):

[0214] 16c was prepared by method A by stirring the reaction mixture at room temperature overnight. Yield 80%, white solid; TIFF2024170457000095.tif27150

[0215] Preparation of (16d):

[0216] tert-Butyl 4-(3-nitrophenyl)piperazine-1-carboxylate (15d * ):

[0217] To a solution of Boc anhydride (790 mg, 3.62 mmol) in DCM (10 mL) was added DMAP (60 mg, 0.49 mmol) and the solution was stirred for 5 min. Then 1-(3-nitrophenyl)piperazine (500 mg, 2.41 mmol) was added to the above solution and stirred for 20 h at room temperature. After completion, the reaction mixture was concentrated and purified by column chromatography on silica gel (30% EtOAc / Hexane) to give 15d * (700 mg, 94%) was obtained as a yellow solid. TIFF2024170457000096.tif27150

[0218] tert-Butyl 4-(3-aminophenyl)piperazine-1-carboxylate (15d):

[0219] CH 3 15d in OH (15 mL) * (700 mg, 2.27 mmol) was added to an excess of 10% Pd / C (200 mg) and heated with H 2 (g) was passed through a latex bulb bladder overnight. The reaction mixture was then filtered through Celite and concentrated to give 15d (600 mg, 95%) as a brown viscous liquid, which was used in the next step without purification.

[0220] tert-Butyl 4-(3-formamidophenyl)piperazine-1-carboxylate (16d):

[0221] Method B: 15d (200 mg, 0.72 mmol) and ethyl formate (1.16 mL, 14.4 mmol) were added to a round bottom flask equipped with a reflux condenser. TEA (0.15 mL, 1.08 mmol) was added to the above mixture and heated under reflux. After stirring overnight, the solvent was evaporated and the mixture was dissolved in DCM and extracted with water and brine solution. The extract was concentrated and purified by column chromatography on silica gel (30% EtOAc / Hexane) to give 16d (100 mg, 45%) as a colorless liquid.

[0222] N-(pyridin-2-yl)formamide (16e):

[0223] Method C: Formic acid (0.28 mL, 7.43 mmol) was added dropwise to acetic anhydride (0.60 mL, 6.37 mmol) kept at 0° C. The mixture was heated to reflux at 60° C. for 2 h to generate acetic formic anhydride reagent. The mixture was cooled to room temperature and 2 mL of THF was added. 15e (200 mg, 2.12 mmol) dissolved in THF (1 mL) was added to the acetic formic anhydride mixture and refluxed for another 2 h. After completion, the solvent was evaporated and extracted with EtOAc / water system. The extract was concentrated and purified by column chromatography on silica gel (3% MeOH / DCM) to give 16e (173 mg, 67%) as a white solid.

[0224] N-(pyridin-3-yl)formamide (16f):

[0225] 16f was prepared by Method C. Yield 75%, white solid.

[0226] General procedure for preparation of 17a-s:

[0227] Figure 17 shows a synthesis scheme of compounds 17a-s (UH15), which are exemplary inhibitors of RIPK3. Figures 18-21 show structures of exemplary inhibitors of RIPK3 according to preferred embodiments.

[0228] To a solution of 16a (30 mg, 0.13 mmol) in THF (0.5 mL) and DMF (0.5 mL) was added 60% NaH (8 mg, 0.33 mmol) at 0° C. and the mixture was stirred at room temperature for 30 min under argon. The mixture was then cooled to 0° C. and 14a (25 mg, 0.06 mmol) was added and stirred at room temperature for 2 h. The reaction mixture was quenched by the addition of ice and NaOH (0.5 mL, 2N) solution and then partitioned between water and EtOAc. Anhydrous Na 2 SO 4 After filtration through HCl and concentration, the crude mixture was purified by column chromatography on silica gel (5% MeOH / DCM) to give 17a (UH15_1) as a yellow solid.

[0229] 6-(2,4-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (17a) UH15_1:

[0230] 74% yield, yellow solid: TIFF2024170457000097.tif47150 Purity 95.6%(t R 21.56 minutes).

[0231] 6-(2-chlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (17b) UH15_2:

[0232] 17b was made using 16a (40 mg, 0.17 mmol) and 14b (89 mg, 0.25 mmol).

[0233] Yield 62%, yellow solid; TIFF2024170457000098.tif47150 Purity 99.15%(t R 19.93 minutes).

[0234] 2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-methyl-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (17c) UH15_3:

[0235] 17c was made using 16a (40 mg, 0.17 mmol) and 14c (20 mg, 0.06 mmol).

[0236] Yield 64%, yellow solid; TIFF2024170457000099.tif47150 Purity 99.4%(t R 19.75 minutes).

[0237] 2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-ethyl-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (17i) UH15_4:

[0238] 17i was made using 16a (30 mg, 0.13 mmol) and 14i (40 mg, 0.12 mmol).

[0239] Yield 54%, yellow solid; TIFF2024170457000100.tif34150 Purity 95.8%(t R 20.50 minutes).

[0240] 2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-(2-hydroxyethyl)-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (17j) UH15_5:

[0241] 17j was made using 16a (30 mg, 0.13 mmol) and 14j (44 mg, 0.13 mmol).

