Kinase inhibitors

Abstract

A class of kinase inhibitors is disclosed. Related pharmaceutical compositions and methods of making and using kinase inhibitors are also disclosed.

Description

[0001] Statement Regarding Federally Sponsored Research or Development

[0002] This invention was made with government support under National Institutes of Health, National Cancer Institute, grant number Z01ZIABC011744 and Cancer Moonshot NCI Program for Natural Product Discovery, grant number Z01ZIABC011854. The government has certain rights in this invention.

[0003] Cross - Reference to Related Applications

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 389,937, filed Jul. 17, 2022, which is hereby incorporated by reference in its entirety. Incorporation by Reference of Electronic Submissions

[0005] A computer - readable nucleotide / amino acid sequence listing, submitted concurrently herewith, is hereby incorporated by reference in its entirety and is identified as follows: a 1,926 - byte Extensible Markup Language (XML) file named "767696.xml", created on Jul. 12, 2023. Background of the Invention

[0007] Mammals have enzymes called kinases that are associated with cellular functions such as cell signaling, metabolism, and division. Some kinases have been found to be more active in certain types of cancer. Blocking kinases associated with cancer growth can provide a therapeutic advantage for cancer patients. Given that cancer is a major health concern currently and there is a lack of effective treatments for all cancers, there is an urgent need to identify new kinase inhibitors for treating cancer. Additionally, there is an urgent need to identify kinase inhibitors associated with non - cancer pathologies (e.g., infections) for treating conditions and disorders related to non - cancer pathologies.

[0008] Brief Summary of the Invention

[0009] One aspect of the invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof

[0010]

[0011] wherein X 1 、X 2 、X 3 、 R 1, A, D, and E are as defined herein.

[0012] Another aspect of the invention provides a pharmaceutical composition comprising a compound of an aspect of the invention.

[0013] Another aspect of the invention provides a method of inhibiting kinase activity in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of an aspect of the invention.

[0014] Another aspect of the invention provides a method of inhibiting the immune system in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of an aspect of the invention.

[0015] Another aspect of the invention provides a method of preventing organ rejection in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of an aspect of the invention.

[0016] Another aspect of the invention provides a method of treating cancer in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of an aspect of the invention.

[0017] Another aspect of the invention provides a method of treating diabetic neuropathic pain in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of an aspect of the invention.

[0018] Another aspect of the invention provides a method of treating malaria in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of an aspect of the invention.

[0019] Another aspect of the invention provides a method of treating protozoan-related infections in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of an aspect of the invention.

[0020] Another aspect of the invention provides a method of preparing a compound of an aspect of the invention.

[0021] Brief Description of the Several Views of the Drawings

[0022] Figure 1A is a graph showing the percentage of normalized activity of PKADJ and wild-type PKA (WT) treated with Aplithianine A (183A034G PKADJ and 183A034GWT) and Aplithianine B (183A034H PKADJ and 183A034H WT).

[0023] Figure 1B is a graph showing the percentage of normalized activity of PKADJ treated with Aplithianine A (Compound 1) and Aplithianine B (Compound 2).

[0024] Figure 2A It is a graph showing the percentage of the activity of Aplithianine A (183A034G) in a luciferase assay with or without (only luciferase) PKA as the target receptor.

[0025] Figure 2B It is a competitive kinetic study on how Aplithianine A interacts with the PKA protein.

[0026] Figure 3A An example shown by the Ribbon display of the co-crystal structure of Compound 1 and PKADJ.

[0027] Figure 3B An example showing the ATP binding pocket in the PKADJ crystal structure (PDB#: 4WB7).

[0028] Figure 3C An example showing the aplithianine A binding pocket in the co-crystal structure of aplithianine A and PKADJ.

[0029] Figure 3D An example showing the aplithianine B binding pocket in the co-crystal structure of aplithianine B and PKADJ.

[0030] Figure 4A It is a graph showing the normalized activities of PKADJ and wild-type PKA (WT) treated with Aplithianine A1 (183A041B).

[0031] Figure 4B It is a graph showing the normalized activities of PKADJ and wild-type PKA (WT) treated with Aplithianine A2 (183A041E).

[0032] Figure 4C It is a graph showing the normalized activities of PKADJ and wild-type PKA (WT) treated with Aplithianine A3 (183A041D).

[0033] Figure 4D It is a graph showing the normalized activities of PKADJ and wild-type PKA (WT) treated with Aplithianine A4 (183A041C).

[0034] Figures 5A to 5CThis is an example of kinase profiling of the compounds of the present invention. Larger circles indicate greater potency. Aplithianine A (1) / 183A034G was tested against a panel of 370 human protein kinases at two concentrations (2 μM and 50 nM). The top 50 hits from the 50 nM test were highlighted on a human kinome tree generated using the web application CORAL. The node for PKG1 represents data for PKG1α. Data for PKG1β are not shown.

[0035] Figure 6A This is a graph showing the percentage of normalized activity of 10 selected kinases treated with Aplithianine A (183A034G).

[0036] Figure 6B This is a graph showing the IC 50 of Aplithianine A (183A034G) and Aplithianine A1 (183A041B) against ten selected kinases.

[0037] Figure 6C This is another graph showing the IC 50 of Aplithianine A (183A034G) and Aplithianine A1 (183A041B) against four selected kinases.

[0038] Figure 7 This is an example of a synthetic scheme for the total synthesis of aplithianine A.

[0039] Figure 8 This is another example of a synthetic scheme for the total synthesis of aplithianine A.

[0040] Figure 9A This is the structure of compound 183A041B.

[0041] Figure 9B This is the structure of compound 183A041C.

[0042] Figure 9C This is the structure of compound 183A041D.

[0043] Figure 9D This is the structure of compound 183A041E.

[0044] Figure 9E This is a graph showing the percentage of normalized activity of PKADJ and wild-type PKA (WT) treated with native aplithianine A.

[0045] Figure 9FIt is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with synthetic aplithianine A for display.

[0046] Figure 9G It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A041B.

[0047] Figure 9H It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A041C.

[0048] Figure 9I It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A041D.

[0049] Figure 9J It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A041E.

[0050] Figure 10A It is the structure of a compound according to one aspect of the present invention.

[0051] Figure 10B It is the structure of a compound according to one aspect of the present invention.

[0052] Figure 10C It is the structure of a compound according to one aspect of the present invention.

[0053] Figure 10D It is the structure of a compound according to one aspect of the present invention.

[0054] Figure 10E It is the structure of a compound according to one aspect of the present invention.

[0055] Figure 10F It is the structure of a compound according to one aspect of the present invention.

[0056] Figure 10G It is the structure of a compound according to one aspect of the present invention.

[0057] Figure 10H It is the structure of a compound according to one aspect of the present invention.

[0058] Figure 10I It is the structure of a compound according to one aspect of the present invention.

[0059] Figure 10J It is the structure of a compound according to one aspect of the present invention.

[0060] Figure 10K It is the structure of a compound according to one aspect of the present invention.

[0061] Figure 10L It is the structure of the compound of one aspect of the present invention.

[0062] Figure 10M It is the structure of the compound of one aspect of the present invention.

[0063] Figure 10N It is the structure of the compound of one aspect of the present invention.

[0064] Figure 10O It is the structure of the compound of one aspect of the present invention.

[0065] Figure 10P It is the structure of the compound of one aspect of the present invention.

[0066] Figure 11A It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A046A.

[0067] Figure 11B It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A046F.

[0068] Figure 11C It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A046A.

[0069] Figure 11D It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A046B.

[0070] Figure 11E It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A046C.

[0071] Figure 11F It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A047A.

[0072] Figure 11G It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A047C.

[0073] Figure 11H It is a graph showing the percentage of the normalized activity of PKADJ and wild-type PKA (WT) treated with 183A047F.

[0074] Figure 11IIt is a graph showing the percentage of normalized activity of PKADJ and wild-type PKA (WT) treated with 183A049B.

[0075] Figure 11J It is a graph showing the percentage of normalized activity of PKADJ and wild-type PKA (WT) treated with 183A049C.

[0076] Figure 11K It is a graph showing the percentage of normalized activity of PKADJ and wild-type PKA (WT) treated with 183A049E.

[0077] Figure 11L It is a graph showing the percentage of normalized activity of PKADJ and wild-type PKA (WT) treated with 183A049F.

[0078] Figure 11M It is a graph showing the percentage of normalized activity of PKADJ and wild-type PKA (WT) treated with 183A050B.

[0079] Figure 11N It is a graph showing the percentage of normalized activity of PKADJ and wild-type PKA (WT) treated with 183A050C.

[0080] Figure 11O It is a graph showing the percentage of normalized activity of PKADJ and wild-type PKA (WT) treated with 183A050D.

[0081] Figure 11P It is a graph showing the percentage of normalized activity of PKADJ and wild-type PKA (WT) treated with 183A050E.

[0082] Figure 12A It is the structure of a compound of one aspect of the present invention.

[0083] Figure 12B It is the structure of a compound of one aspect of the present invention.

[0084] Figure 12C It is the structure of a compound of one aspect of the present invention.

[0085] Figure 12D It is the structure of a compound of one aspect of the present invention.

[0086] Figure 13 It is a graph showing the percentage of normalized activity of PKADJ treated with a compound of one aspect of the present invention.

[0087] Figure 14It is a graph showing the percentage of normalized activity of 20 selected kinases treated with aplithianine A1.

[0088] Figure 15 It is an example of kinase mapping.

[0089] Figure 16 It is Figure 15 an example of several branches of kinase mapping.

[0090] Figure 17 It is Figure 15 an example of several branches of kinase mapping.

[0091] Figure 18 It is Figure 15 an example of several branches of kinase mapping.

[0092] Figure 19 It is a graph showing the percentage of normalized activity of 20 selected kinases treated with the compound aplithianine A of one aspect of the present invention.

[0093] Figure 20 It is a graph showing the IC 50 values of 20 selected kinases treated with aplithianine A.

[0094] Figure 21 It is a graph showing the PKG1a differential IC 50 values of 20 selected kinases treated with aplithianine A.

[0095] Figure 22 It shows the structures of Compounds 1 to 5 and semi-synthetic analogs 1a to 1d.

[0096] Figure 23 It shows the selected 1 H- 1 H COSY and HMBC correlations of Compound 1 and Compound 3.

[0097] Figure 24 It shows the 1 H NMR spectrum of Compound 1 in DMSO-d6.

[0098] Figure 25 It shows the 13 C NMR spectrum of Compound 1 in DMSO-d6.

[0099] Figure 26 It shows the 1 H- 1 H COSY spectrum of Compound 1 in DMSO-d6.

[0100] Figure 27 Show the HSQC spectrum of Compound 1 in DMSO-d6.

[0101] Figure 28 Show the HMBC spectrum of Compound 1 in DMSO-d6.

[0102] Figure 29 Show the 1 1H NMR spectrum of Compound 1 in methanol-d4.

[0103] Figure 30 Show the 13 13C NMR spectrum of Compound 1 in methanol-d4.

[0104] Figure 31 Show the 1 1H- 1 1H COSY spectrum of Compound 1 in methanol-d4.

[0105] Figure 32 Show the HSQC spectrum of Compound 1 in methanol-d4.

[0106] Figure 33 Show the HMBC spectrum of Compound 1 in methanol-d4.

[0107] Figure 34 Show the 1 1H NMR spectrum of Compound 2 in DMSO-d6.

[0108] Figure 35 Show the HSQC spectrum of Compound 2 in DMSO-d6.

[0109] Figure 36 Show the HMBC spectrum of Compound 2 in DMSO-d6.

[0110] Figure 37 Show the 1 1H NMR spectrum of Compound 2 in methanol-d4.

[0111] Figure 38 Show the 13 13C NMR spectrum of Compound 2 in methanol-d4.

[0112] Figure 39 Show the 1 1H- 1 1H COSY spectrum of Compound 2 in methanol-d4.

[0113] Figure 40 Show the HSQC spectrum of Compound 2 in methanol-d4.

[0114] Figure 41Show the HMBC spectrum of Compound 2 in methanol-d4.

[0115] Figure 42 Show the 1 1H NMR spectrum of Compound 1a in DMSO-d6.

[0116] Figure 43 Show the 13 13C NMR spectrum of Compound 1a in DMSO-d6.

[0117] Figure 44 Show the 1 1H- 1 1H COSY spectrum of Compound 1a in DMSO-d6.

[0118] Figure 45 Show the HSQC spectrum of Compound 1a in DMSO-d6.

[0119] Figure 46 Show the HMBC spectrum of Compound 1a in DMSO-d6.

[0120] Figure 47 Show the 1 1H NMR spectrum of Compound 1b in DMSO-d6.

[0121] Figure 48 Show the 13 13C NMR spectrum of Compound 1b in DMSO-d6.

[0122] Figure 49 Show the HSQC spectrum of Compound 1b in DMSO-d6.

[0123] Figure 50 Show the HMBC spectrum of Compound 1b in DMSO-d6.

[0124] Figure 51 Show the 1 1H NMR spectrum of Compound 1c in DMSO-d6.

[0125] Figure 52 Show the 13 13C NMR spectrum of Compound 1c in DMSO-d6.

[0126] Figure 53 Show the 1 1H- 1 1H COSY spectrum of Compound 1c in DMSO-d6.

[0127] Figure 54 Show the HSQC spectrum of Compound 1c in DMSO-d6.

[0128] Figure 55 Show the HMBC spectrum of compound 1c in DMSO-d6.

[0129] Figure 56 Show the 1 1H NMR spectrum of compound 1d in DMSO-d6.

[0130] Figure 57 Show the 13 13C NMR spectrum of compound 1d in DMSO-d6.

[0131] Figure 58 Show the 1 1H- 1 1H COSY spectrum of compound 1d in DMSO-d6.

[0132] Figure 59 Show the HSQC spectrum of compound 1d in DMSO-d6.

[0133] Figure 60 Show the HMBC spectrum of compound 1d in DMSO-d6.

[0134] Figure 61 Show the 1 1H NMR spectrum of compound 3 in DMSO-d6.

[0135] Figure 62 Show the 13 13C NMR spectrum of compound 3 in DMSO-d6.

[0136] Figure 63 Show the 1 1H- 1 1H COSY spectrum of compound 3 in DMSO-d6.

[0137] Figure 64 Show the HSQC spectrum of compound 3 in DMSO-d6.

[0138] Figure 65 Show the HMBC spectrum of compound 3 in DMSO-d6.

[0139] Figure 66 Show the 1 1H NMR spectrum of compound 4 in DMSO-d6.

[0140] Figure 67 Show the 13 13C NMR spectrum of compound 4 in DMSO-d6.

[0141] Figure 68 Show the 1 1H- 11H COSY spectrum.

[0142] Figure 69 HSQC spectrum of Compound 4 in DMSO-d6 is shown.

[0143] Figure 70 HMBC spectrum of Compound 4 in DMSO-d6 is shown.

[0144] Figure 71 1H NMR spectrum of Compound 5 in DMSO-d6 is shown. 1 1H NMR spectrum.

[0145] Figure 72 13C NMR spectrum of Compound 5 in DMSO-d6 is shown. 13 13C NMR spectrum.

[0146] Figure 73 1H- 1 H COSY spectrum of Compound 5 in DMSO-d6 is shown. 1 1H COSY spectrum.

[0147] Figure 74 HSQC spectrum of Compound 5 in DMSO-d6 is shown.

[0148] Figure 75 HMBC spectrum of Compound 5 in DMSO-d6 is shown.

[0149] Figure 76A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0150] Figure 76B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0151] Figure 77A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0152] Figure 77B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0153] Figure 78A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0154] Figure 78B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0155] Figure 79A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0156] Figure 79BIt is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0157] Figure 80A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0158] Figure 80B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0159] Figure 81A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0160] Figure 81B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0161] Figure 82A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0162] Figure 82B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0163] Figure 83A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0164] Figure 83B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0165] Figure 84A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0166] Figure 84B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0167] Figure 85A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0168] Figure 85B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0169] Figure 86A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0170] Figure 86B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0171] Figure 87A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0172] Figure 87B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0173] Figure 88A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0174] Figure 88B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0175] Figure 89A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0176] Figure 89B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0177] Figure 90A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0178] Figure 90B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0179] Figure 91A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0180] Figure 92B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0181] Figure 93A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0182] Figure 93B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0183] Figure 94A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0184] Figure 94B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0185] Figure 95A It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0186] Figure 95B It is a graph showing the percentage of enzyme activity (relative to the control) and logarithmic curve fitting.

[0187] Figure 96 1H NMR spectrum of 8 in DMSO-d6 1 1H NMR spectrum.

[0188] Figure 97 1H NMR spectrum of 8 in DMSO-d6 13 13C NMR spectrum.

[0189] Figure 98 1H NMR spectrum of 9 in DMSO-d6 1 1H NMR spectrum.

[0190] Figure 99 1H NMR spectrum of 9 in DMSO-d6 13 13C NMR spectrum.

[0191] Figure 100 1H NMR spectrum of 10 in DMSO-d6 1 1H NMR spectrum.

[0192] Figure 101 1H NMR spectrum of 10 in DMSO-d6 13 13C NMR spectrum.

[0193] Figure 102 1H NMR spectrum of 11 in DMSO-d6 1 1H NMR spectrum.

[0194] Figure 103 1H NMR spectrum of 11 in DMSO-d6 13 13C NMR spectrum.

[0195] Figure 104 1H NMR spectrum of 12 in DMSO-d6 1 1H NMR spectrum.

[0196] Figure 105 1H NMR spectrum of 12 in DMSO-d6 13 13C NMR spectrum.

[0197] Figure 106 is a graph showing the normalized JPKAcα activity % curve of compound 183A056C.

[0198] Figure 107A A graph showing the kinase test results of the compound of one aspect of the present invention at a concentration of 50 nM.

[0199] Figure 107B A graph showing the kinase test results of the compound of one aspect of the present invention at a concentration of 2 μM.

[0200] Figure 108 A graph showing the kinase test results of the compound of one aspect of the present invention at a concentration of 50 nM.

[0201] Figure 109 A graph showing the kinase assay results of a compound of one aspect of the present invention at a concentration of 50 nM.

[0202] Figure 110 A graph showing the kinase assay results of a compound of one aspect of the present invention at a concentration of 50 nM.

[0203] Figure 111 A graph showing the kinase assay results of Compound 1.

[0204] Figure 112 A graph showing the kinase assay results of Compound 1 and Compound 3.

[0205] Detailed Description of the Invention

[0206] The Molecular Targets Program of the National Cancer Institute (NCI) of the United States completed the screening of approximately 150,000 pre-fractionated natural products from the NCI Natural Products Discovery Program (NPNPD) (Thornburg et al., ACS Chem. Biol., 13:2484 - 2497 (2018)). A class of bioactive compounds were isolated from the marine organism Aplidium sp. These compounds, named Aplithianines A & B, were shown to effectively inhibit both (1) the oncogenic gene fusion DNAJB1 - PRKACA (PKADJ) and (2) wild-type protein kinase A (PKA) at nanomolar concentrations. Aplithianine A was shown to effectively and selectively inhibit a broad range of kinases, not just PKADJ or PKA, broadening its potential applications. Further kinetic analysis showed that Aplithianine A is a competitive inhibitor of kinases, competing with ATP for binding to PKA. Additional structural studies showed that aplithianine A binds to the catalytic pocket where ATP normally binds in PKADJ, further demonstrating the competitive mechanism of inhibition and providing structural insights for further synthetic modification of this class of compounds. Additional semi-synthetic work in the MTP has created non-natural aplithianine derivatives, and one of them (the brominated derivative) was found to have equivalent activity compared to the natural product. Additional derivatives were also created, further providing SAR data on the structure-activity relationship for this class of compounds. Two synthetic schemes have been designed and completed for the total synthesis of aplithianine A.

