Kinase inhibitor

Abstract

Disclosed are a group of kinase inhibitors. Also disclosed are related pharmaceutical compositions and methods of making and using the kinase inhibitors.

Description

Technical Field

[0001] Statement Statement Regarding Research and Development Supported by the Federal Government This invention was made with government support under Project No. Z01ZIABC011744 by the National Institutes of Health, National Cancer Institute, and Project No. Z01ZIABC011854 by the Cancer Moonshot NCI Program for Natural Product Discovery. The government has certain rights in this invention. Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 389,937, filed on July 17, 2022, the entire disclosure of which is incorporated herein by reference. Incorporation by Reference of Electronically Submitted Materials

[0003] Incorporated herein by reference is a computer - readable nucleotide / amino acid sequence listing, submitted contemporaneously herewith and identified as follows: a 1,926 - byte Extensible Markup Language (XML) file named "767696.xml", created on July 12, 2023. Background of the Invention

[0004] Mammals have enzymes called kinases that are related to cellular functions such as cell signaling, metabolism, and division. Among kinases, some have been found to be more active in certain cancers. Blocking kinases related to cancer growth may provide therapeutic benefits to people suffering from cancer. Considering that cancer is currently a major health concern and there is no effective treatment for all cancers, the identification of new kinase inhibitors for treating cancer is an urgent task. Also, it is an urgent task to identify kinase inhibitors related to non - cancerous conditions (e.g., infectious diseases) for treating conditions and disorders associated with them.

Summary of the Invention

[0005] One aspect of the present invention provides a compound of formula (I).

[0006]

Chemical formula

[0007] Here, X 1 、X 2 、X 3 、

[0008]

Chemical formula

[0009] 、R 1 、A, D, and E are as defined herein or are pharmaceutically acceptable salts thereof.

[0010] A further aspect of the present invention provides a pharmaceutical composition comprising a compound of an aspect of the present invention.

[0011] Another aspect of the present invention provides a method of inhibiting kinase activity in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of an aspect of the present invention.

[0012] A further aspect of the present invention provides a method of suppressing the immune system of a subject, the method comprising administering to the subject a compound or pharmaceutical composition of an aspect of the present invention.

[0013] A further aspect of the present invention provides a method of preventing organ rejection in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of an aspect of the present invention.

[0014] A further aspect of the present invention provides a method of treating cancer in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of an aspect of the present invention.

[0015] A further aspect of the present invention provides a method for treating painful diabetic neuropathy in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of an aspect of the present invention.

[0016] A further aspect of the present invention provides a method for treating malaria in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of an aspect of the present invention.

[0017] A further aspect of the present invention provides a method for treating protozoa-related infections in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of an aspect of the present invention.

[0018] Another aspect of the present invention provides a method for producing a compound of an aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

FIG. 1A

FIG. 1B

FIG. 2A

FIG. 2B

FIG. 3A

FIG. 3B

FIG. 3C

FIG. 3D

FIG. 4A

FIG. 4B

FIG. 4C

FIG. 4D

FIGS. 5A - 5C

FIG. 6A

FIG. 6B

FIG. 6C

FIG. 7

FIG. 8

FIG. 9A

FIG. 9B

FIG. 9C

FIG. 9D

FIG. 9E

FIG. 9F

FIG. 9G

FIG. 9H

FIG. 9I

FIG. 9J

FIG. 10A

FIG. 10B

FIG. 10C

FIG. 10D

FIG. 10E

FIG. 10F

FIG. 10G

FIG. 10H

FIG. 10I

FIG. 10J

FIG. 10K

FIG. 10L

FIG. 10M

FIG. 10N

FIG. 10O

FIG. 10P

FIG. 11A

FIG. 11B

FIG. 11C

FIG. 11D

FIG. 11E

FIG. 11F

FIG. 11G

FIG. 11H

FIG. 11I

FIG. 11J

FIG. 11K

FIG. 11L

FIG. 11M

FIG. 11N

FIG. 11O

FIG. 11P

FIG. 12A

FIG. 12B

FIG. 12C

FIG. 12D

FIG. 13

FIG. 14

FIG. 15

FIG. 16

FIG. 17

FIG. 18

FIG. 19

FIG. 20

FIG. 21

FIG. 22

FIG. 23

FIG. 24

FIG. 25

FIG. 26

FIG. 27

FIG. 28

FIG. 29

FIG. 30

FIG. 31

FIG. 32

FIG. 33

FIG. 34

FIG. 35

FIG. 36

FIG. 37

FIG. 38

FIG. 39

FIG. 40

FIG. 41

FIG. 42

FIG. 43

FIG. 44

FIG. 45

FIG. 46

FIG. 47

FIG. 48

FIG. 49

FIG. 50

FIG. 51

FIG. 52

FIG. 53

FIG. 54

FIG. 55

FIG. 56

FIG. 57

FIG. 58

FIG. 59

FIG. 60

FIG. 61

FIG. 62

FIG. 63

FIG. 64

FIG. 65

FIG. 66

FIG. 67

FIG. 68

FIG. 69

FIG. 70

FIG. 71

FIG. 72

FIG. 73

FIG. 74

FIG. 75

FIG. 76A

FIG. 76B

FIG. 77A

FIG. 77B

FIG. 78A

FIG. 78B

FIG. 79A

FIG. 79B

FIG. 80A

FIG. 80B

FIG. 81A

FIG. 81B

FIG. 82A

FIG. 82B

FIG. 83A

FIG. 83B

FIG. 84A

FIG. 84B

FIG. 85A

FIG. 85B

FIG. 86A

FIG. 86B

FIG. 87A

FIG. 87B

FIG. 88A

FIG. 88B

FIG. 89A

FIG. 89B

FIG. 90A

FIG. 90B

FIG. 91A

FIG. 92B

FIG. 93A

FIG. 93B

FIG. 94A

FIG. 94B

FIG. 95A

FIG. 95B

FIG. 96

FIG. 97

FIG. 98

FIG. 99

FIG. 100

FIG. 101

FIG. 102

FIG. 103

FIG. 104

FIG. 105

FIG. 106

FIG. 107A

FIG. 107B

FIG. 108

FIG. 109

FIG. 110

FIG. 111

Figure 112

[0020] Detailed Description of the Invention The Molecular Targets Program at the US National Cancer Institute (NCI) completed screening of ~150,000 fractionated crude natural products from the NCI Natural Products Repository and Discovery (NPNPD) program (Thornburg et al., ACS Chem. Biol., 13: 2484-2497 (2018)). A group of identified bioactive compounds were isolated from the marine organism Aplidium sp. These compounds, named aplidine A&B, were shown to potently inhibit both (1) the oncogenic gene fusion DNAJB1-PRKACA (PKADJ) and (2) wild-type protein kinase A (PKA) at nanomolar concentrations. Aplidine A was shown to potently and selectively inhibit a broad range of kinases in addition to PKADJ and PKA, expanding its utility. Further kinetic analysis showed that aplidine A is a competitive inhibitor of kinases and competes with ATP for binding to PKA. As a result of further structural analysis, aplidine A was shown to bind to the catalytic pocket of PKADJ, where ATP normally binds. Further semisynthetic efforts in MTP led to the creation of unnatural aplidine derivatives, one of which (the brominated derivative) was found to have equivalent activity compared to the natural product. Additional derivatives were also created, and SAR data regarding the structure-activity relationship of this class of compounds were obtained. Two synthetic plans were designed and completed for the total synthesis of aplidine A.

[0021] The apritianin structural class is a group of potent kinase inhibitors with the potential for broad application to many important kinases such as cancer chemotherapy. For example, gene fusions (genetic lesions in which two parts of the genome that are not normally adjacent are adjacent to each other) are one of the earliest recognized biomarkers for cancer. Approximately 20% of solid malignancies have at least one identifiable gene fusion. Given the experience with imatinib, a BCR-ABL1 kinase inhibitor (Savage, et al., N. Engl. J. Med., 346(9): 683-93 (2002)), and the continued emphasis on precision medicine, it has been suggested that focusing on drug development related to gene fusions could lead to the generation of 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)). Among liver cancers, FL-HCC is unusually tragic in that its patient population is young (less than 35 years old), there are no successful examples of disease-specific chemotherapy, and the 5-year survival rate is only about 34% (Riggle, et al., Pediatr. Blood Cancer, 63: 1163-7 (2016)). In 2014, it was first shown that all FL-HCC patients have an in-frame chromosomal gene fusion between the first exon of the gene encoding the heat shock protein 40 (HSP40) family member DNAJB1 and the second exon of the gene for the adenosine 3’,5’-monophosphate (cAMP)-dependent PKA catalytic subunit α, PRKACA, advancing the biological understanding of FL-HCC (Honeyman, et al., Science, 343: 1010-14 (2014)). The DNAJB1-PRKACA gene fusion produces an enzymatically active chimeric protein, DNAJ. Studies have shown that PKA activity is required for tumor formation.Expression of the equivalent of PKAcα or the kinase-dead 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)).

[0022] The DNAJ fusion complex may present a novel small molecule binding site that can be utilized 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, a modified sandwich ELISA assay using a biotinylated peptide derived from a PKA substrate was developed (see, for example, Example 4). The reaction and readout components of this assay were further optimized, including the optimal reactant concentrations for a kinase reaction suitable for identifying both inhibitors and activators of PKAJ. The same system was also used to test active samples for inhibition against WT-PKA to identify compounds with selectivity for the fusion protein (see, for example, Example 6).

[0023] Compound In one aspect, the present invention relates to a compound of formula (I)

[0024]

Chemical formula

[0025] where

[0026]

Chemical formula

[0027] is a single bond or a double bond, X 1and X 2 is, independently of one another, 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 C1-C3 alkyl; R 3 is aryl; R 6 is C1-C3 alkyl; A is optionally, when present, -C(O)-, -C(O)O-, -C(O)NH-, -C(O)N(C1-C3-alkyl)-; -C(O)NH-(C1-C6 alkyl)-NHC(O)-, -NH; D is optionally, when present, 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)-, where any of the foregoing alkyl or cycloalkyl groups is optionally substituted with one or more substituents selected from hydroxy, C1-C6 alkyl, amino, C1-C6 alkylamino, or di-C1-C6 alkylamino; -NH-aryl, and combinations thereof; and E is: 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, halo, nitro, hydroxy, amino, C1-C6 alkylamino, di-C1-C6 alkylamino; -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; Amino, C1-C6 alkylamino, or di-C1-C6 alkylamino; or -NH-aryl; 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; C3-C8 cycloalkyl or heterocycloalkyl hydroxy, optionally substituted with one or more substituents selected from 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; -C(O)NH2; -C(O)OH; -C(O)H; -N-(C1-C6 alkyl)-acrylamide; Halogen; or Hydrogen) or a pharmaceutically acceptable salt thereof is provided.

[0028] In some embodiments, the present invention provides a compound of formula (I)

[0029] [Chemical]

[0030] of the compound (where

[0031] [Chemical]

[0032] is a single bond or a double bond, X 1 and X 2 are each independently 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 C1-C3 alkyl; R 3 is aryl; A is optionally, when present, -C(O)-, -C(O)O-, -C(O)NH-, -C(O)N(C1-C3-alkyl)-; -C(O)NH-(C1-C6 alkyl)-NHC(O)-, -NH; D is optionally, when present, 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)-, where any of the aforementioned alkyl groups is optionally substituted with hydroxy; E is: aryl or heteroaryl, optionally substituted with C1-C6 alkyl or alkoxy, halo, nitro, hydroxy, C1-C6 haloalkyl, -CN, -(C1-C3 alkyl)-CN, or carbonyl; amino, C1-C6 alkylamino, or di-C1-C6 alkylamino; or -NH-aryl; C1-C6 alkyl or alkoxy optionally substituted with hydroxy; C3-C8 cycloalkyl or heterocycloalkyl optionally substituted with hydroxy; -C(O)NH2; -C(O)OH; -C(O)H; -N-(C1-C6 alkyl)-acrylamide; halogen; or hydrogen) or a pharmaceutically acceptable salt thereof is provided.

[0033] In one aspect of the present invention,

[0034]

Chemical formula

[0035] is a single bond.

[0036] In one aspect of the present invention,

[0037]

Chemical formula

[0038] is a double bond.

[0039] In one aspect of the present invention, the compound of formula (I) is of formula (Ia):

[0040]

Chemical formula

[0041] or a pharmaceutically acceptable salt thereof.

[0042] In one aspect of the present invention, the compound of formula (I) is of formula (Ib):

[0043]

Chemical formula

[0044] or a pharmaceutically acceptable salt thereof.

[0045] In one embodiment of the present invention, R 1 is H.

[0046] In certain embodiments, (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 halo; or C3-C8 cycloalkyl or heterocycloalkyl optionally substituted with hydroxy; (ii) A is absent; D is C1-C6 alkyl, optionally C1-C3 alkyl; and E is -C(O)NH2 or C3-C8 cycloalkyl or heterocycloalkyl optionally substituted with hydroxy; (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; E is amino, C1-C6 alkylamino, or di-C1-C6 alkylamino; or -NH-aryl; C1-C6 alkyl or alkoxy or C1-C3 alkyl or alkoxy optionally substituted with hydroxy; or C1-C3 alkyl or alkoxy, halo, nitro, hydroxy, C1-C3 haloalkyl, -CN, -(C1-C3 alkyl)-CN, or aryl or heteroaryl optionally substituted with 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)-, any of the foregoing alkyl groups being optionally substituted with hydroxy and any of the foregoing alkyl groups being optionally branched, and E is C1-C6 alkyl or C3-C8 cycloalkyl or heterocycloalkyl optionally substituted with hydroxy; (v) A is -C(O)NH-(C1-C6 alkyl)-NHC(O)-, optionally -C(O)NH-(C1-C3 alkyl)-NHC(O)-; D is absent; E is C1-C6 alkyl or C1-C3 alkyl; or aryl or heteroaryl optionally substituted with C1-C3 alkyl or alkoxy, halo, 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 heterosilalkyl.

[0047] In one embodiment of the present invention, the compound of formula (I) is of formula (Ic):

[0048] [Chemical formula]

[0049] (wherein R 4 and R 5 are the same or different and each is H or halo (e.g., bromine, fluorine, chlorine or iodine)) or a pharmaceutically acceptable salt thereof.

[0050] In one embodiment of the present invention, one of R 4 and R 5 is halo. In one embodiment of the present invention, both of R 4 and R 5 are halo.

[0051] In one embodiment of the present invention, both of R 4 and R 5 are hydrogen. In some embodiments of the foregoing embodiments, the halogen is bromine.

[0052] In one embodiment of the present invention, the compound is not aplidine A (Compound 1).