[0242] Yield 33%, yellow solid; TIFF2024170457000101.tif34150

[0243] 6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-(2-methoxyethyl)pyrido[2,3-d]pyrimidin-7(8H)-one (17k)UH15_6:

[0244] 17k was made using 16a (22 mg, 0.09 mmol) and 14k (20 mg, 0.05 mmol).

[0245] Yield 61%, yellow solid; TIFF2024170457000102.tif47150 Purity 97.4%(t R 20.76 minutes).

[0246] 8-Benzyl-6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17l) UH15_7:

[0247] 17l was made using 16a (16 mg, 0.07 mmol) and 14l (32 mg, 0.07 mmol).

[0248] Yield 68%, yellow solid; TIFF2024170457000103.tif47150 Purity 98.4%(t R 22.59 minutes).

[0249] 6-(4-chlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (17d) UH15_8:

[0250] 17d was made using 16a (19 mg, 0.08 mmol) and 14d (28 mg, 0.08 mmol).

[0251] Yield 45%, yellow solid; TIFF2024170457000104.tif47150 Purity 96.5%(t R 21.33 minutes).

[0252] 6-(4-(tert-butyl)phenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (17e) UH15_9:

[0253] 17e was made using 16a (32 mg, 0.14 mmol) and 14e (50 mg, 0.14 mmol).

[0254] Yield 58%, yellow solid; TIFF2024170457000105.tif47150 Purity 99.8%(t R 23.44 minutes).

[0255] 6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-isobutylpyrido[2,3-d]pyrimidin-7(8H)-one (17m) UH15_10:

[0256] 17m was made using 16a (23 mg, 0.09 mmol) and 14m (42 mg, 0.09 mmol).

[0257] Yield 36%, yellow solid; TIFF2024170457000106.tif54150

[0258] 6-(2,6-dichlorophenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17f) UH15_11:

[0259] 17f was made using 16c (21 mg, 0.11 mmol) and 14f (20 mg, 0.05 mmol).

[0260] Yield 92%, white solid; TIFF2024170457000107.tif41150 Purity 98.2%(t R 22.98 minutes).

[0261] 6-(2,6-dichlorophenyl)-8-methyl-2-((4-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17f * )UH15_12:

[0262] 16b (11 mg, 0.05 mmol) and 14f (10 mg, 0.03 mmol) were used to synthesize 17f * was prepared.

[0263] Yield 75%, white powder; TIFF2024170457000108.tif34150 Purity 97.6%(tR 22.92 minutes).

[0264] 6-(4-chlorophenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17d * )UH15_13:

[0265] 16c (29 mg, 0.15 mmol) and 14d (26 mg, 0.07 mmol) were used for 17d. * was prepared.

[0266] Yield 61%, light yellow powder; TIFF2024170457000109.tif27150

[0267] 6-(4-chlorophenyl)-8-isobutyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17n) UH15_14:

[0268] 17n was made using 16c (15 mg, 0.07 mmol) and 14n (20 mg, 0.05 mmol).

[0269] Yield 33%, pale yellow solid; TIFF2024170457000110.tif33150

[0270] 6-(2-chlorophenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17b * )UH15_15:

[0271] 16c (34 mg, 0.17 mmol) and 14b (50 mg, 0.14 mmol) were used to prepare 17b. * was prepared.

[0272] Yield 72%, white solid; TIFF2024170457000111.tif27150 Purity 98.1%(tR 22.36 minutes).

[0273] 6-(2,4-dichlorophenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17a * )UH15_16:

[0274] 16c (8 mg, 0.04 mmol) and 14a (12 mg, 0.03 mmol) were used to synthesize 17a * was prepared.

[0275] Yield 63%, pale yellow solid; TIFF2024170457000112.tif41150 Purity 99.5%(t R 24.04 minutes).

[0276] 6-(2,3-dichlorophenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17g) UH15_17:

[0277] 17g was made using 16c (16 mg, 0.08 mmol) and 14g (25 mg, 0.06 mmol).

[0278] Yield 68%, pale yellow solid; TIFF2024170457000113.tif47150 Purity 98.5%(t R 23.50 minutes).

[0279] 6-(2-chloro-4-fluorophenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17h) UH15_18:

[0280] 17h was made using 16c (11 mg, 0.05 mmol) and 14h (20 mg, 0.04 mmol).

[0281] Yield 60%, white solid; TIFF2024170457000114.tif47150 Purity 97.6%(t R 22.72 minutes).

[0282] 6-(2,5-dichlorophenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17o) UH15_19:

[0283] 17o was prepared using 16c (6 mg, 0.03 mmol) and 14o (10 mg, 0.026 mmol).

[0284] Yield 92%, white solid; TIFF2024170457000115.tif27150 Purity 97.4%(t R 23.76 minutes).

[0285] 6-(4-hydroxyphenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17p) UH15_21:

[0286] 17p was made using 16c (22 mg, 0.11 mmol) and 14p (40 mg, 0.09 mmol).

[0287] Yield 79%, pale yellow solid; TIFF2024170457000116.tif27150

[0288] 2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-6-(4-hydroxyphenyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (17p * )UH15_22:

[0289] 16a (20 mg, 0.081 mmol) and 14p (30 mg, 0.067 mmol) were used to * was prepared.