[0207] The aplithianine structural class is a group of potent kinase inhibitors with broad potential applicability to many important kinases (e.g., for cancer chemotherapy). For example, gene fusions (genetic lesions that join two normally non-adjacent parts of the genome together) are among the earliest recognized cancer biomarkers. In all solid malignancies, approximately 20% of solid malignancies have at least one identifiable gene fusion. The experience with the BCR-ABL1 kinase inhibitor imatinib (Savage et al., N. Engl. J. Med., 346(9):683-93 (2002)) and the continued emphasis on precision medicine suggest that focusing on drug development related to gene fusions can yield disease-specific drugs. One such fusion is the recently identified PKADJ oncogenic gene fusion associated with fibrolamellar hepatocellular carcinoma (FL-HCC) (Honeyman et al., Science, 343:1010-14 (2014) and Kastenhuber et al., PNAS USA, 114:13076-84 (2017)). In liver cancer, FL-HCC is particularly unfortunate because its patient population is young (<35 years old) and lacks any successful disease-specific chemotherapy regimens, with a 5-year survival rate of only approximately 34% (Riggle et al., Pediatr. Blood Cancer, 63:1163-7 (2016)). In 2014, the biological understanding of FL-HCC was improved, and it was first discovered that all FL-HCC patients carry an in-frame intrachromosomal gene fusion between the first exon of the gene encoding a member of the heat shock protein 40 (HSP40) family, DNAJB1, and the second exon of the gene for the adenosine 3’,5’-monophosphate (cAMP)-dependent PKA catalytic subunit α, PRKACA (Honeyman et al., Science, 343:1010-14 (2014)). The DNAJB1-PRKACA gene fusion produces an enzymatically active chimeric protein, DNAJ. Studies have demonstrated that PKA activity is required for tumor formation. Equivalent expression of PKAcα or expression of a kinase-dead apoptotic version of the oncogenic fusion protein is not sufficient for transformation, and the tumorigenicity of PKAJ depends on its kinase activity (Kastenhuber et al., PNAS USA, 114:13076-84 (2017)).

[0208] The DNAJ fusion complex may provide new small molecule binding sites that can be exploited for the treatment of FL-HCC (Tomasini et al., Scientific Reports, 8:720 (2018); Cheung et al., PNAS USA, 112:1374-79 (2015); and Averill et al., J. Cell Biochem., 120:13783-91 (2019)). Accordingly, an improved sandwich ELISA assay was developed using a biotinylated peptide derived from a PKA substrate (see, for example, Example 4). The reaction and read components of this assay were then optimized, including the optimal reactant concentrations for kinase reactions suitable for identifying both inhibitors and activators of PKAJ. The same system was also used to test samples for inhibiting the activity of WT-PKA to identify any compounds selective for the fusion protein (see, for example, Example 6).

[0209] Compound

[0210] In one aspect, the present invention provides a compound of formula (I)

[0211]

[0212] Wherein

[0213] is a single bond or a double bond,

[0214] X 1 and X 2 are each independently CH, CR 6 or N;

[0215] X 3 is S, S=O or S(=O)2;

[0216] R 1 is H or -NR 2 R 3 ;

[0217] R 2 is H or C1-C3 alkyl;

[0218] R 3 is aryl;

[0219] R 6 is C1-C3 alkyl;

[0220] A is optional, and when present, A is –C(O)-, -C(O)O-, –C(O)NH-, -C(O)N(C1-C3-alkyl)-; -C(O)NH-(C1-C6 alkyl)-NHC(O)-, -NH;

[0221] D is optional and when present, D is C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, -(C1-C6 alkyl)-O-, -(C1-C6 alkyl)-NH-, -(C1-C3 alkyl)-O-(C1-C3 alkyl)- or -(C1-C3 alkyl)-NH-(C1-C3 alkyl)-, wherein the alkyl group or cycloalkyl / heterocycloalkyl group in any of the foregoing is optionally substituted with one or more substituents selected from hydroxy, C1-C6 alkyl, amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; -NH-aryl and combinations thereof; and

[0222] E is:

[0223] aryl or heteroaryl, optionally substituted with one or more substituents selected from C1-C6 alkyl or alkoxy, -(C1-C6 alkyl)-OH, -(C1-C6 alkyl)-COOH, -(C1-C6 alkyl)-NH2, halogen, nitro, hydroxy, amino, C1-C6 alkylamino, di-C1-C6 alkyl-amino; -NH-aryl, C1-C6 haloalkyl, C3-C8 cycloalkyl or heterocycloalkyl, fused C3-C8 cycloalkyl or heterocycloalkyl, aryl or heteroaryl, fused aryl or heteroaryl, -CN, -(C1-C3 alkyl)-CN, carbonyl and combinations thereof;

[0224] amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; or -NH-aryl;

[0225] C1-C6 alkyl or alkoxy, optionally substituted with one or more substituents selected from hydroxy, C1-C6 alkyl, amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; -NH-aryl and combinations thereof;

[0226] C3-C8 cycloalkyl or heterocycloalkyl, optionally substituted with one or more substituents selected from hydroxy, C1-C6 alkyl, amino, C1-C6 alkylamino, di-C1-C6 alkyl-amino; -NH-aryl, C3-C8 cycloalkyl or heterocycloalkyl, fused C3-C8 cycloalkyl or heterocycloalkyl, aryl or heteroaryl, fused aryl or heteroaryl and combinations thereof;

[0227] -C(O)NH2;

[0228] -C(O)OH;

[0229] -C(O)H;

[0230] -N-(C1-C6 alkyl)-acrylamide;

[0231] halogen; or

[0232] hydrogen;

[0233] or a pharmaceutically acceptable salt thereof.

[0234] In some embodiments, the present invention provides a compound of formula (I)

[0235]

[0236] wherein

[0237] is a single bond or a double bond,

[0238] X 1 and X 2 are each independently CH or N;

[0239] X 3 is S, S═O or S(═O)2;

[0240] R 1 is H or -NR 2 R 3 ;

[0241] R 2 is H or C1-C3 alkyl;

[0242] R 3 is aryl;

[0243] A is optional, and when present, A is –C(O)-, -C(O)O-, –C(O)NH-, -C(O)N(C1-C3-alkyl)-; -C(O)NH-(C1-C6 alkyl)-NHC(O)-, -NH;

[0244] D is optional, and when present, D is C1-C6 alkyl, -(C1-C6 alkyl)-O-, -(C1-C6 alkyl)-NH-, -(C1-C3 alkyl)-O-(C1-C3 alkyl)- or -(C1-C3 alkyl)-NH-(C1-C3 alkyl)-, wherein the alkyl groups in any of the foregoing are optionally substituted with hydroxy; and

[0245] E is:

[0246] aryl or heteroaryl, optionally substituted with C1-C6 alkyl or alkoxy, halogen, nitro, hydroxy, C1-C6 haloalkyl, -CN, -(C1-C3 alkyl)-CN or carbonyl;

[0247] amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; or -NH-aryl;

[0248] C1-C6 alkyl or alkoxy, optionally substituted with hydroxy;

[0249] A C3-C8 cycloalkyl or heterocyclic hydrocarbon group, optionally substituted with a hydroxyl group;

[0250] -C(O)NH2;

[0251] -C(O)OH;

[0252] -C(O)H;

[0253] -N-(C1-C6 alkyl)-acrylamide;

[0254] a halogen; or

[0255] hydrogen;

[0256] or a pharmaceutically acceptable salt thereof.

[0257] In one aspect of the present invention, is a single bond.

[0258] In one aspect of the present invention, is a double bond.

[0259] In one aspect of the present invention, the compound of formula (I) is a compound of formula (Ia):

[0260]

[0261] or a pharmaceutically acceptable salt thereof.

[0262] In one aspect of the present invention, the compound of formula (I) is a compound of formula (Ib):

[0263]

[0264] or a pharmaceutically acceptable salt thereof.

[0265] In one aspect of the present invention, R 1 is H.

[0266] In some aspects,

[0267] (i) A and D are absent, and E is a halogen; -C(O)OH; -C(O)H; an aryl or heteroaryl group, optionally substituted with a C1-C3 alkyl or halogen; or a C3-C8 cycloalkyl or heterocyclic hydrocarbon group, optionally substituted with a hydroxyl group;

[0268] (ii) A is absent; D is a C1-C6 alkyl, optionally a C1-C3 alkyl; and E is -C(O)NH2 or a C3-C8 cycloalkyl or heterocyclic hydrocarbon group, optionally substituted with a hydroxyl group;

[0269] (iii) A is -NH-, -C(O)NH- or -C(O)N(C1-C3-alkyl)-; D is absent or is C1-C6 alkyl, optionally C1-C3 alkyl; and E is amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; or -NH-aryl; C1-C6 alkyl or alkoxy or C1-C3 alkyl or alkoxy, optionally substituted by hydroxy; or aryl or heteroaryl, optionally substituted by C1-C3 alkyl or alkoxy, halogen, nitro, hydroxy, C1-C3 haloalkyl, -CN, -(C1-C3 alkyl)-CN or carbonyl;

[0270] (iv) A is absent, D is -(C1-C6 alkyl)-O-, -(C1-C6 alkyl)-NH-, -(C1-C3 alkyl)-O-(C1-C3 alkyl)- or -(C1-C3 alkyl)-NH-(C1-C3 alkyl)-, wherein the alkyl groups in any of the foregoing are optionally substituted by hydroxy and wherein the alkyl groups in any of the foregoing are optionally branched; and E is C1-C6 alkyl or C3-C8 cycloalkyl or heterocyclic hydrocarbyl, optionally substituted by hydroxy;

[0271] (v) A is -C(O)NH-(C1-C6 alkyl)-NHC(O)-, optionally -C(O)NH-(C1-C3 alkyl)-NHC(O)-; D is absent; and E is C1-C6 alkyl or C1-C3 alkyl; or aryl or heteroaryl, optionally substituted by C1-C3 alkyl or alkoxy, halogen, nitro, hydroxy, C1-C6 haloalkyl, -CN, -(C1-C3 alkyl)-CN or carbonyl;

[0272] (vi) A is -C(O)O-; D is absent; and E is C1-C6 alkyl; or

[0273] (vii) A is -C(O)-; D is absent; and E is heterocyclic hydrocarbyl.

[0274] In one aspect of the invention, the compound of formula (I) is a compound of formula (Ic) or a pharmaceutically acceptable salt thereof:

[0275]

[0276] wherein R 4 and R 5 are the same or different and each is H or halogen (e.g., bromine, fluorine, chlorine or iodine).

[0277] In one aspect of the invention, one of R 4 and R 5 is halogen. In one aspect of the invention, both R 4 and R 5 are halogen.

[0278] In one aspect of the present invention, R 4 and R 5 are both hydrogen. In some embodiments of the foregoing aspect, the halogen is bromine.

[0279] In one aspect of the present invention, the compound is not aplithianine A (Compound 1)

[0280]

[0281] One aspect of the present invention provides the following compounds:

[0282]

[0283]

[0284]

[0285]

[0286] or a pharmaceutically acceptable salt thereof. One aspect of the present invention provides the following compounds:

[0287]

[0288] or a pharmaceutically acceptable salt thereof.

[0289] One aspect of the present invention provides the following compounds:

[0290]

[0291] or a pharmaceutically acceptable salt thereof.

[0292] One aspect of the present invention provides Figures 9A to 9J the compound of.

[0293] One aspect of the present invention provides Figures 10A to 10P the compound of.

[0294] One aspect of the present invention provides Figures 12A to 12D the compound of.

[0295] Other aspects of the present invention provide enantiomers of the compounds disclosed herein.

[0296] In any of the above aspects, the term "alkyl" means a straight or branched alkyl substituent containing, for example, from about 1 to about 6 carbon atoms, such as from about 1 to about 4 carbon atoms. Examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, and the like. This definition also applies when "alkyl" appears as part of a group, such as C3-C6 cycloalkylalkyl, hydroxyalkyl, haloalkyl (e.g., mono-haloalkyl, di-haloalkyl, and tri-haloalkyl), cyanoalkyl, aminoalkyl, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, arylcarbonylalkyl (-(alkyl)C(O)aryl), arylalkyl, etc. The alkyl can be substituted or unsubstituted as described herein. Even when the alkyl is an alkylene chain (e.g., -(CH2) n -), the alkyl can be substituted or unsubstituted.

[0297] In any of the above aspects, the term "alkenyl" as used herein means a straight-chain alkenyl substituent containing, for example, from about 2 to about 6 carbon atoms (branched alkenyl being from about 3 to about 6 carbon atoms), such as from about 3 to about 5 carbon atoms (branched alkenyl being from about 3 to about 6 carbon atoms). According to one aspect of the invention, the alkenyl is C2-C4 alkenyl. Examples of alkenyl include vinyl, allyl, 2-propenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, and the like. The alkenyl can be substituted or unsubstituted as described herein.

[0298] In any of the above aspects, the term "cycloalkyl" as used herein means a cyclic alkyl moiety containing, for example, 3 to 6 carbon atoms or 5 to 6 carbon atoms. Examples of such moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The cycloalkyl can contain a carbonyl group such that an exocyclic (=O) group is present. The cycloalkyl can be substituted or unsubstituted as described herein. The cycloalkyl can also be fused to an adjacent substituent (e.g., cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), i.e., sharing two atoms and bonds with the adjacent substituent.

[0299] In any of the above aspects, the term "aryl" refers to a monocyclic, bicyclic or tricyclic carbocyclic ring system having one, two or three aromatic rings, e.g., phenyl, naphthyl, anthryl or biphenyl. The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic moiety as commonly understood in the art and includes monocyclic and polycyclic aromatic groups, e.g., phenyl, biphenyl, naphthyl, anthryl, pyrenyl, etc. The aryl moiety typically contains, e.g., 6 to 30 carbon atoms, 6 to 18 carbon atoms, 6 to 14 carbon atoms or 6 to 10 carbon atoms. It is understood that the term aryl includes carbocyclic moieties that are planar and contain 4n + 2 π electrons according to Hückel's rule, where n = 1, 2 or 3. This definition also applies when "aryl" appears as part of a group, e.g., in groups such as haloaryl (e.g., mono-haloaryl, di-haloaryl and tri-haloaryl), arylalkyl, etc. The aryl can be substituted or unsubstituted as described herein. The aryl can also be fused to an adjacent substituent (e.g., cycloalkyl, heterocycloalkyl, aryl or heteroaryl), i.e., sharing two atoms and bonds with the adjacent substituent.

[0300] In any of the above aspects, the term "heteroaryl" refers to an aromatic 5- or 6-membered monocyclic group, 9- or 10-membered bicyclic group and 11- to 14-membered tricyclic group, said groups having at least one heteroatom (O, S or N) in at least one ring. Each ring of the heteroaryl containing a heteroatom can contain one or two oxygen or sulfur atoms and / or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less and each ring has at least one carbon atom. The fused rings completing the bicyclic and tricyclic groups can contain only carbon atoms and can be saturated, partially saturated or unsaturated. The nitrogen and sulfur atoms can be optionally oxidized and the nitrogen atoms can be optionally quaternized. The heteroaryl which is bicyclic or tricyclic must include at least one fully aromatic ring, but the other fused ring or other multiple fused rings can be aromatic or non-aromatic. The heteroaryl can be attached at any available nitrogen or carbon atom of any ring. Exemplary instances of heteroaryl are pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl, (1,2,3)- and (1,2,4)-triazolyl, pyrazolyl, tetrazolyl, furyl, pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl and oxadiazolyl. The heteroaryl can be substituted or unsubstituted as described herein. The heteroaryl can also be fused to an adjacent substituent (e.g., cycloalkyl, heterocycloalkyl, aryl or heteroaryl), i.e., sharing two atoms and bonds with the adjacent substituent.

[0301] The term "heterocyclic hydrocarbyl" means a stable, saturated or partially unsaturated monocyclic, bicyclic and spirocyclic system containing 3 to 7 ring members, said ring members being carbon atoms and other atoms selected from nitrogen, sulfur and / or oxygen. In one aspect, the heterocyclic hydrocarbyl is a 5-, 6- or 7-membered monocyclic ring and contains one, two or three heteroatoms selected from nitrogen, oxygen and sulfur. The heterocyclic hydrocarbyl can be attached to the parent structure through a carbon atom or through any heteroatom of the heterocyclic hydrocarbyl, thereby generating a stable structure. Optionally, or alternatively, the heterocyclic hydrocarbyl can contain a carbonyl group such that an exocyclic (=O) group is present. Examples of such heterocyclic hydrocarbyl rings are isoxazolyl, thiazolinyl, imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolyl, pyrrolinyl, pyrazolyl, pyranyl, piperidinyl, oxazolyl and morpholinyl. The heterocyclic hydrocarbyl can be substituted or unsubstituted as described herein.

[0302] In any of the above aspects, the term "hydroxyl" refers to the group -OH.

[0303] In any of the above aspects, the term "cyano" refers to the group -CN, and the term "thiocyanato" refers to -SCN.

[0304] In any of the above aspects, the terms "alkoxy" and "cycloalkyloxy" include a straight-chain or branched-chain alkyl group and a cycloalkyl group attached to a divalent oxygen, respectively. The alkyl group and the cycloalkyl group are the same as those described herein.

[0305] In any of the above aspects, the term "halo" refers to a halogen selected from fluorine, chlorine, bromine and iodine.

[0306] In any of the above aspects, the term "carboxylate" refers to the group -C(O)OH.

[0307] In any of the above aspects, the term "amino" refers to the group -NH2. The term "alkylamino" refers to -NHR, and the term "dialkylamino" refers to -NRR'. R and R' are the same or different and each is a substituted or unsubstituted alkyl group as described herein.

[0308] In any of the above aspects, the term "amido" refers to the group -C(O)NRR', where R and R' are the same or different and each is hydrogen or a substituted or unsubstituted alkyl group as described herein.

[0309] In any of the above aspects, the term "phosphonyl" refers to the group -P(O)(OR)2, where R is hydrogen or a substituted or unsubstituted alkyl group as described herein.

[0310] In other aspects, any substituent that is not hydrogen (e.g., C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl or heterocycloalkylalkyl) can be an optionally substituted moiety. The substituted moiety typically contains at least one substituent (e.g., 1, 2, 3, 4, 5, 6, etc.) at any suitable position (e.g., 1-, 2-, 3-, 4-, 5- or 6-position, etc.). When an aryl is substituted with a substituent (e.g., halogen, amino, alkyl, OH, alkoxy, etc.), the aromatic ring hydrogen is replaced by the substituent, and this can occur at any available hydrogen (e.g., 2, 3, 4, 5 and / or 6 positions, where the 1-position is the point of attachment of the aryl in the compounds of the present invention). Suitable substituents include, for example, halogen, alkyl, alkenyl, hydroxy, nitro, cyano, amino, alkylamino, alkoxy, aryloxy, aralkyloxy, carboxyl, carboxyalkyl, carboxyalkoxy, acylamino, alkylacylamino, haloalkylacylamino, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, fused aryl, fused heteroaryl, fused heterocycloalkyl and fused cycloalkyl, each of which is described herein. In some cases, the substituent is at least one alkyl, halogen and / or haloalkyl (e.g., 1 or 2).

[0311] In any of the above aspects, whenever a range of the number of atoms in a structure is specified (e.g., C 1-12 , C 1-8 , C 1-6 , C 1-4 , etc.), it is specifically contemplated that carbon atoms falling within any sub-range or individual value within the specified range can also be used. Thus, for example, the expression of a range of 1 to 8 carbon atoms (e.g., C1-C8), 1 to 6 carbon atoms (e.g., C1-C6), 1 to 4 carbon atoms (e.g., C1-C4), 1 to 3 carbon atoms (e.g., C1-C3) or 2 to 8 carbon atoms (e.g., C2-C8) used with respect to any chemical group mentioned herein (e.g., alkyl, cycloalkyl, etc.) encompasses and specifically describes 1, 2, 3, 4, 5, 6, 7 and / or 8 carbon atoms, as appropriate, and any sub-range thereof (e.g., 1 to 2 carbon atoms, 1 to 3 carbon atoms, 1 to 4 carbon atoms, 1 to 5 carbon atoms, 1 to 6 carbon atoms, 1 to 7 carbon atoms, 1 to 8 carbon atoms, 2 to 3 carbon atoms, 2 to 4 carbon atoms, 2 to 5 carbon atoms, 2 to 6 carbon atoms, 2 to 7 carbon atoms, 2 to 8 carbon atoms, 3 to 4 carbon atoms, 3 to 5 carbon atoms, 3 to 6 carbon atoms, 3 to 7 carbon atoms, 3 to 8 carbon atoms, 4 to 5 carbon atoms, 4 to 6 carbon atoms, 4 to 7 carbon atoms, 4 to 8 carbon atoms, etc., as appropriate).