[0053] [Chemical formula]

[0054] Aspects of the present invention relate to the following compounds:

[0055] [Chemical formula]

[0056] [Chemical formula]

[0057] [Chemical formula]

[0058] [Chemical formula]

[0059] [Chemical formula]

[0060] or a pharmaceutically acceptable salt thereof.

[0061] Aspects of the present invention relate to the following compounds:

[0062] [Chemical formula]

[0063] or a pharmaceutically acceptable salt thereof.

[0064] Aspects of the present invention relate to the following compounds:

[0065] [Chemical formula]

[0066] Alternatively, a pharmaceutically acceptable salt thereof is provided.

[0067] Aspects of the present invention provide the compounds of FIGS. 9A-9J.

[0068] Aspects of the present invention provide the compounds of FIGS. 10A-10P.

[0069] Aspects of the present invention provide the compounds of FIGS. 12A-12D.

[0070] A further aspect of the present invention provides enantiomers of the compounds disclosed herein.

[0071] In any of the above aspects, the term "alkyl" means, for example, a straight or branched alkyl substituent containing from about 1 to about 6 carbon atoms, such as from about 1 to about 4 carbon atoms. Examples of alkyl groups 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., monohaloalkyl, dihaloalkyl, trihaloalkyl), cyanoalkyl, aminoalkyl, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, arylcarbonylalkyl (-(alkyl)C(O)aryl), arylalkyl, and the like. Alkyl can be substituted or unsubstituted as described herein. Even when alkyl is an alkylene chain (e.g., -(CH2) n -), the alkyl group can be substituted or unsubstituted.

[0072] In any of the above aspects, the term "alkenyl", as used herein, means, for example, a linear alkenyl substituent containing from about 2 to about 6 carbon atoms (branched alkenyl contains from about 3 to about 6 carbon atoms), for example, from about 3 to about 5 carbon atoms (branched alkenyl contains from about 3 to about 6 carbon atoms). According to one aspect of the present invention, the alkenyl group is C2-C4 alkenyl. Examples of alkenyl groups include ethenyl, allyl, 2-propenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, and the like. Alkenyl can be substituted or unsubstituted as described herein.

[0073] In any of the above aspects, the term "cycloalkyl", as used herein, means, for example, a cyclic alkyl moiety containing 3 to 6 carbon atoms or 5 to 6 carbon atoms. Examples of such moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Cycloalkyl may contain a carbonyl such that an exocyclic (=O) group is present. Cycloalkyl can be substituted or unsubstituted as described herein. Cycloalkyl may also be fused to an adjacent substituent (e.g., cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), i.e., it may share two atoms and bonds with an adjacent substituent.

[0074] 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, such as, for example, phenyl, naphthyl, anthracenyl, or biphenyl. The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic moiety, as generally understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl, biphenyl, naphthyl, anthracenyl, pyrenyl, etc. An aryl moiety generally contains, for example, 6 to 30 carbon atoms, 6 to 18 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. The term aryl is understood to include a carbocyclic moiety that is planar and contains 4n + 2 π electrons in accordance with Hückel's rule, where n = 1, 2, or 3. This definition also applies when "aryl" appears as part of a group, such as, for example, haloaryl (e.g., mono-haloaryl, di-haloaryl, tri-haloaryl), arylalkyl, etc. An aryl can be substituted or unsubstituted as described herein. An aryl may also be fused to adjacent substituents (e.g., cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), i.e., it may share two atoms and bonds with adjacent substituents.

[0075] In any of the above aspects, the term "heteroaryl" refers to an aromatic 5- or 6-membered monocyclic group, a 9- or 10-membered bicyclic group, and an 11- to 14-membered tricyclic group having at least one heteroatom (O, S, or N) in at least one of the rings. Each ring of a heteroaryl group 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 may contain only carbon atoms and may be saturated, partially saturated, or unsaturated. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atoms may optionally be quaternized. A heteroaryl group that is bicyclic or tricyclic must contain at least one fully aromatic ring, although the other fused rings or rings may be aromatic or non-aromatic. The heteroaryl group may be attached to any available nitrogen or carbon atom of any ring. Exemplary embodiments of heteroaryl groups are pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl, (1,2,3)- and (1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl. Heteroaryl can be substituted or unsubstituted as described herein. Heteroaryl may also be fused to an adjacent substituent (e.g., cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), i.e., share two atoms and bonds with the adjacent substituent.

[0076] The term "heterocycloalkyl" means a stable, saturated, or partially unsaturated monocyclic, bicyclic, and spiro ring system containing 3 to 7 ring members of carbon atoms and other atoms selected from nitrogen, sulfur, and / or oxygen. In an embodiment, heterocycloalkyl is a 5-, 6-, or 7-membered monocyclic ring containing 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, and sulfur. Heterocycloalkyl may be attached to the parent structure via a carbon atom or via any heteroatom of the heterocycloalkyl that results in a stable structure. Alternatively, or additionally, heterocycloalkyl may contain a carbonyl such that an exocyclic (=O) group is present. Examples of such heterocycloalkyl rings are isoxazolyl, thiazolinyl, imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolyl, pyrrolinyl, pyrazolyl, pyranyl, piperidyl, oxazolyl, and morpholinyl. Heterocycloalkyl can be substituted or unsubstituted as described herein.

[0077] In any of the above embodiments, the term "hydroxy" refers to the group -OH.

[0078] In any of the above embodiments, the term "cyano" refers to the group -CN and the term "thiocyano" refers to -SCN.

[0079] In any of the above embodiments, the terms "alkoxy" and "cycloalkyloxy" each encompass a straight-chain or branched alkyl group and a cycloalkyl group bonded to a divalent oxygen. The alkyl group and the cycloalkyl group are as described herein.

[0080] In any of the above embodiments, the term "halo" refers to a halogen selected from fluorine, chlorine, bromine, and iodine.

[0081] In any of the above embodiments, the term "carboxylato" refers to the group -C(O)OH.

[0082] In any of the above aspects, the term "amino" refers to the group -NH2. The term "alkylamino" refers to -NHR, while the term "dialkylamino" refers to -NRR'. R and R' are the same or different, each being a substituted or unsubstituted alkyl group, as described herein.

[0083] In any of the above aspects, the term "amide" refers to the group -C(O)NRR', where R and R' are the same or different, each being hydrogen or a substituted or unsubstituted alkyl group, as described herein.

[0084] In any of the above aspects, the term "phosphonato" refers to the group -P(O)(OR)2, where R is hydrogen or a substituted or unsubstituted alkyl group, as described herein.

[0085] In other embodiments, 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., the 1-position, 2-position, 3-position, 4-position, 5-position, or 6-position, etc.). When an aryl group is substituted with a substituent, such as halo, amino, alkyl, OH, alkoxy, etc., the aromatic ring hydrogen is substituted with the substituent, which can occur at any of the available hydrogens, e.g., the 2-, 3-, 4-, 5-, and / or 6-positions where the 1-position is the attachment point of the aryl group in the compounds of the present invention. Suitable substituents include, for example, halo, alkyl, alkenyl, hydroxy, nitro, cyano, amino, alkylamino, alkoxy, aryloxy, aralkoxy, carboxyl, carboxyalkyl, carboxyalkyloxy, amide, alkylamide, haloalkylamide, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, fused aryl, fused heteroaryl, fused heterocycloalkyl, and fused cycloalkyl, each as described herein. In some embodiments, the substituent is at least one alkyl, halo, and / or haloalkyl (e.g., 1 or 2).

[0086] Whenever the range of the number of atoms in the structure is indicated in any of the above embodiments (e.g., C 1-12 、C 1-8 、C 1-6 、C 1-4It is particularly contemplated that any subrange or individual number of carbon atoms falling within the indicated ranges, such as etc., may also be used. Thus, for example, 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., as described herein, C2-C8 used with respect to any chemical group (e.g., alkyl, cycloalkyl, etc.) appropriately includes 1, 2, 3, 4, 5, 6, 7, and / or 8 carbon atoms, as well as any subrange thereof (e.g., as follows), specifically described as 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.).

[0087] In any of the above aspects, the terms "salt" or "pharmaceutically acceptable salt" are intended to include non-toxic salts synthesized by conventional chemical methods from parent compounds containing basic or acidic moieties. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of an appropriate base or acid in water, an organic solvent, or a mixture thereof. For example, inorganic acids (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, 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, methylsulfonic 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. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, acetonitrile, etc. are representative. A list of suitable salts can be found in Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977). For example, it can be a salt of an alkali metal (e.g., sodium or potassium), an alkaline earth metal (e.g., calcium), or ammonium of the salt. In one aspect, the salt is a trifluoroacetate salt.

[0088] Pharmaceutical composition One aspect of the present invention provides a pharmaceutical composition comprising a compound of the present invention. The pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0089] One aspect of the present invention provides a pharmaceutical composition comprising a compound of Formula I.

[0090] One aspect of the present invention provides a pharmaceutical composition comprising at least about 80% purity of

[0091] [Chemical formula]

[0092] and a pharmaceutical carrier.

[0093] In one aspect, the purity of the compound is 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%).

[0094] The pharmaceutical composition may include one or more other pharmaceutically active agents or drugs, such as chemotherapeutic agents, such as topoisomerase I inhibitors, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0095] Preferably, the carrier is a pharmaceutically acceptable carrier. For a pharmaceutical composition, the carrier can be any of those conventionally used and is limited only by such chemical-physical considerations as solubility and lack of reactivity with the active compound(s) and the route of administration. The pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are generally readily available. The pharmaceutically acceptable carrier is preferably chemically inert to the active agent(s) and has no harmful side effects or toxicity under the conditions of use.

[0096] The choice of carrier is determined in part by the particular compound and the particular method used to administer the compound. Accordingly, there are a variety of suitable formulations for the pharmaceutical compositions of the present invention. The compound, its pharmaceutically acceptable salts, can be administered by any suitable method (e.g., oral, intravenous, intramuscular, intrathecal, subcutaneous, sublingual, buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, topical, systemic, transdermal, or combinations thereof). In one embodiment, the pharmaceutical composition of the present invention is administered orally.

[0097] The following dosage formulations are exemplary and are in no way limiting. One or more routes can be used to administer the compound and, in certain embodiments, a particular route can provide a more immediate and more effective response than other routes.

[0098] Suitable formulations 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, and aqueous and non-aqueous sterile suspensions may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. The compounds may be in a pharmaceutically acceptable diluent in a pharmaceutical carrier such as water, physiological saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol and hexadecyl alcohol, glycols such as propylene glycol and polyethylene glycol, dimethyl sulfoxide, glycerol, ketals such as 2,2-dimethyl-1,3-dioxolan-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or a sterile liquid or mixture of liquids such as acetylated fatty acid glycerides, a pharmaceutically acceptable surfactant such as soap or detergent, pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose or other suspending agents, or emulsifying agents and pharmaceutical adjuvants may be administered with or without addition.

[0099] Oils that can be used in the formulation include petroleum oil, animal oil, vegetable oil, synthetic oil, etc. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, mineral, etc. Fatty acids suitable for use in the formulation include oleic acid, stearic acid, isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0100] Soaps suitable for use in the formulation include fatty alkali metal salts, ammonium salts, and triethanolamine salts. Suitable detergents include (a) cationic detergents such as, for example, dimethyldialkylammonium halides and alkylpyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and sulfosuccinates, ethers, monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-β-aminopropionates and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0101] The formulation usually contains from about 0.5% to about 25% by weight of the compound in solution. Preservatives and buffers may be used. To minimize or remove irritation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17. The amount of surfactant in such formulations will typically range 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 and propylene glycol. The formulation can be provided in unit dose or multi-dose sealed containers such as ampoules and vials, and can be stored in a lyophilized (freeze-dried) state that requires only the addition of a sterile liquid excipient for injection, such as water, immediately prior to use. Injectable solutions and suspensions can be prepared from sterile powders, granules, tablets as described above. The requirements for an effective pharmaceutical carrier for the composition are well known to those skilled in the art (see, for example, Lloyd et al. (eds.), Remington: The Science and Practice of Pharmacy, 22nd Ed., Pharmaceutical Press (2012)).

[0102] In addition to the pharmaceutical compositions described above, it will be understood by those skilled in the art that the compounds of the present invention can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or as liposomes.

[0103] For the purposes of the present invention, the amount or dosage of the compound administered should be sufficient to produce the desired response, e.g., a therapeutic or prophylactic response, in a mammal over a reasonable time frame. For example, the dosage of the compound should be sufficient to inhibit the growth of target cells or to treat and prevent cancer for a period of about 2 hours or more from the time of administration, e.g., for a period of 12 to 24 hours or more. In certain embodiments, the period can be even longer. The dosage is determined by the effectiveness of the particular compound and the condition of the mammal (e.g., human), as well as the weight of the mammal being treated (e.g., human).

[0104] Many assays for determining the dosage to be administered are known in the art. The dosage can be determined in vitro (e.g., cell culture) or in vivo (e.g., animal studies). For example, the dosage can be determined in cell culture and / or animal studies by determining the IC 50 (the dose to achieve 50% maximal inhibition of symptoms), LD 50 (the lethal dose for 50% of the population), ED 50 (the therapeutically effective dose for 50% of the population), and the therapeutic index. The therapeutic index is the ratio of LD 50 to ED 50 (i.e., LD 50 / ED 50 .

[0105] Also, the dosage of the compound is determined by the presence, nature, and extent of any potential side effects associated with the administration of the particular compound. Typically, the attending physician determines the dosage of the compound to be administered to an individual patient, taking into account various factors such as age, weight, general health, diet, sex, the compound to be administered, the route of administration, and the severity of the condition to be treated. By way of example and not by way of limitation of the present invention, the dosage of the compound can be about 0.001 to about 1000 mg / day, about 0.01 to about 10 mg / kg / day, about 0.01 mg to about 1 mg / kg body weight / day, about 1 to about 1000 mg / kg body weight / day, about 5 to about 500 mg / kg body weight / day, about 10 to about 250 mg / kg body weight / day, about 25 to about 150 mg / kg body weight / day, or about 10 mg / kg body weight / day per kg of the body weight of the subject being treated.

[0106] 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 concentration of the compound that naturally exists in the natural environment (e.g., the corpus cavernosum).

[0107] Method of Use In one aspect, the present invention provides a method for inhibiting kinase activity in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of the present invention. As used herein, "inhibit" does not necessarily 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%.

[0108] In one aspect of the present invention, the kinase is PKA, PKG, PKC, STK, CLK, DYRK, or LATS.

[0109] 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.

[0110] In one aspect of the present invention, the kinase is PKA, PKA / DNAJ, or cAMP-PKA.