[0290] Yield 64%, pale yellow solid; TIFF2024170457000117.tif34150

[0291] 6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-(3-(methylsulfonyl)benzyl)pyrido[2,3-d]pyrimidin-7(8H)-one (17q) UH15_26:

[0292] Yield 84%, pale yellow solid; TIFF2024170457000118.tif34150 Purity 95.6%(t R 21.35 minutes).

[0293] 6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-(4-(methylsulfonyl)benzyl)pyrido[2,3-d]pyrimidin-7(8H)-one (17r) UH15_27:

[0294] Yield 70%, pale yellow solid; TIFF2024170457000119.tif34150 Purity 95.6%(t R 21.24 minutes).

[0295] 6-(4-Methoxyphenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17s) UH15_29:

[0296] 17s was made using 16c (12 mg, 0.06 mmol) and 14s (20 mg, 0.05 mmol).

[0297] Yield 95%, yellow solid; TIFF2024170457000120.tif26150

[0298] 2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-6-(4-methoxyphenyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (17s * )UH15_30:

[0299] 16a (8 mg, 0.03 mmol) and 14s (14 mg, 0.03 mmol) were used to prepare 17s. * was prepared.

[0300] Yield 66%, yellow solid; TIFF2024170457000121.tif33150

[0301] Preparation of UH15_32:

[0302] FIG. 22 shows a synthetic scheme for compound UH15_32, an exemplary inhibitor of protein kinase, according to a preferred embodiment.

[0303] Ethyl 6-chloro-4-(methylamino)nicotinate (52):

[0304] To a solution of ethyl 4,6-dichloronicotinate 51 (100 mg, 0.45 mmol) in THF (3 mL) was added aqueous methylamine (0.4 mL) at 0° C. and the mixture was stirred at the same temperature for 30 min. After 30 min, the reaction mixture was stirred at room temperature for 2 h. After completion, the mixture was concentrated and purified by column chromatography on silica gel (10% EtOAc / Hexane) to give 52 (65 mg, 67%) as a white solid. TIFF2024170457000122.tif20151

[0305] (6-chloro-4-(methylamino)pyridin-3-yl)methanol (53):

[0306] A solution of 52 (100 mg, 0.46 mmol) in THF (2 mL) was added dropwise to a suspension of LAH (27 mg, 0.69 mmol) in THF (5 mL) at 0° C. and stirred at room temperature for 30 min. The reaction mixture was then cooled at 0° C. and 15% NaOH (2 mL) and water (4 mL) were added dropwise. The reaction mixture was stirred for 1 h, filtered and washed with EtOAc. The solvent was evaporated and the extract was purified by column chromatography on silica gel (2.5% MeOH / DCM) to give 53 (77 mg, 96%) as a white solid. TIFF2024170457000123.tif20150

[0307] 6-Chloro-4-(methylamino)nicotinaldehyde (54):

[0308] A solution of 53 (495 mg, 2.86 mmol) in DCM (10 mL) was diluted with MnO 2 (1496 mg, 17.20 mmol) was added and the mixture was stirred at room temperature overnight. The reaction mixture was then filtered, concentrated to remove DCM and purified by column chromatography on silica gel (2% MeOH / DCM) to give 54 (453 mg, 93%) as a white solid. TIFF2024170457000124.tif13150

[0309] 7-Chloro-3-(2-chlorophenyl)-1-methyl-1,6-naphthyridin-2(1H)-one (56):

[0310] A stirred solution of 54 (300 mg, 1.75 mmol) and methyl 2-(2-chlorophenyl)acetate 55 (325 mg, 1.75 mmol) in dry DMA (4 mL) was diluted with KF / Al 2 O 3 (1800 mg, 40 wt%) was added and the reaction mixture was stirred at room temperature for 2 h. Upon completion, the reaction mixture was filtered through Celite, the remaining solid was washed with DCM and the filtrate was concentrated. The residue was purified by column chromatography on silica gel (20% EtOAc / Hexanes) to give 56 (400 mg, 75%) as a white solid. TIFF2024170457000125.tif20150

[0311] 3-(2-chlorophenyl)-1-methyl-7-((3-(methylsulfonyl)phenyl)amino)-1,6-naphthyridin-2(1H)-one (57) UH15_32:

[0312] In a preheated round-bottom flask, add 56 (30 mg, 0.09 mmol), 15c (20 mg, 0.11 mmol), and Pd 2 (dba) 3 (9 mg, 0.009 mmol), XPhos Pd G1 (9 mg, 0.02 mmol) and sodium tert-butoxide (28 mg, 0.29 mmol) were added and flushed with argon for 10 min. Dioxane (1.5 mL) was added to the mixture and flushed again for 5 min, then heated at 80 °C overnight. Upon completion, the reaction mixture was partitioned between ethyl acetate and water, filtered, concentrated and purified by column chromatography on silica gel (2% MeOH / DCM) to give 57 (UH15_32) (20 mg, 47%) as a white solid. TIFF2024170457000126.tif41150

[0313] Preparation of UH15_33

[0314] FIG. 23 shows a synthetic scheme for compound UH15_33, an exemplary inhibitor of protein kinase, according to a preferred embodiment.

[0315] 4-(3-((6-(2-chlorophenyl)-8-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)phenyl)piperazine-1-carboxylate tert-butyl (58).

[0316] 58 was made using 14b (62 mg, 0.17 mmol) and 16d (54 mg, 0.17 mmol).