[0312] In any of the foregoing aspects, the phrase "salt" or "pharmaceutically acceptable salt" is intended to include non-toxic salts synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. For example, inorganic acids (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid), organic acids (e.g., oxalic acid, malonic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, acetic acid, trifluoroacetic acid, gluconic acid, ascorbic acid, methanesulfonic acid, or benzenesulfonic acid), inorganic bases (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or ammonium hydroxide), organic bases (e.g., methylamine, diethylamine, triethylamine, triethanolamine, ethylenediamine, tris(hydroxymethyl)methylamine, guanidine, choline, or cinchonine), or amino acids (e.g., lysine, arginine, or alanine) can be used. Typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical. A list of suitable salts can be found in Remington’s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Easton, PA, 1990, p. 1445 and Journal of Pharmaceutical Science, 66, 2-19 (1977). For example, they can be salts of an alkali metal (e.g., sodium or potassium), an alkaline earth metal (e.g., calcium), or an ammonium salt in the salt. In one aspect, the salt is a trifluoroacetate salt.

[0313] Pharmaceutical composition

[0314] One aspect of the present invention provides a pharmaceutical composition comprising a compound of the present invention. The pharmaceutical composition contains a pharmaceutically acceptable carrier.

[0315] One aspect of the present invention provides a pharmaceutical composition comprising a compound of Formula I.

[0316] One aspect of the present invention provides a pharmaceutical composition comprising aplithianine A (Compound 1) having a purity of at least about 80% aplithianine B (Compound 2) aplidipurinide A (Compound 3) aplidipurinide B (Compound 4) or aplidipurinide C (Compound 5) and a pharmaceutical carrier.

[0317] In one aspect, the compound has a purity of at least about 85% (e.g., at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%).

[0318] The pharmaceutical composition may comprise a combination of a compound of the invention with one or more other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., topoisomerase I inhibitors, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0319] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemical-physical factors such as solubility and lack of reactivity with the active compound, as well as by the route of administration. Pharmaceutically acceptable carriers described herein, e.g., vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and readily available to the public. Preferred pharmaceutically acceptable carriers are carriers that are chemically inert to the active agent and that have no adverse side effects or toxicity under the conditions of use.

[0320] The choice of carrier will be determined in part by the particular compound and the particular method for administering the compound. Accordingly, there are various suitable formulations of the pharmaceutical compositions of the invention. The compound, its pharmaceutically acceptable salts can be administered in any suitable manner (e.g., orally, intravenously, intramuscularly, intrathecally, subcutaneously, sublingually, buccally, rectally, vaginally, by the ocular route, by the otic route, nasally, by inhalation, by nebulization, topically, systemically, transdermally, or a combination thereof). In an embodiment, the pharmaceutical composition of the invention is administered orally.

[0321] The following formulations for administration are exemplary and in no way limiting. More than one route can be used to administer the compound, and in certain cases, a particular route may provide a more immediate and more effective response than another route.

[0322] Formulations suitable for administration include aqueous and non-aqueous, isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compounds may be administered in a physiologically acceptable diluent in a pharmaceutical carrier such as a sterile liquid or mixture of liquids including water, saline, dextrose aqueous solution and related sugar solutions, alcohols such as ethanol or cetyl alcohol, glycols such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without a pharmaceutically acceptable surfactant such as a soap or detergent, suspending agent such as pectin, carbomer, methyl cellulose, hydroxypropyl methyl cellulose or carboxymethyl cellulose, or emulsifying agent and other pharmaceutical adjuvants.

[0323] Oils that may be used in the formulations include petroleum, animal, vegetable or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and mineral oil. Suitable fatty acids for use in the formulations include oleic acid, stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0324] Suitable soaps for use in the formulations include alkali metal salts of fatty acids, ammonium salts and triethanolamine salts, and suitable detergents include (a) cationic detergents such as dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic detergents such as alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates and sulfosuccinates; (c) nonionic detergents such as fatty amine oxides, fatty acid alkanolamides and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary salts; and (e) mixtures thereof.

[0325] The formulation typically contains from about 0.5% to about 25% by weight of the compound in solution. Preservatives and buffers can be used. To minimize or eliminate irritation at the injection site, such compositions can contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is typically from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters such as sorbitan monooleate and high molecular weight adducts of ethylene oxide with hydrophobic bases formed by the condensation of propylene oxide with propylene glycol. The formulation can be present in unit dose or multi-dose sealed containers such as ampoules and vials and can be stored under lyophilized (freeze-dried) conditions, requiring only the immediate addition of a sterile liquid excipient (e.g., water) for injection immediately prior to use. Solutions and suspensions for parenteral injection can be prepared from sterile powders, granules, and tablets of the foregoing types. The requirements for an effective pharmaceutical carrier for use in the compositions are well known to those of ordinary skill in the art (see, e.g., Lloyd et al. (eds.), Remington: The Science and Practice of Pharmacy, 22nd Ed., Pharmaceutical Press (2012)).

[0326] Those skilled in the art will understand that, in addition to the above pharmaceutical compositions, the compounds of the invention can be formulated as inclusion complexes such as cyclodextrin inclusion complexes or liposomes.

[0327] For the purposes of the present invention, the amount or dose of the compound administered should be sufficient to effect the desired response, e.g., a therapeutic or prophylactic response, in a mammal within a reasonable time frame. For example, the dose of the compound should be sufficient to inhibit the growth of target cells or to treat or prevent cancer over a period of time of about 2 hours or more from the time of administration, e.g., 12 to 24 hours or more hours. In certain aspects, the time period can even be longer. The dose will be determined by the efficacy of the particular compound and the condition of the mammal (e.g., human) to be treated as well as the weight of the mammal (e.g., human) to be treated.

[0328] Many assays for determining the administered dose are known in the art. The administered dose can be determined in vitro (e.g., cell culture) or in vivo (e.g., animal studies). For example, the administered dose can be determined by measuring the IC 50 (dose for 50% maximum inhibition of symptoms), LD 50 (dose lethal to 50% of the population), ED 50 (dose therapeutically effective in 50% of the population) and the therapeutic index. The therapeutic index is the ratio of LD 50 to ED 50 (i.e., LD50 / ED 50 )。

[0329] The dosage of the compound will also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular compound. Typically, the attending physician will consider various factors such as age, weight, general health, diet, gender, the compound to be administered, the route of administration, and the severity of the condition being treated to determine the dosage of the compound for each individual patient. By way of example and not intended to limit the invention, the dosage of the compound can be about 0.001 to about 1000 mg / kg of the body weight of the individual being treated per day, about 0.01 to about 10 mg / kg of body weight per day, about 0.01 mg to about 1 mg / kg of body weight per day, about 1 to about 1000 mg / kg of body weight per day, about 5 to about 500 mg / kg of body weight per day, about 10 to about 250 mg / kg of body weight per day, about 25 to about 150 mg / kg of body weight per day, or about 10 mg / kg of body weight per day.

[0330] In one aspect, the concentration of the compound in the pharmaceutical composition is at least 0.05 mg / ml (e.g., at least about 0.1 mg / ml, at least about 0.2 mg / ml, at least about 0.5 mg / ml, or at least about 1 mg / ml). This concentration is greater than the naturally occurring concentration of the compound in its natural environment (e.g., in a sponge).

[0331] Method of Use

[0332] In one aspect, the present invention provides a method of inhibiting kinase activity in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of the present invention. As used herein, "inhibit" need not mean a 100% reduction in activity, but can mean a reduction in activity of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

[0333] In one aspect of the present invention, the kinase is PKA, PKG, PKC, STK, CLK, DYRK, or LATS.

[0334] In one aspect of the present invention, the kinase is PKA, PKA / DNAJ, PKG1a, PKG1b, PKG2, PKC-θ, PKC-nu, PKC-d, PKC-eta, PKC-g, STK39, CLK1, CLK2, CLK3, CLK4, DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4, LATS1, or LATS2.

[0335] In one aspect of the present invention, the kinase is PKA, PKA / DNAJ, or cAMP-PKA.

[0336] In one aspect of the present invention, the kinase is protein kinase A (PKA). In one aspect, PKA is inhibited, thereby producing a therapeutic benefit to an individual. In one aspect, PKA is inhibited, thereby achieving the treatment of cancer. In one aspect, PKA is inhibited, thereby achieving the treatment of liver cancer, such as hepatocellular carcinoma (HCC) and fibrolamellar hepatocellular carcinoma. In one aspect, PKA is inhibited, thereby achieving the treatment of diabetic neuropathic pain (Ma et al., Neuroscience Letters, 750: 135763 (2021)).

[0337] In one aspect of the present invention, the kinase is PKA / DNAJ. In one aspect, PKA / DNAJ is inhibited, thereby producing a therapeutic benefit to an individual. In one aspect, PKA / DNAJ is inhibited, thereby achieving the treatment of cancer. In one aspect, PKA / DNAJ is inhibited, thereby achieving the treatment of liver cancer, such as hepatocellular carcinoma (HCC) and fibrolamellar hepatocellular carcinoma. In one aspect, PKA / DNAJ is inhibited, thereby achieving the treatment of diabetic neuropathic pain.

[0338] In one aspect of the present invention, the kinase is cyclic adenosine monophosphate-protein kinase A (cAMP-PKA). In one aspect, cAMP-PKA is inhibited, thereby producing a therapeutic benefit to an individual. In one aspect, cAMP-PKA is inhibited, thereby achieving the treatment of cancer. In one aspect, cAMP-PKA is inhibited, thereby achieving the treatment of liver cancer, such as hepatocellular carcinoma (HCC) and fibrolamellar hepatocellular carcinoma. In one aspect, cAMP-PKA is inhibited, thereby achieving the treatment of diabetic neuropathic pain.

[0339] In one aspect, the kinase inhibited by the compound of one aspect of the present invention is protein kinase G (PKG). In one aspect, PKG is inhibited, thereby producing a therapeutic benefit to an individual. In one aspect, PKG is inhibited, thereby achieving the treatment of cancer, such as gastric cancer or colon cancer (Wu et al., Molecular Medicine Reports, 14: 1849-1856 (2016); Islam et al., Carcinogenesis, 43(6): 584–593 (2022)). In one aspect, PKG is inhibited, thereby achieving the treatment and / or prevention of infection, such as parasitic infection, such as malaria (i.e., infection caused by Plasmodium) (Eck et al., ChemBioChem, 23(7): 1-8 (2022)).

[0340] In one aspect of the present invention, the kinase is PKG1a, PKG1b, PKG2 or PfPKG.

[0341] In one aspect of the present invention, the kinase is PKG1a. In one aspect, PKG1a is inhibited to provide a therapeutic benefit to an individual. In one aspect, PKG1a is inhibited to effect the treatment of cancer, such as gastric cancer or colon cancer. In one aspect, PKG1a is inhibited to effect the treatment and / or prevention of an infection, such as malaria.

[0342] In one aspect of the present invention, the kinase is PKG1b. In one aspect, PKG1b is inhibited to provide a therapeutic benefit to an individual. In one aspect, PKG1b is inhibited to effect the treatment of cancer, such as gastric cancer or colon cancer. In one aspect, PKG1b is inhibited to effect the treatment and / or prevention of an infection, such as malaria.

[0343] In one aspect of the present invention, the kinase is PKG2. In one aspect, PKG2 is inhibited to provide a therapeutic benefit to an individual. In one aspect, PKG2 is inhibited to effect the treatment of cancer, such as gastric cancer or colon cancer. In one aspect, PKG2 is inhibited to effect the treatment and / or prevention of an infection, such as malaria.

[0344] In one aspect of the present invention, the kinase is PfPKG. In one aspect, PfPKG is inhibited to provide a therapeutic benefit to an individual. In one aspect, PfPKG is inhibited to effect the treatment and / or prevention of an infection, such as malaria.

[0345] In one aspect of the present invention, the kinase is protein kinase C (PKC).

[0346] In one aspect, PKC is inhibited to provide a therapeutic benefit to an individual. In one aspect, PKC is inhibited to effect the treatment of cancer.

[0347] In one aspect of the present invention, the kinase is PKC-θ, PKC-nu, PKC-d, PKC-eta or PKC-g. In one aspect of the present invention, the kinase is PKC-θ. In one aspect of the present invention, the kinase is PKC-nu. In one aspect of the present invention, the kinase is PKC-d. In one aspect of the present invention, the kinase is PKC-eta. In one aspect of the present invention, the kinase is PKC-g.

[0348] In one aspect of the present invention, the kinase is serine / threonine kinase (STK). In one aspect, STK is inhibited to provide a therapeutic benefit to an individual. In one aspect, STK is inhibited to effect the treatment of cancer, such as breast cancer.

[0349] In one aspect, the kinase is STK39. In one aspect, STK39 is inhibited, thereby conferring a therapeutic benefit to an individual. In one aspect, STK39 is inhibited, thereby achieving the treatment of cancer, such as breast cancer.

[0350] In one aspect, the kinase inhibited by the compound of one aspect of the present invention is dual-specificity tyrosine-regulated kinase (DYRK). In one aspect, DYRK is inhibited, thereby conferring a therapeutic benefit to an individual. In one aspect, DYRK is inhibited, thereby achieving the treatment of cancer, such as gastric cancer or colon cancer (Boni et al., Cancers, 12: 1-26 (2020); Henderson et al., J. Med. Chem., 64: 11709-11728 (2021)). In one aspect, DYRK is inhibited, thereby achieving the treatment and / or prevention of an infection, such as an infection caused by protozoa ( et al., Mar. Drugs, 15(316): 1-15 (2017)) or parasites (e.g., Trypanosoma brucei; Cayla et al., eLife, 1-34 (2020)).

[0351] In one aspect of the present invention, the kinase is DYRK1A, DYRK1B, DYRK2, DYRK3 or DYRK4.

[0352] In one aspect of the present invention, the kinase is DYRK1A. In one aspect, DYRK1A is inhibited, thereby conferring a therapeutic benefit to an individual. In one aspect, DYRK1A is inhibited, thereby achieving the treatment of cancer, such as gastric cancer or colon cancer. In one aspect, DYRK1A is inhibited, thereby achieving the treatment and / or prevention of an infection, such as an infection caused by protozoa or parasites.

[0353] In one aspect of the present invention, the kinase is DYRK1B. In one aspect, DYRK1B is inhibited, thereby conferring a therapeutic benefit to an individual. In one aspect, DYRK1B is inhibited, thereby achieving the treatment of cancer, such as gastric cancer or colon cancer. In one aspect, DYRK1B is inhibited, thereby achieving the treatment and / or prevention of an infection, such as an infection caused by protozoa or parasites.

[0354] In one aspect of the present invention, the kinase is DYRK2. In one aspect, DYRK2 is inhibited, thereby conferring a therapeutic benefit to an individual. In one aspect, DYRK2 is inhibited, thereby achieving the treatment of cancer, such as gastric cancer or colon cancer. In one aspect, DYRK2 is inhibited, thereby achieving the treatment and / or prevention of an infection, such as an infection caused by protozoa or parasites.

[0355] In one aspect of the present invention, the kinase is DYRK3. In one aspect, DYRK3 is inhibited, thereby producing a therapeutic benefit to an individual. In one aspect, DYRK3 is inhibited, thereby achieving the treatment of cancer, such as gastric cancer or colon cancer. In one aspect, DYRK3 is inhibited, thereby achieving the treatment and / or prevention of an infection, such as an infection caused by a protozoan or a parasite.

[0356] In one aspect of the present invention, the kinase is DYRK4. In one aspect, DYRK4 is inhibited, thereby producing a therapeutic benefit to an individual. In one aspect, DYRK4 is inhibited, thereby achieving the treatment of cancer, such as gastric cancer or colon cancer. In one aspect, DYRK4 is inhibited, thereby achieving the treatment and / or prevention of an infection, such as an infection caused by a protozoan or a parasite.

[0357] In one aspect, the kinase inhibited by the compound of one aspect of the present invention is Cdc2-like kinase (CLK). In one aspect, CLK is inhibited, thereby producing a therapeutic benefit to an individual. In one aspect, CLK is inhibited, thereby achieving the treatment of gastric cancer. In one aspect, CLK is inhibited, thereby achieving the prevention of memory impairment and neurotoxicity induced by the administration of oligomeric Aβ25-35 peptide (Naert et al., European Neuropsychopharmacology, 2170–2182 (2015); Tam et al., Cancer Letters, 473: 186–197 (2020); Moyano et al., Int. J. Mol. Sci., 21: 7549 (2020); and Qin et al., J. Med. Chem., 64: 13191-13211 (2021)).

[0358] In one aspect of the present invention, the kinase is CLK1, CLK2, CLK3 or CLK4.

[0359] In one aspect, CLK1 is inhibited, thereby producing a therapeutic benefit to an individual. In one aspect, CLK1 is inhibited, thereby achieving the treatment of cancer, such as gastric cancer. In one aspect, CLK1 is inhibited, thereby achieving the prevention of memory impairment and neurotoxicity induced by the administration of oligomeric Aβ25-35 peptide.

[0360] In one aspect, CLK2 is inhibited, thereby producing a therapeutic benefit to an individual. In one aspect, CLK2 is inhibited, thereby achieving the treatment of cancer, such as gastric cancer. In one aspect, CLK2 is inhibited, thereby achieving the prevention of memory impairment and neurotoxicity induced by the administration of oligomeric Aβ25-35 peptide.

[0361] In one aspect, CLK3 is inhibited, thereby conferring a therapeutic benefit to an individual. In one aspect, CLK3 is inhibited, thereby achieving the treatment of cancer, such as gastric cancer. In one aspect, CLK3 is inhibited, thereby achieving the prevention of memory impairment and neurotoxicity induced by the administration of oligomeric Aβ25-35 peptide.

[0362] In one aspect, CLK4 is inhibited, thereby conferring a therapeutic benefit to an individual. In one aspect, CLK4 is inhibited, thereby achieving the treatment of cancer, such as gastric cancer. In one aspect, CLK4 is inhibited, thereby achieving the prevention of memory impairment and neurotoxicity induced by the administration of oligomeric Aβ25-35 peptide.

[0363] In one aspect, the kinase inhibited by the compound of one aspect of the present invention is LATS (large tumor suppressor kinase). In one aspect, LATS is inhibited, thereby conferring a therapeutic benefit to an individual. In one aspect, LATS is inhibited, thereby achieving the treatment of cancer.

[0364] In one aspect, the kinase inhibited by the compound of one aspect of the present invention is LATS1 (large tumor suppressor kinase 1). In one aspect, LATS1 is inhibited, thereby conferring a therapeutic benefit to an individual. In one aspect, LATS1 is inhibited, thereby achieving the treatment of cancer.

[0365] In one aspect, the kinase inhibited by the compound of one aspect of the present invention is LATS2 (large tumor suppressor kinase 2). In one aspect, LATS2 is inhibited, thereby conferring a therapeutic benefit to an individual. In one aspect, LATS2 is inhibited, thereby achieving the treatment of cancer.

[0366] In one aspect, the present invention provides a method of suppressing the immune system in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention.

[0367] In one aspect, the present invention provides a method of preventing organ rejection in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention.

[0368] In one aspect, the present invention provides a method of treating diabetic neuropathic pain in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention.

[0369] In one aspect, the present invention provides a method of treating malaria in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention.

[0370] In one aspect, the present invention provides a method for treating protozoan-related infections in an individual, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention.

[0371] In one aspect, the present invention provides a method for treating a neurodegenerative disease, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention. In one aspect, the present invention provides a method for treating Down syndrome, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention. In one aspect, the present invention provides a method for treating Alzheimer's disease, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention.

[0372] In one aspect, the present invention provides a method for treating a heart disease, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention. In one aspect, the present invention provides a method for treating heart failure, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention.

[0373] In one aspect, the present invention provides a method for treating Cushing's syndrome, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention.

[0374] In one aspect, the present invention provides a method for treating McCune-Albright syndrome, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention.

[0375] In one aspect, the present invention provides a method for treating Carney syndrome, the method comprising administering to the individual a compound or pharmaceutical composition of one aspect of the present invention.

[0376] One aspect of the present invention provides compounds and pharmaceutical compositions for treating or preventing cancer. Without being bound by a particular theory or mechanism, it is believed that the compounds inhibit kinases.

