Compounds containing 2,4-diaminopyrimidine, methods for producing the same, and pharmaceutical compositions for preventing or treating cancer containing the same as an active ingredient

Abstract

The present invention relates to an isobenzofuran-1(3H)-one derivative having an HPK1 inhibitory activity and an MLK3 inhibitory activity, and a pharmaceutical composition containing the same for preventing or treating cancer, viral infections, Parkinson's disease, non-alcoholic steatohepatitis, or tuberculosis. The above compound can be usefully used as a composition for preventing or treating cancer, including administration in combination with an anticancer drug or a cell therapy drug.

Description

Technical Field

[0001] The present invention relates to isobenzofuran-1(3H)-one derivatives and their uses, and more specifically, to isobenzofuran-1(3H)-one derivatives having HPK1 inhibitory activity and MLK3 inhibitory activity, and pharmaceutical compositions for preventing or treating cancer, viral infectious diseases, Parkinson's disease, non-alcoholic steatohepatitis or tuberculosis containing the same.

Background Art

[0002] Protein kinases are enzymes that catalyze the reaction of transferring the terminal phosphate group of adenosine triphosphate (ATP) to specific residues (tyrosine, serine, threonine) of proteins, and are involved in signals that regulate the activity, growth, and differentiation of cells in response to extracellular mediators and environmental changes.

[0003] Generally, protein kinases are classified into serine and / or threonine kinase groups and tyrosine kinase groups according to the substrates they phosphorylate (Hanks, S.K. et al., FASEB J., 9(8), 576-596, 1995). The serine / threonine kinase group includes protein kinase C isoforms (Newton, A.C., J Biol Chem., 270(48), 28495-28498, 1995), cyclin-dependent kinase groups, and cdc2 (Pines, J., Trends Biochem Sci., 18(6), 197, 1993). The tyrosine kinase group is divided into membrane-spanning growth factor receptors including epidermal growth factor receptor (Iwashita, S. et al., Cell Signal., 4(2), 123-132, 1992), cytoplasmic non-receptor kinases including p56tck, p59fYn, ZAP-70, and C-terminal Src kinase (Chan, A.C. et al., Annu Rev Immunol., 12, 555-592, 1994).

[0004] Undesirably high protein kinase activity is directly or indirectly associated with a number of diseases resulting from abnormal cellular function. For example, diseases can occur due to failure of the appropriate regulatory mechanisms of kinases associated with mutation, overexpression, or inappropriate enzyme activity; or due to over- or under-production of factors participating in upstream or downstream signaling of cytokines or kinases. Therefore, selective inhibition of kinase activity can be a beneficial target for the development of new drugs for the treatment of diseases.

[0005] MLK3 is one of the MLK (mixed lineage kinase) family and can regulate MAP kinases (especially JNK), which are important for cancer cell proliferation and survival, using various signaling systems.

[0006] In addition, in recent years, numerous studies have been conducted to develop therapeutic agents for cancer, viral infectious diseases, Parkinson's disease, non-alcoholic steatohepatitis, etc. by developing compounds that inhibit the enzymatic activity of MLK3 (Chadee, D.N., Can J Physiol Pharmacol., 91(4), 268-274, 2013; Rattanasinchai, C. et al., Cancers (Basel)., 8(5), 51, 2016; Xu, H. et al., J Virol., 93(18), e00758-19, 2019; Saminathan, P. et al., J Neuroimmune Pharmacol., 14(1), 44-51, 2019; Goodfellow, V.S. et al., J Med Chem., 56(20), 8032-8048, 2013; Jiang, J.X. et al., Liver Int., 34(8), 1131-1132, 2014; Tomita, K. et al., JCI Insight., 2(15), 94488, 2017; Ibrahim, S.H. et al., Liver Int., 34(3), 427-437, 2014; Parkinson Study Group PRECEPT Investigators, Neurology, 69(15), 1480-1490, 2007; Kline, E.M. et al., Exp Neurol., 318, 157-164, 2019).