[0111] In one aspect of the present invention, the kinase is protein kinase A (PKA). In one aspect, PKA is inhibited, resulting in a therapeutic benefit to the subject. In one aspect, PKA is inhibited, resulting in the treatment of cancer. In one aspect, PKA is inhibited, resulting in the treatment of liver cancer, such as hepatocellular carcinoma (HCC) and fibrolamellar hepatocellular carcinoma. In one aspect, PKA is inhibited, resulting in the treatment of painful diabetic neuropathy (Ma, et al., Neuroscience Letters, 750: 135763 (2021)).

[0112] In one aspect of the present invention, the kinase is PKA / DNAJ. In one aspect, PKA / DNAJ is inhibited, resulting in a therapeutic benefit to the subject. In one aspect, PKA / DNAJ is inhibited, resulting in the treatment of cancer. In one aspect, PKA / DNAJ is inhibited, resulting in the treatment of liver cancer, such as hepatocellular carcinoma (HCC) and fibrolamellar hepatocellular carcinoma. In one aspect, PKA / DNAJ is inhibited, resulting in the treatment of painful diabetic neuropathy.

[0113] 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, resulting in a therapeutic benefit to the subject. In one aspect, cAMP-PKA is inhibited, resulting in the treatment of cancer. In one aspect, cAMP-PKA is inhibited, resulting in the treatment of liver cancer, such as hepatocellular carcinoma (HCC) and fibrolamellar hepatocellular carcinoma. In one aspect, cAMP-PKA is inhibited, resulting in the treatment of painful diabetic neuropathy.

[0114] In one aspect, the kinase inhibited by the compounds of the aspects of the present invention is protein kinase G (PKG). In one aspect, PKG is inhibited, and as a result, a therapeutic benefit is provided to a subject. In one aspect, PKG is inhibited, and as a result, treatment of cancer, such as gastric cancer or colorectal cancer, is provided (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, and as a result, treatment and / or prevention of an infection, such as a parasitic infection, such as malaria (i.e., an infection caused by Plasmodium), is provided (Eck, et al., ChemBioChem, 23(7): 1-8 (2022)).

[0115] In one aspect of the present invention, the kinase is PKG1a, PKG1b, PKG2, or PfPKG.

[0116] In one aspect of the present invention, the kinase is PKG1a. In one aspect, PKG1a is inhibited, and as a result, a therapeutic benefit is provided to a subject. In one aspect, PKG1a is inhibited, and as a result, treatment of cancer, such as gastric cancer or colorectal cancer, is provided. In one aspect, PKG1a is inhibited, and as a result, treatment and / or prevention of an infectious disease, such as malaria, is provided.

[0117] In one aspect of the present invention, the kinase is PKG1b. In one aspect, PKG1b is inhibited, and as a result, a therapeutic benefit is provided to a subject. In one aspect, PKG1b is inhibited, and as a result, treatment of cancer, such as gastric cancer or colorectal cancer, is provided. In one aspect, PKG1b is inhibited, and as a result, treatment and / or prevention of an infectious disease, such as malaria, is provided.

[0118] In one aspect of the present invention, the kinase is PKG2. In one aspect, PKG2 is inhibited, and as a result, a therapeutic benefit is provided to the subject. In one aspect, PKG2 is inhibited, and as a result, it provides treatment for cancer, such as gastric cancer or colorectal cancer. In one aspect, PKG2 is inhibited, and as a result, it provides treatment and / or prevention of infectious diseases such as malaria.

[0119] In one aspect of the present invention, the kinase is PfPKG. In one aspect, PfPKG is inhibited, and as a result, a therapeutic benefit is provided to the subject. In one aspect, PfPKG is inhibited, and as a result, it provides treatment and / or prevention of infections such as malaria.

[0120] In one aspect of the present invention, the kinase is protein kinase C (PKC).

[0121] In one aspect, PKC is inhibited, and as a result, a therapeutic benefit is provided to the subject. In one aspect, PKC is inhibited, and as a result, it provides treatment for cancer.

[0122] 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.

[0123] In one aspect of the present invention, the kinase is serine / threonine kinase (STK). In one aspect, STK is inhibited, and as a result, a therapeutic benefit is provided to the subject. In one aspect, STK is inhibited, and as a result, it provides treatment for cancer, such as breast cancer.

[0124] In one aspect, the kinase is STK39. In one aspect, STK39 is inhibited, resulting in a therapeutic benefit to the subject. In one aspect, STK39 is inhibited, resulting in the treatment of cancer, such as breast cancer.

[0125] In one aspect, the kinase inhibited by a compound of one aspect of the present invention is dual specificity tyrosine-regulated kinase (DYRK). In one aspect, DYRK is inhibited, resulting in a therapeutic benefit to the subject. In one aspect, DYRK is inhibited, resulting in the treatment of cancer, such as gastric cancer or colorectal 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, resulting in the treatment and / or prevention of an infectious disease, such as an infectious disease caused by a protozoan (Loaec, et al., Mar. Drugs, 15(316): 1-15 (2017)) or parasite (e.g., Trypanosoma brucei; Cayla, et al., eLife, 1-34 (2020)).

[0126] In one aspect of the present invention, the kinase is DYRK1A, DYRK1B, DYRK2, DYRK3, or DYRK4.

[0127] In one aspect of the present invention, the kinase is DYRK1A. In one aspect, DYRK1A is inhibited, resulting in a therapeutic benefit to the subject. In one aspect, DYRK1A is inhibited, resulting in the treatment of cancer, such as gastric cancer or colorectal cancer. In one aspect, DYRK1A is inhibited, resulting in the treatment and / or prevention of an infectious disease, such as an infectious disease caused by a protozoan or parasite.

[0128] In one aspect of the present invention, the kinase is DYRK1B. In one aspect, DYRK1B is inhibited, and as a result, a therapeutic benefit is provided to the subject. In one aspect, DYRK1B is inhibited, and as a result, the treatment outcome of cancer, such as gastric cancer or colorectal cancer, is achieved. In one aspect, DYRK1B is inhibited, and as a result, the treatment and / or prevention of an infectious disease, such as an infectious disease caused by protozoa or parasites, is achieved.

[0129] In one aspect of the present invention, the kinase is DYRK2. In one aspect, DYRK2 is inhibited, and as a result, a therapeutic benefit is provided to the subject. In one aspect, DYRK2 is inhibited, and as a result, the treatment of cancer, such as gastric cancer or colorectal cancer, is achieved. In one aspect, DYRK2 is inhibited, and as a result, the treatment and / or prevention of an infectious disease, such as an infectious disease caused by protozoa or parasites, is achieved.

[0130] In one aspect of the present invention, the kinase is DYRK3. In one aspect, DYRK3 is inhibited, and as a result, a therapeutic benefit is provided to the subject. In one aspect, DYRK3 is inhibited, and as a result, the treatment of cancer, such as gastric cancer or colorectal cancer, is achieved. In one aspect, DYRK3 is inhibited, and as a result, the treatment and / or prevention of an infectious disease, such as an infectious disease caused by protozoa or parasites, is achieved.

[0131] In one aspect of the present invention, the kinase is DYRK4. In one aspect, DYRK4 is inhibited, and as a result, a therapeutic benefit is provided to the subject. In one aspect, DYRK4 is inhibited, and as a result, the treatment of cancer, such as gastric cancer or colorectal cancer, is achieved. In one aspect, DYRK4 is inhibited, and as a result, the treatment and / or prevention of an infectious disease, such as an infectious disease caused by protozoa or parasites, is achieved.

[0132] In one aspect, the kinase inhibited by the compounds of the aspects of the present invention is a Cdc2-like kinase (CLK). In one aspect, the CLK is inhibited, resulting in a therapeutic benefit to the subject. In one aspect, the CLK is inhibited, resulting in the treatment of gastric cancer. In one aspect, the CLK is inhibited, resulting in the prevention of memory impairment and neurotoxicity induced by oligomeric Aβ25-35 peptide administration (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)).

[0133] In one aspect of the present invention, the kinase is CLK1, CLK2, CLK3, or CLK4.

[0134] In one aspect, CLK1 is inhibited, resulting in a therapeutic benefit to the subject. In one aspect, CLK1 is inhibited, resulting in the treatment of cancer, such as gastric cancer. In one aspect, CLK1 is inhibited, resulting in the prevention of memory impairment and neurotoxicity induced by oligomeric Aβ25-35 peptide administration.

[0135] In one aspect, CLK2 is inhibited, resulting in a therapeutic benefit to the subject. In one aspect, CLK2 is inhibited, resulting in the treatment of cancer, such as gastric cancer. In one aspect, CLK2 is inhibited, resulting in the prevention of memory impairment and neurotoxicity induced by oligomeric Aβ25-35 peptide administration.

[0136] In one aspect, CLK3 is inhibited, resulting in a therapeutic benefit to a subject. In one aspect, CLK3 is inhibited, resulting in the treatment of cancer, such as gastric cancer. In one aspect, CLK3 is inhibited, resulting in the prevention of memory impairment and neurotoxicity induced by administration of oligomeric Aβ25-35 peptide.

[0137] In one aspect, CLK4 is inhibited, resulting in a therapeutic benefit to a subject. In one aspect, CLK4 is inhibited, resulting in the treatment of cancer, such as gastric cancer. In one aspect, CLK4 is inhibited, resulting in the prevention of memory impairment and neurotoxicity induced by administration of oligomeric Aβ25-35 peptide.

[0138] 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, resulting in a therapeutic benefit to a subject. In one aspect, LATS is inhibited, resulting in the treatment of cancer.

[0139] 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, resulting in a therapeutic benefit to a subject. In one aspect, LATS1 is inhibited, resulting in the treatment of cancer.

[0140] In one aspect, the kinase inhibited by the compound of the aspect of the present invention is LATS2 (Large Tumor Suppressor Kinase 2). In one aspect, LATS2 is inhibited, resulting in a therapeutic benefit to a subject. In one aspect, LATS2 is inhibited, resulting in the treatment of cancer.

[0141] In one aspect, the present invention provides a method of suppressing the immune system of a subject, the method comprising administering to the subject a compound or pharmaceutical composition of one aspect of the present invention.

[0142] In one aspect, the present invention provides a method of preventing organ rejection in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of one aspect of the present invention.

[0143] In one aspect, the present invention provides a method of treating painful diabetic neuropathy in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of one aspect of the present invention.

[0144] In one aspect, the present invention provides a method of treating malaria in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of one aspect of the present invention.

[0145] In one aspect, the present invention provides a method of treating an infection associated with protozoa in a subject, the method comprising administering to the subject a compound or pharmaceutical composition of one aspect of the present invention.

[0146] In one aspect, the present invention provides a method of treating a neurodegenerative disease, the method comprising administering to the subject a compound or pharmaceutical composition of one aspect of the present invention. In one aspect, the present invention provides a method of treating Down syndrome, the method comprising administering to the subject a compound or pharmaceutical composition of an aspect of the present invention. In one aspect, the present invention provides a method of treating Alzheimer's disease, the method comprising administering to the subject a compound or pharmaceutical composition of an aspect of the present invention.

[0147] In one aspect, the present invention provides a method of treating a heart disease, the method comprising administering to the subject a compound or pharmaceutical composition of one aspect of the present invention. In one aspect, the present invention provides a method of treating heart failure, the method comprising administering to the subject a compound or pharmaceutical composition of an aspect of the present invention.

[0148] In one aspect, the present invention provides a method for treating Cushing's syndrome, the method comprising administering to a subject a compound or pharmaceutical composition of one aspect of the present invention.

[0149] In one aspect, the present invention provides a method for treating McCune-Albright syndrome, the method comprising administering to a subject a compound or pharmaceutical composition of one aspect of the present invention.

[0150] In one aspect, the present invention provides a method for treating Carney complex, the method comprising administering to a subject a compound or pharmaceutical composition of one aspect of the present invention.

[0151] One aspect of the present invention provides compounds and pharmaceutical compositions for use in treating and preventing cancer. Without being bound by a particular theory or mechanism, the compounds are thought to inhibit kinases.

[0152] As used herein, the terms "treat" and "prevent" and terms derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, the degree of treatment or prevention recognized by those of ordinary skill in the art as having a potential benefit or therapeutic effect varies. In this regard, the methods of one aspect of the present invention can provide any amount at any level of treatment or prevention of cancer in a mammal. Further, the treatment or prevention provided by the methods of one aspect of the present invention can include treatment or prevention of one or more conditions or symptoms of the disease being treated and prevented, such as cancer. Also, as used herein, "prevent" can include delaying the onset of a disease, or a symptom or condition thereof.

[0153] Regarding the method of one aspect of the present invention, cancers include adrenal cancer, sarcomas (e.g., synovial sarcoma, osteogenic sarcoma, uterine leiomyosarcoma, angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxosarcoma, rhabdomyosarcoma, fibroma, lipoma, and teratoma), lymphomas (e.g., small lymphocyte lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma), hepatocellular carcinoma, glioma, head cancers (e.g., squamous cell carcinoma), neck cancers (e.g., squamous cell carcinoma), acute lymphocytic cancer, leukemias (e.g., hairy cell leukemia, myeloid leukemia (acute and chronic), lymphocytic leukemia (acute and chronic), prolymphocytic leukemia (PLL), myelomonocytic leukemia (acute and chronic), lymphocytic leukemia (acute and chronic)), bone cancers (osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (exostosis), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, giant cell tumor), brain tumors (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme, anaplastic glioma, schwannoma, retinoblastoma), fallopian tube cancer, breast cancer, anal cancer, anal canal cancer, anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vulvar cancer (e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, etc.), myeloproliferative disorders (e.g., chronic myeloid leukemia), colorectal cancer (e.g., colorectal cancer), esophageal cancer (e.g., squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), cervical cancer (cervical cancer and preinvasive 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, hemangioma), lung cancer (e.g., bronchial cancer (squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar cancer (bronchioloalveolar carcinoma), bronchial adenoma, chondromatous hamartoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, etc.), malignant mesothelioma, skin cancer (e.g., malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi sarcoma, nevus, dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid), multiple myeloma, nasopharyngeal cancer, ovarian cancer (e.g., ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid adenocarcinoma, clear cell adenocarcinoma), granulosa cell tumor, Sertoli-Leydig cell tumor, mixed germ cell tumor, malignant teratoma), pancreatic cancer (e.g., ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, VIPoma),Peritoneal, ovarian, mesenteric cancer, pharyngeal cancer, prostate cancer (e.g., adenocarcinoma and sarcoma), rectal cancer, kidney cancer (e.g., adenocarcinoma, Wilms tumor (nephroblastoma), and renal cell carcinoma), small intestine cancer (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma), soft tissue cancer, stomach cancer (e.g., cancer, lymphoma, leiomyosarcoma), testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig 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 urinary bladder cancer). In one aspect of the invention, the cancer is hepatocellular carcinoma. In one aspect of the invention, the cancer is fibrolamellar hepatocellular carcinoma. In one aspect of the invention, the cancer is liver cancer. In one aspect of the invention, the cancer is breast cancer. In one aspect of the invention, the cancer is stomach cancer. In one aspect of the invention, the cancer is colorectal cancer.,

[0154] In certain aspects of the invention, the compounds or pharmaceutically acceptable salts thereof of the aspects of the invention can be co-administered with an anti-cancer agent (e.g., a chemotherapeutic agent) and / or radiation therapy. In one aspect, the compounds or pharmaceutically acceptable salts thereof of one aspect of the invention are administered in an amount effective to sensitize cancer cells to one or more treatment regimens (e.g., chemotherapy or radiation therapy). The terms "co-administer" or "co-administered" mean administration simultaneously or sequentially. The compounds or pharmaceutically acceptable salts thereof of one aspect of the invention can be administered before, simultaneously with, or after the administration of another anti-cancer agent (e.g., a chemotherapeutic agent).