[0317] Yield 70%, pale yellow solid; TIFF2024170457000127.tif41150

[0318] 6-(2-chlorophenyl)-8-methyl-2-((3-(piperazin-1-yl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (59) UH15_33:

[0319] 58 (67 mg, 0.12 mmol) was taken up in DCM (4.5 mL) and TFA (0.5 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated and the crude mixture was purified by column chromatography on silica gel (4% MeOH / DCM) to give 59 (UH15_33) (55 mg, 96%) as a pale yellow solid. TIFF2024170457000128.tif27150

[0320] 6-(2-chlorophenyl)-8-methyl-2-(pyridin-3-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (UH15_34):

[0321] UH15_34 was made using 14b (15 mg, 0.05 mmol) and 16f (6 mg, 0.05 mmol).

[0322] Yield 67%, white solid; TIFF2024170457000129.tif41151

[0323] 6-(2-chlorophenyl)-8-methyl-2-(pyridin-2-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (UH15_35):

[0324] UH15_35 was made using 14b (20 mg, 0.06 mmol) and 16e (8 mg, 0.06 mmol).

[0325] Yield 62%, pale yellow solid. TIFF2024170457000130.tif27151

[0326] Preparation of UH15PN compound:

[0327] According to a preferred embodiment, Figure 24A shows a synthetic scheme of exemplary inhibitors of protein kinases, and Figure 24B shows structures of intermediate compounds used in the synthesis of these exemplary inhibitors of protein kinases. Figure 25 shows structures of exemplary inhibitors of protein kinases, according to a preferred embodiment.

[0328] 6-Bromo-N-(4-(2-(diethylamino)ethoxy)phenyl)quinazolin-2-amine (19a):

[0329] To a mixture of 6-bromo-2-chloroquinazoline (18) (200 mg, 0.82 mmol) and 15a (207 mg, 0.98 mmol) was added propan-2-ol (4 mL) and the reaction mixture was heated at 100° C. for 2 h. Upon completion, the reaction mixture was cooled to room temperature, concentrated and purified by column chromatography on silica gel (5% MeOH / DCM) to give 19 (203 mg, 61%) as a yellow solid. TIFF2024170457000131.tif41151

[0330] 6-(2-chlorophenyl)-N-(4-(2-(diethylamino)ethoxy)phenyl)quinazolin-2-amine UH15PN1:

[0331] 6-Bromo-N-(4-(2-(diethylamino)ethoxy)phenyl)quinazolin-2-amine (19a) (30 mg, 0.07 mmol), (2-chlorophenyl)boronic acid (20a) (17 mg, 0.11 mmol) and Pd(PPh 3 ) 4 (8.5 mg, 0.007 mmol) was taken in a round-bottom flask (rb) and purged with argon for 10 min. Then DMF (1 mL) and CH 3 A solvent mixture of CN (2 mL) was added to the above reaction and purged again for 10 min. After purging, 1M Na 2 CO 3(150 μl) solution was added dropwise and heating at 90° C. for 5 h was initiated. Upon completion, the reaction mixture was cooled to room temperature and the solvent was evaporated. The residue was partitioned between water and EtOAc, filtered and concentrated to give a crude mixture which was purified by column chromatography on silica gel (5% MeOH / DCM) to give UH15PN1 (23 mg, 70%) as a yellow solid. TIFF2024170457000132.tif47150

[0332] 6-Bromo-N-(3-(methylsulfonyl)phenyl)quinazolin-2-amine (19c):

[0333] To a mixture of 6-bromo-2-chloroquinazoline 18 (100 mg, 0.41 mmol) and 15c (85 mg, 0.49 mmol) was added propan-2-ol (2 mL) and the reaction mixture was heated at 100° C. for 2 h. Upon completion, the reaction mixture was cooled to room temperature, concentrated and purified by column chromatography on silica gel (2% MeOH / DCM) to give 19c (150 mg, 97%) as a pale yellow solid. TIFF2024170457000133.tif20150

[0334] 6-(2-chlorophenyl)-N-(3-(methylsulfonyl)phenyl)quinazolin-2-amine (UH15PN2):

[0335] 6-Bromo-N-(3-(methylsulfonyl)phenyl)quinazolin-2-amine (19c) (50 mg, 0.08 mmol) and (2-chlorophenyl)boronic acid (20a) (31 mg, 0.12 mmol) were used to prepare UH15PN2 (31 mg, 95%) as a yellow solid. TIFF2024170457000134.tif34150

[0336] 4-(2-((3-(methylsulfonyl)phenyl)amino)quinazolin-6-yl)phenol (UH15PN3):

[0337] 19c (30 mg, 0.08 mmol) and (4-hydroxyphenyl)boronic acid (20c) (17 mg, 0.12 mmol) were used to prepare UH15PN3 (22 mg, 71%) as a yellow solid. TIFF2024170457000135.tif27150

[0338] N-(3-(methylsulfonyl)phenyl)-6-(pyridin-2-yl)quinazolin-2-amine (UH15PN5):

[0339] FIG. 26 shows a synthetic scheme for UH15PN5, an exemplary inhibitor of protein kinases, according to a preferred embodiment.

