Protein kinase inhibitors and their use in the 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 on 16 May 2019, entitled "Pyrido[2,3-d]pyrimidin-7-ones and Related Compounds as Inhibitors of RIPK3 to Treat Inflammatory and Degenerative Conditions," and to U.S. Provisional Patent Application No. 62 / 848,719, filed on 16 May 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 to the invention.

[0003] This disclosure relates to compounds exhibiting protein kinase inhibitory activity. Protein kinases are important enzymes in cellular signaling. Abnormal signaling occurs in many pathological conditions. Therefore, protein kinase inhibitors can be used as therapeutic agents for the treatment of various diseases. [Background technology]

[0004] Autoimmune, autoinflammatory, and degenerative conditions in humans involve a continuous loop of cell death and inflammation, leading to tissue damage and dysfunction. This is seen in progressive chronic diseases with inflammatory etiologies, including TNF-driven conditions such as atherosclerosis, rheumatoid arthritis, and ulcerative colitis. It also occurs during adverse acute inflammatory responses, such as those caused by influenza viruses. While treatments exist for some of these conditions, many inflammatory diseases remain poorly controlled, and new therapeutic strategies are urgently needed.

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

[0006] Lung epithelium is the primary site of influenza A virus disease in mammals. Influenza A virus (IAV) is a negative-sense RNA virus belonging to the Orthomyxoviridae family. In waterfowl, the primary natural host of IAV, viral replication occurs in the gastrointestinal tract and is usually asymptomatic. In contrast, mammalian IAV strains replicate in the respiratory tract, causing symptoms ranging from mild "flu" to severe, sometimes fatal illness. IAV infects epithelial cells along the entire respiratory tract. Transmission is associated with upper respiratory tract infection, but severe illness is associated with lower lung infection, and the degree of lung involvement correlates with disease outcomes in human and animal models.

[0007] As a lytic virus, IAV kills most lung cell types in which it replicates. However, not all cell death caused by IAV is pathogenic. While programmed cell death is essential for the early control of IAV replication and the prevention of viral spread throughout the lung, death of the lung epithelial layer is also one of the main causes of morbidity and death associated with IAV infection.10 In particular, loss of type I airway epithelial cells (AEC, which are essential for gas exchange) is strongly correlated with death in IAV-infected mouse models when it exceeds a threshold of about 10%. If cell death is well controlled and apoptotic, this means a host defense mechanism that limits both viral spread and immunopathology. However, if cell death is uncontrolled or primarily necrotizing (i.e., highly pro-inflammatory), this can lead to a hyperinflammatory response, severe deterioration of the airway epithelium, and consequent host death, even if the virus is eliminated. Such severe symptoms are observed in mouse models where airway epithelial destruction 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). Therefore, the ideal treatment for IAV disease should promote death that facilitates clearance while suppressing inflammatory death and viral and secondary bacterial pneumonia that induce exacerbation.

[0008] Receptor-interacting protein kinase 3 (RIPK3) is a multifunctional protein involved in various tissue cell death pathways. Previous studies have demonstrated that loss of RIPK3 kinase activity provides protection in various animal models of inflammatory and degenerative conditions, including sepsis, traumatic brain injury, acute kidney injury, lung injury associated with influenza (IAV) infection, atherosclerosis, and many others. For example, it has recently been revealed that RIPK3 is a targetable signaling pathway that accounts for almost all 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 injury seen during IAV infection. Interestingly, eliminating necroptosis not only significantly reduces lung injury and improves animal survival rates, but does so without interfering with viral clearance. This is because RIPK3 also activates a parallel pathway of non-pathogenic cell death (apoptosis) that mediates viral clearance. Since only necroptosis and not apoptosis depend on RIPK3 kinase activity, inhibitors of RIPK3 kinase function are expected to improve necrotizing lung injury without affecting viral clearance, potentially offering a completely new strategy for treating IAV disease. Similarly, two separate studies have shown that animals lacking the Ripk3 gene are resistant to the development of atherosclerosis by suppressing macrophage cell death and systemic inflammation.

[0009] Selective RIPK3 kinase inhibitors are not in clinical use and have not advanced to clinical trials. In 2014, a panel of RIPK3-selective inhibitors was reported, but these compounds showed only moderate activity intracellularly and unfortunately induced unexpected conformational changes in the structure of RIPK3 that led to toxicity. Current vaccine and antiviral strategies have limited effectiveness or are susceptible to viral resistance and avoidance mechanisms, so identifying new therapeutic entry points for seasonal and virulent IAV lung disease, preferably those targeting pathogenic host signaling pathways, is an urgent challenge. There is a large unmet need for clinically practicable inhibitors of RIPK3 kinase activity.

Summary of the Invention

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

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

[0012] RIPK3 inhibitors potently and specifically inhibit RIPK3-driven necrosis. 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 necrosis in cells compared to previously reported RIPK3 inhibitor compounds. Importantly, compounds that block necrosis without toxicity have been identified.

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

[0014] RIPK3 is the central mediator of epithelial cell death or necroptosis caused by IAV. Necroptosis is activated by viral and microbial infections (including IAV), several innate immune signaling pathways (among others, TNF, interferon, and TLR), certain pro-inflammatory stimuli (e.g., asbestos, oxidized LDL), and genotoxic stress. For decades, cell death associated with acute IAV infection was thought to be apoptosis, autophagy, or simply a passive and unprogrammed consequence of viral infection, with details of which were unclear. However, these conclusions were mainly derived from cell culture studies of transformed cell lines with significantly deficient cell death pathways (e.g., A549 cells) and / or unrelated to IAV biology (e.g., HeLa, MDCK cells). In 2016, a cell death pathway active in lung epithelial cells and accounting for most of the cell death caused by IAV replication in these and other primary cell types was reported. This pathway is initiated when the host protein DNA-dependent activator of interferon regulators (DAIs) 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 a pseudokinase called mixed-lineage kinase domain-like (MLKL). The apoptosis arm requires FAS-associated protein (FADD) with a death domain and caspase-8 and proceeds without requiring RIPK3 kinase activity. From within the necrosome, RIPK3 phosphorylates and activates MLKL, which is then oligomerized and migrates to the cell membrane, where it creates holes in the membrane, causing cell swelling and lysis. Such necroptotic lysis of cells is thought to be highly inflammatory, as it leads to the release of "danger-associated molecular patterns" (DAMPs) into the extracellular space. In certain cases, such as during IAV infection, RIPK3 can also activate a caspase-8-mediated apoptosis parallel pathway that promotes viral clearance.Importantly, this apoptotic pathway does not require the catalytic activity of RIPK3 and can actually be driven by the blockade of RIPK3 kinase.

[0015] Figure 1 shows the RIPK3-mediated cell death or necroptosis pathway, including the pathways activated by tumor necrosis factor (TNF) (left) and IAV (right). Overall, this branching pathway is responsible for almost all cell death induced by IAV replication. 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 went undiscovered for so long.

[0016] Blocking RIPK3 kinase can prevent inflammatory disease progression without affecting viral clearance. Mice lacking the DAI-RIPK3 cell death pathway are unable to control IAV spread in the lungs and succumb to the virus, highlighting the importance of DAI-RIPK3 signaling in anti-IAV host defense. However, downstream necrotic branching of RIPK3 is not only superfluous to viral clearance but also actively promotes epithelial degradation and inflammation. Therefore, selectively blocking downstream necroptosis of RIPK3 is thought to prevent disease progression without affecting beneficial viral clearance that would otherwise proceed normally through caspase-8 mediated apoptosis. Interestingly, since only RIPK3-mediated necroptosis, and not apoptosis, requires RIPK3 kinase activity, it is pharmacologically easily targetable with the use of RIPK3 kinase inhibitors. Clinically viable RIPK3 small molecule inhibitors, as seen on the right side of Figure 1, would represent a promising and novel therapeutic option against IAV-induced necrosis, epithelial degradation, inflammation, and resulting lung damage.

[0017] RIPK3-mediated necroptosis strongly amplifies inflammation in various chronic conditions, including atherosclerosis and many TNF-associated conditions, and underlies lung injury during acute IAV infection and ARDS. Furthermore, RIPK3 can directly activate inflammatory gene expression independently of cell death, highlighting its central role in the host inflammatory process. There is agreement that genetic excision of RIPK3 in mice improves the development of atherosclerotic plaques, almost completely eliminates TNF-induced inflammatory shock, significantly reduces injury resulting from chronically produced TNF, and eliminates IAV-induced necrotizing lung injury without affecting viral clearance. In addition to the above examples, RIPK3 is associated with lipid and lysosomal storage disorders, such as Niemann-Pick disease and Gaucher disease, respectively. RIPK3 also plays a pathogenic role in autoimmune diseases, including multiple sclerosis and lupus. RIPK3 activity has also been linked to neurogenesis in different tissue and disease paradigms, suggesting it is a target for 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, and renal, retinal, and hepatic ischemia.