[0155] One or more, e.g., two, three or more anti-cancer agents can be administered. In this regard, the invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a compound or a pharmaceutically acceptable salt thereof of one aspect of the invention, and at least one anti-cancer agent (e.g., a chemotherapeutic agent).

[0156] Examples of anticancer agents include platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, nitrogen mustard, thiotepa, melphalan, busulfan, procarbazine, streptozocin, temozolomide, dacarbazine, bendamustine), antitumor antibiotics (e.g., antitumor antibiotics (daunorubicin, doxorubicin, idarubicin, epirubicin, mitoxantrone, bleomycin, mitomycin C, plicamycin, dactinomycin), taxane drugs (e.g., paclitaxel, docetaxel), antimetabolites (e.g., 5-fluorouracil, cyclohexane, cyclohexane, cyclohexane, cyclohexane, cyclohexane, cyclohexane, cyclohexane, cyclohexane, cyclohexane, cyclohexane, etc.), 5-fluorouracil, cytarabine, pemetrexed, thioguanine, floxuridine, capecitabine, methotrexate), nucleoside analogs (e.g., fludarabine, clofarabine, cladribine, pentostatin, nelarabine), topoisomerase inhibitors (e.g., topotecan, irinotecan), hypomethylating agents (e.g., azacitidine, decitabine), proteasome inhibitors (e.g., bortezomib), epipodophyllotoxins (e.g., etoposide, teniposide), DNA synthesis inhibitors (e.g., hydroxyurea), vinca alkaloids (e.g., vincristine, vindesine, vinorelbine, 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, lomustine), enzymes (e.g., L-asparaginase), biological agents (e.g., interferon, interleukin), hexamethylmelamine, mitotane, angiogenesis inhibitors (e.g., thalidomide, lenalidomide), steroids (e.g., prednisone, dexamethasone, prednisolone), hormonal agents (e.g., tamoxifen, raloxifene, leuprolide, bicalutamide, granisetron, flutamide), aromatase inhibitors (e.g., letrozole, anastrozole),Arsenic trioxide, tretinoin, 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 combinations thereof. In some embodiments, the anti-cancer agent is cisplatin, cytarabine, methotrexate, doxorubicin, or combinations thereof.,

[0157] In certain embodiments of the invention, the compound of an embodiment of the invention or a pharmaceutically acceptable salt thereof can be conjugated to a targeting molecule. Such targeting molecules include antibodies (in the case of ADCs) and small molecules that target other regions of kinases to enhance selectivity (i.e., a second molecule that binds to the DNAJ domain of the PKADJ fusion protein).

[0158] In certain embodiments of the invention, the compound of an embodiment of the invention or a pharmaceutically acceptable salt thereof can be conjugated to an E3 ligase binding molecule to create a proteolysis targeting chimera (PROTAC).

[0159] As used herein, the term “mammal” refers to any mammal including, but not limited to, rodents such as mice and hamsters, lagomorphs such as rabbits, carnivores such as felines (cats) and canines (dogs), Artiodactyls such as bovines (cows) and swines (pigs), Perissodactyls such as equines (horses), primates, Ceboids or Simoids (monkeys), and Anthropoids (humans and apes). Particularly preferred mammals are humans.

[0160] Preparation method The compounds of the aspects of the present invention can be prepared by any of a number of prior arts.

[0161] In certain aspects, the present invention provides a method for the production of apicitabine A

[0162] [Chemical formula]

[0163] which method comprises coupling a purine-thiazine conjugate having the following structure:

[0164] [Chemical formula]

[0165] with an imidazole having the following structure:

[0166] [Chemical formula]

[0167] to provide apicitabine A.

[0168] In one aspect of the present invention, the coupling is carried out in the presence of a catalyst.

[0169] In certain aspects, the present invention provides a method for the preparation of a compound:

[0170] [Chemical formula]

[0171] (wherein at least one of R 3 or R 4 is halogen), which method comprises the formula:

[0172] [Chemical formula]

[0173] comprises halogenating the compound of

[0174] In one embodiment, compound Ic is aplidine A.

[0175] In one embodiment, aplidine A is brominated using N-bromosuccinimide to provide:

[0176]

Chemical Structure

[0177] Examples of non-limiting embodiments of the present disclosure Embodiments that include one embodiment of the subject matter described herein may be useful, alone or in combination with one or more other embodiments or implementations. Without limiting the foregoing description, specific non-limiting embodiments (numbers 1-38) of the present disclosure are provided below. As will be apparent to those skilled in the art upon reading the present disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered embodiments. This is intended to provide support for all combinations of such embodiments and is not limited to the combinations of embodiments explicitly provided below:

[0178] (1) A compound of formula (I)

[0179]

Chemical Structure

[0180] (wherein

[0181]

Chemical Structure

[0182] is a single bond or a double bond, X 1 and X 2 are each independently 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 C1-C3 alkyl; R 3 is aryl; R 6 is C1-C3 alkyl; A is optionally, when present, -C(O)-, -C(O)O-, -C(O)NH-, -C(O)N(C1-C3-alkyl)-; -C(O)NH-(C1-C6 alkyl)-NHC(O)-, -NH; D is optionally, when present, 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)-, where any of the foregoing alkyl or cycloalkyl groups is optionally substituted with one or more substituents selected from hydroxy, C1-C6 alkyl, amino, C1-C6 alkylamino, or di-C1-C6 alkylamino; -NH-aryl, and combinations thereof; and E is: 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, halo, nitro, hydroxy, amino, C1-C6 alkylamino, di-C1-C6 alkylamino; -NH-aryl, C1-C6 haloalkyl, C3-C8 cycloalkyl or heterocycloalkyl, fused C3-C8 cycloalkyl or heterocycloalkyl, aryl or heteroaryl, -CN, -(C1-C3 alkyl)-CN, carbonyl, and combinations thereof; Amino, C1-C6 alkylamino, or di-C1-C6 alkylamino; or -NH-aryl; 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; 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, aryl or heteroaryl, fused aryl or heteroaryl, and combinations thereof; -C(O)NH2; -C(O)OH; -C(O)H; -N-(C1-C6 alkyl)-acrylamide; Halogen; or Hydrogen) Or a pharmaceutically acceptable salt thereof.

[0183] (2)

[0184]

Chemical formula

[0185] is a single bond or a double bond, X 1 and X 2 are each independently 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 C1-C3 alkyl; R 3 is aryl; A is optionally, when present, -C(O)-, -C(O)O-, -C(O)NH-, -C(O)N(C1-C3-alkyl)-; -C(O)NH-(C1-C6 alkyl)-NHC(O)-, -NH-; D is optionally, when present, 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)-, where any of the foregoing alkyl groups is optionally substituted with hydroxy; E is: aryl or heteroaryl, optionally substituted with C1-C6 alkyl or alkoxy, halo, nitro, hydroxy, C1-C6 haloalkyl, -CN, -(C1-C3 alkyl)-CN, or carbonyl; amino, C1-C6 alkylamino, or di-C1-C6 alkylamino; or -NH-aryl; C1-C6 alkyl or alkoxy optionally substituted with hydroxy; C3-C8 cycloalkyl or heterocycloalkyl optionally substituted with hydroxy; -C(O)NH2; -C(O)OH; -C(O)H; -N-(C1-C6 alkyl)-acrylamide; halogen; or hydrogen, a compound of embodiment 1 or a pharmaceutically acceptable salt thereof.

[0186] (3)

[0187]

Chem.

[0188] A compound of embodiment 1 or 2 wherein is a double bond.

[0189] (4) The compound of formula (I) is of formula (Ia):

[0190]

Chem.

[0191] A compound according to any one of embodiments 1 to 3 or a pharmaceutically acceptable salt thereof.

[0192] (5) The compound of formula (I) is of formula (Ib):

[0193]

Chem.

[0194] A compound according to any one of embodiments 1 to 4 or a pharmaceutically acceptable salt thereof.

[0195] (6) R 1 is H, a compound according to any one of embodiments 1 to 5.

[0196] (7) A compound according to any one of embodiments 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 heterocycloalkyl optionally substituted with hydroxy. (ii) A is absent; D is C1-C6 alkyl, optionally C1-C3 alkyl; and E is -C(O)NH2 or C3-C8 cycloalkyl or heterocycloalkyl optionally substituted with hydroxy; (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 alkylamino; or -NH-aryl; C1-C6 alkyl or alkoxy or C1-C3 alkyl or alkoxy optionally substituted with hydroxy; or C1-C3 alkyl or alkoxy, halo, nitro, hydroxy, C1-C3 haloalkyl, -CN, -(C1-C3 alkyl)-CN, or aryl or heteroaryl optionally substituted with 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)-, where any of the foregoing alkyl groups is optionally substituted with hydroxy and any of the foregoing alkyl groups is optionally branched, and E is C1-C6 alkyl or C3-C8 cycloalkyl or heterocycloalkyl optionally substituted with hydroxy; (v) A is -C(O)NH-(C1-C6 alkyl)-NHC(O)-, optionally -C(O)NH-(C1-C3 alkyl)-NHC(O)-; D is absent; E is C1-C6 alkyl or C1-C3 alkyl; or C1-C3 alkyl or alkoxy, halo, nitro, hydroxy, C1-C6 haloalkyl, -CN, -(C1-C3 alkyl)-CN, or aryl or heteroaryl optionally substituted with carbonyl; (vi) A is -C(O)O-; D is absent; E is C1-C6 alkyl or (vii) A is -C(O)-; D is absent; E is heterosilalkyl.

[0197] (8) The compound of formula (I) is of formula (Ic):

[0198]

Chemical Structure

[0199] (wherein R 4 and R 5 are the same or different and each is H or halo), a compound of embodiment 1 or a pharmaceutically acceptable salt thereof.

[0200] (9) Either R 4 or R 5 is halo, or both R 4 and R 5の are halo, a compound of embodiment 8.

[0201] (10) Both R 4 and R 5 are hydrogen, a compound of embodiment 8.

[0202] (11) The compound is not aplidine A

[0203]

Chemical Structure

[0204] a compound of any one of embodiments 1 to 10.

[0205] (12) The compound of formula (I) is selected from the following, a compound of embodiment 1 or a pharmaceutically acceptable salt thereof.

[0206]

Chemical Structure

[0207]

Chemical Structure

[0208] [Chemistry]

[0209] [Chemistry]

[0210] [Chemistry]

[0211] (13) The compound of formula (I) is as follows

[0212] [Chemistry]

[0213] the compound of embodiment 12 or a pharmaceutically acceptable salt thereof, selected from the following

[0214] (14) Compound

[0215] [Chemistry] or a pharmaceutically acceptable salt thereof.

[0216] (15) A pharmaceutical composition comprising a compound of any one of embodiments 1 to 14 and a pharmaceutical carrier.

[0217] (16) A pharmaceutical composition comprising a

[0218] [Chemistry]

[0219] with a purity of at least 80% and a pharmaceutical carrier.

[0220] (17) A method for inhibiting kinase activity in a subject, the method comprising administering to the subject a compound of any one of aspects 1 to 14, or a pharmaceutical composition of aspect 15 or 16.

[0221] (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.

[0222] (19) The method of aspect 17, wherein the kinase is PKA, PKA / DNAJ, or cAMP-PKA.

[0223] (20) The method of aspect 17, wherein the kinase is PKG1a, PKG1b, PKG2, or PfPKG.

[0224] (21) The method of aspect 17, wherein the kinase is DYRK1A, DYRK1B, DYRK2, DYRK3, or DYRK4.

[0225] (22) The method of aspect 17, wherein the kinase is CLK1, CLK2, CLK3, or CLK4.

[0226] (23) A method for suppressing the immune system of a subject, the method comprising administering to the subject a compound of any one of aspects 1 to 14, or a pharmaceutical composition of aspect 15 or 16.

[0227] (24) A method for preventing organ rejection in a subject, the method comprising administering to the subject a compound of any one of aspects 1 to 14, or a pharmaceutical composition of aspect 15 or 16.

[0228] (25)A method for treating cancer in a subject, the method comprising administering to the subject a compound of any one of aspects 1-14, or a pharmaceutical composition of aspect 15 or 16.

[0229] (26)The method of aspect 25, wherein the cancer is fibrolamellar carcinoma (FLC).

[0230] (27)The method of aspect 25, wherein the cancer is fibrolamellar hepatocellular carcinoma (FL-HCC).

[0231] (28)The method of aspect 25, wherein the cancer is gastric cancer.

[0232] (29)The method of aspect 25, wherein the cancer is colorectal cancer.

[0233] (30)A method for treating painful diabetic neuropathy in a subject, the method comprising administering to the subject a compound of any one of aspects 1-14, or a pharmaceutical composition of aspect 15 or 16.

[0234] (31)A method for treating malaria in a subject, the method comprising administering to the subject a compound of any one of aspects 1-14, or a pharmaceutical composition of aspect 15 or 16.

[0235] (32)The method is a method for treating protozoa-related infectious diseases in a subject, the method comprising administering to the subject a compound of any one of aspects 1-14, or a pharmaceutical composition of aspect 15 or 16.

[0236] (33)Any one of aspects 17-32, wherein the subject is a human.

[0237] (34)Apicitamine A

[0238] [Chemical formula]

[0239] A production method, having the following structure:

[0240]

Chemical formula

[0241] Coupling a purine - thiazine conjugate having the following structure:

[0242]

Chemical formula

[0243] with imidazole having the following structure to provide apithiamine A, including the method.

[0244] (35) The method of embodiment 34, wherein the coupling is carried out in the presence of a catalyst.

[0245] (36) A method for preparing the compound of embodiment 8:

[0246]

Chemical formula

[0247] wherein at least one of R 3 or R 4 is halogen; Formula:

[0248]

Chemical formula

[0249] A method including halogenating the compound of.

[0250] (37) The method of embodiment 36, wherein compound Ic is apithiamine A.

[0251] (38) Brominating apithiamine A using N - bromosuccinimide

[0252]

Chem.