[0377] As used herein, the terms "treat" and "prevention" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Instead, there are varying degrees of treatment or prevention, which are considered by those of ordinary skill in the art to have potential benefits or therapeutic effects. In this regard, the methods of one aspect of the present invention can provide any amount, any level of cancer treatment or prevention in a mammal. In addition, the treatment or prevention provided by the methods of one aspect of the present invention can include the treatment or prevention of one or more conditions or symptoms in the disease being treated or prevented (e.g., cancer). In addition, for the purposes of this disclosure, "prevention" can encompass delaying the onset of a disease or its symptoms or conditions.

[0378] Method of one aspect of the present invention, the cancer can be any cancer, including adrenal cancer, sarcoma (e.g., synovial sarcoma, osteosarcoma, uterine leiomyosarcoma, angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma), lymphoma (e.g., small lymphocytic lymphoma, Hodgkin lymphoma and non-Hodgkin lymphoma), hepatocellular carcinoma, glioma, head cancer (e.g., squamous cell carcinoma), neck cancer (e.g., squamous cell carcinoma), acute lymphocytic carcinoma, leukemia (e.g., hairy cell leukemia, myeloid leukemia (acute and chronic), lymphocytic leukemia (acute and chronic), prolymphocytic leukemia (PLL), myelomonocytic leukemia (acute and chronic) and lymphocytic leukemia (acute and chronic)), bone cancer (osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondral exostosis), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumor), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pinealoma), glioblastoma, oligodendroglioma, schwannoma and retinoblastoma), fallopian tube cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer (e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma and fibrosarcoma), myeloproliferative disorders (e.g., chronic myeloid cancer), colon cancer (e.g., colon cancer), esophageal cancer (e.g., squamous cell carcinoma, adenocarcinoma, leiomyosarcoma and lymphoma), cervical cancer (cervical cancer and pre-invasive cervical dysplasia), gastric cancer, gastrointestinal carcinoid tumor, hypopharyngeal cancer, laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma, fibrolamellar hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma and hemangioma), lung cancer (e.g., bronchogenic carcinoma (squamous cell carcinoma, undifferentiated small cell, undifferentiated large cell and adenocarcinoma), alveolar (bronchioloalveolar) carcinoma, bronchial adenoma, chondromatous hamartoma, small cell lung cancer, non-small cell lung cancer and lung adenocarcinoma), malignant mesothelioma, skin cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi sarcoma, nevus, dysplastic nevus, lipoma, hemangioma, skin fibroma and keloid), multiple myeloma, nasopharyngeal cancer, ovarian cancer (e.g., ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid carcinoma and clear cell adenocarcinoma), granulosa-theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma and malignant teratoma), pancreatic cancer (e.g., ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid and VIPoma), peritoneal, omental, mesenteric cancer, pharyngeal cancer, prostate cancer (e.g., adenocarcinoma and sarcoma), rectal cancer, kidney cancer (e.g.,Any one of adenocarcinoma, Wilms' tumor (nephroblastoma), and renal cell carcinoma, small intestine cancer (adenocarcinoma, lymphoma, carcinoid, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma), soft tissue cancer, gastric cancer (e.g., adenocarcinoma, lymphoma, and leiomyosarcoma), testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell tumor, fibroma, fibroadenoma, adenomatoid tumor, and lipoma), uterine cancer (e.g., endometrial cancer), thyroid cancer, and urothelial cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureteral cancer, and bladder cancer). In one aspect of the present invention, the cancer is hepatocellular carcinoma. In one aspect of the present invention, the cancer is fibrolamellar hepatocellular carcinoma. In one aspect of the present invention, the cancer is liver cancer. In one aspect of the present invention, the cancer is breast cancer. In one aspect of the present invention, the cancer is gastric cancer. In one aspect of the present invention, the cancer is colon cancer.,

[0379] In certain aspects of the present invention, the compound of one aspect of the present invention or a pharmaceutically acceptable salt thereof can be co-administered with an anti-cancer agent (e.g., a chemotherapeutic agent) and / or radiotherapy. In one aspect, the compound of one aspect of the present invention or a pharmaceutically acceptable salt thereof is administered in an amount effective to render cancer cells sensitive to one or more treatment regimens (e.g., chemotherapy or radiotherapy). The terms "co-administered" or "co-administration" refer to administration either simultaneously or sequentially. The compound of one aspect of the present invention or a pharmaceutically acceptable salt thereof can be administered before, simultaneously with, or after the administration of another anti-cancer agent (e.g., a chemotherapeutic agent).

[0380] One or more anti-cancer agents can be administered, e.g., two, three, or more. In this regard, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a combination of the compound of one aspect of the present invention or a pharmaceutically acceptable salt thereof with at least one anti-cancer agent (e.g., a chemotherapeutic agent).

[0381] Examples of anti-cancer agents include platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, mechlorethamine, thiotepa, melphalan, busulfan, procarbazine, streptozotocin, temozolomide, dacarbazine, bendamustine), anti-tumor antibiotics (e.g., daunorubicin, doxorubicin, idarubicin, epirubicin, mitoxantrone, bleomycin, mitomycin C, plicamycin, actinomycin D), taxanes (e.g., paclitaxel and docetaxel), anti-metabolites (e.g., 5-fluorouracil, cytarabine, pemetrexed, thioguanine, floxuridine, capecitabine, and methotrexate), nucleoside analogs (e.g., fludarabine, clofarabine, cladribine, pentostatin, nelarabine), topoisomerase inhibitors (e.g., topotecan and irinotecan), demethylating agents (e.g., azacitidine and decitabine), proteasome inhibitors (e.g., bortezomib), epipodophyllotoxins (e.g., etoposide and teniposide), DNA synthesis inhibitors (e.g., hydroxyurea), vinca alkaloids (e.g., vincristine, vindesine, vinorelbine, and vinblastine), tyrosine kinase inhibitors (e.g., imatinib, dasatinib, nilotinib, sorafenib, sunitinib), monoclonal antibodies (e.g., rituximab, cetuximab, panitumumab, tositumomab, trastuzumab, alemtuzumab, gemtuzumab ozogamicin, bevacizumab), nitrosoureas (e.g., carmustine, fotemustine, and lomustine), enzymes (e.g., L-asparaginase), biologic agents (e.g., interferon and interleukin), hexamethylmelamine, mitotane, angiogenesis inhibitors (e.g., thalidomide, lenalidomide), steroids (e.g., prednisone, dexamethasone, and prednisolone), hormonal agents (e.g., tamoxifen, raloxifene, leuprolide, bicalutamide, granisetron, flutamide), aromatase inhibitors (e.g., letrozole and anastrozole), arsenic trioxide, retinoic acid, non-selective cyclooxygenase inhibitors (e.g., non-steroidal anti-inflammatory agents, salicylates, aspirin, piroxicam, ibuprofen, indomethacin, naproxen, diclofenac, tolmetin, ketoprofen, nabumetone, oxaprozin), selective cyclooxygenase-2 (COX-2) inhibitors, cellular immunotherapies (e.g., chimeric antigen receptor T cell therapy, tumor infiltrating lymphocyte therapy), or any combination thereof. In some aspects, the anti-cancer agent is cisplatin, cytarabine, methotrexate, doxorubicin, or a combination thereof.

[0382] In certain aspects of the invention, a compound of an aspect of the invention or a pharmaceutically acceptable salt thereof can be linked to a targeting molecule. Such targeting molecules include antibodies (for ADCs) and small molecules that target other regions of kinases to provide higher selectivity (i.e., a second molecule that binds to the DNAJ domain of the PKADJ fusion protein).

[0383] In certain aspects of the present invention, a compound of an aspect of the present invention or a pharmaceutically acceptable salt thereof can be linked to an E3 ligase binding molecule to prepare a proteolysis targeting chimera (PROTAC).

[0384] As used herein, the term "mammal" refers to any mammal, including but not limited to mammals of the order Rodentia (including mice and hamsters), mammals of the order Lagomorpha (including rabbits), mammals of the order Carnivora (including felines (cats) and canines (dogs)), mammals of the order Artiodactyla (including bovines (cows) and suids (pigs)), mammals of the order Perissodactyla (including equines (horses)), mammals of the order Primates, Ceboids or Simoids (monkeys) and mammals of the order Anthropoids (humans and great apes). Particularly preferred mammals are humans.

[0385] Preparation method

[0386] The compounds of an aspect of the present invention can be prepared by any of a number of conventional techniques.

[0387] In certain aspects, the present invention provides a method for preparing aplithianine A

[0388]

[0389] which comprises coupling a purine-thiazine conjugate having the following structure:

[0390]

[0391] with an imidazole having the following structure:

[0392]

[0393] to provide apithianine A.

[0394] In one aspect of the present invention, the coupling is carried out in the presence of a catalyst. In certain aspects, the present invention provides a method for preparing a compound as follows:

[0395]

[0396] wherein R 3 or R 4 at least one of which is a halogen;

[0397] The method includes halogenating a compound of the following formula:

[0398]

[0399] In one aspect, compound Ic is aplithianine A.

[0400] In one aspect, aplithianine A is brominated using N-bromosuccinimide to provide:

[0401]

[0402] Examples of non-limiting aspects of the present disclosure

[0403] Aspects (including embodiments) of the subject matter of the invention described herein can be individually beneficial or can be beneficial in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure numbered 1 to 38 are provided below. It will be apparent to those skilled in the art upon reading the present disclosure that each individually numbered aspect can be used or combined with any one of the individually numbered aspects before or after it. This is intended to provide support for all such combinations of aspects and is not limited to the combinations of aspects explicitly provided below:

[0404] (1) A compound of formula (I)

[0405]

[0406] wherein

[0407] is a single bond or a double bond,

[0408] X 1 and X 2 are each independently CH, CR 6 or N;

[0409] X 3 is S, S=O or S(=O)2;

[0410] R 1 is H or -NR 2 R 3 ;

[0411] R 2 is H or C1-C3 alkyl;

[0412] R 3 is aryl;

[0413] R 6 is C1-C3 alkyl;

[0414] A is optional, and when present, A is –C(O)-, -C(O)O-, –C(O)NH-, -C(O)N(C1-C3-alkyl)-; -C(O)NH-(C1-C6 alkyl)-NHC(O)-, -NH;

[0415] D is optional, and when present, D is C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 heterocyclic hydrocarbyl, -(C1-C6 alkyl)-O-, -(C1-C6 alkyl)-NH-, -(C1-C3 alkyl)-O-(C1-C3 alkyl)- or -(C1-C3 alkyl)-NH-(C1-C3 alkyl)-, wherein the alkyl group or cycloalkyl / cycloalkenyl group in any of the foregoing is optionally substituted by one or more substituents selected from hydroxy, C1-C6 alkyl, amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; -NH-aryl and combinations thereof; and

[0416] E is:

[0417] aryl or heteroaryl, optionally substituted by one or more substituents selected from C1-C6 alkyl or alkoxy, -(C1-C6 alkyl)-OH, -(C1-C6 alkyl)-COOH, -(C1-C6 alkyl)-NH2, halogen, nitro, hydroxy, amino, C1-C6 alkylamino, di-C1-C6 alkyl-amino; -NH-aryl, C1-C6 haloalkyl, C3-C8 cycloalkyl or heterocyclic hydrocarbyl, fused C3-C8 cycloalkyl or heterocyclic hydrocarbyl, aryl or heteroaryl, -CN, -(C1-C3 alkyl)-CN, carbonyl and combinations thereof;

[0418] amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; or -NH-aryl;

[0419] C1-C6 alkyl or alkoxy, optionally substituted by one or more substituents selected from hydroxy, C1-C6 alkyl, amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; -NH-aryl and combinations thereof;

[0420] C3-C8 cycloalkyl or heterocyclic hydrocarbyl, optionally substituted by one or more substituents selected from hydroxy, C1-C6 alkyl, amino, C1-C6 alkylamino, di-C1-C6 alkyl-amino; -NH-aryl, aryl or heteroaryl, fused aryl or heteroaryl and combinations thereof;

[0421] -C(O)NH2;

[0422] -C(O)OH;

[0423] -C(O)H;

[0424] -N-(C1-C6 alkyl)-acrylamide;

[0425] halogen; or

[0426] hydrogen;

[0427] or a pharmaceutically acceptable salt thereof.

[0428] (2) The compound of aspect 1,

[0429] wherein

[0430] is a single bond or a double bond,

[0431] X 1 and X 2 are each independently CH or N;

[0432] X 3 is S, S=O or S(=O)2;

[0433] R 1 is H or -NR 2 R 3 ;

[0434] R 2 is H or C1-C3 alkyl;

[0435] R 3 is aryl;

[0436] A is optional, and when present, A is –C(O)-, -C(O)O-, –C(O)NH-, -C(O)N(C1-C3-alkyl)-; -C(O)NH-(C1-C6 alkyl)-NHC(O)-, -NH-;

[0437] D is optional, and when present, D is C1-C6 alkyl, -(C1-C6 alkyl)-O-, -(C1-C6 alkyl)-NH-, -(C1-C3 alkyl)-O-(C1-C3 alkyl)- or -(C1-C3 alkyl)-NH-(C1-C3 alkyl)-, wherein the alkyl groups in any of the foregoing are optionally substituted by hydroxy; and

[0438] E is:

[0439] aryl or heteroaryl, optionally substituted by C1-C6 alkyl or alkoxy, halogen, nitro, hydroxy, C1-C6 haloalkyl, -CN, -(C1-C3 alkyl)-CN or carbonyl;

[0440] amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; or -NH-aryl;

[0441] A C1-C6 alkyl or alkoxy group, optionally substituted with a hydroxyl group;

[0442] A C3-C8 cycloalkyl or heterocyclic hydrocarbon group, optionally substituted with a hydroxyl group;

[0443] -C(O)NH2;

[0444] -C(O)OH;

[0445] -C(O)H;

[0446] -N-(C1-C6 alkyl)-acrylamide;

[0447] A halogen; or

[0448] Hydrogen;

[0449] Or a pharmaceutically acceptable salt thereof.

[0450] (3) The compound of aspect 1 or 2, wherein Is a double bond.

[0451] (4) The compound of any one of aspects 1 to 3, wherein the compound of formula (I) is a compound of formula (Ia):

[0452]

[0453] Or a pharmaceutically acceptable salt thereof.

[0454] (5) The compound of any one of aspects 1 to 4, wherein the compound of formula (I) is a compound of formula (Ib):

[0455]

[0456] Or a pharmaceutically acceptable salt thereof.

[0457] (6) The compound of any one of aspects 1 to 5, wherein R 1 Is H.

[0458] (7) The compound of any one of aspects 1 to 6, wherein:

[0459] (i) A and D are absent, and E is a halogen; -C(O)OH; -C(O)H; an aryl or heteroaryl group, optionally substituted with a C1-C3 alkyl or halogen; or a C3-C8 cycloalkyl or heterocyclic hydrocarbon group, optionally substituted with a hydroxyl group;

[0460] (ii) A is absent; D is a C1-C6 alkyl group, optionally a C1-C3 alkyl group; and E is -C(O)NH2 or a C3-C8 cycloalkyl or heterocyclic hydrocarbon group, optionally substituted with a hydroxyl group;

[0461] (iii) A is -NH-, -C(O)NH- or -C(O)N(C1-C3-alkyl)-; D is absent or is C1-C6 alkyl, optionally C1-C3 alkyl; and E is amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; or -NH-aryl; C1-C6 alkyl or alkoxy or C1-C3 alkyl or alkoxy, optionally substituted by hydroxy; or aryl or heteroaryl, optionally substituted by C1-C3 alkyl or alkoxy, halogen, nitro, hydroxy, C1-C3 haloalkyl, -CN, -(C1-C3 alkyl)-CN or carbonyl;

[0462] (iv) A is absent, D is -(C1-C6 alkyl)-O-, -(C1-C6 alkyl)-NH-, -(C1-C3 alkyl)-O-(C1-C3 alkyl)- or -(C1-C3 alkyl)-NH-(C1-C3 alkyl)-, wherein the alkyl groups in any of the foregoing are optionally substituted by hydroxy and wherein the alkyl groups in any of the foregoing are optionally branched; and E is C1-C6 alkyl or C3-C8 cycloalkyl or heterocyclic hydrocarbyl, optionally substituted by hydroxy;

[0463] (v) A is -C(O)NH-(C1-C6 alkyl)-NHC(O)-, optionally -C(O)NH-(C1-C3 alkyl)-NHC(O)-; D is absent; and E is C1-C6 alkyl or C1-C3 alkyl; or aryl or heteroaryl, optionally substituted by C1-C3 alkyl or alkoxy, halogen, nitro, hydroxy, C1-C6 haloalkyl, -CN, -(C1-C3 alkyl)-CN or carbonyl;

[0464] (vi) A is -C(O)O-; D is absent; and E is C1-C6 alkyl; or

[0465] (vii) A is -C(O)-; D is absent; and E is heterocyclic hydrocarbyl.

[0466] (8) The compound of aspect 1, wherein the compound of formula (I) is a compound of formula (Ic) or a pharmaceutically acceptable salt thereof:

[0467]

[0468] wherein R 4 and R 5 are the same or different and each is H or halogen.

[0469] (9) The compound of aspect 8, wherein one of R 4 and R 5 is halogen, or wherein both R 4 and R 5 are halogen.

[0470] (10) A compound of aspect 8, wherein R 4 and R 5 are both hydrogen.

[0471] (11) A compound of any one of aspects 1 to 10, wherein the compound is not aplithianine A

[0472] (12) A compound of aspect 1, wherein the compound of formula (I) is selected from

[0473]

[0474]

[0475]

[0476]

[0477] or a pharmaceutically acceptable salt thereof.

[0478] (13) A compound of aspect 12, wherein the compound of formula (I) is selected from

[0479] or a pharmaceutically acceptable salt thereof.

[0480] (14) A compound

[0481]

[0482] or a pharmaceutically acceptable salt thereof.

[0483] (15) A pharmaceutical composition comprising a compound of any one of aspects 1 to 14 and a pharmaceutical carrier.

[0484] (16) A pharmaceutical composition comprising

[0485] aplithianine A (Compound 1) with a purity of at least 80%

[0486] aplithianine B (Compound 2)

[0487] aplidipurinide A (Compound 3),

[0488] aplidipurinide B (Compound 4)

[0489] or aplidipurinide C (Compound 5)

[0490] and a pharmaceutical carrier.

[0491] (17) A method of inhibiting kinase activity in an individual, the method comprising administering to the individual a compound of any one of aspects 1 to 14 or a pharmaceutical composition of aspect 15 or 16.

[0492] (18) The method of aspect 17, wherein the kinase is PKA, PKA / DNAJ, cAMP-PKA, PKG1a, PKG1b, PKG2, PfPKG, PKC-θ, PKC-nu, PKC-d, PKC-eta, PKC-g, STK39, CLK1, CLK2, CLK3, CLK4, DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4, LATS1 or LATS2.

[0493] (19) The method of aspect 17, wherein the kinase is PKA, PKA / DNAJ or cAMP-PKA.

[0494] (20) The method of aspect 17, wherein the kinase is PKG1a, PKG1b, PKG2 or PfPKG.

[0495] (21) The method of aspect 17, wherein the kinase is DYRK1A, DYRK1B, DYRK2, DYRK3 or DYRK4.

[0496] (22) The method of aspect 17, wherein the kinase is CLK1, CLK2, CLK3 or CLK4.

[0497] (23) A method of inhibiting the immune system in an individual, the method comprising administering to the individual a compound of any one of aspects 1 to 14 or a pharmaceutical composition of aspect 15 or 16.

[0498] (24) A method of preventing organ rejection in an individual, the method comprising administering to the individual a compound of any one of aspects 1 to 14 or a pharmaceutical composition of aspect 15 or 16.

[0499] (25) A method of treating cancer in an individual, the method comprising administering to the individual a compound of any one of aspects 1 to 14 or a pharmaceutical composition of aspect 15 or 16.

[0500] (26) The method of aspect 25, wherein the cancer is fibrolamellar carcinoma (FLC).

[0501] (27) The method of aspect 25, wherein the cancer is fibrolamellar hepatocellular carcinoma (FL-HCC).

[0502] (28) The method of aspect 25, wherein the cancer is gastric cancer.