[0018] This disclosure relates to a novel panel of RIPK3 inhibitors sometimes referred to as the UH15 series. These compounds are based on pyrido[2,3-d]pyrimidine scaffolds that are structurally different from conventional RIPK3 inhibitor molecules. Members of this series exhibit approximately 10 times stronger necroptosis blockade compared to other disclosed RIPK3 inhibitors. Interestingly, these UH15 compounds block RIPK3 without inducing apoptotic activity, and consequently without the toxicity associated with other RIPK3 inhibitors. For most indications (e.g., TNF conditions), blocking RIPK3 necroptosis without simultaneously activating apoptosis is ideal. However, for IAV, a RIPK3 inhibitor that can efficiently block necroptosis while simultaneously enhancing apoptosis in infected cells would not only halt harmful necrotizing lung injury but also accelerate viral clearance. Of course, such apoptosis induction must be limited to infected cells (i.e., stimulus-specific), otherwise toxicity would occur (as seen with previous compounds).

[0019] In particular, this RIPK3 inhibitor targets RIPK3 necroptosis as a novel therapeutic entry point for IAV disease. Seasonal and generalized strains of IAV cause necrotizing lung injury underlying both ARDS and viral / bacterial pneumonia, each of which remains a major cause of morbidity and death, with few effective treatment options available. The ideal treatment for severe IAV would block RIPK3-dependent necroptosis but not interfere with (or even potentially promote) non-inflammatory viral clearance via RIPK3-dependent apoptosis.

[0020] In addition, this disclosure supports RIPK3 blockade as a fundamental and novel approach to acute and chronic inflammation. Evidence from genetic models demonstrates that RIPK3 plays a central role in multiple inflammatory diseases, in addition to its role in IAV infection. These include both acute and chronic inflammatory conditions known to be TNF-driven, such as colitis and rheumatoid arthritis. Furthermore, while RIPK3 is essential in all known pathways of necroptosis, the similar kinase RIPK1 (currently the subject of research by others) is involved only in a subset of necroptosis pathways, e.g., those activated by TNF (see the right side of Figure 1). Thus, RIPK3 inhibitors would have merit in a broader range of acute and chronic inflammatory conditions than those that function as anti-TNF approaches or RIPK1 inhibitors. [Invention 1001] Compounds exhibiting protein kinase inhibitory activity and having the following structure: TIFF0007897681000001.tif23128In formula, A is N or CH, R1 is Me, The file is TIFF0007897681000002.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 filename is TIFF0007897681000003.tif13128. [Invention 1002] A pharmaceutical composition comprising a therapeutically effective amount of the compound of the present invention 1001 and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer, stabilizer, or mixture thereof. [Invention 1003] Steps for administering the pharmaceutical composition of the present invention 1002. A method for treating a disease or condition related to protein kinases, including [specific example of protein kinases]. [Invention 1004] Steps for administering the pharmaceutical composition of the present invention 1002. A method for treating a disease or condition involving the activity of receptor-interacting kinase 2 (RIPK2), activin-like kinase 2 (ALK2), or receptor-interacting kinase 3 (RIPK3), including the above. [Invention 1005] Steps for administering the pharmaceutical composition of the present invention 1002. A method for treating an inflammatory or degenerative disease or condition involving the activity of receptor-interacting kinase 3 (RIPK3), including [specific example]. [Invention 1006] Compound 1001 of the present invention having the following structure: In formula TIFF0007897681000004.tif123128, R2 is Et, Et-O-Me, or isobutyl, and X is F or Me. [Invention 1007] A pharmaceutical composition comprising a therapeutically effective amount of the compound of the present invention 1006 and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer, stabilizer, or mixture thereof. [Invention 1008] Steps for administering the pharmaceutical composition of the present invention 1007. A method for treating a disease or condition related to protein kinases, including [specific example of protein kinases]. [Invention 1009] Steps for administering the pharmaceutical composition of the present invention 1007. A method for treating a disease or condition involving the activity of receptor-interacting kinase 2 (RIPK2), activin-like kinase 2 (ALK2), or receptor-interacting kinase 3 (RIPK3), including the above. [Invention 1010] Steps for administering the pharmaceutical composition of the present invention 1007. A method for treating an inflammatory or degenerative disease or condition involving the activity of receptor-interacting kinase 3 (RIPK3), including [specific example]. [Invention 1011] A method for treating an inflammatory or degenerative disease or condition involving receptor interaction kinase 3 (RIPK3) inhibitory activity, comprising the following steps: The following structure: TIFF0007897681000005.tif36128[In the formula, In one available ring position, R is H, or R is The file is TIFF0007897681000006.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, R1 is either H, or R1 is C-Cl, CF, C-OCH3, CC(CH3)3, or C-OH, and XY is C=C or The file is TIFF0007897681000007.tif10128, where R2 is H, alkyl, alkylhydroxyl, alkylalkoxyl, or alkylaryl. A step of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound having and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer, stabilizer, or mixture thereof. [Invention 1012] The compound has the following structure: The method of the present invention 1011, having TIFF0007897681000008.tif174128. [Invention 1013] The method of the present invention 1011, wherein the inflammatory or degenerative disease or condition involving RIPK3 activity is influenza A virus (IAV). [Brief explanation of the drawing]

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

[0022] Detailed description of preferred embodiments This disclosure relates to protein kinase inhibitors and their uses.

[0023] The following figure shows schematic structures of preferred embodiments of compounds that inhibit protein kinases, including receptor interaction kinase 2 (RIPK2), activin-like kinase 2 (ALK2), and receptor interaction kinase 3 (RIPK3). TIFF0007897681000009.tif23128

[0024] In the above structure, A may be N or CH. R1 is Me, It may also be TIFF0007897681000010.tif23128, where X is F or Me, where Me is methyl. R2 may be Me, Et, Et-O-Me, or isobutyl. R3 is It could also be TIFF0007897681000011.tif13128, where Et is ethyl.

[0025] Further preferred embodiments of compounds that inhibit RIPK2, ALK2, and RIPK3 are shown in Figure 2.

[0026] Further preferred embodiments of compounds that inhibit RIPK2, ALK2, and RIPK3 are shown below. In formula TIFF0007897681000012.tif129128, R2 is Et(UH15-4), Et-O-Me(UH15-6), or isobutyl(UH15-10), and X is F(UH15-18) or Me(UH15-20).

[0027] The following figure shows a schematic structure of a further preferred embodiment of a compound that inhibits RIPK3. TIFF0007897681000013.tif36128

[0028] In the above structure, R may be H, or R may be at any one available position on the phenyl ring. The substituent may be TIFF0007897681000014.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. R1 may be H, or R1 may be C-Cl, CF, C-OCH3, CC(CH3)3, or C-OH at any one available position on the ring. XY may be C=C, or they may be It may also be TIFF0007897681000015.tif10128, where R2 is H, an alkyl group containing methyl, ethyl or isobutyl indefinitely, an alkylhydroxyl group containing 2-hydroxyethyl indefinitely, an alkylalkoxyl group containing 2-methoxyethyl indefinitely, or an alkylaryl group containing benzyl or phenethyl indefinitely.

[0029] The following figure shows a schematic structure of a further preferred embodiment of a compound that inhibits RIPK3. TIFF0007897681000016.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 It may also be TIFF0007897681000017.tif10128, where R2 is H, or an alkyl containing methyl, ethyl or isobutyl indefinitely, an alkylhydroxyl containing 2-hydroxyethyl indefinitely, an alkylalkoxyl containing 2-methoxyethyl indefinitely, or an alkylaryl containing benzyl or phenethyl indefinitely. R is H, It may also be TIFF0007897681000018.tif12128, where Me is methyl and Et is ethyl. R1 may be any alkyl group that non-limitingly includes methyl, ethyl, or propyl, or R1 may be any aryl group that non-limitingly includes naphthyl, thienyl, indoyl, etc. R3 may be H, or R3 may be C-Cl, CF, C-OCH3, CC(CH3)3, or C-OH at any one available position on the ring.

[0031] Further preferred embodiments of compounds that inhibit RIPK3 are shown in Figure 3.