[0340] 6-Bromo-N-(3-(methylsulfonyl)phenyl)quinazolin-2-amine (19c) (20 mg, 0.05 mmol), pyridin-2-ylboronic acid (20e) (22 mg, 0.10 mmol), Pd(dppf)Cl 2 (4 mg, 0.005 mmol), CuBr (12 mg, 0.05 mmol) and Cs 2 CO 3 (69 mg, 0.21 mmol) was taken in a round bottom flask and purged with argon for 10 minutes. DMF (2 mL) was then added to the above reaction and purged again for 10 minutes. After purging, the contents were heated at 100° C. for 16 hours. Upon completion, the reaction mixture was cooled to room temperature and the solvent was evaporated. The residue was partitioned between water and EtOAc, filtered and concentrated to give a crude mixture which was purified by column chromatography on silica gel (2.5% MeOH / DCM) to give UH15PN5 (15 mg, 60%) as a light brown solid. TIFF2024170457000136.tif27150

[0341] N-(3-(methylsulfonyl)phenyl)-6-(pyridin-3-yl)quinazolin-2-amine (UH15PN6):

[0342] 19c (30 mg, 0.08 mmol) and pyridin-3-ylboronic acid (20d) (15 mg, 0.12 mmol) were used to prepare UH15PN6 (15 mg, 50%) as a yellow solid. TIFF2024170457000137.tif26150

[0343] Preparation of UH15_25:

[0344] FIG. 27 shows a synthetic scheme for UH15_25, an exemplary inhibitor of protein kinase, according to a preferred embodiment.

[0345] 4-(3-((3-(2-chlorophenyl)-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)amino)phenyl)piperazine-1-carboxylate tert-butyl (60):

[0346] In a preheated round-bottom flask, add 56 (20 mg, 0.065 mmol), 15d (22 mg, 0.078 mmol), and Pd 2 (dba) 3 (6 mg, 0.006 mmol), Xantphos (8 mg, 0.013 mmol) and cesium carbonate (43 mg, 0.13 mmol) were added and flushed with argon for 10 minutes. Dioxane (1.5 ml) was added to the mixture and flushed again for 5 minutes and then heated at 80° C. overnight. The reaction mixture was then partitioned between ethyl acetate and water and anhydrous Na 2 SO 4 The mixture was dried at rt, filtered, concentrated and purified by column chromatography on silica gel (1.5% MeOH / DCM) to give 60 (20 mg, 57%) as a pale yellow solid.

[0347] 3-(2-chlorophenyl)-1-methyl-7-((3-(piperazin-1-yl)phenyl)amino)-1,6-naphthyridin-2(1H)-one (61)UH15_25:

[0348] 60 (20 mg, 0.036 mmol) was taken up in DCM (2 mL) and TFA (0.2 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated and the crude mixture was purified by column chromatography on silica gel (7% MeOH / DCM) to give 61 (UH15_25) (12 mg, 75%) as a pale yellow solid. TIFF2024170457000138.tif27150

[0349] Preparation of UH15_36:

[0350] FIG. 28 shows a synthetic scheme for UH15_36, an exemplary inhibitor of protein kinase, according to a preferred embodiment.

[0351] tert-Butyl 4-(5-nitropyridin-3-yl)piperazine-1-carboxylate (69):

[0352] Add Pd to a preheated round-bottom flask. 2 (dba) 3 (125 mg, 0.13 mmol), Xantphos (155 mg, 0.26 mmol) and potassium carbonate (740 mg, 5.36 mmol) were added and flushed with argon for 10 min. DMF (4 mL) was added to the mixture and flushed for an additional 5 min, followed by addition of 67 (500 mg, 2.68 mmol) and 68 (545 mg, 2.68 mmol) and then refluxed for 24 h. The reaction mixture was then partitioned between ethyl acetate and water and anhydrous Na 2 SO 4 The mixture was dried at rt, filtered, concentrated and purified by column chromatography on silica gel (15% EtOAc / DCM) to give 69 (250 mg, 30%) as a pale yellow solid. TIFF2024170457000139.tif13140

[0353] tert-Butyl 4-(5-aminopyridin-3-yl)piperazine-1-carboxylate (70):

[0354] CH 3To a solution of 69 (250 mg, 0.81 mmol) in OH (15 mL) was added 10% Pd / C (50 mg) and the reaction was incubated at room temperature for 30 min with H 2 (g) (1 atm) for 4 h. The reaction mixture was then filtered through Celite and concentrated to give 70 (220 mg, 97%) as a brown solid, which was used in the next step without purification.

[0355] 4-(tert-butyl 5-((3-(2-chlorophenyl)-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)amino)pyridin-3-yl)piperazine-1-carboxylate (71):

[0356] In a preheated round-bottom flask, add 56 (42 mg, 0.13 mmol), 70 (39 mg, 0.13 mmol), and Pd 2 (dba) 3 (13 mg, 0.013 mmol), Xantphos (16 mg, 0.027 mmol) and cesium carbonate (90 mg, 0.27 mmol) were added and flushed with argon for 10 minutes. Dioxane (3 ml) was added to the mixture and flushed again for 5 minutes and then heated at 80° C. overnight. The reaction mixture was then partitioned between ethyl acetate and water and anhydrous Na 2 SO 4 The mixture was dried at rt, filtered, concentrated and purified by column chromatography on silica gel (2.5% MeOH / DCM) to give 71 (26 mg, 35%) as a light brown solid.