[0253] The method of embodiment 37 that provides.

[0254] The following examples further illustrate the present invention, but of course, should not be construed as limiting its scope.

Example

[0255] UV data was measured with a VARIAN CARY TM 50 UV-Vis Spectrophotometer. The IR spectrum was recorded with a Bruker ALPHA II FT-IR spectrometer. The NMR data was obtained with a Bruker Avance III NMR spectrometer equipped with a 3 mm cryoprobe ( 1 H at 600 MHz, 13 C at 150 MHz). The HRESIMS data was collected with an Agilent Technology 6530 Accurate-mass Q-TOF LC / MS. The HPLC separation was performed on a Shimadzu system equipped with a CBM-40 controller, an SPD-M40 PDA detector, and two LC-20AR pumps.

[0256] All solvents used were of LC-MS grade or higher.

[0257] Specimens of Aplidium sp. sea squirts collected from the coral reefs of South Africa in September 2000 were stored frozen until extraction. The collection was made by the Coral Reef Research Foundation under contract with the Natural Products Branch of the US National Cancer Institute. Voucher specimens (voucher ID # 0CDN7423) were deposited at the Smithsonian Museum (Washington D.C.). The animal specimens (234 g, wet weight) were ground by the standard NCI method for marine samples (McCloud, Molecules, 15: 4526-4563 (2010)) to obtain 3.27 g of an organic extract (NSC # C020725) and 16.2 g of an aqueous extract (NSC # C020724).

[0258] Example 1 This example demonstrates that the compounds of the present invention can be extracted and purified.

[0259] The organic crude Aplidium sp. extract (NSC # C020725, 1 g, described above) was subjected to a C8 solid-phase extraction (SPE) process (loading 250 mg of the extract onto each of two 2 g C8 SPE cartridges and eluting with a gradient of 5%, 20%, 40%, 60%, 80%, 100% MeOH in H2O and 50% MeOH in MeCN), generating seven fractions. The active fraction Fr.5 was further separated by prep-HPLC using a Kinetex 5μm EVO C18 column (110Å, 250×21.2 mm) at a flow rate of 10 mL / min (eluting with 5% - 100% MeCN containing 0.1% TFA), yielding 20 fractions. Fractions Fr.5-14 were further purified by semi-prep HPLC using a Kinetex 5μm F5 column (110Å, 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). Fractions Fr.5-15 were purified by semi-prep HPLC using a Kinetex 5μm F5 column (110Å, 250×10 mm) at a flow rate of 4 mL / min (eluting with 12% MeCN containing 0.1% TFA) to obtain compounds 1 (6.9 mg), 3 (0.8 mg), 4 (2.6 mg), and 5 (1.9 mg). The structures of compounds 1-5 are shown in Figure 22.

[0260] To accumulate active compound 1 for chemical modification, 1.38 g of organic extract (NSC # C020725) and 15.4 g of aqueous extract (NSC # C020724) were obtained from the NCI Natural Products Repository. The aqueous extract was first desalted by HP20ss VLC (washed with H2O and eluted with MeOH). The fraction eluted with MeOH (660 mg) was further purified by prep-HPLC using a Kinetex 5μm F5 column (110Å, 250×21.2 mm) at a flow rate of 10 mL / min (eluted with 15% MeCN containing 0.1% TFA) to obtain 66 mg of 1.

[0261] Apricitianin A (1): white solid; UV (MeOH) λ max (log ε) 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] + (calcd for C 13 H 14 N7S, 300.1031).

[0262] Apricitianin B (2): white solid; UV (MeOH) λ max (log ε) 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] + (calcd for C 13 H 14 N7OS, 316.0981).

[0263] Apridiprinide A (3): white solid; UV (MeOH) λ max (log ε) 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] + (calcd for C 14 H 17 N 10 O2S2, 421.0977).

[0264] Apridiprinide B (4): white solid; UV (MeOH) λ max (log ε) 212 (4.41), 271 (4.38); IR (neat) 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] + (calcd for C 14 H 17 N 10 S2, 389.1079).

[0265] Apridopride C(5):; UV (MeOH) λ max (log ε) 211(4.56), 273 (4.11); IR (neat) 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] + (calcd for C 14 H 17 N 10 OS2, 405.1028).

[0266]

Table 1

[0267]

Table 2

[0268]

Table 3

[0269] Structure elucidation of Compounds 1 - 5 (Figure 22)

[0270] Compound 1 was isolated as a white solid. From the analysis of HRESIMS and 1 H and 13 C NMR data, the molecular formula was determined to be C 13 H 13 N7S (Tables 1 and 2). When the NMR data were collected in DMSO-d6, methine (δ H 9.00, δ C 128.7) and methylene (δ H 4.61, δ Cbroad and weak at 42.8 1 H and 13 C signals were observed and the two-dimensional NMR (HSQC and HMBC) correlations were insufficient (Figs. 24 - 28). This problem was overcome by changing the solvent to methanol-d (4) and the structure of 1 could be clearly elucidated (Fig. 22). 1 H and 13 From the analysis of the H and H C NMR and HSQC data, the presence of five aromatic methines, two linked methylenes, and an N-methyl group was revealed (δ C 4.02, δ 1 35.1). By comprehensively analyzing the HMBC correlations (Fig. 23) and 13 comparing the chemical shifts of H and H C with the reported data, three heterocyclic ring systems containing two common natural product components, 6-substituted purines (δ C 8.45, 8.18; δ H 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 C 8.88, 7.62, 4.02; δ H 137.7, 134.5, 120.0, 35.1) (Savage, et al., N. Engl. J. Med., 346:683 - 93 (2002)) were established. The remaining part of the structure was constructed as a disubstituted dihydro-1,4-thiazine moiety evidenced by the major H H-H 1 COSY correlation between 2H-5 (δ 1 4.71) and 2H-6 (δ H 3.38), and the HMBC correlations from H-3 (δ C 9.00) to C-2 (δ C 97.6) and C-5 (δ H7.62) to C-2 via a single bond supported by HMBC correlations. C 134.5) to the N-Me imidazole moiety. Finally, H-3 and 2H-5 to C-6'' (δ C Based on the predominant HMBC correlation to N-4 and C-6'' (δ 151.8), the dihydro-1,4-thiazine moieties were C The structural elucidation of 1 was completed by linking the purine moiety between the aryl groups (Figure 29-33). Compound 1, named aplichianin A, is the first example of a new class of purine-thiazine-imidazole interconnected alkaloids.

[0271] Compound 2 was isolated as a white solid. HRESIMS data indicated the molecular formula C 13 H 13 N7OS was determined. 1 H and 13 C, Tables 1 and 2) and two-dimensional ( 1 H- 1 Comparison of the H COSY, HSQC, HMBC, and NMR data (Fig. 2) revealed that the structure was the same except for a slight change in the structure of the purine moiety. H 8.18, δ C 141.7) is the delta of 2 C The carbonyl was converted to 2 at 151.8. 1 NH-7'' (δ H 11.1) and NH-9''(δ H This assignment was supported by the presence of an exchangeable proton at C-8' (Figure 34-41) and the major HMBC correlations from these two protons to C-8'. Thus, the structure of 2, which was given the uninteresting name of aplychianin B, was determined as the 8''-oxopurine analogue of 1.

[0272] The molecular formula of 3 is C, based on the interpretation of the HRESIMS data. 14 H 16 N 10 It was determined to be O2S2. 1 H and13 The 13C NMR spectrum showed only eight protons and seven carbons, suggesting that this structure is a homodimer. 1 H and 13 13C NMR data (Table 3) and 1 H, 1 H-COSY and HMBC correlation analyses immediately revealed the presence of the 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)). The 8-oxopurine moiety (δ H 8.05, 9.98, 11.35, and δ C 104.7, 145.3, 147.3, 150.7, 152.7) was the same as that in structure 2 (Tables 1, 2). Based on the major HMBC correlation from NH-10 (δ H 6.73) and 2H-11 (δ H 3.72) to C-6 (δ C 145.3), the two moieties were linked between N-10 and C-6. Since NMR analysis established the structure of half of the molecule occupying C7H8N5OS, the homodimer was formed only by the two half-structures linked via a disulfide bond (Figs. 61-65). Thus, the structure of 3 was determined as a new nucleic acid base homodimer with a disulfide bond and named as aplydiprinide A.

[0273] Compounds 4 and 5 showed very similar UV spectra (UV maxima at ~211 nm and 273 nm) and molecular formulas (4 is C 14 H 16 N 10 S2, 5 is C 14 H 16 N 10 OS2) compared with that of 3, indicating that they are structural analogs. The 1 H and 13The C NMR spectrum (Table 3) also showed signals for only half of the molecule, and the nucleobase moiety had been changed to a 6-N-purine moiety. There was no oxygen atom in the molecule, and the 8-methine signal (δ H 8.42, δ C 142.3) is evident from the existence of 5, in contrast to 3 and 4. 1 H and 13 The C NMR data (Table 3) showed two sets of signals, similar to the NMR features of 3 and 4, respectively. Thus, compound 4 was determined to be a homodimeric analog of 3 with the nucleobase moiety changed to 6-N-purine, while the structure of 5 was predicted to be a heterodimer constructed by linking half structures of 3 and 4 via a disulfide bond (Figure 22). This structure determination was based on a two-dimensional ( 1 H, 1 This was supported by the analysis of H-COSY, HSQC, and HMBC NMR correlations (Figures 66-75). Compound 4 was first reported as a synthetic compound (Kastenhuber, et al., PNAS USA, 114: 13076-84 (2017)), but was never found in natural sources. Compounds 4 and 5 were therefore determined as new natural analogues of 3 and were named apridiprinides B and C, respectively.

[0274] Example 2 This example demonstrates that the compounds of the present invention can be synthesized.

[0275] Chemical investigation of a bulk extract of Aplidium sp. accumulated 116 mg of 1, allowing the production of semisynthetic aplichianin analogues and providing preliminary insight into structure-activity relationships. Given the limited supply of naturally occurring 1, the initial design of the semisynthesis focused solely on optimizing certain robust reactions expected to yield simple product profiles.

[0276] Total synthesis of aplichianin A.

[0277] [ka]

[0278] To a mixture of ethyl 3,4-dihydro-2H-1,4-thiazine-6-carboxylate (6, 1 equiv., 100 mg) and 6-bromopurine (7, 1.5 equiv., 170 mg) were added Xantphos Pd G3 (10 mol%, 55 mg), Cs2CO3 (3 equiv., 560 mg), DMF (5 mL), and 4 Å molecular sieves. The reaction vial was filled with N2 and sealed. The reaction mixture was stirred vigorously at 110 °C overnight and then dried under vacuum. The residue was redissolved in DMSO and purified by preparative HPLC using a Gemini 5 μm NX-C18 column (110 Å, 250 × 21.2 mm) with a flow rate of 10 mL / min (eluting with 10% - 100% MeCN in 0.1% TFA), to give 8 (61 mg, 36% yield).

[0279] Ethyl 4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxylate (8): Pale orange solid; 1 H NMR (600 MHz, DMSO-d (6 )): 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.

[0280]

Chemical Structure

[0281] Compound 8 (61 mg) was dissolved in 2 M NaOH (3.5 mL) and THF (3.5 mL), and stirred overnight at room temperature. The reaction solution was acidified with 2 M HCl (5 mL) and dried under reduced pressure under vacuum. The crude product was washed and desalted with H2O (1 mL x 3 times) to obtain 9 (49 mg, 90% yield). Figure 96 shows the 1 1H NMR spectrum of 8 in DMSO-d6. Figure 97 shows the 13 13C NMR spectrum of 8 in DMSO-d6.

[0282] 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-d (6 6): 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 shows the 1 1H NMR spectrum of 9 in DMSO-d6. Figure 99 shows the 13 13C NMR spectrum of 9 in DMSO-d6. Route 1

[0283]

Chemical Structure

[0284] 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 4 Å MS. The reaction mixture was stirred vigorously overnight at room temperature and then dried under vacuum. The residue was redissolved in DMSO and purified by preparative HPLC using a Gemini 5 μm NX-C18 column (110 Å, 250 × 21.2 mm) at a flow rate of 10 mL / min (eluting with 30% MeCN in 0.1% TFA) to give 10 (40 mg, 93% yield).

[0285] N-Methoxy-N-methyl-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (10): Pale orange solid; 1 H NMR (600 MHz, DMSO-d (6) ): 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 C 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 is the 1 1H NMR spectrum of 10 in DMSO-d6. Figure 101 is the 13 13C NMR spectrum of 10 in DMSO-d6.

[0286]

Chem.

[0287] Compound 10 (1 equiv., 40 mg) was stirred in THF (5 mL) at 0 °C, followed by 2M LiAlH in THF (4)(4 equiv., 260 μL) was added. The reaction was stopped after 1 hour by the addition of H2O (1 mL). The reaction mixture was dried under vacuum. The residue was redissolved in DMSO and purified by preparative HPLC using a Gemini 5 μm NX-C18 column (110 Å, 250 × 21.2 mm) (flow rate 10 mL / min, eluting with 30% MeCN in 0.1% TFA) to give 11 (22 mg, 68% yield).

[0288] 4-(7H-Purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carbaldehyde (11): Pale orange solid; 1 H NMR (600 MHz, DMSO-d (6 )): 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 C 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 is the 1 1H NMR spectrum of 11 in DMSO-d6. Figure 103 is the 13 13C NMR spectrum of 11 in DMSO-d6.

[0289]

Chem.

[0290] Compound 11 (1 equiv., 22 mg) was stirred in 33% methylamine in EtOH (5 mL) in the presence of 4 Å molecular sieves at room temperature for 4 h. After drying the reaction mixture under vacuum, tosylmethyl isocyanide (2 equiv., 55 mg), K2CO3 (2 equiv., 23 mg), MeOH (5 mL) and 4 Å molecular sieves were added. The reaction mixture was stirred at 60 °C overnight and then dried under vacuum. The residue was redissolved in DMSO and purified by preparative HPLC using a Synergi 5 μm Hydro-RP column (110 Å, 250 × 21.2 mm) at a flow rate of 10 mL / min (eluting with 19% MeCN in 0.1% TFA) to give 1 (4 mg, yield 15%). Route 2

[0291] [Chemical formula]

[0292] A reaction vial containing a solution of 9 (1 equiv., 49 mg) in DMF (5 mL) with 4 Å molecular sieves 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, then warmed to room temperature and stirring was continued overnight. The reaction mixture was dried under vacuum. The residue was redissolved in DMSO and purified by preparative HPLC using a Gemini 5 μm NX-C18 column (110 Å, 250 × 21.2 mm) at a flow rate of 10 mL / min (eluting with 10 - 100% MeCN in 0.1% TFA) to give 12 (25 mg, yield 45%).