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

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

[0034] In another aspect of the present invention, a pharmaceutical composition is provided comprising a compound that inhibits protein kinase activity as defined above a therapeutically effective dose, and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer, or stabilizer. “Therapeutally effective dose” should be understood as a sufficient amount of exemplary protein kinase inhibitor compound to exhibit an inhibitory effect on protein kinase activity. The actual dose, rate, and course of administration will vary depending on the nature and severity of the disease being treated. Prescription of treatment is the responsibility of general practitioners and other physicians. Pharmaceutically acceptable excipients, adjuvants, carriers, buffers, or stabilizers are preferably non-toxic and do not interfere with the efficacy of the active ingredient. The exact properties of the carrier or other material will vary depending on the route of administration, whether orally, by injection such as skin, 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 a solid carrier or adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain saline, dextrose, or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. Capsules may contain a solid carrier 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 pyrogenic and has a suitable pH, isotonicity, and stability. Those skilled in the art can easily prepare suitable solutions using isotonic vehicles such as sodium chloride solution, Ringer's solution, or lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0036] In another context, the use of protein kinase inhibitor compounds, defined above a therapeutically effective dose, in the manufacture of drugs for administration to a target is provided.

[0037] The term “pharmacologically acceptable salt” as used throughout this specification should be interpreted to mean any acid or base derivative 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, etc., and any acid or base derivative salt formed from potassium carbonate, sodium hydroxide or potassium, ammonia, triethylamine, triethanolamine, etc.

[0038] The term "prodrug" refers to a pharmacological substance that is administered in an inactive or significantly less active form. Once administered, prodrugs are metabolized in vivo to active metabolites.

[0039] The term "therapeutic dose" refers to a sufficient amount of drug to provide the desired therapeutic effect without being toxic. The "effective" dose will vary from subject to subject, depending on the individual's age and overall health, the specific concentration and composition administered, and other factors. Therefore, it is not always possible to specify an exact effective dose. However, the appropriate effective dose in any given individual case can be determined by those skilled in the art using standard experimental methods. Furthermore, the effective dose is a concentration within a range sufficient to allow for the immediate application of the formulation in order to deliver a therapeutically effective amount of drug.

[0040] Further aspects of the present invention will become apparent from the following description, which is given only as an example. [Examples]

[0041] Example 1. Synthesis Figure 4 shows the overall schematic synthetic scheme of exemplary preferred compounds disclosed herein as inhibitors of protein kinase activity, generally referred to as UH15 analogs or UH15. In particular, in the following discussion, the compounds 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] Unless otherwise specified, all reactions were carried out under anhydrous conditions using dry solvents in an argon atmosphere. Unless otherwise specified, all commercially available chemicals and reagent-grade solvents were used directly without further purification. Reactions were monitored by thin-layer chromatography (TLC) on Baker-flex® silica gel plates (IB2-F) using UV light (254 and 365 nm) as a visualization agent and either an ethanol solution of phosphomolybdic acid or a ninhydrin solution as a developer, along with heat. Flash chromatography was performed on silica gel (230-400 mesh) using Teledyne ISCO CombiFlash® Rf. NMR spectra were recorded at room temperature using a JEOL ECA-500 (1H NMR and 13C NMR at 400, 500, and 600 MHz) with tetramethylsilane (TMS) as an internal standard. Chemical shifts (δ) are given in parts per million (ppm) with reference to the solvent signal [¹H-NMR: CDCl3 (7.26 ppm), CD3OD (3.30 ppm), DMSO-d6 (2.49 ppm); ¹³C-NMR: CDCl3 (77.0 ppm), CD3OD (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 at the Mass Spectroscopy Facility of the Department of Chemistry, University of Texas at Austin, using an Agilent 6530 Q-TOF instrument. Electrospray ionization (ESI) is used as the ionization source, and the spectrum is obtained as molecular [M] or [M+H]. +The ionic species were reported as m / z (relative intensity). The purity of the compound 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. The injection volume was 15 μL. The HPLC gradient was from 2% acetonitrile / 98% water to 90% acetonitrile / 10% water (both solvents contained 0.1% trifluoroacetic acid), with a total run time of 30 minutes 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 WO2018 / 213219 by Cuny et al.

[0044] 2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-ethyl-6-phenylpyrido[2,3-d]pyrimidine-7(8H)-one(UH15-4): TIFF0007897681000019.tif28128

[0045] Yellow solid (yield 54%): TIFF0007897681000020.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]pyrimidine-7(8H)-one(UH15-6): TIFF0007897681000021.tif33128

[0047] Yellow solid (61% yield): TIFF0007897681000022.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]pyrimidine-7(8H)-one(UH15-10): TIFF0007897681000023.tif30128

[0049] Yellow solid (yield 36%): TIFF0007897681000024.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]pyrimidine-7(8H)-one(UH15-18): TIFF0007897681000025.tif28128

[0051] White solid (60% yield): TIFF0007897681000026.tif48150 Purity 97.6%(t R 22.72 minutes).MP248~250℃.

[0052] Synthesis of UH15-20: TIFF0007897681000027.tif26128

[0053] The synthesis of UH15-20 proceeded as shown in Figure 5. Reagents and conditions: (a) NaH, THF, reflux, 2.5 hours, 42%; (b) Pd(PPh3)4, Na2CO3, 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]pyrimidine-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 under argon at room temperature for 10 minutes. A solution of 2-chloro-2-(diethoxyphosphoryl)ethyl acetate A18 (42 mg, 0.16 mmol) in THF (0.5 mL) was added dropwise to the above mixture and refluxed for 2.5 hours. The reaction mixture was cooled to room temperature, concentrated, and extracted with ethyl acetate and water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a residue, which was purified by silica gel column chromatography (30% ethyl acetate / hexane) to obtain A19 as a white solid (yield 42%). TIFF0007897681000028.tif20150

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

[0057] A19 (24 mg, 0.11 mmol), (2-chloro-4-methylphenyl)boronic acid (28 mg, 0.16 mmol), and Pd(PPh3)4 (13 mg, 0.011 mmol) were placed in a round-bottom flask and purged with argon for 10 minutes. DMF (1 mL) and CH3CN (2 mL) were added to the mixture and purged for 10 minutes. A 1 M Na2CO3 (23 mg, 0.21 mmol) (220 μL) solution was added dropwise, and the reaction mixture was heated at 90°C for 5 hours. The reaction mixture was cooled to room temperature, the solvent was evaporated, and the residue was separated into water and ethyl acetate. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated to obtain the residue, which was purified by silica gel column chromatography (10% ethyl acetate / DCM) to obtain A20 as a white solid (yield 57%). TIFF0007897681000029.tif27151

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

[0059] A21 was synthesized by adapting the method described in WO2018 / 213219 by Cuny et al. White solid (yield 68%): TIFF0007897681000030.tif27150

[0060] Synthesis of A16c:

[0061] Formic acid (1 mL) was added to 3-(methylsulfonyl)aniline in a round-bottom flask containing a molecular sieve (4 Å, 8-12 mesh). The reaction mixture was heated at 60°C for 6 hours, and then separated into a saturated solution of NaHCO3 and RINKAN. The organic layer was then washed with brine and concentrated to obtain A16c, which was used without purification.

[0062] N-(3-(methylsulfonyl)phenyl)formamide (A16c) TIFF0007897681000031.tif27128

[0063] White solid (80% yield): TIFF0007897681000032.tif20150

[0064] 6-(2-chloro-4-methylphenyl)-8-methyl-2-((3-(methylsulfonyl)phenyl)amino)pyrido[2,3-d]pyrimidine-7(8H)-one(UH15-20): TIFF0007897681000033.tif27128

[0065] UH15-20 was prepared from A21 and A16c by adapting the method described in Cuny et al. WO2018 / 213219. White solid (yield 75%): TIFF0007897681000034.tif34151 Purity 99.6%(t R 23.51 minutes). MP 182~184℃.

[0066] Synthesis of UH15-28: TIFF0007897681000035.tif29128

[0067] The synthesis of UH15-28 proceeded as shown in Figure 6. Reagents and conditions: (a) CH3NH2(aq), THF, 0°C to room temperature, 2 hours, 67%; (b) LAH, THF, 0°C to room temperature, 1.5 hours, 96%; (c) MnO2, DCM, room temperature, overnight, 93%; (d) KF / Al2O3, DMA, room temperature, 2 hours, 75%; (e) Pd2(dba)3, xanthophos, K2CO3, DMF, reflux, 24 hours, 37%; (f) Pd / C, H2, MeOH, room temperature, overnight, 94%; (g) Pd2(dba)3, xanthophos, Cs2CO3, dioxane, 80°C, overnight, 36%.