[0357] 3-(2-chlorophenyl)-1-methyl-7-((5-(piperazin-1-yl)pyridin-3-yl)amino)-1,6-naphthyridin-2(1H)-one (72) UH15_36:

[0358] 71 (20 mg, 0.036 mmol) was taken up in DCM (2 mL) and TFA (0.2 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated and the crude mixture was purified by reverse phase preparative HPLC (95% H with 0.1% TFA). 2 O / CH 3 CN ~ 95% CH containing 0.1% TFA 3CN / H 2 O) to afford 72 (UH15_36) (12 mg, 75%) as a pale yellow solid. TIFF2024170457000140.tif34150

[0359] Preparation of 34 (UH15_20):

[0360] FIG. 29 shows a synthetic scheme for UH15_20, an exemplary inhibitor of protein kinase, according to a preferred embodiment.

[0361] 6-Chloro-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (31):

[0362] A mixture of 7 (20 mg, 0.11 mmol) and NaH (8 mg, 0.33 mmol) was added to THF (1.5 mL) and stirred at room temperature for 10 min under argon. A solution of ethyl 2-chloro-2-(diethoxyphosphoryl)acetate 30 (42 mg, 0.16 mmol) in THF (0.5 mL) was added dropwise and the mixture was refluxed for 2.5 h. After completion, the reaction mixture was cooled to room temperature, concentrated and extracted with EtOAc and water system. The organic solvent was evaporated and the residue was purified by column chromatography on silica gel (30% EtOAc / Hexane) to give 31 (10 mg, 42%) as a white solid. TIFF2024170457000141.tif20150

[0363] 6-(2-chloro-4-methylphenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (32):

[0364] 31 (24 mg, 0.11 mmol), (2-chloro-4-methylphenyl)boronic acid (28 mg, 0.16 mmol) and Pd(PPh 3 ) 4 (13 mg, 0.011 mmol) was taken in a round bottom flask (rb) and purged with argon for 10 min. DMF (1 mL) and CH 3CN (2 mL) was added to the above reaction and the reaction mixture was purged again for 10 min. After purging, 1M Na 2 CO 3 A solution of (23 mg, 0.21 mmol) (220 μL) was added dropwise and heating at 90° C. for 5 h was initiated. Upon completion, the reaction mixture was cooled to room temperature and the solvent was evaporated. The residue was partitioned between water and EtOAc, filtered and concentrated to give a crude mixture which was purified by column chromatography on silica gel (10% EtOAc / DCM) to give 32 (20 mg, 57%) as a white solid. TIFF2024170457000142.tif27150

[0365] 6-(2-chloro-4-methylphenyl)-8-methyl-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (33):

[0366] Yield 68%, white solid; TIFF2024170457000143.tif27150

[0367] 6-(2-chloro-4-methylphenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (34) UH15_20:

[0368] Yield 75%, white solid; TIFF2024170457000144.tif26150 Purity 99.6%(t R 23.51 minutes).

[0369] Preparation of UH15-38:

[0370] Figure 30 shows a synthesis scheme of UH15_20, an exemplary inhibitor of protein kinase, according to a preferred embodiment. In Figure 30, reagents and conditions: (a) TBSCl, imidazole, DMAP, DMF, 0°C to room temperature, 4 hours, 83%; (b) KF / Al 2 O 3 , DMA, room temperature, 2 h, 67%; (c)Pd2 (dba) 3 , Xantphos, CS 2 CO 3 , 1,4-dioxane, 80°C, 16h, 47%.

[0371] 2-(3-((tert-butyldimethylsilyl)oxy)phenyl)methyl acetate (2):

[0372] To a solution of 1 (250 mg, 1.504 mmol) in dry DMF (3 mL) was added catalytic amounts of DMAP (15 mg) and imidazole (295 mg, 1.953 mmol) in one portion, followed by TBSCl (255 mg, 3.75 mmol) in one portion at 0° C. The resulting mixture was then stirred at room temperature for 4 h. After completion of the reaction, the mixture was quenched by the addition of water (10 mL) and diluted with ethyl acetate (20 mL). The resulting mixture was washed multiple times with water, brine, and anhydrous NaCl. 2 SO 4 The mixture was dried at 40° C. and concentrated in vacuo. The crude compound was purified by Combiflash chromatography (0-5% ethyl acetate in hexanes) to give 2 (353 mg, 83%) as a colorless liquid. TIFF2024170457000145.tif27150

[0373] 7-Chloro-3-(3-hydroxyphenyl)-1-methyl-1,6-naphthyridin-2(1H)-one (4):

[0374] A solution of 2 (190 mg, 0.677 mmol) and 3 (115 mg, 0.674 mmol) in dry DMA (3 mL) was diluted with KF / Al 2 O 3 (1.14 g) was added in one portion. The resulting mixture was stirred at room temperature for 2 hours. After completion of the reaction, the mixture was filtered through a small pad of Celite and washed with ethyl acetate (50 mL). The resulting mixture was washed multiple times with water, brine, and anhydrous Na 2 SO 4 The crude material was purified by Combiflash chromatography (CH 2 Cl 2Purification by elution with 0% methanol in water) to give 4 (130 mg, 67%) as a white solid. TIFF2024170457000146.tif21150

[0375] 3-(3-hydroxyphenyl)-1-methyl-7-((3-(4-methylpiperazin-1-yl)phenyl)amino)-1,6-naphthyridin-2(1H)-one (6;UH15-38):