[0503] (29) The method of aspect 25, wherein the cancer is colon cancer.

[0504] (30) A method of treating diabetic neuropathic pain in an individual, the method comprising administering to the individual a compound of any one of aspects 1 to 14 or a pharmaceutical composition of aspect 15 or 16.

[0505] (31) A method of treating malaria in an individual, the method comprising administering to the individual a compound of any one of aspects 1 to 14 or a pharmaceutical composition of aspect 15 or 16.

[0506] (32) A method of treating protozoan-related infections in an individual, the method comprising administering to the individual a compound of any one of aspects 1 to 14 or a pharmaceutical composition of aspect 15 or 16.

[0507] (33) The method of any one of aspects 17 to 32, wherein the individual is a human.

[0508] (34) A method for preparing aplithianine A

[0509]

[0510] which comprises coupling a purine-thiazine conjugate having the following structure:

[0511]

[0512] with an imidazole having the following structure:

[0513]

[0514] to provide aplithianine A.

[0515] (35) The method of aspect 34, wherein the coupling is carried out in the presence of a catalyst.

[0516] (36) A method for preparing the compound of aspect 8:

[0517]

[0518] wherein R 3 or R 4 at least one of which is a halogen;

[0519] The method comprises halogenating a compound of the following formula:

[0520]

[0521] (37) The method of aspect 36, wherein compound Ic is aplithianine A.

[0522] (38) The method of aspect 37, wherein aplithianine A is brominated using N-bromosuccinimide to provide:

[0523]

[0524] The following examples further illustrate the invention by way of example and, of course, should not be construed as limiting its scope in any way. Example

[0525] UV data was measured using a VARIAN CARY TM 50UV-Vis spectrophotometer. IR spectra were recorded using a Bruker ALPHA II FT-IR spectrometer. NMR data was obtained on a Bruker Avance III NMR spectrometer equipped with a 3mm cryoprobe (600 MHz for 1 H and 150 MHz for 13 C). HRESIMS data was collected on an Agilent Technology 6530 accurate mass Q-TOF LC / MS. HPLC separations were performed on a Shimadzu system equipped with a CBM-40 controller, an SPD-M40 PDA detector, and two LC-20AR pumps.

[0526] All solvents were LC-MS grade or better.

[0527] In September 2000, a sample of the tunicate Aplidium sp. was collected from a reef in South Africa and kept frozen until extraction. The collection was made by the Coral Reef Research Foundation under contract with the Natural Products Branch of the National Cancer Institute of the United States. Voucher specimens (voucher ID#0CDN7423) were deposited at the Smithsonian Institution, Washington, D.C. The animal material (234 g, wet weight) was ground and processed using standard NCI methods for marine samples (McCloud, Molecules, 15:4526-4563 (2010)), providing 3.27 g of an organic extract (NSC#C020725) and 16.2 g of an aqueous extract (NSC#C020724).

[0528] Example 1

[0529] This example demonstrates that the compounds of the present invention can be extracted and purified.

[0530] The organic crude Aplidium sp. extract (NSC#C020725, 1 g, as described above) was subjected to a C8 solid-phase extraction (SPE) process (loading 250 mg of the extract onto each 2 g C8 SPE column and eluting with a gradient of H2O solutions of 5%, 20%, 40%, 60%, 80%, and 100% MeOH and a MeCN solution of 50% MeOH) to yield 7 fractions. The active fraction Fr.5 was further separated by preparative HPLC using a Kinetex 5μm EVO C18 column ( 250×21.2 mm) at a flow rate of 10 mL / min (eluting with 5% to 100% MeCN containing 0.1% TFA) to obtain 20 fractions. Fraction Fr.5-14 was further purified by semi-preparative HPLC using a Kinetex 5μm F5 column ( 250×10 mm) at a flow rate of 4 mL / min (eluting with 8% MeCN containing 0.1% TFA) to obtain compound 2 (0.7 mg). Fraction Fr.5-15 was purified by semi-preparative HPLC using a Kinetex 5μm F5 column ( 250×10 mm) at a flow rate of 4 mL / min (eluting with 12% MeCN containing 0.1% TFA) to obtain compound 1 (6.9 mg), compound 3 (0.8 mg), compound 4 (2.6 mg), and compound 5 (1.9 mg). The structures of compounds 1 to 5 are provided in Figure 22 as follows.

[0531] To accumulate the active compound 1 for chemical modification, 1.38 g of the organic extract (NSC#C020725) and 15.4 g of the aqueous extract (NSC#C020724) were requested from the NCI Natural Products Repository. A similar separation procedure was performed on the organic extract to obtain 46 mg of 1. The aqueous extract was first desalted by HP20ss VLC (washed with H2O and then eluted with MeOH). The fraction eluted with MeOH (660 mg) was further purified by preparative HPLC using a Kinetex 5μm F5 column ( 250×21.2 mm) at a flow rate of 10 mL / min (eluting with 15% MeCN containing 0.1% TFA) to obtain 66 mg of 1.

[0532] Aplithianine A (1): white solid; UV (MeOH) λmax (logε) 242 (3.98), 333 (4.23); IR (neat) ν max 3093, 2974, 2919, 2849, 2828, 1685, 1573, 1452, 1415, 1359, 1330, 1288, 1253, 1209, 1183, 1129, 1027, 975, 937, 912, 844, 803, 724, 643 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 2; HRESIMS m / z 300.1028, [M + H] + (C 13 H 14 N7S theoretical value, 300.1031).

[0533] Aplithianine B (2): White solid; UV (MeOH) λ max (logε) 250 (3.82), 326 (4.08); IR (neat) ν max 2919, 2850, 1718, 1682, 1595, 1413, 1295, 190, 1132, 1074, 1060, 1033, 940, 832, 797, 762, 720, 570, 534 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 2; HRESIMS m / z 316.0974, [M + H] + (C 13 H 14 N7OS theoretical value, 316.0981).

[0534] Aplidipurinide A (3): White solid; UV (MeOH) λ max (logε) 212 (4.40), 277 (4.23); IR (neat) ν max 3217, 2922, 1694, 1638, 1489, 1441, 1350, 1209, 1185, 1137, 842, 805, 725, 650, 606 cm -1 ; 1 H and 13 C NMR data, see Table 3; HRESIMS m / z 421.0981, [M + H] + (C 14 H 17 N 10Theoretical value of O2S2, 421.0977).

[0535] Aplidipurinide B(4): White solid; UV(MeOH) λ max (logε) 212(4.41), 271(4.38); IR(pure) ν max 2976, 2849, 1678, 1488, 1435, 1402, 1352, 1286, 1204, 1136, 1054, 1032, 842, 801, 724, 644 cm -1 ; 1 H and 13 C NMR data, see Table 3; HRESIMS m / z 389.1077, [M+H] + (C 14 H 17 N 10 S2 theoretical value, 389.1079).

[0536] Aplidipurinide C(5): White solid; UV(MeOH) λ max (logε) 211(4.56), 273(4.11); IR(pure) ν max 2922, 2852, 1683, 1615, 1585, 1443, 1368, 1296, 1209, 1183, 1138, 1032, 844, 804, 771, 725, 647 cm -1 ; 1 H and 13 C NMR data, see Table 3; HRESIMS m / z 405.1025, [M+H] + (C 14 H 17 N 10 OS2 theoretical value, 405.1028).

[0537] Table 1. 13 C NMR(150 MHz, δ ppm) data of Compound 1, Compound 2 and Compound 1a to Compound 1d.

[0538] Number <![CDATA[1 a > <![CDATA[1 b > <![CDATA[2 b > <![CDATA[1a a > <![CDATA[1b a > <![CDATA[1c a > <![CDATA[1d a > 2 95.4 97.6 95.3 97.5 97.0 103.1 102.8 3 128.7 130.4 129.0 129.0 129.4 138.3 140.9 5 42.8 44.6 46.3 43.0 42.7 31.3 42.4 6 25.3 26.8 26.7 25.5 25.3 43.2 47.5 2’ 137.0 137.7 137.4 138.5 119.9 137.1 138.1 4’ 118.6 120.0 119.8 115.4 114.7 120.1 124.1 5’ 132.0 134.5 134.1 127.2 130.6 130.4 123.3 6’ 34.3 35.1 34.8 32.7 33.7 34.0 33.4 2” 151.4 152.7 151.4 151.5 151.4 151.4 151.3 4” 152.6 153.6 152.6 152.4 152.5 153.4 153.4 5” 119.8 121.6 110.0 119.7 119.7 120.5 120.5 6” 149.6 151.8 144.2 149.7 149.6 149.0 148.8 8” 141.1 141.7 155.6 140.7 140.9 142.7 142.7

[0539] a Measured in DMSO-d6; b Measured in methanol-d4.

[0540] Table 2. 11H NMR (600 MHz, δ ppm, J in Hz) data.

[0541]

[0542] a Measured in DMSO-d6; b Measured in methanol-d4.

[0543] Table 3. 1H and 1 13C NMR data of Compounds 3 to 5 in DMSO-d6 (600 MHz for 13 1H and 150 MHz for 1 13C, δ ppm), respectively. 13

[0544]

[0545] Structure elucidation of Compounds 1 to 5 ( Figure 22 )

[0546] Compound 1 was isolated as a white solid. Based on the analysis of HRESIMS and 1 1H and 13 13C NMR data (Tables 1 and 2), the molecular formula was determined to be C 13 12H 13 11N7S. When collecting NMR data in DMSO-d6, broad and weak H 1H and C 13C signals were observed for the methine (δ H 9.00, δ C 128.7) and methylene (δ 1 4.61, δ 13 42.8), which provided insufficient 2D NMR (HSQC and HMBC) correlations ( Figures 24 to 28 ). This problem was overcome by changing the solvent to methanol-d4, enabling the structure of Compound 1 to be unambiguously elucidated ( Figure 22 ). 1 1H and 13 13C NMR and HSQC data analysis revealed the presence of 5 aromatic methines, 2 connected methylenes, and an N-methyl (δ H 4.02, δ C 35.1). Through comprehensive analysis of HMBC correlations ( Figure 23 ) and comparison of 1 1H and 13 13C chemical shifts with reported data, three heterocyclic systems were established, including two common natural product building blocks, 6-substituted purines (δ H 8.45, 8.18; δC (153.6, 152.7, 151.8, 141.7, 121.6) (Schram et al., Nature Reviews Clinical Oncology, 14:735 - 48 (2017)) and 5 - substituted N - Me imidazole (δ H 8.88, 7.62, 4.02; δ C 137.7, 134.5, 120.0, 35.1) (Savage et al., N. Engl. J. Med., 346:683 - 93 (2002)). The remainder of the structure was constructed as a disubstituted dihydro - 1,4 - thiazine moiety, which was demonstrated by the key H H - H H COSY correlation between 2H - 5 (δ 1 H - 1 and 2H - 6 (δ H 9.00) to C - 2 (δ C 97.6) and C - 5 (δ C 44.6) and the HMBC correlations from H - 3 to C - 2 and C - 5 and from 2H - 6 to C - 2. The dihydro - 1,4 - thiazine moiety was connected to the N - Me imidazole moiety via a single bond between C - 2 and C - 5' (δ C 134.5), and this connection was supported by the HMBC correlations from H - 3 to C - 5' and from H - 4' (δ H 7.62) to C - 2. Finally, based on the key HMBC correlations of H - 3 and 2H - 5 to C - 6'', the dihydro - 1,4 - thiazine moiety was connected to the purine moiety between N - 4 and C - 6'' (δ C 151.8) to complete the structure elucidation of 1 ( Figures 29 to 33 ). Compound 1, named aplithianine A, represents the first example of a new class of purine - thiazine - imidazole - linked alkaloids.

[0547] Compound 2 was isolated as a white solid. The molecular formula C 13 H 13 N7OS was determined based on HRESIMS data. Comparison of the 1D ( 1 H and 13 C, Tables 1 and 2) and 2D ( 1 H - 1 H COSY, HSQC and HMBC, Figure 2) NMR data of compound 2 and compound 1 revealed an identical structural backbone except for minor alterations in the structure of the purine moiety. The C - 8'' methine of compound 1 (δ H 8.18, δ C141.7) is changed to compound 2 in δ C This assignment yielded the carbonyl group at 151.8 for compound 2 measured in DMSO-d6. 1 The H NMR data (Table 2) show that there is a H 11.1) and NH-9”(δ H 11.9) are exchangeable protons, and the key HMBC correlations of these two protons to C-8' support ( Figures 34 to 41 ). Therefore, the structure of compound 2 (given the experimental name aplithianine B) was determined to be the 8"-oxopurine analog of compound 1.

[0548] The molecular formula of compound 3 was determined to be C by interpretation of HRESIMS data. 14 H 16 N 10 O2S2. 1 H and 13 The C NMR spectrum showed only eight protons and seven carbons, implying the homodimeric nature of the structure. 1 H and 13 C NMR data (Table 3) and 1 H, 1 Analysis of H-COSY and HMBC correlations quickly revealed the presence of a cysteamine moiety (-S-CH2-, δ H 2.96 and δ C 37.3; -NH-CH2-,δ H 3.72, 6.73 and δ C 39.0) (Honeyman et al., Science, 343:1010-14 (2014)) and 8-oxopurine moiety (δ H 8.05, 9.98, 11.35 and δ C 104.7, 145.3, 147.3, 150.7, 152.7), which is the same as the part in the structure of compound 2 (Table 1 and Table 2). H 6.73) and 2H-11(δ H 3.72) to C-6 (δ C 145.3), the two parts are connected between N-10 and C-6. Since NMR analysis determined that the half structure of the molecule is C7H8N5OS, the homodimer can only be formed by connecting the two half structures via a disulfide bond ( Figures 61 to 65 ). Therefore, the structure of compound 3 was determined to be a new disulfide-bonded nucleobase homodimer and was named aplidipurinide A.

[0549] Compared with the UV spectrum and molecular formula of compound 3, both compound 4 and compound 5 have very similar UV spectra (UV maxima at ~211 and 273 nm) and molecular formulas (compound 4 is C 14 H 16 N 10 S2 and compound 5 is C 14 H 16 N 10 OS2), indicating that they are structural analogs. The 1 H and 13 C NMR spectra of compound 4 (Table 3) also show signals for only half of the molecule, and the nucleobase moiety is altered to a 6-N-purine moiety, which is demonstrated by the absence of an oxygen atom in the molecule and the presence of an 8-methine signal (δ H 8.42, δ C 142.3). In contrast to compounds 3 and 4, the 1 H and 13 C NMR data of compound 5 (Table 3) show two sets of signals that are similar to the NMR characteristics of both compounds 3 and 4, respectively. Thus, compound 4 was determined to be a homodimeric analog of compound 3 in which the nucleobase moiety was altered to 6-N-purine, while the structure of compound 5 was deduced to be a heterodimer constructed via a disulfide bond linking the half-structures of compound 3 and compound 4 ( Figure 22 ). Structure determination was supported by analysis of 2D ( 1 H, 1 H-COSY, HSQC, and HMBC) NMR correlations ( Figures 66 to 75 ). Compound 4 was first reported as a synthetic compound (Kastenhuber et al., PNAS USA, 114:13076 - 84 (2017)), but has never been found in natural sources. Thus, compounds 4 and 5 were determined to be new natural analogs of compound 3 and were named aplidipurinides B and aplidipurinides C, respectively.

[0550] Example 2

[0551] This example confirmed that the compounds of the present invention can be synthesized.

[0552] Chemical studies of a large number of Aplidium sp. extracts led to the accumulation of 116 mg of compound 1, enabling the generation of semi-synthetic aplithianine analogs to obtain some preliminary insights into structure-activity relationships. Considering the limited supply of compound 1 from natural sources, the initial design of the semi-synthesis focused only on optimizing certain robust reactions that were expected to produce a simple product profile.

[0553] Total Synthesis of Aplithianine A

[0554]

[0555] To a mixture of ethyl 3,4-dihydro-2H-1,4-thiazine-6-carboxylate (6, 1.0 equiv, 100 mg) and 6-bromopurine (7, 1.5 equiv, 170 mg) was added Xantphos Pd G3 (10 mol%, 55 mg), Cs2CO3 (3 equiv, 560 mg), DMF (5 mL), and molecular sieve. The reaction vial was filled with N2 and tightly capped. The reaction mixture was then stirred vigorously at 110 °C overnight and then dried under vacuum. The residue was redissolved in DMSO and subsequently purified by preparative HPLC using a Gemini 5 μm NX-C18 column ( 250 × 21.2 mm) at a flow rate of 10 mL / min (eluting with 10% to 100% MeCN containing 0.1% TFA) to afford 8 (61 mg, 36% yield).

[0556] Ethyl 4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxylate (8): Pale orange solid; 1 1H NMR (600 MHz, DMSO-d6): δ 13.56 (s, 1H), 9.77 (s, 1H), 8.52 (s, 1H), 8.44 (s, 1H), 4.55 (br s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.16 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H); 13 13C NMR (150 MHz, DMSO-d6): δ 164.7, 153.0, 151.3, 149.5, 141.7, 134.6, 120.4, 102.1, 60.5, 43.5, 23.8, 14.4.

[0557]

[0558] Compound 8 (61 mg) was dissolved in 2 M NaOH (3.5 mL) and THF (3.5) and stirred at room temperature overnight. The reaction solution was acidified with 2 M HCl (5 mL) and then dried under vacuum. The crude product was then washed with H2O (1 mL × 3 times) and desalted to afford 9 (49 mg, 90% yield). Figure 96 1H NMR spectrum of 8 in DMSO-d6 1 is shown. Figure 97 1H NMR spectrum of 8 in DMSO-d613 13C NMR spectrum

[0559] 4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxylic acid (9): pale orange solid; 1 1H NMR (600 MHz, DMSO-d6): δ 13.53 (s, 1H), 12.56 (brs, 1H), 9.78 (s, 1H), 8.50 (s, 1H), 8.43 (s, 1H), 4.51 (br s, 2H), 3.13 (m, 2H); 13 13C NMR (150 MHz, DMSO-d6): δ 166.3, 152.9, 151.3, 149.5, 141.6, 134.3, 120.2, 103.2, 43.1, 23.7. Figure 98 1H NMR spectrum of 9 in DMSO-d6 1 1H NMR spectrum Figure 99 1H NMR spectrum of 9 in DMSO-d6 13 13C NMR spectrum

[0560] Route 1

[0561]

[0562] To a mixture of 9 (1 equiv, 37 mg) and N,O-dimethylhydroxylamine hydrochloride (3 equiv, 40 mg) was added HATU (1.5 equiv, 71 mg), DIPEA (10 equiv, 228 μL), DMF (5 mL) and MS. The reaction mixture was stirred vigorously overnight at room temperature and then dried under vacuum. The residue was redissolved in DMSO and subsequently purified by preparative HPLC using a Gemini 5 μm NX-C18 column ( 250 × 21.2 mm) at a flow rate of 10 mL / min (eluting with 30% MeCN containing 0.1% TFA) to afford 10 (40 mg, 93% yield).

[0563] N-methoxy-N-methyl-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (10): pale orange solid; 1 1H NMR (600 MHz, DMSO-d6): δ 13.50 (s, 1H), 9.68 (s, 1H), 8.49 (s, 1H), 8.40 (s, 1H), 4.52 (br s, 2H), 3.70 (s, 3H), 3.17 (s, 3H), 3.08 (m, 2H); 1313C NMR (150 MHz, DMSO-d6): δ 166.5, 152.8, 151.3, 149.8, 141.3, 133.2, 120.2, 104.4, 61.1, 44.8, 33.6, 24.5. Figure 100 10 in DMSO-d6 1 1H NMR spectrum. Figure 101 10 in DMSO-d6 13 13C NMR spectrum.

[0564]

[0565] Compound 10 (1 equiv, 40 mg) was stirred in THF (5 mL) at 0 °C, and then a THF solution of 2 M LiAlH4 (4 equiv, 260 μL) was added. The reaction was stopped after 1 h by adding H2O (1 mL). The reaction mixture was dried under vacuum. The residue was redissolved in DMSO, and then preparative HPLC purification was carried out using a Gemini 5 μm NX-C18 column ( 250 × 21.2 mm) at a flow rate of 10 mL / min (eluted with 30% MeCN containing 0.1% TFA) to give 11 (22 mg, 68% yield).