[0068] 6-Chloro-4-(methylamino)nicotinate ethyl(D2):

[0069] A solution of D1 (100 mg, 0.45 mmol) in THF (2 mL) was mixed with aqueous methylamine solution (0.4 mL) at 0°C. The mixture was stirred at the same temperature for 30 minutes, then at room temperature for 2 hours. After removing the THF by evaporation under vacuum, the crude mixture was then divided into H2O and siRNA. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (10% siRNA / hexane) to obtain D2 (65 mg, 67%) as a white solid. TIFF0007897681000036.tif21150

[0070] (6-Chloro-4-(methylamino)pyridine-3-yl)methanol(D3):

[0071] A suspension of LAH (27 mg, 0.69 mmol) in THF (2 mL) was cooled to 0°C, and 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 minutes. 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 hour, filtered, and washed with ethyl acetate. The residue was obtained by evaporation of ethyl acetate under vacuum, and this was purified by silica gel column chromatography (5% MeOH / DCM) to obtain D3 (77 mg, 96%) as a white solid. TIFF0007897681000037.tif20150

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

[0073] A solution of D3 (495 mg, 2.86 mmol) in DCM (10 mL) was mixed with MnO2 (1496 mg, 17.20 mmol), 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 obtain D4 (453 mg, 93%) as a white solid. TIFF0007897681000038.tif20150

[0074] 7-Chloro-3-(2-chlorophenyl)-1-methyl-1,6-naphthyridine-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 mixed with KF / Al2O3 (1800 mg, 40 wt%), and the reaction mixture was stirred under argon at room temperature for 2 hours. After 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% Â / hexane to obtain D6 (400 mg, 75%) as a white solid. TIFF0007897681000039.tif13128

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

[0077] Pd2(dba)3 (68 mg, 0.073 mmol), xanthophos (85 mg, 0.147 mmol), and potassium carbonate (408 mg, 2.95 mmol) were added to a preheated round-bottom flask and flushed with argon for 10 minutes. DMF (4 mL) was added to the mixture and flushed for a further 5 minutes, followed by the addition of D12 (200 mg, 1.47 mmol) and D13 (356 mg, 1.77 mmol), and the mixture was refluxed for 24 hours. The reaction mixture was then separated into ethyl acetate and water, dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (1.5% MeOH / DCM) to obtain D14 (140 mg, 37%) as a pale yellow solid. TIFF0007897681000040.tif13128

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

[0079] A solution of D14 (140 mg, 0.54 mmol) in CH3OH (10 ml) was mixed with 10% Pd / C (50 mg), and the reaction was stirred at room temperature in H2 (g) (1 atm) for 4 hours. The reaction mixture was then filtered through Celite and concentrated to obtain 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-dioxidethiomorpholino)phenyl)amino)-1-methyl-1,6-naphthyridine-2(1H)-one(UH15-28):

[0081] D6 (40 mg, 0.13 mmol), D15 (30 mg, 0.13 mmol), Pd2(dba)3 (12 mg, 0.013 mmol), xanthophos (15 mg, 0.026 mmol), and cesium carbonate (86 mg, 0.26 mmol) were added to a preheated round-bottom flask and flushed with argon for 10 minutes. Dioxane (3 ml) was added to the mixture and flushed again for 5 minutes, then heated overnight at 80°C. The reaction mixture was then separated into ethyl acetate and water, dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (20% Â / DCM) to obtain Nap-5 (23 mg, 36%) as a pale yellow solid. TIFF0007897681000041.tif47150

[0082] Synthesis of UH15-37: TIFF0007897681000042.tif30128

[0083] The synthesis of UH15-37 proceeded as shown in Figure 7. Reagents and conditions: (a) Pd2(dba)3, xanthophos, Cs2CO3, dioxane, 80°C, overnight, 47%.

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

[0085] In a preheated and dried round-bottom flask, D6 (30 mg, 0.098 mmol), 3-(4-methylpiperazin-1-yl)aniline (19 mg, 0.098 mmol), Pd2(dba)3 (9 mg, 0.009 mmol), xanthophos (12 mg, 0.019 mmol), and cesium carbonate (64 mg, 0.19 mmol) were added, and the mixture was flushed with argon for 10 minutes. Dry dioxane (1.5 mL) was added to the mixture, and after flushing for 10 minutes, 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 separated into water and ethyl acetate. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated to obtain the residue, which was purified by silica gel column chromatography (5% MeOH / DCM) to obtain UH15-37 (21 mg, 47%) as a yellow solid. TIFF0007897681000043.tif47150

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

[0087] 4-amino-2-(methylthio)pyrimidine-5-carboxylate ethyl(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), triethylamine (0.2 mL, 1.29 mmol) and ammonium hydroxide (0.5 mL) were added. The resulting mixture was stirred at room temperature for 2 hours until complete. After removing the THF by evaporation under vacuum, the crude mixture was divided into H2O and siRNA. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (15% siRNA / hexane) to obtain 2 (90 mg, 98%) as a white solid. TIFF0007897681000044.tif26150

[0089] 4-(methylamino)-2-(methylthio)pyrimidine-5-carboxylate ethyl(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), aqueous methylamine solution (aq) (6 mL) was added. The resulting mixture was stirred at room temperature for 2 hours until complete. After removing the THF by evaporation under vacuum, the crude mixture was then divided into H2O and siRNA. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (10% siRNA / hexane) to obtain 3 (2500 mg, 85%) as a white solid. TIFF0007897681000045.tif20150

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

[0092] A suspension of LiAlH4 (120 mg, 3.16 mmol) in THF (4 mL) was cooled to 0°C. A solution of 2 (450 mg, 2.11 mmol) in THF (2 mL) was added dropwise under argon, and the mixture was stirred at room temperature for 30 minutes. 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 hour, filtered, and washed with ethyl acetate. Evaporation under vacuum was used to remove ethyl acetate, and 4 (220 mg, 61%) was obtained as a pale yellow solid, which was used in the next step without purification. TIFF0007897681000046.tif13130

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

[0094] A suspension of LiAlH4 (626 mg, 16.50 mmol) in THF (10 mL) was cooled to 0°C. A solution of 3 (2500 mg, 11.00 mmol) in THF (5 mL) was added dropwise under argon, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was then cooled to 0°C, and 15% NaOH (2 mL) and water (4 mL) were added dropwise. The reaction mixture was stirred for 1 hour, filtered, and washed with ethyl acetate. Evaporation under vacuum was used to remove ethyl acetate, and 5 (1790 mg, 95%) was obtained as a pale yellow solid, which was used in the next step without purification. TIFF0007897681000047.tif13129

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

[0096] To a solution of 4 (220 mg, 1.28 mmol) in DCM (5 mL), MnO2 (670 mg, 7.71 mmol) was added, and the mixture was stirred under argon at room temperature overnight. The reaction mixture was then filtered, concentrated to remove the DCM, and purified by silica gel column chromatography (30% siRNA / hexane) to obtain 6 (180 mg, 83%) as a pale yellow solid. TIFF0007897681000048.tif19150

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

[0098] To a solution of 5 (1795 mg, 10.489 mmol) in DCM (15 mL), MnO2 (5471 mg, 62.93 mmol) was added, and the mixture was stirred under argon at room temperature overnight. The reaction mixture was then filtered, concentrated to remove the DCM, and purified by silica gel column chromatography (20% siRNA / hexane) to obtain 7 (1470 mg, 77%) as a pale yellow solid. TIFF0007897681000049.tif20150

[0099] Basic procedure for preparing 9a-c:

[0100] A mixture of 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 K2CO3 (944 mg, 6.82 mmol) was mixed with DMF (4 mL) under argon, and the solution was refluxed at 105°C for 18 hours. After completion, the reaction mixture was separated into water and ethyl acetate, and the organic layer was washed with brine. The residue was removed by evaporation under vacuum to obtain the residue, which was purified by silica gel column chromatography (5% MeOH / DCM) to obtain 9a.

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

[0102] Yield 63%, pale red solid; TIFF0007897681000050.tif20150

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

[0104] 9b was prepared using 7 (500 mg, 2.73 mmol) and 8b (621 mg, 4.10 mmol), and this was used in the next step without purification.

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

[0106] Formula 9c was prepared using 7 (600 mg, 3.28 mmol) and 8c (576 mg, 4.91 mmol).

[0107] Yield 54%, pale red solid; TIFF0007897681000051.tif13132

[0108] Basic procedure for preparing 13a-c:

[0109] A suspension of 9a (200 mg, 0.57 mmol) in acetic anhydride (3 mL) was refluxed at 139°C for 30 minutes. The residue was obtained by evaporation of the solvent under vacuum, and this was treated with concentrated HCl (2 mL) and refluxed at 100°C for 5 minutes. The reaction mixture was then neutralized with a saturated solution of NaHCO3 and separated into water and toluene. The organic layer was then washed with brine, concentrated, and the residue was purified by silica gel column chromatography (30% toluene / hexane) to obtain 13a.

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

[0111] Yield 65%, pale yellow solid; TIFF0007897681000052.tif13132

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

[0113] Formula 13b was prepared using 9b (350 mg, 1.10 mmol).

[0114] Yield 61%, pale yellow solid; TIFF0007897681000053.tif27150

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

[0116] 13c was prepared using 9c (200 mg, 0.71 mmol).