[0376] 4 (30 mg, 0.104 mmol), 3-(4-methylpiperazin-1-yl)aniline (5, 20 mg, 0.104 mmol), Xantphos (12 mg, 0.021 mmol), and CS in dioxane (2 mL). 2 CO 3 (68 mg, 0.208 mmol) in a solution of 10 mol% Pd 2 (dba) 3 The reaction mixture was purged with Ar gas for 15 min and refluxed at 80° C. for 16 h. After completion of the reaction, the mixture was filtered through a small pad of Celite, water (5 mL) was added, and the organic compounds were extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with brine and anhydrous Na 2 SO 4 The crude compound was purified by Combiflash chromatography (CH 2 Cl 2 Purification by elution with 0% methanol in water) gave the product 6 (22 mg, 47%) as a yellow solid. TIFF2024170457000147.tif40150

[0377] Example 2. Evaluation and Classification Active: Table 1 below shows the activity of selected UH15 analogs. IC50 values ​​were calculated for inhibition of recombinant RIPK3, inhibition of necroptosis in LPS / IDN-treated murine RAW264.7 macrophages and TNF / IDN-treated human HeLa cells expressing RIPK3, RIPK3 binding by inhibitors in live cells (NanoBRET assay), and induction of RIPK3-dependent apoptosis (i.e., on-target toxicity). 50 / LD 50 Values ​​were determined. Assays were performed using ADPGlo (kinase assay), CellTiter-Glo (viability), and NanoBRET (target binding) assays. GSK'872 values ​​shown are from Mandal et al., 2014.

[0378] [Table 1] NT - Not Tested * Values ​​given in literature

[0379] For the ADP-Glo ​​assay, recombinant RIPK3 protein (20 ng per reaction) is diluted in reaction buffer consisting of 50 mM HEPES, pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.05% bovine serum albumin (BSA), 0.02% CHAPS. The diluted protein is added (2 μL / well) to a low volume white 384-well plate. Inhibitors are diluted in reaction buffer (25% DMSO final) and 1 μL is added to each well and incubated at room temperature for 5 min. The reaction is started by the addition of 2 μL of 100 μM ATP to the reaction buffer. The plate is sealed with a plastic coverslip and incubated at room temperature for 4 h. The reaction is stopped by the addition of 5 μL of ADP-Glo ​​reagent (Promega) and the ADP generation reaction is carried out for 40 min at room temperature. 10 μL of Kinase Detection Reagent (Promega) is added and luminescence signal is generated for 30 min at room temperature. Luminescent signals are determined using an appropriate luminescence plate reader (typical integration times 0.3-1 sec). To calculate percent inhibition, the average background signal is subtracted from the test wells and from the maximum signal well. Inhibition (%) = (1-(test signal / maximum signal))*100 Nonlinear regression in GraphPad Prism software was used to determine the percentage of inhibition at a specified concentration based on the inhibitor concentration over a dose range, or IC 50 For the CellTiter-Glo assay, cells are treated in 96-well plates for 6–24 h with or without 10 ng / ml hTNFα (Peprotech, Hela-R3 cells) or E. coli LPS (Sigma, RAW264.7 cells) in combination with 10 μM IDN6556 (MedKoo) to induce necroptosis. After incubation with the drugs, 15 μL of CellTiter-Glo reagent is added to the wells and incubated for 10 min. Luminescent signals are determined using an appropriate luminescence plate reader (typical integration time 0.3 s). To calculate the viability, the following: Viability (%) = (1-(test signal / maximum viability in untreated wells))*100

[0380] Phosphorylation of RIPK3 and MLKL: Figure 31 shows that selected UH15 compounds and GSK'872 block phosphorylation of RIPK3 and MLKL in RAW264.7 macrophages. Necroptosis was induced by a combination of LPS (10 ng / ml) and the pan-caspase inhibitor IDN6556 (20 μM). Note that LPS, like TNF, is a TLR4 ligand that can activate RIPK3 (and necroptosis) in the presence of caspase inhibitors. Asterisks represent nonspecific bands occasionally seen in immunoblots of pMLKL. Samples are subjected to SDS-PAGE gel electrophoresis and western blotting with RIPK3, phospho-Thr231 / Ser232-RIPK3, MLKL and phospho-Ser345-MLKL antibodies (Abcam).

[0381] RIPK3-dependent toxicity: Table 2 shows the RIPK3-dependent toxicity of selected UH15 analogs, both stimulus-dependent (with TNF, 10 ng / ml) and independent (without TNF). Cell death was assessed by CellTiter-Glo assay. Toxicity was compared in Hela cells in which RIPK3 was deleted and in cells in which RIPK3 was re-expressed by lentivirus (Hela-RIPK3 cells).