[0566] 4-(7H-Purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carbaldehyde (11): pale orange solid; 1 1H NMR (600 MHz, DMSO-d6): δ 13.69 (s, 1H), 9.61 (s, 1H), 9.32 (s, 1H), 8.60 (s, 1H), 8.52 (s, 1H), 4.64 (m, 2H), 3.18 (m, 2H); 13 13C NMR (150 MHz, DMSO-d6): δ 187.4, 153.2, 151.3, 149.1, 144.0, 142.4, 120.7, 115.4, 44.8, 22.9. Figure 102 11 in DMSO-d6 1 1H NMR spectrum. Figure 103 11 in DMSO-d6 13 13C NMR spectrum.

[0567]

[0568] Compound 11 (1 equiv, 22 mg) was in Stirred in a solution of 33% methylamine in EtOH (5 mL) in the presence of molecular sieve at room temperature for 4 h. The reaction mixture was dried under vacuum, and then tosylmethyl isocyanide (2 equiv, 55 mg), K2CO3 (2 equiv, 23 mg), MeOH (5 mL) and molecular sieve were added. The reaction mixture was stirred overnight at 60 °C and then dried under vacuum. The residue was redissolved in DMSO, and then purified by preparative HPLC using a Synergi 5 μm Hydro-RP column ( 250×21.2 mm) at a flow rate of 10 mL / min (eluted with 19% MeCN containing 0.1% TFA) to give 1 (4 mg, 15% yield).

[0569] Route 2

[0570]

[0571] A reaction vial containing 9 (1 equiv, 49 mg) in DMF (5 mL) solution with molecular sieve was filled with N2 and cooled in an ice bath. A solution of N-bromosuccinimide (1.2 equiv, 3.8 mL) in MeCN at 10 mg / mL was added dropwise to the reaction vial. The reaction mixture was stirred at 0 °C for 2 h and then warmed to room temperature and stirred overnight. The reaction mixture was dried under vacuum. The residue was redissolved in DMSO, and then purified by preparative HPLC using a Gemini 5 μm NX-C18 column ( 250×21.2 mm) at a flow rate of 10 mL / min (eluted with 10% to 100% MeCN containing 0.1% TFA) to give 12 (25 mg, 45% yield).

[0572] 6-Bromo-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine (12): Pale orange solid; 1 1H NMR (600 MHz, DMSO-d6): δ 13.36 (s, 1H), 8.98 (s, 1H), 8.37 (s, 1H), 8.31 (s, 1H), 4.56 (br s, 2H), 3.32 (m, 2H); 13 13C NMR (150 MHz, DMSO-d6): δ 152.3, 151.3, 149.0, 140.4, 125.7, 119.3, 91.1, 41.7, 28.2. Figure 104 Showing the 1 1H NMR spectrum of 12 in DMSO-d6. Figure 105 Showing the 1313C NMR spectrum.

[0573]

[0574] To a mixture of 12 (1 equiv, 14.5 mg) and 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-imidazole (4 equiv, 58 mg) was added Xantphos Pd G3 (20 mol%, 8.7 mg), Cs2CO3 (3 equiv, 560 mg), DMF (3 mL), H2O (0.3 mL), and molecular sieve. The reaction vial was filled with N2 and tightly capped. The reaction mixture was then stirred vigorously at 95 °C overnight and then dried under vacuum. The residue was redissolved in DMSO and subsequently purified by preparative HPLC using a Gemini 5μm NX-C18 column ( 250×21.2 mm) at a flow rate of 10 mL / min (eluting with 10% to 100% MeCN containing 0.1% TFA) and a Synergi 5μm Hydro-RP column ( 250×21.2 mm) at a flow rate of 10 mL / min (eluting with 19% MeCN containing 0.1% TFA) to afford 1 (9.8 mg, 67% yield).

[0575] To continue the development of aplithianine A (1) as a drug candidate, a stable supply of the molecule needed to be addressed. Therefore, two overlapping synthetic routes were designed for the total synthesis of aplithianine A (1) (Scheme 1, Figure 7)。Both routes start with the Buchwald-Hartwig coupling of the commercially available precursors ethyl 3,4-dihydro-2H-1,4-thiazine-6-carboxylate (6) and 6-bromopurine (7), followed by alkaline hydrolysis of the resulting ester 8 to quantitatively generate the carboxylic acid 9. The yield of the Buchwald–Hartwig coupling reaction was brought to approximately 36% through extensive optimization of the reaction conditions, which included the catalyst / precatalyst [e.g., Pd2(dba)3, Pd(OAc)2, XantPhos Pd G3, XantPhos Pd G4, XPhos Pd G4, and P(t-Bu)3Pd G4], ligand (e.g., Xantphos, BINAP, JohnPhos, DavePhos, XPhos, RuPhos), base (e.g., Cs2CO3, sodium tert-butoxide, and LiHMDS), solvent (e.g., DMF, THF, toluene, and MeCN), and temperature (e.g., 25 to 130 °C). The combination of the precatalyst XantPhos Pd G3 with the base Cs2CO3 in DMF as the solvent provided the best yield at 110 °C compared to most of the other tested conditions (different combinations of the aforementioned reaction factors) that typically produced 1 in less than 5% yield. For Route 1, the carboxylic acid 9 was converted to its Weinreb amide 10 via a typical HATU / DIPEA / DMF amidation system. Reduction of 10 with LiAlH4 gave the aldehyde 11, which was further subjected to the Van Leusen imidazole synthesis in two steps to afford the final product 1. Unfortunately, mainly due to the low yield (15% yield) of the last-step Van Leusen imidazole synthesis, the overall yield of 1 from the six-step Route 1 was only approximately 3%. Therefore, a second, shorter synthetic route was designed by directly coupling the purine-thiazine conjugate with the imidazole unit. The carboxylic acid 9 was converted to the brominated 12 by decarboxylative bromination with NBS / DMF. A catalytic system similar to that of Step 1 (the Buchwald-Hartwig coupling of 6 and 7) (i.e., XantPhos Pd G3 / Cs2CO3 / DMF) was applied to directly conjugate the brominated 12 with the boronate 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-imidazole (13) to provide 1, with an improved yield of 10% over a total of 4 reaction steps (see Figure 8 ).

[0576] Example 3

[0577] This example confirmed that the compounds of the present invention can be brominated.

[0578] Bromination of Aplithianine A (1)

[0579]

[0580] To a dry mixture of N-bromosuccinimide (5 mg, 2 equiv) and compound 1 (4 mg, 1 equiv) was added 2 mL of anhydrous DMF. The resulting solution was stirred overnight at room temperature. The reaction solution was dried under vacuum and the residue was redissolved in DMSO. The product was purified by semi-preparative HPLC using a Synergy 5 μm Polar-RP column ( 250×10 mm) at a flow rate of 4 mL / min (eluting with 20% to 100% MeCN containing 0.1% TFA) to afford 1a (1.3 mg) and 1b (0.7 mg).

[0581] Aplithianine A1 (1a): White solid; UV (MeOH) λ max (logε) 240 (3.84), 329 (4.15); IR (neat) ν max 3066, 3007, 2921, 2847, 1607, 1571, 1498, 1451, 1373, 1291, 1191, 1139, 1076, 1034, 934, 855, 793, 776, 571, 553, 534 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 2 and Figures 42 to 46 ; HRESIMS m / z 378.0128, [M + H] + (C 13 H 13 BrN7S calcd, 378.0137).

[0582] Aplithianine A2 (1b): White solid; UV (MeOH) λ max (logε) 242 (4.14), 329 (4.42); IR (neat) ν max 3074, 3009, 2923, 2850, 1608, 1572, 1505, 1453, 1404, 1364, 1330, 1294, 1192, 1141, 1077, 1062, 1033, 934, 867, 793, 778, 642, 571, 554, 535 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 2 and Figures 47 to 50 ; HRESIMS m / z 455.9238, [M + H] + (C 13 H12 The theoretical value of Br2N7S is 455.9242).

[0583] The reaction of 1 with 1 and 2 equivalents of N-bromosuccinimide (NBS) produces the monobrominated product 1a and the dibrominated product 1b that are brominated only at the imidazole moiety.

[0584] Example 4

[0585] This example confirmed that different methods can be used to oxidize the compounds of the present invention.

[0586] Oxidation of aplithianine A (1) with SELECTFLUOR

[0587]

[0588] SELECTFLUOR (4.4 mg, 1 equivalent) and compound 1 (3.7 mg, 1 equivalent) were stirred in 2 mL of anhydrous DMF at room temperature for 2 h. The reaction solution was dried under vacuum, and the residue was redissolved in MeOH. The product was purified by semi-preparative HPLC using a Synergy 5 μm Polar-RP column ( 250×10 mm) at a flow rate of 4 mL / min (eluted with 10% MeCN containing 0.1% TFA) to obtain 1c (1.4 mg).

[0589] Aplithianine A3 (1c): white solid; UV (MeOH) λ max (logε) 219 (3.87), 322 (4.06); IR (neat) ν max 3116, 3057, 2963, 2920, 2849, 1681, 1632, 1596, 1569, 1452, 1414, 1367, 1288, 1258, 1203, 1182, 1128, 1050, 1031, 935, 837, 799, 721, 644 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 2 and Figures 51 to 55 ; HRESIMS m / z 316.0975, [M + H] + (C 13 H 14 The theoretical value of N7OS is 316.0981).

[0590] Oxidation of Aplithianine A (1) with H2O2

[0591]

[0592] To a solution of compound 1 (4 mg) in acetic acid (4 mL) was added 1 mL of 30% H2O2 solution. The reaction mixture was dried overnight at room temperature. The reaction solution was dried under vacuum, and the residue was redissolved in MeOH. The product was purified by semi-preparative HPLC using a Kinetex 5 μm EVO C18 column ( 250×10 mm) at a flow rate of 4 mL / min (eluted with 7% MeCN containing 0.1% TFA) to give 1d (1.5 mg).

[0593] Aplithianine A4 (1d): white solid; UV (MeOH) λ max (logε) 220 (4.00), 312 (4.22); IR (neat) ν max 3117, 3010, 2921, 2826, 1674, 1640, 1598, 1571, 1454, 1413, 1366, 1328, 1290, 1194, 1176, 1120, 1077, 1040, 934, 840, 795, 719, 643, 571, 534 cm -1 ; 1 1H and 13 13C NMR data, see Tables 1 and 2 and Figures 56 to 60 ; HRESIMS m / z 332.0923, [M+H] + (C 13 15 14 H

[0594] Efforts to generate fluorinated analogues using 1 equivalent of SELECTFLUOR led to the complete conversion of 1 to the partially oxidized sulfoxide analogue 1c, while the complete oxidation of 1 in H2O2 / AcOH gave the sulfone analogue 1d.

[0595] Example 5

[0596] This example demonstrates the activity of the compounds of the present invention against PKADJ / PKA.

[0597] To evaluate the activity of compounds 1 to 5 against PKADJ, they were tested in a modified sandwich ELISA assay. The PKADJ holoenzyme was treated with the test compound in the reaction step, where the holoenzyme was dissociated to release the activated catalytic unit (PKADJc). The activity of the dissociated PKADJc was quantified by measuring the phosphorylation of the biotinylated peptide substrate (KRREILSRRPSYR (SEQ ID NO: 1)) by immunofluorescence. Compound 1 showed effective inhibition of PKADJ activity, IC 50The value is 1.1 μM (see Figures 1A to 1B ). In contrast, analogue 2 only weakly inhibits PKADJ (IC 50 = 69 μM), with a potency more than 60-fold lower than that of 1. The nucleobase dimers 3 to 5 are inactive in this assay, with IC 50 > 90 μM.

[0598] Both 1 and 2 were further evaluated for their activity against wild-type PKA (wt-PKA) using the same assay. When compared with their inhibition of PKADJ, both compounds showed almost identical potencies against PKA (the IC 50 value of 1 was 1.64 μM and the IC 50 value of 2 was 45 μM). Despite the lack of selectivity for PKADJ, aplithianines represent a new class of kinase inhibitors of natural origin with an unprecedented structural backbone. Further studies on the mechanism of action and structure-activity relationship of this new class of kinase inhibitors are necessary (see Figures 11A to 11P ).

[0599] Example 6

[0600] This example demonstrates that the compounds of the present invention inhibit PKA activity.

[0601] To study the mechanism of action of aplithianine A (1), a luciferase assay was first performed to confirm whether 1 truly inhibits PKA activity without interfering with the assay. Compound 1 effectively inhibits PKA activity with an IC 50 value of 0.4 μM, and no activity was observed when PKA was absent from the assay. Therefore, competitive kinetic studies were further performed to investigate how 1 interacts with the PKA protein. At concentrations from 0 to 25 nM, compound 1 competitively inhibits PKA activity induced by 0 to 100 μM ATP in a dose-dependent manner, with K iapp and r 2 values of 10.4 nM and 0.91, respectively. Therefore, all the results support that 1 may act as an ATP-competitive inhibitor by directly interacting with the catalytic unit of PKA (see Figures 2A to 2B ).

[0602] To further investigate the mechanism of the binding of aplithianines to PKA, both 1 and 2 were subjected to co-crystallization experiments with the DNAJ-PKAc fusion protein. X-ray diffraction experiments on the co-crystals of 1 and 2 with DNAJ-PKAc respectively revealed different binding modes. Compound 1 partially occupies the ATP-binding pocket, and it is predicted that there are three H-bonds from the N-3″, NH-9″, and N-7″ of the purine moiety to the PKAc residues Val178, Glu176, and Thr238 respectively, and there is another H-bond between the imidazole N-3′ and Lys127. In contrast, the adenine moiety of ATP is repositioned in the binding pocket, where N-1, NH2-6, and N-7 coordinate with Val178, Glu176, and Thr238 respectively. Unexpectedly, compared with the binding mode of 1 in the ATP-binding pocket, the oxidation of C-8″ in the structure of 2 results in a completely opposite binding mode. Contrary to 1, the structure of 2 flips around the C-3 / C-6 axis, and both the purine and imidazole rings rotate around the N-4 / C-6″ and C-2 / C-5′ bonds respectively, providing two H-bonds between the purine N-3″ and Lys127 and between the imidazole N-3′ and Val178 respectively. Thus, both 1 and 2 may inhibit PKAc activity by competitively binding to the ATP pocket. However, the slight modification of purine C-8″ completely reverses their binding postures, which may lead to different binding affinities for PKAc and different potencies for PKAc inhibition (see Figures 3A to 3D ).

[0603] Example 7

[0604] This example confirmed the activities of analogs 1a to 1d.

[0605] Analogs 1a to 1d (Examples 3 and 4) were tested in the same manner as compound 1 described in Example 5. Among the four semi-synthetic analogs 1a, 1b, 1c, and 1d (Examples 3 and 4 above), only 1a showed almost equivalent potencies against both PKADJ (IC 50 = 1.05 μM) and WT-PKA (IC 50 = 1.22 μM) compared to 1. Analogs 1b to 1d were inactive against PKADJ or WT-PKA, with IC 50 values > 90 μM. Further co-crystallization and X-ray diffraction experiments also revealed almost identical binding modes of 1a and 1, consistent with their equivalent potencies against PKADJ and PKA (see Figures 4A to 4D ).

[0606] Example 8

[0607] This example confirmed that the compounds of the present invention inhibit kinases.

[0608] Dose-response studies of compounds 1 and 1a were performed against 10 kinases belonging to different phylogenetic groups to investigate their selectivity and potency against the human kinome. For two compounds against 10 kinases, considerable potency and selectivity were shown, with PKA, GSK3β, and AKT1 being the most potent targets (IC 50 values between 43 and 189 nM). Lower but still significant inhibition of PKCa, CK1a1, and ERK1 was observed (IC 50 values between 0.8 and 6.6 μM), while only moderate activity was shown against VEGFR1, MEK1, HER2, and CAMK1d (IC 50 values between 18 and 65 μM). Further extended kinase profiling of the semi-synthetic analogue 1a was performed against a panel of 370 kinases. At 2 μM, 1a potently inhibited 101 kinases with >50% inhibition, while it was completely inactive against 65 kinases (<5% inhibition). At 50 nM, 29 kinases emerged as more potent targets than PKA (78% inhibition), with the most potent inhibition (>50% inhibition) observed for eight kinases, including PKG1a, STK39 / STLK3, PKG1b, PKC-θ, CLK1, DYRK1 / DYRK1A, DYRK2, and LATS1 (belonging mainly to the AGC and CMGC kinase groups) (see Figures 5, Figures 6A to 6C and Figures 13 to 21 ).

[0609] Further dose-response studies of 1 were performed against 19 kinases most sensitive to 1a, revealing similar selectivity, with nine more potent targets than PKA, including PKG1a, PKG1b, Pkg2, PKCθ, STK39, CLK1, LATS1, LATS2, and CLK2 (see Table 4). Curve fitting is shown in Figures 76A to 95B . Curve fitting was performed where the enzyme activity at the highest compound concentration was less than 65%. The differential was calculated as follows: IC 50 (kinase) / IC 50 (PKG1a).

[0610] Table 4

[0611]

[0612]

[0613] As the data confirm, the compounds of one aspect of the present invention are active kinase inhibitors.

[0614] Example 9

[0615] This example provides an exemplary synthesis of the exemplary thiazinecarboxamide disclosed herein.

[0616] Synthesis of Acid Skeleton - A. Acid Skeleton - A is synthesized from commercially available H - 1 and H - 2. Specifically, H - 1 and H - 2 are reacted in the presence of Xantphos Pd G3 and Cs2CO3 to obtain H - 3, and H - 3 is reacted with KOH to obtain Acid Skeleton - A, as illustrated in Scheme 2.

[0617] Scheme 2: Preparation of Acid Scaffold - A

[0618]

[0619] Preparation of Ethyl 4-(7H - pyrrolo[2,3 - d]pyrimidin - 4 - yl)-3,4 - dihydro - 2H - 1,4 - thiazine - 6 - carboxylate (H - 3): To a mixture of ethyl 3,4 - dihydro - 2H - 1,4 - thiazine - 6 - carboxylate (H - 1, 1 equiv, 100 mg) and 4 - bromo - 7H - pyrrolo[2,3 - d]pyrimidine (H - 2, 1.1 equiv, 125 mg) was added Xantphos Pd G3 (10 mol%, 55 mg), Cs2CO3 (3 equiv, 560 mg), and DMF (5 mL). The reaction vial was filled with N2 and tightly capped. Then the reaction mixture was stirred vigorously at 110 °C overnight and then dried under vacuum. The residue was redissolved in DMSO and subsequently purified by preparative HPLC using a Gemini 5μm NX - C18 column ( 250×21.2 mm) at a flow rate of 10 mL / min (eluting with 10% to 100% MeCN containing 0.1% TFA) to obtain H - 3 (109 mg, 65% yield) as a pale white solid. 1 H NMR (600 MHz, DMSO - d6): δ12.21 (s, 1H), 8.84 (s, 1H), 8.44 (s, 1H), 7.49 (s, 1H), 6.67 (s, 1H), 4.35 (br s, 2H), 4.20 (br s, 2H), 3.13 (br s, 2H), 1.24 (br s, 3H); 13 C NMR (150 MHz, DMSO - d6): δ164.7, 152.8, 152.6, 149.9, 133.3, 125.0, 104.5, 100.7, 100.1, 60.4, 45.3, 23.9, 14.3; HRESIMSm / z 291.0910, [M + H] + (C 13 H 15 N4O2S calcd, 291.0916).

[0620] Preparation of 4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxylic acid (acid skeleton-A): H-3 (109 mg) was reacted with KOH (3 equivalents, 62 mg) in MeOH (5 mL) and H2O (5 mL), and stirred at 95 °C for 2 h. The reaction solution was acidified with 2 M HCl (1.4 mL), and then dried under vacuum. Then the crude product was washed with H2O (1 mL × 3 times) and desalted to obtain acid skeleton-A (80 mg, 81% yield) as a pale white solid. 1 H NMR (600 MHz, DMSO-d6): δ 12.18 (s, 1H), 8.79 (s, 1H), 8.43 (s, 1H), 7.47 (s, 1H), 6.65 (s, 1H), 4.33 (br s, 2H), 3.12 (br s, 2H); 13 C NMR (150 MHz, DMSO-d6): δ 166.2, 152.7, 152.6, 149.9, 132.9, 124.8, 104.4, 101.7, 100.1, 44.9, 24.0; HRESIMS m / z 263.0608, [M+H] + (C 11 H 11 N4O2S calcd, 263.0603).