[0117] Yield 55%, yellow solid; TIFF0007897681000054.tif27150

[0118] Basic procedure for preparing 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 mixed with KF / Al2O3 (76 mg, 40 wt%), and the reaction mixture was stirred under argon at room temperature for 24 hours. After 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% siRNA / hexane to obtain 13d as a pale yellow solid.

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

[0121] Yield 58%, pale yellow solid; TIFF0007897681000055.tif27150

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

[0123] Yield 41%, yellow solid; TIFF0007897681000056.tif27150

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

[0125] Yield 30%, white solid; TIFF0007897681000057.tif27150

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

[0127] Yield 38%, white solid; TIFF0007897681000058.tif13139

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

[0129] Yield 35%, white solid; TIFF0007897681000059.tif34150

[0130] Preparation of (13o):

[0131] 2-(2,5-dichlorophenyl)methyl acetate (110):

[0132] 110 in methanol (3 mL) * To a stirred solution of (100 mg, 0.537 mmol), thionyl chloride (0.5 mL) was added dropwise at 0°C. The reaction mixture was maintained at the same temperature for 5 hours, then stirred overnight at room temperature. After completion, the reaction mixture was concentrated and diluted with ethyl acetate. The organic layer was then washed with sodium bicarbonate and brine solution, concentrated, and the residue was purified by silica gel column chromatography (20% ethyl acetate / hexane) to obtain 110. Yield 59%, colorless liquid: TIFF0007897681000060.tif20150

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

[0134] Yield 23%, white solid; TIFF0007897681000061.tif13128

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

[0136] Yield 55%, pale yellow solid; TIFF0007897681000062.tif20150

[0137] Basic procedure for preparing 12i, 12k, and 12n:

[0138] A stirred solution of 6 (37 mg, 0.22 mmol) and 2-phenylmethyl acetate (11i) (30 mg, 0.20 mmol) in dry DMA (2.0 mL) was mixed with KF / Al2O3 (187 mg, 40 wt%), and the reaction mixture was stirred under argon at room temperature for 24 hours. After 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% siRNA / hexane to obtain 12i as a white solid.

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

[0140] Yield 51%, white solid; TIFF0007897681000063.tif27150

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

[0142] Yield 28%, pale yellow solid; TIFF0007897681000064.tif27150

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

[0144] Yield 40%, white solid; TIFF0007897681000065.tif27150

[0145] Basic procedure for preparing 13i-r:

[0146] A mixture of 12i (20 mg, 0.07 mmol) and NaH (3 mg, 0.11 mmol) was mixed with DMF (1.5 mL) under argon, followed by dropwise addition of iodoethane (10 μl, 0.11 mmol). The reaction mixture was heated at 50°C for 1 hour, after which it was separated into water and ethyl acetate. The organic layer was then washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (10% ethyl acetate / hexane) to obtain 13i as a colorless liquid.

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

[0148] 74% yield, colorless liquid; TIFF0007897681000066.tif27150

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

[0150] Purification by silica gel column chromatography (40% toluene / hexane) yielded 13j (48% yield), a pale yellow, viscous liquid. TIFF0007897681000067.tif27150

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

[0152] Yield 44%, colorless liquid; TIFF0007897681000068.tif33150

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

[0154] Yield 29%, white semi-solid; TIFF0007897681000069.tif33150

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

[0156] Yield 28%, white solid; TIFF0007897681000070.tif34150

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

[0158] Yield 73%, yellow solid; TIFF0007897681000071.tif20150

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

[0160] Yield 36%, pale yellow solid; TIFF0007897681000072.tif20150

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

[0162] Yield 53%, pale yellow solid; TIFF0007897681000073.tif20150

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

[0164] To a solution of 13p (20 mg, 0.06 mmol) in acetone (2 mL), anhydrous potassium carbonate (14 mg, 0.10 mmol) and iodomethane (10 μl, 0.10 mmol) were added, and the mixture was refluxed for 8 hours. After completion, the crude mixture was extracted with ethyl acetate and water, concentrated, and purified by column chromatography using silica gel (20% siRNA / hexane) to obtain 13s (20 mg, 95%) as a pale yellow solid. TIFF0007897681000074.tif27150

[0165] Basic procedure for preparing 14a-s:

[0166] A solution of 6-(2,4-dichlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]pyrimidine-7(8H)-one (13a) (38 mg, 0.108 mmol) in DCM (2 mL) was mixed with mCPBA (85 mg, 55%) and stirred for 3 hours. The reaction mixture was then separated into water and DCM, and the organic layer was washed with brine. After concentration, the residue was purified by column chromatography using silica gel (40% siRNA / hexane) to obtain 14a as a white powder.

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

[0168] Yield 63%, white solid; TIFF0007897681000075.tif26150

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

[0170] Yield 45%, white solid; TIFF0007897681000076.tif27150

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

[0172] Yield 54%, pale yellow solid; TIFF0007897681000077.tif27150

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

[0174] Yield 72%, white solid; TIFF0007897681000078.tif27151

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

[0176] Yield 81%, white solid; TIFF0007897681000079.tif27151

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

[0178] Yield 93%, white solid; TIFF0007897681000080.tif13145

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

[0180] Yield 65%, pale yellow solid; TIFF0007897681000081.tif27150

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

[0182] Yield 40%, white solid; TIFF0007897681000082.tif41150

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

[0184] Yield 55%, white solid; TIFF0007897681000083.tif20150

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

[0186] The yield was 81%, and the resulting product was a 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]pyrimidine-7(8H)-one(14k):

[0188] Yield 58%, white solid; TIFF0007897681000084.tif33150

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

[0190] Yield 61%, white solid; TIFF0007897681000085.tif27150

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

[0192] Yield 87%, white solid; TIFF0007897681000086.tif34150

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

[0194] The yield was 66%, and a light brown solid was obtained, which was used in the next step without purification.

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

[0196] Yield 75%, white solid; TIFF0007897681000087.tif27150

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

[0198] To a solution of 13p (160 mg, 0.53 mmol) in DMF (2 mL), imidazole (109 mg, 1.60 mmol) and DMAP (catalytic amount 3 mg) were added, 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 overnight at room temperature. After completion, the reaction was separated into ethyl acetate and water, the organic layer was filtered and concentrated, and purified by column chromatography using silica gel (5% ethyl acetate / DCM) to obtain 13p.* (194 mg, 88%) was obtained as a pale yellow solid. TIFF0007897681000088.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; TIFF0007897681000089.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; TIFF0007897681000090.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; TIFF0007897681000091.tif14148

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

[0206] Yield 97%, yellow solid; TIFF0007897681000092.tif20150

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

[0208] An excess of 10% Pd / C (125 mg) was added to a solution of N,N - diethyl - 2 - (4 - nitrophenoxy)ethanamine (490 mg, 2.06 mmol) in CH3OH (10 mL), and H2(g) was passed through a latex valve 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. TIFF0007897681000093.tif27150

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

[0210] Method A: Formic acid (1 mL) was added to 15a (100 mg, 0.48 mmol) in a round - bottom flask containing molecular sieves. The reaction mixture was heated at 60 °C for 6 h and then partitioned between saturated NaHCO3 solution and EtOAc. The organic layer was 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 with stirring the reaction mixture at room temperature overnight. Yield 80%, 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 with stirring the reaction mixture at room temperature overnight. Yield 80%, white solid; TIFF0007897681000094.tif27150

[0215] Preparation of (16d):

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

[0217] To a solution of Boc anhydrous (790 mg, 3.62 mmol) in DCM (10 mL), DMAP (60 mg, 0.49 mmol) was added, and the solution was stirred for 5 minutes. Then, 1-(3-nitrophenyl)piperazine (500 mg, 2.41 mmol) was added to the above solution, and the mixture was stirred at room temperature for 20 hours. After completion, the reaction mixture was concentrated and purified by column chromatography using silica gel (30% Â / hexane) and 15d * (700 mg, 94%) was obtained as a yellow solid. TIFF0007897681000095.tif27150

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

[0219] 15d in CH3OH (15 mL) * To a solution of (700 mg, 2.27 mmol), an excess of 10% Pd / C (200 mg) was added, and the mixture was passed through a latex valve bladder overnight with H2 (g). The reaction mixture was then filtered through Celite and concentrated to obtain 15d (600 mg, 95%) as a brown, viscous liquid, which was used in the next step without purification.

[0220] 4-(3-formamidophenyl)piperazine-1-carboxylate tert-butyl(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 mixture and heated under reflux. After stirring overnight, the solvent was evaporated, the mixture was dissolved in DCM, and extracted with water and brine solution. The extract was concentrated and purified by column chromatography using silica gel (30% HCl / hexane) to obtain 16d (100 mg, 45%) as a colorless liquid.