[0293] 6-Bromo-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine (12): pale orange solid; 1 H NMR (600 MHz, DMSO-d (6) ): 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); 1313C 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 is the 1H NMR spectrum of 12 in DMSO-d6 1 Figure 105 is the 13C NMR spectrum of 12 in DMSO-d6 13 Figure 105 is the 13C NMR spectrum of 12 in DMSO-d6

[0294] [Chemical formula]

[0295] To 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) were added Xantphos Pd G3 (20 mol%, 8.7 mg), Cs2CO3 (3 equiv., 560 mg), DMF (3 mL), H2O (0.3 mL), and 4 Å molecular sieves. The reaction vial was filled with N2 and sealed. The reaction mixture was vigorously stirred at 95 °C overnight and then dried under vacuum. The residue was redissolved in DMSO and purified by preparative HPLC continuously at a flow rate of 10 mL / min using a Gemini 5 μm NX-C18 column (110 Å, 250 × 21.2 mm) (0.1% TFA), and further purified using a Synergi 5 μm Hydro-RP column (110 Å, 250 × 21.2 mm), eluting at a flow rate of 10 mL / min (eluting with 19% MeCN in 0.1% TFA) to obtain 1 (9.8 mg, yield 67%).

[0296] In order to continue the development of aplidine A (1) as a drug candidate, it was necessary to address the stable supply of the molecule. Therefore, two synthetic routes were designed by overlapping for the total synthesis of aplidine A (1) (Scheme 1, Figure 7). Both routes started 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), and the resulting ester 8 was subjected to basic hydrolysis to quantitatively obtain the carboxylic acid 9. By optimizing a wide range of reaction conditions such as catalysts / precatalysts [e.g., Pd2(dba)3, Pd(OAc)2, XantPhos Pd G3, XantPhos Pd G4, XPhos Pd G4, P(t-Bu)3Pd G4], ligands (e.g., Xantphos, B-Bu)3Pd G4), bases (Cs2CO3, sodium tert-butoxide, LiHMDS, etc.), solvents (DMF, THF, toluene, MeCN, etc.), and temperature (25 - 130 °C, etc.), the yield of the Buchwald-Hartwig coupling reaction reached approximately 36%. The combination of the precatalyst XantPhos Pd G3 and the base Cs2CO3 with DMF as the solvent provided an optimal yield at 110 °C compared to most other test conditions (different combinations of the aforementioned reaction factors) where the yield of 1 was generally less than 5%. 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 aldehyde 11, which was further subjected to a two-step Van Leusen imidazole synthesis to obtain the final product 1. Unfortunately, the overall yield of 1 from the six-step Route 1 was only about 3% mainly due to the low yield (15%) of the final-step Van Leusen imidazole synthesis. Therefore, a shorter second synthetic route was designed by directly coupling the purine-thiazine conjugate and the imidazole unit. The carboxylic acid 9 was converted to bromide 12 by decarboxylative bromination with NBS / DMF.Using the same catalyst system (i.e., XantPhos Pd G3 / Cs2CO3 / DMF), brominated 12 was directly conjugated with the boronic ester 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-imidazole (13), and 1 was obtained in a yield of 10% through a total of four steps of reaction (see Figure 8).

[0297] Example 3 This example shows that the compound of the present invention can be brominated.

[0298] Bromination of aplidicatin A (1)

[0299]

Chemical Structure

[0300] To a dry mixture of N-bromosuccinimide (5 mg, 2 equiv.) and compound 1 (4 mg, 1 equiv.), 2 mL of anhydrous DMF was added. The resulting solution was stirred at room temperature overnight. The reaction solution was dried under reduced pressure in vacuo, and the residue was redissolved in DMSO. The product was purified by semi-prep HPLC using a Synergy 5μm Polar-RP column (110 Å, 250×10 mm) at a flow rate of 4 mL / min (eluting with 20 - 100% MeCN in 0.1% TFA), and 1a (1.3 mg) and 1b (0.7 mg) were obtained.

[0301] Aplidicatin A1 (1a): white solid; UV (MeOH) λ max (log ε) 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 1313C NMR data, see Tables 1 and 2 and Figures 42-46; HRESIMS m / z 378.0128, [M+H] + (calcd for C 13 H 13 BrN7S, 378.0137).

[0302] Apristianin A2 (1b): white solid; UV (MeOH) λ max (log ε) 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 1H and 13 13C NMR data, see Tables 1 and 2 and Figures 47-50; HRESIMS m / z 455.9238, [M+H] + (calcd for C 13 H 12 Br2N7S, 455.9242).

[0303] When 1 was reacted with 2 equivalents of N-bromosuccinimide (NBS), the monobrominated product 1a and the dibrominated product 1b in which bromination occurred only at the imidazole moiety were obtained.

[0304] Example 4 This example shows that the compounds of the present invention can be oxidized by different methods.

[0305] Oxidation of Apristianin A (1) with SELECTFLUOR.

[0306]

Chemical Structure

[0307] SELECTFLUOR (4.4 mg, 1 equiv.) and compound 1 (3.7 mg, 1 equiv.) 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-prep HPLC using a Synergy 5 μm Polar-RP column (110 Å, 250 × 10 mm) at a flow rate of 4 mL / min (eluting with 10% MeCN in 0.1% TFA), and 1c (1.4 mg) was obtained.

[0308] Apristianin A3 (1c): white solid; UV (MeOH) λ max (log ε) 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 - 55; HRESIMS m / z 316.0975, [M + H] + (calcd for C 13 H 14 N7OS, 316.0981).

[0309] Oxidation of Apristianin A (1) with H2O2

[0310]

Chem.

[0311] A solution of compound 1 (4 mg) in acetic acid (4 mL) was added with 1 mL of 30% H2O2 solution. The reaction mixture was dried overnight at room temperature. The reaction solution was dried under reduced pressure under vacuum, and the residue was redissolved in MeOH. The product was purified by semi-prep HPLC using a Kinetex 5μm EVO C18 column (110 Å, 250×10 mm) at a flow rate of 4 mL / min (eluted with 7% MeCN in 0.1% TFA), and 1d (1.5 mg) was obtained.

[0312] Apritianin A4 (1d): white solid; UV (MeOH) λ max (log ε) 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 H and 13 C NMR data, see Tables 1 and 2 and Figures 56 - 60; HRESIMS m / z 332.0923, [M+H] + (calcd for C 13 H 14 N7O2S, 332.0930).

[0313] The effort to generate fluorinated analogs using 1 equiv. of SELECTFLUOR, while completely oxidizing 1 to the sulfone analog 1d with H2O2 / AcOH, resulted in the complete conversion of 1 to the partially oxidized sulfoxide analog 1c.

[0314] Example 5 This example shows that the compounds of the present invention are active against PKADJ / PKA.

[0315] To evaluate the activity of Compounds 1-5 against PKADJ, it was tested by an improved sandwich ELISA assay. A reaction step was performed in which the PKADJ holoenzyme was treated with the test compound to dissociate the holoenzyme and 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 had an IC 50 value of 1.1 μM and showed strong inhibition against the activity of PKADJ (see Figures 1A - 1B). In contrast, Analog 2 only weakly inhibited PKADJ (IC 50 = 69 μM) and was not more than 60-fold more potent than 1. The nucleobase dimers 3 - 5 were inactive in this assay with IC 50 > 90 μM.

[0316] 1 and 2 were further evaluated for their activity against wild-type PKA (wt-PKA) using the same assay method. Both compounds showed almost equivalent potency against PKA (IC 50 values were 1.64 μM for 1 and 45 μM for 2) compared to the inhibition against PKADJ. Despite the lack of selectivity for PKADJ, the apritianins were a new class of naturally derived kinase inhibitors with an unprecedented structural skeleton. Further studies on the mechanism of action and structure-activity relationship of this new class of kinase inhibitors are needed (see Figures 11A - 11P).

[0317] Example 6 This example shows that the compounds of the present invention inhibit PKA activity.

[0318] To investigate the mechanism of action of apritianin A (1), first a luciferase assay was performed to confirm whether 1 truly inhibits PKA activity without interfering with the assay. Compound 1 had an IC 50It strongly inhibited PKA activity at a value of 0.4 μM, but no activity was observed when PKA was absent from the assay. Therefore, to investigate how 1 interacts with the PKA protein, further competitive kinetic studies were conducted. At concentrations of 0 - 25 nM, compound 1 competitively inhibited the ATP-induced PKA activity dose-dependently and competitively with ATP at concentrations of 0 - 100 μM, and the values of K iapp and r 2 were 10.4 nM and 0.91, respectively. Therefore, all results supported that 1 is likely to function as an ATP-competitive inhibitor by directly interacting with the PKA catalytic unit (see Figures 2A - 2B).

[0319] To further investigate the binding mechanism between aplidine and PKA, co-crystallization experiments of 1 and 2 with the DNAJ-PKAc fusion protein were performed. Different binding modes were revealed from the X-ray diffraction experiments of the co-crystals of 1 and 2 with DNAJ-PKAc, respectively. For compound 1, three H-bonds from the N-3'', NH-9'', N-7'' of the purine moiety to the PKAc residues of Val178, Glu176, Thr238, respectively, and another H-bond between the N-3' of imidazole and Lys127 were predicted, and it partially occupied the ATP-binding pocket. In contrast, the adenine moiety of ATP was rearranged within the binding pocket, and N-1, NH2-6, N-7 were bound to Val178, Glu176, Thr238, respectively. Surprisingly, in the structure of 2, C-8 was oxidized, and the binding mode in the ATP-binding pocket was completely reversed compared to 1. In contrast to 1, the structure of 2 was inverted around the C-3 / C-6 axis, and the purine ring and imidazole ring were rotated around the N-4 / C-6'' bond and C-2 / C-5' bond, respectively, resulting in two H-bonds between purine N-3'' and Lys127 and between imidazole N-3' and Val178. Therefore, both 1 and 2 may inhibit PKAc activity by competitively binding to the ATP pocket. Nevertheless, a slight modification of purine C-8'' apparently changed the binding affinity for PKAc and resulted in a difference in the efficacy of PKAc inhibition, thereby completely reversing their binding poses (see Figures 3A - 3D).

[0320] Example 7 This example shows the activity of analogs 1a - 1d.

[0321] Analogs 1a - 1d (Examples 3 and 4) were tested in the same way as compound 1, as 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 potency compared to 1 against both PKADJ (IC 50 = 1.05 μM) and WT - PKA (IC 50 = 1.22 μM). Analogs 1b - 1d were inactive against both PKADJ or WT - PKA, and the IC 50 values were 90 μM or higher. Further co - crystallization and X - ray diffraction experiments revealed almost the same binding mode of 1a and 1, consistent with their equivalent potencies against PKADJ and PKA (see Figures 4A - 4D).

[0322] Example 8 This example shows that the compounds of the present invention inhibit kinases.

[0323] To examine the selectivity and potency against the human kinome, dose - response tests for compound 1 and 1a were performed against 10 kinases belonging to divergent phylogenetic groups. Both compounds showed comparable potency and selectivity against the 10 kinases, with PKA, GSK3β, and AKT1 being the most potent targets (IC 50 values were 43 - 189 nM). They showed moderate activity against VEGFR1, MEK1, HER2, and CAMK1d (IC 50 values were 18 - 65 μM), and although not very potent, significant inhibition was observed against PKCα, CK1α1, and ERK1 (IC 50The value was 0.8 - 6.6 μM). Further, kinase profiling of the semi-synthetic analog 1a was performed against the entire kinase panel of 370. At 2 μM, 1a potently inhibited 101 kinases with an inhibition rate of 50% or more, but was completely inactive against 65 kinases (inhibition rate less than 5%). At 50 nM, 29 kinases emerged as more potent targets compared to PKA (78% inhibition), and the most potent inhibition (inhibition of 50% or more) was observed in 8 kinases including PKG1a, STK39 / STLK3, PKG1b, PKC-θ, CLK1, DYRK1 / DYRK1A, DYRK2, and LATS1, which mainly belong to the AGC and CMGC groups of kinases (see FIGS. 5, 6A - 6C, and 13 - 21).

[0324] For 19 kinases most sensitive to 1a, an additional dose-response study was performed, and similar selectivity was shown for 9 targets more potent compared to PKA, including PKG1a, PKG1b, PKG2, PKCtheta, STK39, CLK1, LATS1, LATS2, and CLK2 (see Table 4). Curve fits are shown in FIGS. 76A - 95B. Curve fitting was performed when the enzyme activity at the highest concentration of the compound was less than 65%. The differential was calculated as follows: IC 50 (kinase) / IC 50 (PKG1a).

[0325] [Table 4]

[0326] As the data show, the compounds of the embodiments of the present invention are active kinase inhibitors.

[0327] Example 9 This example provides an exemplary synthesis of an exemplary thiazine carboxamide disclosed herein.

[0328] Synthesis of Acid Scaffold-A. Acid Scaffold-A was synthesized from commercially available H-1 and H-2. Specifically, H-1 was reacted with H-2 in the presence of Xantphos Pd G3 and Cs2CO3 to obtain H-3, which was then reacted with KOH to obtain Acid Scaffold-A as shown in Scheme 2. Scheme 2: Preparation of Acid Scaffold -A

[0329]

Chem.

[0330] 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), Xantphos Pd G3 (10 mol%, 55 mg), Cs2CO3 (3 equiv., 560 mg) and DMF (5 mL) were added. The reaction vial was filled with N2 and sealed. The reaction mixture was stirred vigorously at 110 °C overnight and then dried under vacuum. The residue was redissolved in DMSO and purified by preparative HPLC using a Gemini 5μm NX-C18 column (110 Å, 250×21.2 mm) at a flow rate of 10 mL / min (eluting with 10% - 100% MeCN in 0.1% TFA), and H-3 (109 mg, 65% yield) was obtained as a pale white solid. 1 H NMR (600 MHz, DMSO-d (6 )): δ 12.21 (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-d (6)): δ 164.7, 152.8, 152.6, 149.9, 133.3, 125.0, 104.5, 100.7, 100.1, 60.4, 45.3, 23.9, 14.3; HRESIMS m / z 291.0910, [M+H] + (calcd for C 13 H 15 N4O2S, 291.0916).

[0331] Preparation of 4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxylic acid (acid scaffold-A): H-3 (109 mg) was reacted with KOH (3 equiv., 62 mg) in MeOH (5 mL) and H2O (5 mL) and stirred at 95 °C for 2 h. The crude product was washed and desalted with H2O (1 mL x 3 times) to obtain acid scaffold-A (80 mg, yield 81%) 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] + (calcd for C 11 H 11 N4O2S, 263.0603).

[0332] Synthesis of acid scaffold-B. Acid scaffold-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, which was then reacted with KOH to obtain acid scaffold-B as shown in Scheme 3. Scheme 3: Preparation of Acid Scaffold -B

[0333]

Chem.