[0621] Synthesis of acid skeleton-B. Acid skeleton-B was synthesized from commercially available H-1 and H-4. Specifically, H-1 was reacted with H-4 in the presence of Xantphos Pd G3 and Cs2CO3 to obtain H-5, and H-5 was reacted with KOH to obtain acid skeleton-B, as illustrated in Scheme 3.

[0622] Scheme 3: Preparation of Acid Scaffold - B

[0623]

[0624] Preparation of ethyl 4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxylate (H-5): To a mixture of ethyl 3,4-dihydro-2H-1,4-thiazine-6-carboxylate (H-1, 1 equivalent, 100 mg) and 6-bromopurine (H-4, 1.5 equivalents, 170 mg) was added Xantphos Pd G3 (10 mol%, 55 mg), Cs2CO3 (3 equivalents, 560 mg) and DMF (5 mL). The reaction vial was filled with N2 and tightly capped. Then the reaction mixture was stirred vigorously at 110 °C overnight, and then dried under vacuum. The residue was redissolved in DMSO, followed by using a Gemini 5 μm NX-C18 column ( Purification by preparative HPLC was carried out at a flow rate of 10 mL / min (eluting with 10% to 100% MeCN containing 0.1% TFA) for (250×21.2 mm) to obtain H-5 (61 mg, 36% yield), as a pale white solid. 1 H NMR (600 MHz, DMSO-d6): δ 13.56 (s, 1H), 9.77 (s, 1H), 8.52 (s, 1H), 8.44 (s, 1H), 4.55 (br s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.16 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H); 13 C NMR (150 MHz, DMSO-d6): δ 164.7, 153.0, 151.3, 149.5, 141.7, 134.6, 120.4, 102.1, 60.5, 43.5, 23.8, 14.4; HRESIMS m / z 292.0865, [M+H] + (C 12 H 14 N5O2S calculated value, 292.0863).

[0625] Preparation of 4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxylic acid (acid skeleton - B): Dissolve H-5 (61 mg) in 2M NaOH (3.5 mL) and THF (3.5 mL) and stir overnight at room temperature. Acidify the reaction solution with 2M HCl (5 mL), then dry under vacuum. Then wash the crude product with H2O (1 mL × 3 times) and desalt to obtain acid skeleton - B (49 mg, 90% yield), as a pale orange solid. 1 H NMR (600 MHz, DMSO-d6): δ 13.53 (s, 1H), 12.56 (br s, 1H), 9.78 (s, 1H), 8.50 (s, 1H), 8.43 (s, 1H), 4.51 (br s, 2H), 3.13 (m, 2H); 13 C NMR (150 MHz, DMSO-d6): δ 166.3, 152.9, 151.3, 149.5, 141.6, 134.3, 120.2, 103.2, 43.1, 23.7; HRESIMS m / z 264.0554, [M+H] + (C 10 H 10 N5O2S calculated value, 264.0550).

[0626] Synthesis of Acid Scaffold-C. Acid Scaffold-C was synthesized from commercially available H-6 and H-4. Specifically, H-6 reacted with H-4 in the presence of diisopropylethylamine (DIPEA) to give H-7, and H-7 reacted with KOH to give Acid Scaffold-C, as illustrated in Scheme 4.

[0627] Scheme 4: Preparation of Acid Scaffold - C

[0628]

[0629] Preparation of ethyl 4-(7H-purin-6-yl)thiomorpholine-2-carboxylate (H-7): To a mixture of morpholine-2-carboxylic acid ethyl ester (H-6, 1 equiv, 100 mg), 6-bromopurine (H-4, 1.1 equiv, 113 mg), MS, DIPEA (3 equiv, 375 μL) and EtOH (5 mL) were added. The reaction mixture was then stirred vigorously at 95 °C overnight and then dried under vacuum. The residue was redissolved in DMSO and subsequently purified by preparative HPLC using a Gemini 5 μm NX-C18 column ( 250×21.2 mm) at a flow rate of 10 mL / min (eluted with 25% MeCN containing 0.1% TFA) to give H-7 (150 mg, 90% yield) as a white solid. 1 H NMR (600 MHz, methanol-d4): δ 8.40 (s, 1H), 8.20 (s, 1H), 5.23 (br s, 1H), 5.02 (br s, 1H), 4.48 (d, J = 13.6 Hz, 1H), 4.25 (br s, 1H), 4.03 (m, 2H), 3.71 (dd, J = 3.3, 5.2 Hz, 1H), 3.18 (m, 1H), 2.76 (m, 1H), 1.09 (t, J = 7.1 Hz, 3H); 13 C NMR (150 MHz, methanol-d4): δ 172.2, 154.1, 149.0, 148.6, 141.1, 119.5, 62.5, 50.2, 49.4, 40.7, 26.5, 14.2; HRESIMS m / z 294.1020, [M+H] + (C 12 H 16 N5O2S calcd, 294.1025).

[0630] Preparation of 4-(7H-purin-6-yl)thiomorpholine-2-carboxylic acid (acid backbone-C): Dissolve H-7 (150 mg) in 2 M NaOH (5 mL) and THF (5 mL) and stir overnight at room temperature. Acidify the reaction solution with 2 M HCl (6 mL) and then dry under vacuum. Then wash the crude product with H2O (1 mL × 3 times) and desalt to obtain acid backbone-C (120 mg, 88% yield) as a white solid. 1 H NMR (600 MHz, methanol-d4): δ 8.24 (s, 1H), 8.02 (s, 1H), 5.03 (d, J = 13.3 Hz, 1H), 4.86 (br s, 1H), 4.50 (br s, 1H), 4.31 (m, 1H), 3.66 (dd, J = 3.2, 7.5 Hz, 1H), 2.97 (m, 1H), 2.78 (m, 1H); 13 C NMR (150 MHz, methanol-d4): δ 173.8, 155.1, 153.0, 152.3, 139.4, 120.5, 50.1, 48.6, 42.1, 27.1; HRESIMS m / z 266.0714, [M+H] + (C 10 H 12 N5O2S calcd, 266.0712).

[0631] Synthesis of thiazine carboxamides. Each of the following compounds was synthesized by the general procedure for amide coupling described in Scheme 5.

[0632] Scheme 5: General Procedure for Amide Coupling

[0633]

[0634] Add HATU (1.5 equiv), DIPEA (10 equiv) and DMF to a mixture of acid backbone-A, B or C (1 equiv) and a primary / secondary amine or N-Boc-ethylenediamine (1.5 equiv). Then stir the reaction mixture vigorously overnight at room temperature and then dry it under vacuum. Redissolve the residue in DMSO and then perform preparative HPLC purification using a Gemini 5 μm NX-C18 column ( 250 × 21.2 mm) at a flow rate of 10 mL / min (eluting with 10% to 100% MeCN containing 0.1% TFA) to obtain the formamide or Boc-protected amide product. Deprotect the purified Boc-protected amide product in DCM:TFA (2:1) and then use a Synergi 5 μm Hydro-RP column ( HPLC purification was carried out at a flow rate of 10 mL / min (eluting with 10% to 100% MeCN containing 0.1% TFA) for (250×21.2 mm) to obtain the deprotected amide product.

[0635] Preparation of N-(2-aminoethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (TFA salt) (183A049E):

[0636]

[0637] N-(2-aminoethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (TFA salt) (183A049E) was obtained (33% yield) as an off-white solid. 1 HNMR (600 MHz, DMSO-d6): δ 13.50 (s, 1H), 9.45 (br s, 1H), 8.49 (s, 1H), 8.40 (s, 1H), 7.88 (t, J = 5.7 Hz, 1H), 7.79 (br s, 3H), 4.61 (br s, 2H), 3.40 (m, 2H), 3.17 (m, 2H), 2.92 (m, 2H); 13 C NMR (150 MHz, DMSO-d6): δ 165.3, 152.9, 151.4, 149.8, 141.3, 130.4, 120.2, 105.6, 44.1, 38.9, 37.4, 24.2; HRESIMS m / z 306.1133, [M + H] + (C 12 H 16 N7OS calcd, 306.1137).

[0638] Preparation of N-(2-aminoethyl)-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (TFA salt) (183A056C):

[0639]

[0640] N-(2-aminoethyl)-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (TFA salt) (183A056C) was obtained (70% yield) as a colorless solid. 1HNMR(600MHz, DMSO-d6): δ 12.18 (s, 1H), 8.67 (s, 1H), 8.42 (s, 1H), 7.90 (t, J = 5.7 Hz, 1H), 7.82 (br s, 3H), 7.46 (br s, 1H), 6.71 (br s, 1H), 4.36 (m, 2H), 3.41 (m, 2H), 3.13 (m, 2H), 2.93 (m, 2H); 13 C NMR(150MHz, DMSO-d6): δ 165.3, 153.0, 152.5, 149.9, 129.7, 124.6, 104.6, 104.4, 100.5, 45.4, 38.9, 37.3, 24.4; HRESIMS m / z 305.1182, [M + H] + (C 13 H 17 N6OS theoretical value, 305.1179).

[0641] Preparation of N-(2-aminoethyl)-4-(7H-purin-6-yl)thiomorpholine-2-carboxamide (TFA salt) (183A051C):

[0642]

[0643] Obtained N-(2-aminoethyl)-4-(7H-purin-6-yl)thiomorpholine-2-carboxamide (TFA salt) (183A051C) (87% yield), as an off-white solid. 1 H NMR(600MHz, methanol-d4): δ 8.25 (s, 1H), 8.06 (s, 1H), 4.91 (m, 1H), 4.80 (m, 1H), 4.60 (m, 1H), 4.38 (m, 1H), 3.65 (dd, J = 3.1, 7.3 Hz, 1H), 3.49 (m, 1H), 3.35 (m, 1H), 3.02 (m, 2H), 3.00 (m, 1H), 2.81 (m, 1H); 13 C NMR(150MHz, methanol-d4): δ 173.7, 155.0, 152.6, 152.2, 139.7, 120.1, 50.3, 48.9, 43.0, 40.7, 38.4, 27.3; HRESIMS m / z 308.1289, [M + H] + (C 12 H 18 N7OS theoretical value, 308.1294).

[0644] Preparation of N-methoxy-N-methyl-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A047C):

[0645]

[0646] N-methoxy-N-methyl-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A047C) was obtained (93% yield) as a pale orange solid. 1 1H NMR (600 MHz, DMSO-d6): δ 13.50 (s, 1H), 9.68 (s, 1H), 8.49 (s, 1H), 8.40 (s, 1H), 4.52 (br s, 2H), 3.70 (s, 3H), 3.17 (s, 3H), 3.08 (m, 2H); 13 13C NMR (150 MHz, DMSO-d6): δ 166.5, 152.8, 151.3, 149.8, 141.3, 133.2, 120.2, 104.4, 61.1, 44.8, 33.6, 24.5; HRESIMS m / z 307.0974, [M + H]+ + (C 12 H 15 6N2O2S calcd, 307.0972).

[0647] Preparation of N-((1-methyl-1H-imidazol-2-yl)methyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A046B):

[0648]

[0649] N-((1-methyl-1H-imidazol-2-yl)methyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A046B) was obtained (32% yield) as an off-white solid. 1 1H NMR (600 MHz, DMSO-d6): δ 13.54 (br s, 1H), 9.54 (s, 1H), 8.51 (t, J = 5.1 Hz, 1H), 8.50 (s, 1H), 7.63 (d, J = 1.9 Hz, 1H), 7.58 (d, J = 1.9 Hz, 1H), 4.61 (d, J = 5.1 Hz, 2H), 4.59 (m, 2H), 3.84 (s, 3H), 3.17 (m, 2H); 1313C NMR (150 MHz, DMSO-d6): δ 165.8, 153.3, 151.7, 150.2, 144.7, 141.8, 131.8, 124.0, 120.6, 118.7, 105.0, 44.4, 34.7, 34.5, 24.5; HRESIMS m / z 357.1240, [M+H] + (C 15 H 17 N8OS calcd for 357.1246).

[0650] (4-(7H-Purin-6-yl)-3,4-dihydro-2H-1,4-thiazin-6-yl)(morpholino)methanone (183A047F) Preparation:

[0651]

[0652] (4-(7H-Purin-6-yl)-3,4-dihydro-2H-1,4-thiazin-6-yl)(morpholino)methanone (183A047F) was obtained (60% yield) as an off-white solid. 1 1H NMR (600 MHz, DMSO-d6): δ 9.04 (br s, 1H), 8.47 (s, 1H), 8.41 (s, 1H), 4.83 (m, 2H), 3.63 (m, 4H), 3.58 (m, 4H), 3.21 (m, 2H); 13 13C NMR (150 MHz, DMSO-d6): δ 166.9, 152.1, 151.1, 149.8, 141.2, 128.3, 119.7, 106.2, 66.4 (2C), 45.6 (2C), 43.0, 24.5; HRESIMS m / z 333.1138, [M+H] + (C 14 H 17 N6O2S calcd for 333.1134).

[0653] N-(2-Acetamidoethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050B) Preparation:

[0654]

[0655] N-(2-Acetamidoethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050B) was obtained (79% yield) as an off-white solid. 11H NMR (600 MHz, DMSO-d6): δ 9.38 (br s, 1H), 8.48 (s, 1H), 8.40 (s, 1H), 7.97 (t, J = 5.6 Hz, 1H), 7.75 (t, J = 5.6 Hz, 1H), 4.58 (br s, 2H), 3.21 (m, 2H), 3.16 (m, 2H), 3.15 (m, 2H), 1.80 (s, 3H); 13 13C NMR (150 MHz, DMSO-d6): δ 169.6, 164.6, 152.8, 151.3, 149.9, 141.2, 129.8, 120.1, 106.4, 43.9, 39.3, 38.4, 24.2, 22.7; HRESIMS m / z 348.1246, [M + H] + (C 14 H 18 N7O2S calcd, 348.1243).

[0656] Preparation of N-(2-methoxyethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050C):

[0657]

[0658] N-(2-methoxyethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050C) was obtained (99% yield) as an off-white solid. 1 1H NMR (600 MHz, DMSO-d6): δ 9.39 (br s, 1H), 8.48 (s, 1H), 8.40 (s, 1H), 7.61 (t, J = 5.6 Hz, 1H), 4.58 (br s, 2H), 3.39 (m, 2H), 3.33 (m, 2H), 3.25 (s, 3H), 3.16 (m, 2H); 13 13C NMR (150 MHz, DMSO-d6): δ 164.5, 152.8, 151.3, 149.9, 141.2, 129.9, 120.1, 106.2, 70.5, 58.0, 43.9, 38.9, 24.2; HRESIMS m / z 321.1139, [M + H] + (C 13 H 17 N6O2S calcd, 321.1134).

[0659] Preparation of N-(2-Hydroxyethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050D):

[0660]

[0661] N-(2-Hydroxyethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050D) was obtained (84% yield) as an off-white solid. 1 1H NMR (600 MHz, DMSO-d6): δ 9.39 (br s, 1H), 8.48 (s, 1H), 8.39 (s, 1H), 7.54 (t, J = 5.6 Hz, 1H), 4.58 (br s, 2H), 3.44 (t, J = 6.4 Hz, 2H), 3.23 (m, 2H), 3.16 (m, 2H); 13 13C NMR (150 MHz, DMSO-d6): δ 164.5, 152.8, 151.3, 149.9, 141.2, 129.8, 120.1, 106.2, 59.8, 43.9, 42.1, 24.2; HRESIMS m / z 307.0982, [M+H]+ + (C 12 H 15 6N2O2S calcd, 307.0977).

[0662] Preparation of N-(2-(Methylamino)ethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (TFA salt) (183A050E):

[0663]

[0664] N-(2-(Methylamino)ethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (TFA salt) (183A050E) was obtained (45% yield) as an off-white solid. 1 1H NMR (600 MHz, DMSO-d6): δ 9.47 (br s, 1H), 8.50 (s, 1H), 8.41 (s, 1H), 8.30 (br s, 2H), 7.91 (t, J = 5.6 Hz, 1H), 4.61 (br s, 2H), 3.44 (m, 2H), 3.18 (m, 2H), 3.02 (m, 2H), 2.58 (t, J = 5.4 Hz, 3H); 1313C NMR (150 MHz, DMSO-d6): δ 165.4, 152.8, 151.3, 149.8, 141.4, 130.5, 120.1, 105.6, 48.5, 44.1, 36.1, 32.9, 24.2,; HRESIMS m / z 320.1290, [M+H] + (C 13 H 18 N7OS calcd for 320.1294).

[0665] Preparation of N-methyl-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A046C): N-methyl-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A046C) was prepared as described in Scheme 6.

[0666] Scheme 6. Preparation of 183A046C

[0667]

[0668] H-5 (6 mg) was stirred overnight at room temperature in 2M NH2Me / MeOH solution (5 mL) in the presence of MS, and then dried under vacuum. The residue was redissolved in DMSO, and then purified by semi-preparative HPLC using a Gemini 5μm NX-C18 column ( 250×10 mm) at a flow rate of 4 mL / min (eluted with 20% MeCN containing 0.1% TFA) to give 183A046C (4.5 mg). N-methyl-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A046C) was obtained (71% yield) as an off-white solid. 1 1 1H NMR (600 MHz, DMSO-d6): δ 13.48 (brs, 1H), 9.36 (s, 1H), 8.47 (s, 1H), 8.39 (s, 1H), 7.64 (q, J = 4.6 Hz, 1H), 7.58 (d, J = 1.9 Hz, 1H), 4.57 (m, 2H), 4.59 (m, 2H), 3.15 (m, 2H), 2.67 (d, J = 4.6 Hz, 3H); 13 13C NMR (150 MHz, DMSO-d6): δ 164.9, 152.8, 151.3, 149.9, 141.1, 129.4, 120.0, 106.6, 43.7, 26.5, 24.2; HRESIMS m / z 277.0870, [M+H] +(C 11 H 13 N6S theoretical value, 277.0872).

[0669] Preparation of N-(2-(pyridinecarboxamido)ethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A049C): Prepare N-(2-(pyridinecarboxamido)ethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A049C) as described in Scheme 7.

[0670] Scheme 7. Preparation of 183A049C

[0671]

[0672] Add HATU (1.5 eq, 93 mg), DIPEA (10 eq, 283 μL), and DMF (2 mL) to a mixture of pyridine-2-carboxylic acid (H-9, 1 eq, 20 mg) and ethylenediamine (H-10, 10 eq, 108 μL). Then stir the reaction mixture overnight at room temperature and then dry it under vacuum. Redissolve the residue in DMSO and then purify it by semi-preparative HPLC using a Syergy 5 μm Polar-RP column ( 250×10 mm) at a flow rate of 4 mL / min (eluting with 5% MeCN containing 0.1% TFA) to obtain H-11 (13.6 mg, 50% yield). Add HATU (1.5 eq, 10 mg), DIPEA (10 eq, 30 μL), and DMF (0.5 mL) to a mixture of acid backbone-B (1 eq, 4.7 mg) and H-11 (1.5 eq, 6.0 mg). Then stir the reaction mixture vigorously overnight at room temperature and then dry it under vacuum. Redissolve the residue in DMSO and then purify it by semi-preparative HPLC using a Gemini 5 μm NX-C18 column ( 250×10 mm) at a flow rate of 4 mL / min (eluting with 23% MeCN containing 0.1% TFA) to obtain 183A049C (3.7 mg). Obtain N-(2-(pyridinecarboxamido)ethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A049C) (50% yield) as an off-white solid. 11H NMR (600 MHz, DMSO-d6): δ 9.37 (br s, 1H), 8.97 (t, J = 5.8 Hz, 1H), 8.65 (d, J = 4.8 Hz, 1H), 8.48 (s, 1H), 8.39 (s, 1H), 8.05 (d, J = 7.7 Hz, 1H), 8.01 (t, J = 7.7 Hz, 1H), 7.88 (t, J = 5.4 Hz, 1H), 7.61 (dd, J = 4.8, 7.7 Hz, 1H), 4.59 (br s, 2H), 3.45 (m, 2H), 3.38 (m, 2H), 3.16 (m, 2H); 13 13C NMR (150 MHz, DMSO-d6): δ 164.8, 164.2, 152.7, 151.2, 149.8 (2C), 148.4, 141.2, 138.1, 129.7, 126.7, 122.1, 120.0, 106.6, 43.9, 39.4, 39.0, 24.2; HRESIMS m / z 411.1356, [M+H] + (C 18 H 19 8N8O2S calcd, 411.1351).