[0382] [Table 2]

[0383] Receptor interacting protein kinase 2 (RIPK2) enzyme assay: Recombinant RIPK2 protein (20 ng per reaction) was incubated in 40 mM Tris (pH 7.5); 20 mM MgCl 2 The proteins are diluted in reaction buffer consisting of; 0.1 mg / mL BSA; 50 μM DTT. The diluted proteins are added (2 μL / well) to a low volume white 384-well plate. Inhibitors are diluted in reaction buffer (25% DMSO final) and 1 μL is added to each well and incubated at room temperature for 5 min. The reaction is initiated by the addition of 2 μL of 100 μM ATP and 1 mg / mL RS repeat peptide (SignalChem) to the reaction buffer. The plate is sealed with a plastic coverslip and incubated at room temperature for 2 h. The reaction is stopped by the addition of 5 μL of ADP-Glo ​​reagent (Promega) and the ADP generation reaction is carried out at room temperature for 40 min. 10 μL of Kinase Detection Reagent (Promega) is added and the luminescent signal is generated at room temperature for 30 min. The luminescent signal is determined using an appropriate luminescence plate reader (typical integration time 0.3-1 sec). To calculate the percent inhibition, the average background signal is subtracted from the test wells and the maximum signal well. Inhibition (%) = (1-(test signal / maximum signal))*100 Nonlinear regression in GraphPad Prism software was used to determine the percentage of inhibition at a specified concentration based on the inhibitor concentration over a dose range, or IC 50 Calculate the value.

[0384] Activin-Like Kinase 2 (ALK2) Enzyme Assay: Enzyme inhibitory activity was determined by inhibiting human ALK2 with the protein substrates casein (1 mg / mL) and γ- 33 This was assessed in a standard kinase enzyme assay by incubation with ATP (10 μM). After 30 min, 33 The amount of P-casein was determined. A plot of inhibitor concentration vs. % activity was constructed and the IC 50 value was determined.

[0385] NOD2 signaling assay: HEK-Blue cells expressing human NOD2 and NFkB-SAEP reporter (Invivogen) were plated at 7.5 × 10 cells per well in 96-well clear plates in 100 μL of DMEM medium supplemented with 10% FBS and 1% antibiotic-antimycotic mixture. 3 The cells were incubated at 37°C in 5% CO 2 Allow to attach for 48 h in a tissue culture incubator. On the morning of the experiment, replace the medium in the wells with 100 μL of HEK-Blue detection medium (Invivogen). Incubate the cells with inhibitors diluted in DMSO (0.5 μL per well) for 15 min at 37 °C in 5% CO. 2 The cells are then stimulated by the addition of 1 ng / well of L18-MDP (Invivogen). The cells are incubated at 37°C in 5% CO 2 After 8 hours of incubation in a tissue culture incubator, the absorbance corresponding to SEAP in the medium is determined using a Wallac3V plate reader (Perkin Elmer). Inhibition (%) = (1-((Sample signal-unstimulated and DMSO treated cells) / (L18-MDP stimulated and DMSO treated cells-unstimulated and DMSO treated cells)))*100 IC based on inhibitor concentrations in a dose range using nonlinear regression in GraphPad Prism software 50 Calculate the value.

[0386] Inhibition of RIPK2 and ALK2 enzyme activity and NOD2 cell signaling by compounds: The prepared compounds were evaluated for their ability to inhibit RIPK2 and ALK2 enzyme activity and NOD2 cell signaling using the methods described above. The inhibition of RIPK2 enzyme and NOD2 cell signaling by the compounds was reported as percent inhibition or IC at the specified concentration. 50 The values ​​are shown in Table 3 below. IC for inhibition of ALK2 enzyme activity by compounds 50 The values ​​are also shown in Table 3.

[0387] [Table 3]

[0388] References The following documents are incorporated herein by reference: TIFF2024170457000151.tif197150TIFF2024170457000152.tif220150TIFF2024170457000153.tif81150

Claims

1. A pharmaceutical composition for use in the treatment of an inflammatory or degenerative disease or condition involving receptor interacting kinase 3 (RIPK3) activity, the composition exhibiting protein kinase inhibitory activity and comprising the following structure: (a) [In the formula, At one available ring position, R is H or R is where Me is methyl and Et is ethyl; A and D are independently N or CH; E is N, CH, or CR; B and C are independently N, CH, or C-Cl; At one available ring position, R 1 is H or R 1 C-Cl, CF, C-OCH 3 , CC(CH 3 ) 3 or C—OH, and XY is C=C or where R 2 is H, alkyl, alkylhydroxyl, alkylalkoxyl, or alkylaryl; or (b) [In the formula, A is N or CH; R1 is Me, where X is F or Me, where Me is methyl; R2 is Me, Et, Et-O-Me, or isobutyl, where Et is ethyl; and R3 is is] or a pharma- ceutical acceptable salt thereof.

2. The compound of claim 1, wherein the compound has the following structure:

2. The pharmaceutical composition of claim 1, comprising:

3. The compound of claim 2, wherein the compound has the following structure:

3. The pharmaceutical composition of claim 2, comprising:

4. The compound of claim 1, wherein the compound has the structure:

2. The pharmaceutical composition of claim 1, comprising:

5. The compound of claim 1, wherein the compound has the following structure: wherein R2 is Et, Et-O-Me, or isobutyl, and wherein X is F or Me.

5. The pharmaceutical composition of claim 4, comprising:

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the inflammatory or degenerative disease or condition in which RIPK3 activity is implicated is influenza A virus (IAV) infection.

7. A pharmaceutical composition described in any one of claims 1 to 5, wherein the inflammatory or degenerative disease or condition in which RIPK3 activity is involved is lung injury.

8. The pharmaceutical composition of claim 7, wherein the lung injury is necrotizing lung injury.

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