[0334] 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 equiv, 100 mg) and 6-bromopurine (H-4, 1.5 equiv., 170 mg) were 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 sealed. The reaction mixture was stirred vigorously at 110 °C overnight and then dried under vacuum. The residue was redissolved in DMSO and purified by preparative HPLC using a Gemini 5μm NX-C18 column (110 Å, 250×21.2 mm) at a flow rate of 10 mL / min (eluting with 10% - 100% MeCN in 0.1% TFA), and H-5 (61 mg, 36% yield) was obtained as a pale white solid. 1 H NMR (600 MHz, DMSO-d (6 )): 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-d (6 )): 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] + (calcd for C 12 H 14 N5O2S, 292.0863).

[0335] Preparation of 4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxylic acid (acid scaffold-B): H-5 (61 mg) was dissolved in 2 M NaOH (3.5 mL) and THF (3.5 mL), and stirred at room temperature overnight. The reaction solution was acidified with 2 M HCl (5 mL) and dried under vacuum. The crude product was washed and desalted with H2O (1 mL x 3 times), and acid scaffold-B (49 mg, 90% yield) was obtained as a pale orange solid. 1 H 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 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] + (calcd for C 10 H 10 N5O2S, 264.0550).

[0336] Synthesis of acid scaffold-C. Acid scaffold-C was synthesized from commercially available H-6 and H-4. Specifically, H-6 was reacted with H-4 in the presence of diisopropylethylamine (DIPEA) to obtain H-7, which was then reacted with KOH to obtain acid scaffold-C as shown in Scheme 4. Scheme 4: Preparation of Acid Scaffold C

[0337]

Chemical formula

[0338] Preparation of Ethyl 4-(7H-purin-6-yl)thiomorpholine-2-carboxylate (H-7): To a mixture of ethyl morpholine-2-carboxylate (H-6, 1 equiv., 100 mg), 6-bromopurine (H-4, 1.1 equiv., 113 mg), and 4 Å MS, DIPEA (3 equiv., 375 μL) and EtOH (5 mL) were added. The reaction mixture was stirred vigorously at 95 °C overnight and then dried under vacuum. The residue was redissolved in DMSO and then purified by preparative HPLC using a Gemini 5 μm NX-C18 column (110 Å, 250 × 21.2 mm) at a flow rate of 10 mL / min (eluting with 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] + (calcd for C 12 H 16 N5O2S, 294.1025).

[0339] Preparation of 4-(7H-purin-6-yl)thiomorpholine-2-carboxylic acid (Acid Scaffold-C): H-7 (150 mg) was dissolved in 2M NaOH (5 mL) and THF (5 mL) and stirred at room temperature overnight. The reaction solution was acidified with 2M hydrochloric acid (6 mL) and dried under reduced pressure under vacuum. The crude product was washed and desalted with H2O (1 mL x 3 times) to give Acid Scaffold-C (120 mg, 88% yield) as a white solid.1 1H 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 13C 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]+ (calcd for C11H11N5O2S, 266.0712). + (calcd for C 10 H 12 N5O2S, 266.0712).

[0340] Synthesis of thiazine carboxamide. Each of the following compounds was synthesized according to the general procedure of amide coupling shown in Scheme 5. Scheme 5: General Procedure for Amide Coupling

[0341]

Chemical formula

[0342] To a mixture of acid scaffold - A, B, or C (1 equiv.) and primary / secondary amine or N - Boc - ethylenediamine (1.5 equiv.), HATU (1.5 equiv.), DIPEA (10 equiv.), and DMF were added. The reaction mixture was vigorously stirred overnight at room temperature and then dried under vacuum. The residue was redissolved in DMSO and then purified by preparative HPLC using a Gemini 5μm NX - C18 column (110Å, 250×21.2 mm) at a flow rate of 10 mL / min (eluting with 10% - 100% MeCN in 0.1% TFA) to obtain the carboxamide or Boc - protected amide product. The purified Boc - protected amide product was deprotected in DCM:TFA (2:1) and then purified by HPLC using a Synergi 5μm Hydro - RP column (110Å, 250×21.2 mm) at a flow rate of 10 mL / min (eluting with 10 - 100% MeCN in 0.1% TFA) to obtain the deprotected amide product.

[0343] Preparation of N-(2 - aminoethyl)-4-(7H - purin - 6 - yl)-3,4 - dihydro - 2H - 1,4 - thiazine - 6 - carboxamide (TFA salt) (183A049E):

[0344]

Chemical Structure

[0345] N-(2 - aminoethyl)-4-(7H - purin - 6 - yl)-3,4 - dihydro - 2H - 1,4 - thiazine - 6 - carboxamide (TFA salt) (183A049E) was obtained as an off - white solid (yield 33%). 1 H NMR (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); 1313C NMR (150 MHz, DMSO-d6): δ165.3, 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] + (calcd for C 12 H 16 N7OS, 306.1137).

[0346] 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):

[0347]

Chem.

[0348] 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 as a colorless solid (yield 70%). 1 1H NMR (600 MHz, 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 13C NMR (150 MHz, DMSO-d6): δ165.3, 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] + (calcd for C 13 H 17N6OS, 305.1179).

[0349] Preparation of N-(2-aminoethyl)-4-(7H-purin-6-yl)thiomorpholine-2-carboxamide (TFA salt) (183A051C):

[0350]

Chem.

[0351] N-(2-aminoethyl)-4-(7H-purin-6-yl)thiomorpholine-2-carboxamide (TFA salt) (183A051C) was obtained as an off-white solid (yield 87%). 1 H NMR (600 MHz, 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).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 (150 MHz, 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] + (calcd for C 12 H 18 N7OS, 308.1294).

[0352] Preparation of N-methoxy-N-methyl-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A047C):

[0353]

Chem.

[0354] N-Methoxy-N-methyl-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A047C) was obtained as a pale orange solid (yield 93%). 1 H NMR (600 MHz, DMSO-d (6 )): 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 C 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] + (calcd for C 12 H 15 N6O2S, 307.0972).

[0355] 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):

[0356]

Chemical Structure

[0357] 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 as an off-white solid (yield 32%). 1 H NMR (600 MHz, DMSO-d (6)): δ 13.54 (broad singlet, 1H), 9.54 (singlet, 1H), 8.51 (triplet, J = 5.1 Hz, 1H), 8.50 (singlet, 1H), 7.63 (doublet, J = 1.9 Hz, 1H), 7.58 (doublet, J = 1.9 Hz, 1H), 4.61 (doublet, J = 5.1 Hz, 2H), 4.59 (multiplet, 2H), 3.84 (singlet, 3H), 3.17 (multiplet, 2H); 13 C NMR (150 MHz, DMSO-d (6 )): 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] + (calculated for C 15 H 17 N8OS, 357.1246).

[0358] (4-(7H-Purin-6-yl)-3,4-dihydro-2H-1,4-thiazin-6-yl)(morpholino)methanone (183A047F) Preparation:

[0359] [Chemical Structure]

[0360] (4-(7H-Purin-6-yl)-3,4-dihydro-2H-1,4-thiazin-6-yl)(morpholino)methanone (183A047F) was obtained as an off-white solid (yield 60%). 1 H NMR (600 MHz, DMSO-d (6 )): δ 9.04 (broad singlet, 1H), 8.47 (singlet, 1H), 8.41 (singlet, 1H), 4.83 (multiplet, 2H), 3.63 (multiplet, 4H), 3.58 (multiplet, 4H), 3.21 (multiplet, 2H); 1313C 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] + (calcd for C 14 H 17 N6O2S, 333.1134).

[0361] Preparation of N-(2-Acetamidoethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050B):

[0362]

Chemical Structure

[0363] N-(2-Acetamidoethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050B) was obtained as an off-white solid (yield 79%). 1 1H 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): δ169.6, 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] + (calcd for C 14 H 18 N7O2S, 348.1243).

[0364] Preparation of N-(2-methoxyethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050C):

[0365]

Chemical formula

[0366] N-(2-methoxyethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050C) was obtained as an off-white solid (yield 99%). 1 H NMR (600 MHz, DMSO-d (6 )): 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 C 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] + (calcd for C 13 H 17 N6O2S, 321.1134).

[0367] Preparation of N-(2-hydroxyethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050D):

[0368]

Chemical formula

[0369] N-(2-Hydroxyethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A050D) was obtained as an off-white solid (yield 84%). 1 H 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 C 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] + (calcd for C 12 H 15 N6O2S, 307.0977).

[0370] Preparation of N-(2-(methylamino)ethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (TFA salt) (183A050E):

[0371]

Chem.

[0372] N-(2-(methylamino)ethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (TFA salt) (183A050E) was obtained as an off-white solid (yield 45%). 11H 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); 13 13C NMR (150 MHz, DMSO-d (6 6): δ165.4, 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] + (calcd for C 13 15 18 H17N7OS, 320.1294).

[0373] 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. Scheme 6. Preparation of 183A046C

[0374]

Chem.

[0375] H-5 (6 mg) was stirred overnight at room temperature in 2 M NH2Me / MeOH solution (5 mL) in the presence of 4 Å MS and then dried under vacuum. The residue was redissolved in DMSO and subsequently purified by semi-preparative HPLC using a Gemini 5 μm NX-C18 column (110 Å, 250 × 10 mm) at a flow rate of 4 mL / min (eluted with 20% MeCN in 0.1% TFA) to obtain 183A046C (4.5 mg). N-Methyl-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A046C) was obtained as an off-white solid (yield 71%). 1 H NMR (600 MHz, DMSO-d6): δ 13.48 (br s, 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), 4.9 Hz, 1H), 4.57 (m, 2H), 4.59 (m, 2H), 3.15 (m, 2H), 2.67 (d, J = 4.6 Hz, 3H); 13 C NMR (150 MHz, DMSO-d (6 )): 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] + (calcd for C 11 H 13 N6S, 277.0872).

[0376] Preparation of N-(2-(picolinamido)ethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A049C): N-(2-(picolinamido)ethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A049C) was prepared as described in Scheme 7. Scheme 7. Preparation of 183A049C

[0377] [Chem.]

[0378] To a mixture of pyridine-2-carboxylic acid (H-9, 1 equiv., 20 mg) and ethylenediamine (H-10, 10 equiv., 108 μL), HATU (1.5 equiv., 93 mg), DIPEA (10 equiv., 283 μL) and DMF (2 mL) were added. The reaction mixture was stirred overnight at room temperature and then dried under vacuum. The residue was redissolved in DMSO and purified by semi-preparative HPLC using a Synergy 5μm Polar-RP column (110 Å, 250×10 mm) at a flow rate of 4 mL / min (eluting with 5% MeCN in 0.1% TFA) to give H-11 (13.6 mg, 50% yield). To a mixture of acid scaffold -B (1 equiv., 4.7 mg) and H-11 (1.5 equiv., 6.0 mg), HATU (1.5 equiv., 10 mg), DIPEA (10 equiv., 30 μL), and DMF (0.5 mL) were added. The reaction mixture was vigorously stirred overnight at room temperature and then dried under vacuum. The residue was redissolved in DMSO and purified by semi-preparative HPLC using a Gemini 5μm NX-C18 column (110 Å, 250×10 mm) at a flow rate of 4 mL / min (eluting with 23% MeCN in 0.1% TFA) to give 183A049C (3.7 mg). N-(2-(Picolinamido)ethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A049C) was obtained as an off-white solid (50% yield). 1 H NMR (600 MHz, DMSO-d (6)): δ 9.37 (broad singlet, 1H), 8.97 (triplet, J = 5.8 Hz, 1H), 8.65 (doublet, J = 4.8 Hz, 1H), 8.48 (singlet, 1H), 8.39 (singlet, 1H), 8.05 (doublet, J = 7.7 Hz, 1H), 8.01 (triplet, J = 7.7 Hz, 1H), 7.88 (triplet, J = 5.4 Hz, 1H), 7.61 (doublet of doublets, J = 4.8, 7.7 Hz, 1H), 4.59 (broad singlet, 2H), 3.45 (multiplet, 2H), 3.38 (multiplet, 2H), 3.16 (multiplet, 2H); 13 C NMR (150 MHz, DMSO-d (6 )): 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] + (calcd for C 18 H 19 N8O2S, 411.1351).

[0379] 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. Scheme 8. Preparation of 183A049F

[0380]

Chemical Structure

[0381] To a mixture of benzoic acid (H-12, 1 equiv., 20 mg) and ethylenediamine (H-10, 3 equiv., 32 μL) were added HATU (1.5 equiv., 93 mg), DIPEA (10 equiv., 283 μL) and DMF (2 mL). The reaction mixture was stirred overnight at room temperature and then dried under vacuum. The residue was redissolved in DMSO and purified by semi-preparative HPLC using a Synergy 5 μm Polar-RP column (110 Å, 250×10 mm) at a flow rate of 4 mL / min (eluting with 10% MeCN in 0.1% TFA) to give H-13 (8.7 mg, 33% yield). To a mixture of acid scaffold-B (1 equiv., 3.7 mg) and H-13 (8.7 mg) were added HATU (1.5 equiv., 8 mg), DIPEA (10 equiv., 24 μL), and DMF (0.4 mL). The reaction mixture was stirred vigorously overnight at room temperature and then dried under vacuum. The residue was redissolved in DMSO and purified by semi-preparative HPLC using a Gemini 5 μm NX-C18 column (110 Å, 250×10 mm) at a flow rate of 4 mL / min (eluting with 30% MeCN in 0.1% TFA) to give 183A049F (2.5 mg). N-(2-Benzamidoethyl)-4-(7H-purin-6-yl)-3,4-dihydro-2H-1,4-thiazine-6-carboxamide (183A049F) was obtained as an off-white solid (43% yield). 1 H NMR (600 MHz, DMSO-d6): δ 9.38 (br s, 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 C NMR (150 MHz, DMSO-d (6)): 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] + (calcd for C 19 H 20 N7O2S, 410.1399).

[0382] Example 10 This example provides the IC 50 value of the inhibitory activity against the enzyme-active chimeric protein, JPKAcα, which is observed in almost all FLHCC patients, where the expression level of the fusion kinase in tumor cells is more than 10-fold higher than that of wild-type PKA (wt-PKA) in adjacent normal liver tissue.