[0673] Preparation of N-(2-benzamidoethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A049F): N-(2-benzamidoethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A049F) was prepared as described in Scheme 8.

[0674] Scheme 8. Preparation of 183A049F

[0675]

[0676] To a mixture of benzoic acid (H-12, 1 equiv, 20 mg) and ethylenediamine (H-10, 3 equiv, 32 μL) was added HATU (1.5 equiv, 93 mg), DIPEA (10 equiv, 283 μL), and DMF (2 mL). The reaction mixture was then stirred overnight at room temperature and then dried under vacuum. The residue was redissolved in DMSO and subsequently purified by semi-preparative HPLC using a Syergy 5 μm Polar-RP column ( 250 × 10 mm) at a flow rate of 4 mL / min (eluting with 10% MeCN containing 0.1% TFA) to afford H-13 (8.7 mg, 33% yield).

[0677] To a mixture of acid scaffold - B (1 equiv, 3.7 mg) and H - 13 (8.7 mg) was added HATU (1.5 equiv, 8 mg), DIPEA (10 equiv, 24 μL) and DMF (0.4 mL). The reaction mixture was then stirred vigorously at room temperature overnight and then dried under vacuum. The residue was redissolved in DMSO and subsequently purified by semi - preparative HPLC using a Gemini 5μm NX - C18 column ( 250×10 mm) at a flow rate of 4 mL / min (eluting with 30% MeCN containing 0.1% TFA) to afford 183A049F (2.5 mg). N - (2 - Benzamidoethyl) - 4 - (7H - purin - 6 - yl) - 3,4 - dihydro - 2H - 1,4 - thiazine - 6 - carboxamide (183A049F) was obtained (43% yield) as an off - white solid. 1 1H NMR (600 MHz, DMSO - d6): δ 9.38 (brs, 1H), 8.57 (t, J = 5.4 Hz, 1H), 8.48 (s, 1H), 8.39 (s, 1H), 7.87 (t, J = 5.4 Hz, 1H), 7.85 (d, J = 7.5 Hz, 2H), 7.46 (t, J = 7.5 Hz, 2H), 7.52 (t, J = 7.5 Hz, 1H), 4.58 (br s, 2H), 3.38 (m, 4H), 3.16 (m, 2H); 13 13C NMR (150 MHz, DMSO - d6): δ 166.6, 164.8, 152.7, 151.2, 149.9, 141.3, 134.5, 131.2, 129.7, 128.4 (2C), 127.3 (2C), 120.0, 106.6, 43.9, 39.3 (2C), 24.2; HRESIMS m / z 410.1395, [M + H] + (C 19 H 20 N7O2S calcd, 410.1399).

[0678] Example 10

[0679] This example provides the IC 50 value for the inhibitory activity against the chimeric protein JPKAcα with enzymatic activity, which is found in almost all FLHCC patients and has an over - expression in tumor cells more than 10 - fold compared to the wild - type PKA (wt - PKA) expression in adjacent normal liver tissues.

[0680] Test compounds to determine the IC of inhibitory activity against the RIα2:JPKAcα2 chimeric kinase holoenzyme50 Values. A 3-fold dose-response curve (all final concentrations) was established in the range of 0 to 10 μM final compound concentration in 100 mM Tris-HCl pH 7.5 containing 1 μM cAMP, 50 μM ATP and 0.4% DMSO. A cAMP / ATP-free control was also included for background normalization. Using a 12-channel multi-channel pipette, quadruplicate reactions were initiated by adding 20 μL of the compound / cAMP / ATP solution to reaction wells containing 40 μL of 1.5-fold concentrated PKA holoenzyme (chimeric) and biotinylated substrate protein (0.5 nM chimeric kinase holoenzyme or 0.66 nM wt kinase holoenzyme, 50 μM biotinylated substrate, in kinase buffer). After initiation by addition, the reactions were allowed to proceed for 45 minutes, then 15 μL of 0.5 M EDTA was added to quench the reactions. The quenched reactions were then transferred to the prepared assay binding plates and ELISA was performed as described above for the primary screening assay. For each reaction well, the observed RFU was converted to a normalized % activity measurement using the cAMP / ATP-free well as the low control and the vehicle control (0 μM) as the high control as described above. The JPKAcα % activity curve for compound 183A056C is shown in Figure 106 .

[0681] Then the normalized % activity measurements were fitted to the following equation using non-linear regression least squares fitting with variable slope (GraphPad Prism Software, San Diego, CA), and the IC 50 values were calculated using the following formula.

[0682]

[0683] The averaged (from three dose-response curves) JPKAcα IC 50 values are listed in Table 5.

[0684] Table 5. JPKAcα IC 50 values

[0685]

[0686] *A < 0.1 μM; B is 0.1 μM – 1 μM; C is 1.01 μM – 10 μM; D is > 10 μM

[0687] Example 11

[0688] This example provides the results of the kinase profiling and dose-response testing of the compounds of one aspect of the present invention.

[0689] HOTSPOT using radiometric measurement by Reaction Biology Corp. (Malvern, PA, USA) TM Kinase assays were performed for a pan - human kinome profiling (370 kinases) and subsequent 10 - point dose - response testing for a set of 30 selected kinases (Anastassiadis et al., Nat. Biotechnol., 29(11):1039 - U117(2011)). All kinase reactions were carried out at 10 μM ATP. Compounds 1 and 3 were tested at two concentrations (2 μM and 50 nM) in the pan - human kinome profiling, and for the 10 - point dose - response testing of Compound 1, 3 - fold serial dilutions starting from 20 μM were used. A complete method description and kinome composition are available from Reaction Biology Corporation. Graphical representation of the inhibitory activities of Compounds 1 and 3 across the tested kinome was accomplished using the CORAL software package (Metz et al., Cell Syst., 7(3):347 - 350e1(2018)) (see Figures 5A to 5C and Figures 15 to 18 ).

[0690] Kinome profiling revealed potent inhibition of selected serine / threonine kinases in the CLK, DYRK, and PKG families, with IC 50 values for Compound 1 of approximately 11 to 90 nM. Kinome profiling also revealed the kinase - selectivity profiles of Compounds 1 and 3, with the PKG, CLK, and DYRK families being the most sensitive target classes( Figure 111 and Figure 112 ). DYRK / CLK kinases belong to the CMGC group of serine / threonine kinases. These kinases are involved in a variety of pathological processes such as neurodegenerative diseases (e.g., Down syndrome, Alzheimer's disease), diabetes, solid cancers (e.g., glioblastoma, breast cancer, and pancreatic cancer), leukemia, and infections caused by viruses and parasites. cGMP - dependent protein kinase (PKG) is an important regulator of the cellular nitric oxide (NO) signaling pathway. Dysregulation of PKG signaling is associated with most forms of heart disease, including heart failure. In addition, PKG from Plasmodium falciparum has also been identified and validated as a target for antimalarial chemotherapy.

[0691] Surprisingly, monobromination of the imidazole moiety increased the potency of Compound 3 against DYRK kinases by at least 2 - fold in tests at 50 nM, although the overall selectivity profiles between Compound 1 and Compound 3 across the 370 - kinase panel were very similar( Figure 111, (Table 6 to Table 8 below). Table 6 shows the kinome profiling of Compound 1 against a panel of 370 human protein kinases. When Compound 1 was tested at 50 nM, the top 50 hit data are shown in Table 6. The average inhibition rate was obtained from a single experiment performed in duplicate. Table 7 shows the kinome profiling of Compound 3 against a panel of 370 human protein kinases. When Compound 3 was tested at 50 nM, the top 50 hit data are shown in Table 7. The average inhibition rate was obtained from a single experiment performed in duplicate. Table 8 shows the concentration (nM) of Aplithianine analogs that caused a 50% reduction in the catalytic activity of 20 selected kinases (IC 50 ). The average IC 50 values were obtained from a single experiment performed in duplicate or from two independent studies.

[0692] Table 6

[0693]

[0694] Staurosporine as control; GW5074 as control

[0695] Table 7

[0696]

[0697] a Staurosporine as control; b GW5074 as control; c JNK-IN-7 as control

[0698] Table 8

[0699]

[0700] a Staurosporine as control; b GW5074 as control; c SCH772984 as control.

[0701] Figure 107A and Figure 107B are graphs showing the percentage of kinase activity at 50 nM ( Figure 107A ) and 2 μM ( Figure 107B ). The difference in activity between compounds is more significant at the 2 μM concentration. Figure 108 shows the top 10 kinases with the highest activity (bar graphs of Compound 183A034G / Aplithianine A(1), 183A041B / Aplithianine A1(1a), and 183A049E from left to right for each listed kinase). Figure 109 and Figure 110Also shown are the percentages of kinase activities of 183A034G / Aplithianine A(1), 183A041B / Aplithianine A1(1a), and 183A049E against several kinases.

[0702] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0703] In the context of describing the present invention (especially in the context of the following claims), the use of the terms “a,” “an,” “the,” “at least one,” and similar referents should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The phrase “at least one” followed by a list of one or more items (e.g., “at least one of A and B”) should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by the context. The terms “comprising,” “having,” “including,” and “containing” should be construed as open-ended terms (i.e., meaning “including but not limited to”), unless otherwise specified. References to ranges of values herein are merely intended as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., “such as”) provided herein is merely intended to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0704] Preferred embodiments and aspects of the invention are described herein, including the best mode known to the inventors for practicing the invention. Variations of these preferred embodiments will be apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. In addition, the invention includes any combination of the above elements in all possible variations thereof, unless otherwise indicated herein or clearly contradicted by the context.

Claims

1. Compounds of formula (I) wherein is a single bond or a double bond, X 1 and X 2 each independently is CH, CR 6 or N; X 3 is S, S=O or S(=O)2; R 1 is H or -NR 2 R 3 ; R 2 is H or a C1-C3 alkyl group; R 3 is an aryl group; R 6 is a C1-C3 alkyl group; A is optional, and when present, A is –C(O)-, -C(O)O-, –C(O)NH-, -C(O)N(C1-C3-alkyl)-; -C(O)NH-(C1-C6alkyl)-NHC(O)-, -NH; D is optional, and when present, D is C1-C6alkyl, C3-C8cycloalkyl, C3-C8heterocycloalkyl, -(C1-C6alkyl)-O-, -(C1-C6alkyl)-NH-, -(C1-C3alkyl)-O-(C1-C3alkyl)- or -(C1-C3alkyl)-NH-(C1-C3alkyl)-, wherein the alkyl group or cycloalkyl / heterocycloalkyl group in any of the foregoing is optionally substituted with one or more substituents selected from hydroxy, C1-C6alkyl, amino, C1-C6alkylamino or di-C1-C6alkyl-amino; -NH-aryl and combinations thereof; and E is: aryl or heteroaryl, optionally substituted with one or more substituents selected from C1-C6alkyl or alkoxy, -(C1-C6alkyl)-OH, -(C1-C6alkyl)-COOH, -(C1-C6alkyl)-NH2, halogen, nitro, hydroxy, amino, C1-C6alkylamino, di-C1-C6alkyl-amino; -NH-aryl, C1-C6haloalkyl, C3-C8cycloalkyl or heterocycloalkyl, fused C3-C8cycloalkyl or heterocycloalkyl, aryl or heteroaryl, fused aryl or heteroaryl, -CN, -(C1-C3alkyl)-CN, carbonyl and combinations thereof; amino, C1-C6alkylamino or di-C1-C6alkyl-amino; or -NH-aryl; C1-C6alkyl or alkoxy, optionally substituted with one or more substituents selected from hydroxy, C1-C6alkyl, amino, C1-C6alkylamino or di-C1-C6alkyl-amino; -NH-aryl and combinations thereof; C3-C8cycloalkyl or heterocycloalkyl, optionally substituted with one or more substituents selected from hydroxy, C1-C6alkyl, amino, C1-C6alkylamino, di-C1-C6alkyl-amino; -NH-aryl, C3-C8cycloalkyl or heterocycloalkyl, fused C3-C8cycloalkyl or heterocycloalkyl, aryl or heteroaryl, fused aryl or heteroaryl and combinations thereof; -C(O)NH2; -C(O)OH; -C(O)H; -N-(C1-C6alkyl)-acrylamide; halogen; or hydrogen; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein is a single bond or a double bond, X 1 and X 2 each independently is CH or N; X 3 is S, S=O or S(=O)2; R 1 is H or -NR 2 R 3 ; R 2 is H or a C1-C3 alkyl group; R 3 is an aryl group; A is optional, and when present, A is –C(O)-, -C(O)O-, –C(O)NH-, -C(O)N(C1-C3-alkyl)-; -C(O)NH-(C1-C6alkyl)-NHC(O)-, -NH; D is optional, and when present, D is C1-C6 alkyl, -(C1-C6 alkyl)-O-, -(C1-C6 alkyl)-NH-, -(C1-C3 alkyl)-O-(C1-C3 alkyl)- or -(C1-C3 alkyl)-NH-(C1-C3 alkyl)-, wherein the alkyl group in any of the foregoing is optionally substituted with a hydroxyl group; and E is: aryl or heteroaryl, optionally substituted with C1-C6 alkyl or alkoxy, halogen, nitro, hydroxyl, C1-C6 haloalkyl, -CN, -(C1-C3 alkyl)-CN or carbonyl; amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; or -NH-aryl; C1-C6 alkyl or alkoxy, optionally substituted with a hydroxyl group; C3-C8 cycloalkyl or heterocyclic hydrocarbyl, optionally substituted with a hydroxyl group; -C(O)NH2; -C(O)OH; -C(O)H; -N-(C1-C6 alkyl)-acrylamide; halogen; or hydrogen; or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 or 2, wherein is a double bond.

4. The compound according to any one of claims 1 to 3, wherein the compound of formula (I) is a compound of formula (Ia): or a pharmaceutically acceptable salt thereof.

5. The compound according to any one of claims 1 to 4, wherein the compound of formula (I) is a compound of formula (Ib): or a pharmaceutically acceptable salt thereof.

6. The compound according to any one of claims 1 to 5, wherein R 1 is H.

7. The compound according to any one of claims 1 to 6, wherein: (i) A and D are absent, and E is halogen; -C(O)OH; -C(O)H; aryl or heteroaryl, optionally substituted with C1-C3 alkyl or halogen; or C3-C8 cycloalkyl or heterocyclic hydrocarbyl, optionally substituted with a hydroxyl group; (ii) A is absent; D is C1-C6 alkyl, optionally C1-C3 alkyl; and E is -C(O)NH2 or C3-C8 cycloalkyl or heterocyclic hydrocarbyl, optionally substituted with a hydroxyl group; (iii) A is -NH-, -C(O)NH- or -C(O)N(C1-C3-alkyl)-; D is absent or D is C1-C6 alkyl, optionally C1-C3 alkyl; and E is amino, C1-C6 alkylamino or di-C1-C6 alkyl-amino; or -NH-aryl; C1-C6 alkyl or alkoxy or C1-C3 alkyl or alkoxy, optionally substituted with a hydroxyl group; or aryl or heteroaryl, optionally substituted with C1-C3 alkyl or alkoxy, halogen, nitro, hydroxyl, C1-C3 haloalkyl, -CN, -(C1-C3 alkyl)-CN or carbonyl; (iv) A is absent, D is -(C1-C6 alkyl)-O-, -(C1-C6 alkyl)-NH-, -(C1-C3 alkyl)-O-(C1-C3 alkyl)- or -(C1-C3 alkyl)-NH-(C1-C3 alkyl)-, wherein the alkyl group in any of the foregoing is optionally substituted with a hydroxyl group, and wherein the alkyl group in any of the foregoing is optionally branched; and E is C1-C6 alkyl or C3-C8 cycloalkyl or heterocyclic hydrocarbyl, optionally substituted with a hydroxyl group; (v) A is -C(O)NH-(C1-C6 alkyl)-NHC(O)-, optionally C(O)NH-(C1-C3 alkyl)-NHC(O)-; D is absent; and E is C1-C6 alkyl or C1-C3 alkyl; or aryl or heteroaryl, optionally substituted by C1-C3 alkyl or alkoxy, halogen, nitro, hydroxy, C1-C6 haloalkyl, -CN, -(C1-C3 alkyl)-CN or carbonyl; (vi) A is -C(O)O-; D is absent; and E is C1-C6 alkyl; or (vii) A is -C(O)-; D is absent; and E is heterocyclic hydrocarbyl.

8. The compound according to claim 1, wherein the compound of formula (I) is a compound of formula (Ic), or a pharmaceutically acceptable salt thereof: wherein R 4 and R 5 are the same or different and each is H or a halogen.

9. The compound according to claim 8, wherein one of R 4 and R 5 is a halogen, or wherein both R 4 and R 5 are halogens.

10. The compound according to claim 8, wherein R 4 and R 5 are both hydrogen.

11. The compound according to any one of claims 1 to 10, wherein the compound is not aplithianine A:

12. The compound according to claim 1, wherein the compound of formula (I) is selected from or a pharmaceutically acceptable salt thereof.

13. The compound according to claim 12, wherein the compound of formula (I) is selected from or a pharmaceutically acceptable salt thereof.

14. A compound or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition comprising the compound according to any one of claims 1 to 14 and a pharmaceutical carrier.

16. A pharmaceutical composition comprising aplithianine A (Compound 1) with a purity of at least 80% aplithianine B (Compound 2) aplidipurinide A (Compound 3) aplidipurinide B (Compound 4) or aplidipurinide C (Compound 5) and a pharmaceutical carrier.

17. A method of inhibiting kinase activity in an individual, the method comprising administering to the individual the compound according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15 or 16.

18. The method according to claim 17, wherein the kinase is PKA, PKA / DNAJ, cAMP-PKA, PKG1a, PKG1b, PKG2, PfPKG, PKC-θ, PKC-nu, PKC-d, PKC-eta, PKC-g, STK39, CLK1, CLK2, CLK3, CLK4, DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4, LATS1 or LATS2.

19. The method according to claim 17, wherein the kinase is PKA, PKA / DNAJ or cAMP-PKA.

20. The method according to claim 17, wherein the kinase is PKG1a, PKG1b, PKG2 or PfPKG.

21. The method according to claim 17, wherein the kinase is DYRK1A, DYRK1B, DYRK2, DYRK3 or DYRK4.

22. The method according to claim 17, wherein the kinase is CLK1, CLK2, CLK3 or CLK4.

23. A method of inhibiting the immune system in an individual, the method comprising administering to the individual the compound according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15 or 16.

24. A method of preventing organ rejection in an individual, the method comprising administering to the individual the compound according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15 or 16.

25. A method for treating cancer in an individual, the method comprising administering to the individual a compound according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 or 16.

26. The method according to claim 25, wherein the cancer is fibrolamellar carcinoma (FLC).

27. The method according to claim 25, wherein the cancer is fibrolamellar-like hepatocellular carcinoma (FL-HCC).

28. The method according to claim 25, wherein the cancer is gastric cancer.

29. The method according to claim 25, wherein the cancer is colon cancer.

30. A method for treating diabetic neuropathic pain in an individual, the method comprising administering to the individual a compound according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 or 16.

31. A method for treating malaria in an individual, the method comprising administering to the individual a compound according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 or 16.

32. A method for treating protozoan-related infections in an individual, the method comprising administering to the individual a compound according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 or 16.

33. The method according to any one of claims 17 to 32, wherein the individual is a human.

34. A method for preparing aplithianine A which comprises coupling a purine-thiazine conjugate having the following structure: with an imidazole having the following structure: to provide apithianine A.

35. The method according to claim 34, wherein the coupling is carried out in the presence of a catalyst.

36. A method for preparing the compound according to claim 8: wherein R 3 or R 4 at least one of which is a halogen; the method comprising halogenating a compound of the following formula:

37. The method according to claim 36, wherein compound Ic is aplithianine A.

38. The method according to claim 37, wherein aplithianine A is brominated using N-bromosuccinimide to provide:

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