[0007] Hematopoietic progenitor kinase 1 (HPK1), originally cloned from hematopoietic progenitor cells, belongs to a large family of mitogen-activated protein kinase kinase kinase kinases (MAP4Ks), which includes MAP4K1 / HPK1, MAP4K2 / GCK, MAP4K3 / GLK, MAP4K4 / HGK, MAP4K5 / KHS, and MAP4K6 / MINK (Hu, M.C., et al., Genes Dev, 1996. 10(18): p. 2251-64). HPK1 is of particular interest because it is predominantly expressed in hematopoietic cells such as T cells, B cells, macrophages, dendritic cells, neutrophils, and mast cells (Hu, M.C., et al., Genes Dev, 1996. 10(18): p. 2251-64; Kiefer, F., et al., EMBO J, 1996. 15(24): p. 7013-25). HPK1 kinase activity has been shown to be induced upon activation of the T cell receptor (TCR) (Liou, J., et al., Immunity, 2000. 12(4): p. 399-408), B cell receptor (BCR) (Liou, J., et al., Immunity, 2000. 12(4): p. 399-408), transforming growth factor receptor (TGF-βR) (Wang, W., et al., J Biol Chem, 1997. 272(36): p. 22771-5; Zhou, G., et al., J Biol Chem, 1999. 274(19): p. 13133-8), or Gs-coupled PGE2 receptors (EP2 and EP4) (Ikegami, R., et al., J Immunol, 2001. 166(7): p. 4689-96). Thus, HPK1 regulates diverse functions of various immune cells.

[0008] HPK1 is important in regulating the functions of various immune cells and is involved in autoimmune diseases and antitumor immunity (Shui, J.W., et al., Nat Immunol, 2007.8(1): p. 84-91; Wang, X., et al., J Biol Chem, 2012.287(14): p. 11037-48). HPK1 knockout mice are more sensitive to the induction of experimental autoimmune encephalomyelitis (EAE) (Shui, J.W., et al., Nat Immunol, 2007.8(1): p. 84-91). In humans, HPK1 was downregulated in peripheral blood mononuclear cells of psoriatic arthritis patients and in T cells of systemic lupus erythematosus (SLE) patients (Batliwalla, F.M., et al., Mol Med, 2005.11(1-12): p. 21-9). According to these observations, attenuation of HPK1 activity may be attributed to the patients' autoimmunity. Also, HPK1 can control antitumor immunity through a T cell-dependent mechanism. In a PGE2-producing lung cancer tumor model, tumors developed relatively slower in HPK1 knockout mice compared to wild-type mice (see US2007 / 0087988). Also, it was shown that adoptive transfer of HPK1-deficient T cells was more effective in controlling tumor growth and metastasis compared to wild-type T cells (Alzabin, S., et al., Cancer Immunol Immunother, 2010.59(3): p. 419-29). Similarly, BMDCs from HPK1 knockout mice were more effective than wild-type BMDCs in increasing the T cell response to eradicate Lewis lung carcinoma (Alzabin, S., et al., J Immunol, 2009.182(10): p. 6187-94).In addition, although it has been reported that cancer patients with high expression of HPK1 have a lower survival rate compared to cancer patients showing low expression, if the expression of HPK1 in cancer cells themselves is suppressed, cancer growth is suppressed, and in various CAR-T cells such as CD19CAR-T, Her2CAR-T, and BCMA (B cell maturation antigen) CAR-T (Chimeric Antigen Receptor-modified T cell), which are anti-cancer immune cells, suppressing HPK1 expression recently reported that the anti-cancer immune ability of CAR-T increases and effectively inhibits cancer cell proliferation and growth (Si, J., et al., Cancer Cell, 2020, 38(4): p. 551-66). These data suggest that, along with the restricted expression of HPK1 in hematopoietic cells and the lack of effect on the normal development of immune cells, HPK1 is an excellent drug target for enhancing anti-tumor immunity. In immunotherapy using T cells, the most important is ex vivo culture, which is a technique for rapidly growing a large number of high-quality T cells on a culture vessel (Dudley, ME., et al., Nat. Rev. Cancer, 2003, 3(9): p. 666-75). When a technique capable of regulating the differentiation and proliferation of hematopoietic cells in a culture vessel is used in combination, a new T cell culture technique for immunotherapy can be developed. Therefore, there is a need for a new compound that regulates HPK1 activity.