[0383] IC of the inhibitory activity against the RIα2:JPKAcα2 chimeric kinase holoenzyme 50The compound was tested to determine the value. Three × dose-response curves were set over a final compound concentration range of 0 - 10 μM in 100 mM Tris-HCl pH 7.5 containing 1 μM cAMP, 50 μM ATP, and 0.4% DMSO (all final concentrations). A cAMP / ATP-free control was also added for background normalization. Using a 12-channel multi-channel pipette, 20 μL of the compound / cAMP / ATP solution was added to the reaction wells containing 40 μL of 1.5× concentration 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) to initiate quadruplicate reactions. After initiation by addition, the reaction was allowed to proceed for 45 minutes before adding 15 μL of 0.5 M EDTA to quench the reaction. The quenched reaction was then transferred to an assay-binding plate prepared, and ELISA was developed as described above for the primary screening assay. For each reaction well, the observed RFU was converted to the % activity measurement value normalized as above using the cAMP / ATP-free well as the low control and the vehicle control (0 μM) as the high control. The %JPKAcα activity curve of compound 183A056C is shown in Figure 106.

[0384] Next, the normalized activity measurement values were fitted to the following equation using non-linear regression least squares method with variable slope (GraphPad Prism Software, San Diego, CA), and the IC 50 value was calculated by the following equation.

[0385]

Equation

[0386] JPKAcα IC 50 The average value (from three dose-response curves) is shown in Table 5.

[0387]

Table 5

[0388] Example 11 This example provides the results of the kinome profiling and dose response testing of the compound of one aspect of the present invention.

[0389] The pan-human kinome profiling (370 kinases), followed by a 10-point dose response test against a 30-kinase selected panel, was performed by Reaction Biology Corp. (Malvern, PA, USA) using the radiometric HOTSPOT TM kinase assay (Anastassiadis, et al. Biotechnol., 29(11): 1039-U117 (2011)). All kinase reactions were performed with 10 μM ATP. Compounds 1 and 3 were tested at two concentrations (2 μM and 50 nM) in the pan-human kinome profiling and with 3-fold serial dilutions starting from 20 μM in the 10-point dose response test of Compound 1. The description of this method and details of the kinome composition are available from Reaction Biology Corporation. The graphical representation of the inhibitory activities of Compounds 1 and 3 across the tested kinome is shown in the CORAL software package (Metz, et al., Cell Syst., 7(3): 347-350 e1 (2018)) (see FIGS. 5A-5C and 15-18).

[0390] By kinase profiling, the IC50 value of Compound 1 was ~11~90 nM, revealing potent inhibition of selective serine / threonine kinases of the CLK, DYRK, and PKG families. Kinase 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 (Figures 111 and 112). DYRK / CLK kinases belong to the CMGC group of serine / threonine kinases. These kinases are involved in various pathological processes such as neurodegenerative diseases (Down syndrome, Alzheimer's disease, etc.), diabetes, solid cancers (glioblastoma, breast cancer, pancreatic cancer, etc.), leukemia, and infections by viruses and parasites. Cyclic GMP-dependent protein kinase (PKG) is an important regulator of the intracellular nitric oxide (NO) signaling pathway. Dysregulation of PKG signaling is associated with most heart diseases including heart failure. Furthermore, PKG derived from the malaria parasite has also been identified and validated as a target for antimalarial chemotherapy.

[0391] Modification of the imidazole moiety by monobromination improved the potency of Compound 3 against DYRK kinase by at least two-fold when tested at 50 nM, but unexpectedly, the overall selectivity profile across the 370 kinase panel was very similar between Compound 1 and Compound 3 (Figure 111, Tables 6 - 8 below). Table 6 shows the kinome profiling of Compound 1 against a panel of 370 human protein kinases. Data for the top 50 hits when Compound 1 was tested at 50 nM are shown in Table 6. The mean inhibition rate was obtained from a single experiment performed in duplicate. Table 7 is the kinome profiling of Compound 3 against a panel of 370 human protein kinases. Data for the top 50 hits when Compound 3 was tested at 50 nM are shown in Table 7. The mean inhibition rate was obtained from a single experiment performed in duplicate. Table 8 shows the concentration (nM) of apritanin analogs that cause a 50% decrease in the catalytic activity (IC50) of 20 selected kinases. The averaged IC50 values were obtained from a single experiment performed in duplicate or from two independent studies.

[0392]

Table 6

[0393]

Table 7

[0394]

Table 8

[0395] Figures 107A and 107B are graphs showing % kinase activity at 50 nM (Figure 107A) and 2 μM (Figure 107B). The difference in activity between the compounds is more pronounced at the 2 μM concentration. Figure 108 shows the top 10 most active kinases (from left to right for each kinase listed are the bars for compound 183A034G / apritaniacin A(1), 183A041B / apritaniacin A1(1a), and 183A049E). Figures 109 and 110 also show the percentage of kinase activity of 183A034G / apritaniacin A1, 183A041B / apritaniacin A1(1a), and 183A049E against several kinases.

[0396] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0397] The use of the terms "a", "an", "the", "at least one", and similar reference words in the context of describing the present invention (especially in the context of the following claims) is to be construed as covering both the singular and the plural forms, as described herein or as otherwise clearly contradicted by the context. The use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") means, unless otherwise indicated herein or unless clearly contradicted by the context, one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B). The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise specified. References to ranges of values herein are intended to function simply as a shorthand way of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or unless 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 illuminate the present invention and does not limit the scope of the present invention unless otherwise claimed. No language in this specification should be construed as indicating that any element not recited in any one of the claims is essential to the practice of the invention.

[0398] A preferred embodiment and aspect of the present invention are as described herein, including the best mode known to the inventors for carrying out the present invention. Variations of those 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 adopt such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto that are permitted by the applicable law. Further, all possible combinations of the above-described elements in all possible variations thereof are included in the present invention, unless otherwise described herein or clearly inconsistent in context.

Claims

1. A compound of formula (I) 【Chemical 1】 where [Chemical 2] is a single bond or a double bond, and X １ and X ２ are each independently CH, CR ６ or N; X ３ is S, S=O, or S(=O) ２ and; R １ is H or -NR ２ R ３ wherein; R ２ is H or C １ -C ３ is alkyl; R ３ is aryl; R ６ is C １ -C ３ alkyl; A is optionally, when present, -C(O)-, -C(O)O-, -C(O)NH-, -C(O)N(C １ -C ３ -alkyl)-; -C(O)NH-(C １ -C ６ alkyl)-NHC(O)-, -NH; D is optionally, when present, C １ -C ６ alkyl, C ３ -C ８ cycloalkyl, C ３ -C ８ heterocycloalkyl, -(C １ -C ６ alkyl)-O-, -(C １ -C ６ alkyl)-NH-, -(C １ -C ３ alkyl)-O-(C １ -C ３ alkyl)-, or -(C １ -C ３ alkyl)-NH-(C １ -C ３ alkyl)-, where any of the foregoing alkyl or cycloalkyl groups is optionally substituted with one or more substituents selected from hydroxy, C １ -C ６ alkyl, amino, C １ -C ６ alkylamino, or di-C １ -C ６ alkylamino; -NH-aryl, and combinations thereof; and E is:[[]] C １ -C ６ alkyl or alkoxy, -(C １ -C ６ alkyl)-OH, -(C １ -C ６ alkyl)-COOH, -(C １ -C ６ alkyl)-NH ２ , halo, nitro, hydroxy, amino, C １ -C ６ alkylamino, di-C １ -C ６ alkylamino; -NH-aryl, C １ -C ６ haloalkyl, C ３ -C ８ cycloalkyl or heterocycloalkyl, fused C ３ -C ８ cycloalkyl or heterocycloalkyl, aryl or heteroaryl, fused aryl or heteroaryl, -CN, -(C １ -C ３ alkyl)-CN, carbonyl, and one or more substituents selected from the combinations thereof, optionally substituted aryl or heteroaryl; Amino, C １ -C ６ Alkylamino, or di-C １ -C ６ Alkylamino; or -NH-aryl; Hydroxy, C １ -C ６ -alkyl, amino, C １ -C ６ -alkylamino, or di-C １ -C ６ -alkyl-amino; -NH-aryl, and C optionally substituted with one or more substituents selected from combinations thereof １ -C ６ -alkyl or alkoxy; Hydroxy, C １ -C ６ -alkyl, amino, C １ -C ６ -alkylamino, di-C １ -C ６ -alkyl - amino; -NH - aryl, C ３ -C ８ -cycloalkyl or heterocycloalkyl, fused C ３ -C ８ -cycloalkyl or heterocycloalkyl, aryl or heteroaryl, fused aryl or heteroaryl, and optionally substituted with one or more substituents selected from combinations thereof C ３ -C ８ -cycloalkyl or heterocycloalkyl; -C(O)NH ２ ; -C(O)OH; -C(O)H; -N-(C １ -C ６ -alkyl)-acrylamide; halogen; or hydrogen), or a pharmaceutically acceptable salt thereof.

2. 【Fig. 3】 is a single bond or a double bond, and X １ and X ２ are each independently 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 C １ -C ３ is alkyl; R ３ is aryl; A is optionally, when present, -C(O)-, -C(O)O-, -C(O)NH-, -C(O)N(C １ -C ３ -alkyl)-; -C(O)NH-(C １ -C ６ -alkyl)-NH-C(O)-, -NH and is; D is optionally, when present, C １ -C ６ alkyl, -(C １ -C ６ alkyl)-O-, -(C １ -C ６ alkyl)-NH-, -(C １ -C ３ alkyl)-O-(C １ -C ３ alkyl)-, or -(C １ -C ３ alkyl)-NH-(C １ -C ３ alkyl)-, where any of the aforementioned alkyl groups is optionally substituted with hydroxy; E is:[[]] C １ -C ６ alkyl or alkoxy, halo, nitro, hydroxy, C １ -C ６ haloalkyl, -CN, -(C １ -C ３ aryl or heteroaryl optionally substituted with carbonyl; Amino, C １ -C ６ Alkylamino, or di-C １ -C ６ Alkylamino; or -NH-aryl; C optionally substituted with hydroxy １ -C ６ alkyl or alkoxy; C optionally substituted with hydroxy ３ -C ８ cycloalkyl or heterocycloalkyl; -C(O)NH ２ ; -C(O)OH; -C(O)H; -N-(C １ -C ６ alkyl)-acrylamide; halogen; or hydrogen; The compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. 【Fig. 4】 The compound of claim 1 or 2, wherein

4. The compound of formula (I) is of formula (Ia): 【Chemical Formula 5】 The compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

5. The compound of formula (I) is of formula (Ib): 【Chemical Formula 6】 The compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

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

7. (i) A and D are absent, and E is halogen; -C(O)OH; -C(O)H; C １ -C ３ aryl or heteroaryl optionally substituted with alkyl or halo; or C optionally substituted with hydroxy ３ -C ８ cycloalkyl or heterocycloalkyl; (ii) A does not exist; D is C １ -C ６ alkyl, optionally C １ -C ３ alkyl; E is -C(O)NH ２ or C optionally substituted with hydroxy ３ -C ８ cycloalkyl or heterocycloalkyl; (iii) A is -NH-, -C(O)NH- or -C(O)N(C １ -C ３ -alkyl)-; D is absent or C １ -C ６ alkyl, optionally C １ -C ３ alkyl; E is amino, C １ -C ６ alkylamino, or di-C １ -C ６ alkylamino; or -NH-aryl; optionally substituted with hydroxy C １ -C ６ alkyl or alkoxy or C １ -C ３ alkyl or alkoxy; or C １ -C ３ alkyl or alkoxy, halo, nitro, hydroxy, C １ -C ３ haloalkyl, -CN, -(C １ -C ３ alkyl)-CN, or aryl or heteroaryl optionally substituted with carbonyl; (iv) A is absent, and D is -(C １ -C ６ -alkyl)-O-, -(C １ -C ６ -alkyl)-NH-, -(C １ -C ３ -alkyl)-O-(C １ -C ３ -alkyl)-, or -(C １ -C ３ -alkyl)-NH-(C １ -C ３ -alkyl)-, where any of the aforementioned alkyl groups is optionally substituted with hydroxy, and any of the aforementioned alkyl groups is optionally branched; E is C １ -C ６ -alkyl or C ３ -C ８ -cycloalkyl or heterocycloalkyl, which is optionally substituted with hydroxy; (v) A is -C(O)NH-(C １ -C ６ -alkyl)-NHC(O)-, optionally C(O)NH-(C １ -C ３ -alkyl)-NHC(O)-; D is absent; E is C １ -C ６ -alkyl or C １ -C ３ -alkyl; or C １ -C ３ -alkyl or alkoxy, halo, nitro, hydroxy, C １ -C ６ -haloalkyl, -CN, -(C １ -C ３ -alkyl)-CN, or aryl or heteroaryl optionally substituted with carbonyl; (vi) A is -C(O)O-; D is absent; E is C １ -C ６ alkyl; or (vii) A is -C(O)-; D is absent; E is heterosilalkyl, The compound of any one of claims 1 to 6.

8. The compound of formula (I) is of formula (Ic): 【Chemical Formula 7】 (wherein R ４ and R ５ are the same or different and each is H or halo), a compound according to claim 1 or a pharmaceutically acceptable salt thereof.

9. R ４ and R ５ one of which is halo, or R ４ and R ５の both of which are halo, the compound of claim 8

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

11. The compound is not aplidine A: 【Chemical Formula 8】 The compound of any one of claims 1 to 10.

12. The compound of formula (I) is 【Chemical Formula 9】 【Chemical 10】 【Chemical 11】 【Chemical 12】 【Chemical 13】 The compound of claim 1 or a pharmaceutically acceptable salt thereof, which is a compound selected from.

13. The compound of formula (I) is 【Chemical 14】 The compound of claim 12 or a pharmaceutically acceptable salt thereof, which is a compound selected from.

14. Compound: 【Chemical Formula 15】 or a pharmaceutically acceptable salt thereof.

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

16. With a purity of at least 80% or more 【Chemical 16】 And a pharmaceutical composition comprising a pharmaceutical carrier.

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

18. The method of 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 of claim 17, wherein the kinase is PKA, PKA / DNAJ, or cAMP-PKA.

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

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

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

23. A method of suppressing the immune system of a subject, the method comprising administering to the subject a compound of any one of claims 1 to 14 or a pharmaceutical composition of claim 15 or 16.

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

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

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

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

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

29. The method of claim 25, wherein the cancer is colorectal cancer.

30. A method of treating painful diabetic neuropathy in a subject, the method comprising administering to the subject a compound of any one of claims 1 to 14 or a pharmaceutical composition of claim 15 or 16.

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

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

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

34. A method for producing aplidine A 【Chemical 17】 comprising coupling a purine-thiazine conjugate having the following structure: 【Chemical 18】 with an imidazole having the following structure: 【Chemical Formula 19】 including coupling together.

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

36. The compound of claim 8: 【Chemical 20】 (Here, at least one of R ３ or R ４ is halogen), a preparation method of; This method comprises halogenating a compound of the formula: 【Chemical 21】 including.

37. The method of claim 36, wherein compound Ic is aplidine A.

38. The method of claim 37, wherein aplidine A is brominated using N-bromosuccinimide to provide: 【Chemical 22】 ​

Images