[0222] N-(pyridine-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) maintained at 0°C. The mixture was heated under reflux at 60°C for 2 hours to produce formic acetic 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 formic acetic anhydride mixture and refluxed for another 2 hours. After completion, the solvent was evaporated and extracted with an  / Aqueous system. The extract was concentrated and purified by column chromatography using silica gel (3% MeOH / DCM) to obtain 16e (173 mg, 67%) as a white solid.

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

[0225] 16f was prepared by method C. The yield was 75%, and it was a white solid.

[0226] Basic procedure for preparing 17a-s:

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

[0228] A solution of 16a (30 mg, 0.13 mmol) in THF (0.5 mL) and DMF (0.5 mL) was added with 60% NaH (8 mg, 0.33 mmol) at 0 °C, and the mixture was stirred under argon at room temperature for 30 minutes. Then the mixture was cooled to 0 °C, 14a (25 mg, 0.06 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched by the addition of ice and NaOH (0.5 mL, 2 N) solution, and then partitioned between water and EtOAc. After filtration through anhydrous Na2SO4 and concentration, the crude mixture was purified by column chromatography (5% MeOH / DCM) using silica gel to obtain 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] Yield 74%, yellow solid: TIFF0007897681000096.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 prepared using 16a (40 mg, 0.17 mmol) and 14b (89 mg, 0.25 mmol).

[0233] Yield 62%, yellow solid; TIFF0007897681000097.tif47150 purity 99.15% (t R 19.93 minutes).

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

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

[0236] Yield 64%, yellow solid; TIFF0007897681000098.tif47150 Purity 99.4%(t R 19.75 minutes).

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

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

[0239] Yield 54%, yellow solid; TIFF0007897681000099.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]pyrimidine-7(8H)-one(17j)UH15_5:

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

[0242] Yield 33%, yellow solid; TIFF0007897681000100.tif34150

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

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

[0245] Yield 61%, yellow solid; TIFF0007897681000101.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]pyrimidine-7(8H)-one(17l)UH15_7:

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

[0248] Yield 68%, yellow solid; TIFF0007897681000102.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]pyrimidine-7(8H)-one(17d)UH15_8:

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

[0251] Yield 45%, yellow solid; TIFF0007897681000103.tif47150 Purity 96.5%(t R21.33 minutes).

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

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

[0254] Yield 58%, yellow solid; TIFF0007897681000104.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]pyrimidine-7(8H)-one(17m)UH15_10:

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

[0257] Yield 36%, yellow solid; TIFF0007897681000105.tif54150

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

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

[0260] Yield 92%, white solid; TIFF0007897681000106.tif41150 Purity 98.2%(tR 22.98 minutes).

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

[0262] Using 16b (11 mg, 0.05 mmol) and 14f (10 mg, 0.03 mmol), 17f * I made it.

[0263] 75% yield, white powder; TIFF0007897681000107.tif34150 Purity 97.6%(t R 22.92 minutes).

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

[0265] Using 16c (29 mg, 0.15 mmol) and 14d (26 mg, 0.07 mmol) for 17 days * I made it.

[0266] Yield 61%, pale yellow powder; TIFF0007897681000108.tif27150

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

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

[0269] Yield 33%, pale yellow solid; TIFF0007897681000109.tif33150

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

[0271] Using 16c (34 mg, 0.17 mmol) and 14b (50 mg, 0.14 mmol), 17b * I made it.

[0272] Yield 72%, white solid; TIFF0007897681000110.tif27150 Purity 98.1%(t R 22.36 minutes).

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

[0274] Using 16c (8 mg, 0.04 mmol) and 14a (12 mg, 0.03 mmol), 17a * I made it.

[0275] Yield 63%, pale yellow solid; TIFF0007897681000111.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]pyrimidine-7(8H)-one (17g)UH15_17:

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

[0278] Yield 68%, pale yellow solid; TIFF0007897681000112.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]pyrimidine-7(8H)-one(17h)UH15_18:

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

[0281] Yield 60%, white solid; TIFF0007897681000113.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]pyrimidine-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; TIFF0007897681000114.tif27150 Purity 97.4%(t R 23.76 minutes).

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

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

[0287] Yield 79%, pale yellow solid; TIFF0007897681000115.tif27150

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

[0289] Using 16a (20 mg, 0.081 mmol) and 14p (30 mg, 0.067 mmol), 17p * I made it.

[0290] Yield 64%, pale yellow solid; TIFF0007897681000116.tif34150

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

[0292] Yield 84%, pale yellow solid; TIFF0007897681000117.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]pyrimidine-7(8H)-one(17r)UH15_27:

[0294] Yield 70%, pale yellow solid; TIFF0007897681000118.tif34150 Purity 95.6%(t R 21.24 minutes).

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

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

[0297] Yield 95%, yellow solid; TIFF0007897681000119.tif26150

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

[0299] 17s using 16a (8 mg, 0.03 mmol) and 14s (14 mg, 0.03 mmol) * I made it.

[0300] Yield 66%, yellow solid; TIFF0007897681000120.tif33150

[0301] Preparation of UH15_32:

[0302] Figure 22 shows a preferred embodiment of the synthesis scheme for compound UH15_32, an exemplary inhibitor of protein kinase.

[0303] 6-Chloro-4-(methylamino)nicotinate ethyl(52):

[0304] A solution of ethyl 4,6-dichloronicotinate 51 (100 mg, 0.45 mmol) in THF (3 mL) was mixed with aqueous methylamine solution (0.4 mL) at 0°C, and the mixture was stirred at the same temperature for 30 minutes. After 30 minutes, the reaction mixture was stirred at room temperature for 2 hours. After completion, the mixture was concentrated and purified by column chromatography using silica gel (10% Âxane) to obtain 52 (65 mg, 67%) as a white solid. TIFF0007897681000121.tif20151

[0305] (6-Chloro-4-(methylamino)pyridine-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 the mixture was stirred at room temperature for 30 minutes. The reaction mixture was then cooled to 0°C, and 15% NaOH (2 mL) and water (4 mL) were added dropwise. The reaction mixture was stirred for 1 hour, filtered, and washed with ethyl acetate. The solvent was evaporated, and the extract was purified by silica gel column chromatography (2.5% MeOH / DCM) to obtain 53 (77 mg, 96%) as a white solid. TIFF0007897681000122.tif20150

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

[0308] To a solution of 53 (495 mg, 2.86 mmol) in DCM (10 mL), MnO2 (1496 mg, 17.20 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was then filtered and concentrated to remove the DCM, and purified by silica gel column chromatography (2% MeOH / DCM) to obtain 54 (453 mg, 93%) as a white solid. TIFF0007897681000123.tif13150

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

[0310] To a stirred solution of 54 (300 mg, 1.75 mmol) and 2-(2-chlorophenyl)acetate methyl 55 (325 mg, 1.75 mmol) in dry DMA (4 mL), KF / Al2O3 (1800 mg, 40 wt%) was added, and the reaction mixture was stirred at room temperature for 2 hours. After 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 (20% Â / hexane) to obtain 56 (400 mg, 75%) as a white solid. TIFF0007897681000124.tif20150

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

[0312] In a preheated round-bottom flask, 56 (30 mg, 0.09 mmol), 15c (20 mg, 0.11 mmol), Pd2(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 minutes. Dioxane (1.5 mL) was added to the mixture and flushed again for 5 minutes, then heated overnight at 80°C. After completion, the reaction mixture was separated into ethyl acetate and water, filtered, concentrated, and purified by silica gel column chromatography (2% MeOH / DCM) to obtain 57 (UH15_32) (20 mg, 47%) as a white solid. TIFF0007897681000125.tif41150

[0313] Preparation of UH15_33

[0314] Figure 23 shows a preferred embodiment of the synthesis scheme for compound UH15_33, an exemplary inhibitor of protein kinase.

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

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

[0317] Yield 70%, pale yellow solid; TIFF0007897681000126.tif41150

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

[0319] 58 (67 mg, 0.12 mmol) was taken into DCM (4.5 mL), and TFA (0.5 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. The solvent was evaporated, and the crude mixture was purified by silica gel column chromatography (4% MeOH / DCM) to obtain 59 (UH15_33) (55 mg, 96%) as a pale yellow solid. TIFF0007897681000127.tif27150

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

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

[0322] Yield 67%, white solid; TIFF0007897681000128.tif41151

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

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

[0325] Yield 62%, pale yellow solid. TIFF0007897681000129.tif27151

[0326] Preparation of UH15PN compound:

[0327] In a preferred embodiment, Figure 24A shows a synthesis scheme for exemplary protein kinase inhibitors, and Figure 24B shows the structure of an intermediate compound used in the synthesis of these exemplary protein kinase inhibitors. Figure 25 shows the structure of an exemplary protein kinase inhibitor according to a preferred embodiment.