[0009] On the other hand, as a prior art document related to isobenzofuran-1(3H)-one derivatives having HPK1 inhibitory activity and MLK3 inhibitory activity and a pharmaceutical composition containing the same for preventing or treating cancer, viral infectious diseases, Parkinson's disease, non-alcoholic steatohepatitis, or tuberculosis, Korean Registered Patent No. 10-0832602 has disclosed a novel polycyclic compound related to the activity of the MLK3 enzyme and its uses. However, there has been no report yet mentioning the isobenzofuran-1(3H)-one derivatives such as Chemical Formulas 1 and 2 of the present invention, the HPK1 and MLK3 inhibitory activities of the derivatives, and the fact that cancer, viral infectious diseases, Parkinson's disease, non-alcoholic steatohepatitis, or tuberculosis can be treated thereby.

[0010] Therefore, in the process of researching isobenzofuran-1(3H)-one derivatives, the present inventors confirmed all of the HPK1 inhibitory activity and the inhibitory activity against MLK3, and thus completed the present invention.

Prior Art Documents

Patent Documents

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Patent Document 1

Patent Document 2

Non-Patent Documents

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Summary of the Invention

Problems to be Solved by the Invention

[0013] An object of the present invention is to provide an isobenzofuran-1(3H)-one derivative and its use, and more specifically, an isobenzofuran-1(3H)-one derivative having HPK1 inhibitory activity and MLK3 inhibitory activity, and a pharmaceutical composition containing the same for preventing or treating cancer, viral infectious diseases, Parkinson's disease, non-alcoholic steatohepatitis or tuberculosis.

Means for Solving the Problems

[0014] The present invention relates to a compound represented by the following Chemical Formula 1, 2, 3 or 4, or a pharmaceutically acceptable salt thereof.

[0015] The present invention relates to a compound represented by the following Chemical Formula 1 or Chemical Formula 2, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[0016]

Chemical formula

[0017]

Chemical formula

[0018] In the above Chemical Formula 1 or Chemical Formula 2,

[0019] R1 is independently hydrogen, C 1 -C 4 alkyl or C 1 -C 4 alkoxy, or

[0020] alternatively, two R 1 and R 1 together with the adjacent C form a 3- to 7-membered saturated spiro ring;

[0021] R 2 is hydrogen, C 1 -C 4 alkyl or C 1 -C 4 alkoxy;

[0022] R 3 is hydrogen, C 1 -C 4 alkyl, halo C 1 -C 4 alkyl, hydroxy C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halo C 1 -C 4 alkoxy, C 1 -C 4 alkoxy C 1 -C 4 alkoxy, -COR 4 , -CONH 2 , -CONHR4, -CON(R 4 ) 2 , -SO 3 H, or -SO 2 R 4 ;

[0023] R 4 is substituted or unsubstituted 3- to 12-membered cycloalkyl, substituted or unsubstituted 3- to 12-membered heterocycloalkyl, substituted or unsubstituted 4- to 12-membered aryl, substituted or unsubstituted 4- to 12-membered heteroaryl, substituted or unsubstituted 3- to 12-membered cycloalkyl C 1 -C 4 alkyl, substituted or unsubstituted 3- to 12-membered heterocycloalkyl C 1 -C 4 alkyl, substituted or unsubstituted 4- to 12-membered aryl C 1 -C 4 alkyl, substituted or unsubstituted 4- to 12-membered heteroaryl C 1 -C 4 alkyl, substituted or unsubstituted C 1 -C 4 alkyl, halo C 1 -C 4 alkyl, hydroxy C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halo C 1 -C 4 alkoxy, or C 1 -C4 Alkoxy C 1 -C 4 is alkoxy,

[0024] and further, here, the substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, or alkyl is OH, NO 2 , NH 2 , CN, halogen, C 1 -C 4 alkyl, halo C 1 -C 4 alkyl, hydroxy C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halo C 1 -C 4 alkoxy, and C 1 -C 4 alkoxy C 1 -C 4 alkoxy substituted with one or more substituents selected from the group consisting of;

[0025] R 5 is independently hydrogen, C 1 -C 4 alkyl, or C 1 -C 4 alkoxy, or

[0026] alternatively, two Rs 5 and R 5 together with the adjacent C form a 3- to 7-membered saturated spiro ring;

[0027] A is O or NH;

[0028] B is O or S;

[0029] X is halogen; and

[0030] n is an integer from 0 to 6; is.