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

[0329] A mixture of 6-bromo-2-chloroquinazoline (18) (200 mg, 0.82 mmol) and 15a (207 mg, 0.98 mmol) was mixed with propan-2-ol (4 mL), and the reaction mixture was heated at 100°C for 2 hours. After completion, the reaction mixture was cooled to room temperature, concentrated, and purified by silica gel column chromatography (5% MeOH / DCM) to obtain 19 (203 mg, 61%) as a yellow solid. TIFF0007897681000130.tif41151

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

[0331] 6-Bromo-N-(4-(2-(diethylamino)ethoxy)phenyl)quinazoline-2-amine (19a) (30 mg, 0.07 mmol), (2-chlorophenyl)boronic acid (20a) (17 mg, 0.11 mmol), and Pd(PPh3)4 (8.5 mg, 0.007 mmol) were placed in a round-bottom flask (rb) and purged with argon for 10 minutes. Then, a solvent mixture of DMF (1 mL) and CH3CN (2 mL) was added to the above reactants and purged again for 10 minutes. After purging, a 1 M Na2CO3 (150 μl) solution was added dropwise, and heating at 90°C for 5 hours was started. After completion, the reaction mixture was cooled to room temperature and the solvent was evaporated. The residue was separated into water and RINKAN, filtered, and concentrated to obtain a crude mixture. This was purified by silica gel column chromatography (5% MeOH / DCM) to obtain UH15PN1 (23 mg, 70%) as a yellow solid. TIFF0007897681000131.tif47150

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

[0333] A mixture of 6-bromo-2-chloroquinazoline (18) (100 mg, 0.41 mmol) and (15c) (85 mg, 0.49 mmol) was mixed with propan-2-ol (2 mL), and the reaction mixture was heated at 100 °C for 2 hours. After completion, the reaction mixture was cooled to room temperature, concentrated, and purified by silica gel column chromatography (2% MeOH / DCM) to obtain 19c (150 mg, 97%) as a pale yellow solid. TIFF0007897681000132.tif20150

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

[0335] UH15PN2 (31 mg, 95%) was prepared as a yellow solid using 6-bromo-N-(3-(methylsulfonyl)phenyl)quinazoline-2-amine (19c) (50 mg, 0.08 mmol) and (2-chlorophenyl)boronic acid (20a) (31 mg, 0.12 mmol). TIFF0007897681000133.tif34150

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

[0337] UH15PN3 (22 mg, 71%) was prepared as a yellow solid using 19c (30 mg, 0.08 mmol) and (4-hydroxyphenyl)boronic acid (20c) (17 mg, 0.12 mmol). TIFF0007897681000134.tif27150

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

[0339] Figure 26 shows a preferred embodiment of the synthesis scheme for UH15PN5, an exemplary inhibitor of protein kinase.

[0340] 6-Bromo-N-(3-(methylsulfonyl)phenyl)quinazoline-2-amine (19c) (20 mg, 0.05 mmol), pyridine-2-ylboronic acid (20e) (22 mg, 0.10 mmol), Pd(dppf)Cl2 (4 mg, 0.005 mmol), CuBr (12 mg, 0.05 mmol), and Cs2CO3 (69 mg, 0.21 mmol) were placed in a round-bottom flask and purged with argon for 10 minutes. Then, DMF (2 mL) was added to the reaction mixture and purged again for 10 minutes. After purging, the contents were heated at 100°C for 16 hours. After completion, the reaction mixture was cooled to room temperature and the solvent was evaporated. The residue was separated into water and RINKAN, filtered, and concentrated to obtain a crude mixture. This was purified by silica gel column chromatography (2.5% MeOH / DCM) to obtain UH15PN5 (15 mg, 60%) as a light brown solid. TIFF0007897681000135.tif27150

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

[0342] UH15PN6 (15 mg, 50%) was prepared as a yellow solid using 19c (30 mg, 0.08 mmol) and pyridine-3-ylboronic acid (20d) (15 mg, 0.12 mmol). TIFF0007897681000136.tif26150

[0343] Preparation of UH15_25:

[0344] Figure 27 shows a preferred embodiment of the synthesis scheme for UH15_25, an exemplary inhibitor of protein kinase.

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

[0346] 56 (20 mg, 0.065 mmol), 15d (22 mg, 0.078 mmol), Pd2(dba)3 (6 mg, 0.006 mmol), xanthophos (8 mg, 0.013 mmol), and cesium carbonate (43 mg, 0.13 mmol) were added to a preheated round-bottom flask and flushed with argon for 10 minutes. Dioxane (1.5 ml) was added to the mixture and flushed again for 5 minutes, then heated overnight at 80°C. The reaction mixture was then separated into ethyl acetate and water, dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (1.5% MeOH / DCM) to obtain 60 (20 mg, 57%) as a pale yellow solid.

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

[0348] 60 (20 mg, 0.036 mmol) was taken into DCM (2 mL), and TFA (0.2 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. The solvent was evaporated, and the crude mixture was purified by silica gel column chromatography (7% MeOH / DCM) to obtain 61 (UH15_25) (12 mg, 75%) as a pale yellow solid. TIFF0007897681000137.tif27150

[0349] Preparation of UH15_36:

[0350] Figure 28 shows a preferred embodiment of the synthesis scheme for UH15_36, an exemplary inhibitor of protein kinase.

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

[0352] Pd2(dba)3 (125 mg, 0.13 mmol), xanthophos (155 mg, 0.26 mmol), and potassium carbonate (740 mg, 5.36 mmol) were added to a preheated round-bottom flask and flushed with argon for 10 minutes. DMF (4 mL) was added to the mixture and flushed for a further 5 minutes, followed by the addition of 67 (500 mg, 2.68 mmol) and 68 (545 mg, 2.68 mmol), and the mixture was refluxed for 24 hours. The reaction mixture was then separated into ethyl acetate and water, dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (15% siRNA / DCM) to obtain 69 (250 mg, 30%) as a pale yellow solid. TIFF0007897681000138.tif13140

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

[0354] To a solution of 69 (250 mg, 0.81 mmol) in CH3OH (15 mL), 10% Pd / C (50 mg) was added, and the reaction mixture was stirred at room temperature in H2 (g) (1 atm) for 4 hours. The reaction mixture was then filtered through Celite and concentrated to obtain 70 (220 mg, 97%) as a brown solid, which was used in the next step without purification.

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

[0356] 56 (42 mg, 0.13 mmol), 70 (39 mg, 0.13 mmol), Pd2(dba)3 (13 mg, 0.013 mmol), xanthophos (16 mg, 0.027 mmol), and cesium carbonate (90 mg, 0.27 mmol) were added to a preheated round-bottom flask and flushed with argon for 10 minutes. Dioxane (3 ml) was added to the mixture and flushed again for 5 minutes, then heated overnight at 80°C. The reaction mixture was then separated into ethyl acetate and water, dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (2.5% MeOH / DCM) to obtain 71 (26 mg, 35%) as a light brown solid.

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

[0358] 71 (20 mg, 0.036 mmol) was taken into DCM (2 mL), and TFA (0.2 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. The solvent was evaporated, and the crude mixture was purified by reverse-phase preparative HPLC (95% H2O / CH3CN containing 0.1% TFA ~ 95% CH3CN / H2O containing 0.1% TFA) to obtain 72 (UH15_36) (12 mg, 75%) as a pale yellow solid. TIFF0007897681000139.tif34150

[0359] Preparation of 34(UH15_20):

[0360] Figure 29 shows a preferred embodiment of the synthesis scheme for UH15_20, an exemplary inhibitor of protein kinase.

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

[0362] A mixture of 7 (20 mg, 0.11 mmol) and NaH (8 mg, 0.33 mmol) was mixed with THF (1.5 mL) and stirred under argon at room temperature for 10 minutes. A solution of 2-chloro-2-(diethoxyphosphoryl)ethyl acetate 30 (42 mg, 0.16 mmol) in THF (0.5 mL) was added dropwise, and the mixture was refluxed for 2.5 hours. After completion, the reaction mixture was cooled to room temperature, concentrated, and extracted using an ethyl acetate-water system. The organic solvent was evaporated, and the residue was purified by silica gel column chromatography (30% ethyl acetate / hexane) to obtain 31 (10 mg, 42%) as a white solid. TIFF0007897681000140.tif20150

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

[0364] 31 (24 mg, 0.11 mmol), (2-chloro-4-methylphenyl)boronic acid (28 mg, 0.16 mmol), and Pd(PPh3)4 (13 mg, 0.011 mmol) were placed in a round-bottom flask (rb) and purged with argon for 10 minutes. DMF (1 mL) and CH3CN (2 mL) were added to the reaction mixture, and the reaction mixture was purged again for 10 minutes. After purging, a 1 M Na2CO3 (23 mg, 0.21 mmol) (220 μL) solution was added dropwise, and heating at 90°C for 5 hours was started. After completion, the reaction mixture was cooled to room temperature, and the solvent was evaporated. The residue was separated into water and ethyl acetate, filtered, and concentrated to obtain a crude mixture, which was purified by silica gel column chromatography (10% ethyl acetate / DCM) to obtain 32 (20 mg, 57%) as a white solid. TIFF0007897681000141.tif27150

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

[0366] Yield 68%, white solid; TIFF0007897681000142.tif27150

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

[0368] Yield 75%, white solid; TIFF0007897681000143.tif26150 Purity 99.6%(t R 23.51 minutes).

[0369] Preparation of UH15-38:

[0370] Figure 30 shows a preferred embodiment of the synthesis scheme for UH15_20, an exemplary inhibitor of protein kinase. In Figure 30, reagents and conditions are: (a) TBSCl, imidazole, DMAP, DMF, 0°C to room temperature, 4 hours, 83%; (b) KF / Al2O3, DMA, room temperature, 2 hours, 67%; (c) Pd2(dba)3, xanthophos, CS2CO3, 1,4-dioxane, 80°C, 16 hours, 47%.

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

[0372] To a solution of compound 1 (250 mg, 1.504 mmol) in dry DMF (3 mL), catalytic amounts of DMAP (15 mg) and imidazole (295 mg, 1.953 mmol) were added in one step, followed by TBSCl (255 mg, 3.75 mmol) in one step, at 0°C. The resulting mixture was then stirred at room temperature for 4 hours. After the reaction was complete, the mixture was quenched by adding water (10 mL) and diluted with ethyl acetate (20 mL). The resulting mixture was washed multiple times with water and brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The crude compound was purified by combiflash chromatography (0-5% ethyl acetate in hexane) to obtain compound 2 (353 mg, 83%) as a colorless liquid. TIFF0007897681000144.tif27150

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

[0374] KF / Al2O3 (1.14 g) was added all at once to a solution of 2 (190 mg, 0.677 mmol) and 3 (115 mg, 0.674 mmol) in dry DMA (3 mL). The resulting mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was filtered through a small Celite pad and washed with ethyl acetate (50 mL). The resulting mixture was washed multiple times with water and brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The crude material was purified by combiflash chromatography (0-5% methanol in CH2Cl2) to obtain 4 (130 mg, 67%) as a white solid. TIFF0007897681000145.tif21150

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

[0376] To a solution of 4 (30 mg, 0.104 mmol), 3-(4-methylpiperazin-1-yl)aniline (5, 20 mg, 0.104 mmol), xanthophos (12 mg, 0.021 mmol), and CS2CO3 (68 mg, 0.208 mmol) in dioxane (2 mL), 10 mol% Pd2(dba)3 was added. The reaction mixture was purged with Ar gas for 15 minutes and refluxed at 80°C for 16 hours. After the reaction was complete, the mixture was filtered through a small Celite pad, 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, dried over anhydrous Na2SO4, and concentrated under vacuum. The crude compound was purified by combiflash chromatography (0-5% methanol in CH2Cl2) to obtain product 6 (22 mg, 47%) as a yellow solid. TIFF0007897681000146.tif40150

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

[0378] [Table 1] NT - Not tested * Values ​​listed in the literature

[0379] For the ADP-Glo ​​assay, recombinant RIPK3 protein (20 ng per reaction) is diluted in a 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), and 0.02% CHAPS. The diluted protein is added to a low-volume white 384-well plate (2 μL / well). The inhibitor is diluted in the reaction buffer (final 25% DMSO), 1 μL is added to each well, and the mixture is incubated at room temperature for 5 minutes. The reaction is initiated by adding 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 hours. The reaction is stopped by adding 5 μL of ADP-Glo ​​reagent (Promega), and the ADP generation reaction is carried out at room temperature for 40 minutes. 10 μL of kinase detection reagent (Promega) is added, and the mixture is incubated at room temperature for 30 minutes to generate a luminescence signal. The emission signal is determined using a suitable emission plate reader (typical integration time 0.3–1 second). To calculate the inhibition rate, the mean background signal is subtracted from the test well and the maximum signal well. Inhibition (%) = (1 - (Test signal / Maximum signal)) * 100 Using nonlinear regression in GraphPad Prism software, the inhibition rate at a specified concentration is determined based on the inhibitor concentration within the dose range, or IC 50 The values ​​are calculated. For the CellTiter-Glo assay, cells are treated in a 96-well plate for 6–24 hours with or without 10 μM IDN6556 (MedKoo) combined with 10 ng / ml hTNFα (Peprotech, Hela-R3 cells) or E. coli LPS (Sigma, RAW264.7 cells) to induce necroptosis. After culturing with the drug, 15 μL of CellTiter-Glo reagent is added to the wells and cultured for 10 minutes. The luminescence signal is determined using a suitable luminescence plate reader (typical integration time 0.3 seconds). To calculate viability, the following: Survival rate (%) = (1 - (Test signal / Maximum survival rate in untreated well)) * 100

[0380] Phosphorylation of RIPK3 and MLKL: Figure 31 shows that the selected UH15 compound and GSK'872 block the 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 a caspase inhibitor. Asterisks represent nonspecific bands occasionally observed in pMLKL immunoblots. Samples were subjected to SDS-PAGE gel electrophoresis and Western blotting using RIPK3, phospho-Thr231 / Ser232-RIPK3, MLKL, and phospho-Ser345-MLKL antibody (Abcam).

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

[0382] [Table 2]

[0383] Receptor-interacting protein kinase 2 (RIPK2) enzyme assay: Recombinant RIPK2 protein (20 ng per reaction) is diluted in a reaction buffer consisting of 40 mM Tris (pH 7.5); 20 mM MgCl2; 0.1 mg / mL BSA; and 50 μM DTT. The diluted protein is added to a low-volume white 384-well plate (2 μL / well). The inhibitor is diluted in the reaction buffer (final 25% DMSO), 1 μL is added to each well, and the mixture is incubated at room temperature for 5 minutes. The reaction is initiated by adding 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 hours. The reaction is stopped by adding 5 μL of ADP-Glo ​​reagent (Promega), and the ADP generation reaction is carried out at room temperature for 40 minutes. 10 μL of kinase detection reagent (Promega) is added, and the mixture is incubated at room temperature for 30 minutes to generate a luminescence signal. The emission signal is determined using a suitable emission plate reader (typical integration time 0.3–1 second). To calculate the inhibition rate, the mean background signal is subtracted from the test well and the maximum signal well. Inhibition (%) = (1 - (Test signal / Maximum signal)) * 100 Using nonlinear regression in GraphPad Prism software, the inhibition rate at a specified concentration is determined based on the inhibitor concentration within the dose range, or IC 50 Calculate the value.

[0384] Activin-like kinase 2 (ALK2) enzyme assay: Enzyme inhibitory activity was observed in the presence of various concentrations (10 nM to 100 μM) of test compounds, inhibiting human ALK2 in the presence of the protein substrate casein (1 mg / mL) and γ- 33 Evaluation was performed using a standard kinase enzyme assay involving culture with ATP (10 μM). After 30 minutes, 33 The amount of P-casein was determined. A graph of inhibitor concentration versus % activity was created, and IC was derived from this graph. 50 The value was determined.

[0385] NOD2 signaling assay: HEK-Blue cells expressing human NOD2 and NFκB-SAEP reporter (Invivogen) were placed in 100 μL of DMEM medium supplemented with 10% FBS and 1% antibiotic-antifungal mixture in a 96-well clear plate, with 7.5 × 10⁶ cells per well. 3 Seeds are seeded individually. Cells are allowed to adhere for 48 hours in a 5% CO2 tissue culture incubator at 37°C. On the morning of the experiment, the culture medium in the wells is replaced with 100 μL of HEK-Blue detection medium (Invivogen). Cells are treated for 15 minutes in a 5% CO2 tissue culture incubator at 37°C with an inhibitor diluted with DMSO (0.5 μL per well). Subsequently, cells are stimulated by adding 1 ng / well of L18-MDP (Invivogen). Cells are cultured for 8 hours in a 5% CO2 tissue culture incubator at 37°C, and the absorbance corresponding to SEAP in the culture 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 Using nonlinear regression in GraphPad Prism software, IC is calculated based on the inhibitor concentration within the dose range. 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 method described above. The inhibition of RIPK2 enzyme and NOD2 cell signaling by the compounds was assessed by the inhibition rate or IC at specified concentrations. 50 The values ​​are shown in Table 3 below. IC of 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. TIFF0007897681000150.tif197150TIFF0007897681000151.tif220150TIFF0007897681000152.tif81150

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