[0031] The present invention also relates to a compound represented by the following Chemical Formula 3 or Chemical Formula 4, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[0032]

Chem.

[0033]

Chem.

[0034] In the above Chemical Formula 3 or Chemical Formula 4,

[0035] R 1 each independently represents hydrogen, C 1 -C 4 alkyl or C 1 -C 4 alkoxy, or

[0036] alternatively, two Rs 1 and R 1 together with the adjacent C form a 3- to 7-membered saturated spiro ring;

[0037] R 2 represents hydrogen, C 1 -C 4 alkyl, or C 1 -C 4 alkoxy;

[0038] R 3 represents hydrogen, C 1 -C 4 alkyl, halo C 1 -C 4 alkyl, hydroxy C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halo C 1 -C 4 alkoxy, C 1 -C 4 alkoxy C 1 -C 4 alkoxy, -COR4 、 -CONH 2 、 -CONHR 4 、 -CON(R 4 ) 2 、 -SO 3 H, or -SO 2 R 4 ; and

[0039] R 4 is a substituted or unsubstituted 3- to 12-membered cycloalkyl, a substituted or unsubstituted 3- to 12-membered heterocycloalkyl, a substituted or unsubstituted 4- to 12-membered aryl, a substituted or unsubstituted 4- to 12-membered heteroaryl, a substituted or unsubstituted 3- to 12-membered cycloalkylC 1 -C 4 alkyl, a substituted or unsubstituted 3- to 12-membered heterocycloalkylC 1 -C 4 alkyl, a substituted or unsubstituted 4- to 12-membered arylC 1 -C 4 alkyl, a substituted or unsubstituted 4- to 12-membered heteroarylC 1 -C 4 alkyl, a substituted or unsubstituted C 1 -C 4 alkyl, haloC 1 -C 4 alkyl, hydroxyC 1 -C 4 alkyl, C 1 -C 4 alkoxy, haloC 1 -C 4 alkoxy, or C 1 -C 4 alkoxyC 1 -C 4 alkoxy; and

[0040] Further, herein, the substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, or alkyl may have OH, NO 2 、 NH 2 、 CN, halogen, C 1 -C 4Alkyl, halo C 1 -C 4 Alkyl, hydroxy C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy, halo C 1 -C 4 Alkoxy, and C 1 -C 4 Alkoxy C 1 -C 4 substituted with one or more substituents selected from the group consisting of alkoxy;

[0041] R 5 is independently hydrogen, C 1 -C 4 alkyl or C 1 -C 4 alkoxy, or

[0042] alternatively, two Rs 5 and R 5 together with the adjacent C form a 3- to 7-membered saturated spiro ring;

[0043] A is O or NH;

[0044] B is O or S;

[0045] X is halogen; and

[0046] n is an integer from 0 to 6;

[0047] Specifically, the said compound is 5 - ((5 - chloro - 2 - ((1,2,3,4 - tetrahydroisoquinolin - 6 - yl)amino)pyrimidin - 4 - yl)amino)-3,3 - dimethylisobenzofuran - 1(3H)-one (Compound 1);

[0048] 6 - ((5 - chloro - 2 - ((1,2,3,4 - tetrahydroisoquinolin - 6 - yl)amino)pyrimidin - 4 - yl)amino)-3,3 - dimethylisobenzofuran - 1(3H)-one (Compound 2); and

[0049] 5,5’-((5-chloropyrimidine-2,4-diyl)bis(azanediyl))bis(3,3-dimethylisobenzofuran-1(3H)-one) (Compound 3);

[0050] A compound represented by Chemical Formula 1, 2, 3 or 4, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, characterized by being selected from the group consisting of

[0051] In the present invention, the following terms have the following meanings unless otherwise specified. Any term that is not defined has the meaning understood in the art.

[0052] The terms "halo" and "halogen" are used in their ordinary meanings to represent substituents of fluoro, chloro, bromo or iodo.

[0053] The term "alkyl" means a straight-chain or branched-chain hydrocarbon group of a single bond. For example, there are methyl, ethyl, propyl, n-butyl, isobutyl, tert-butyl, 1-methylpropyl, etc.

[0054] The term "alkoxy" means an oxygen group bonded to a straight-chain or branched-chain saturated hydrocarbon of a single bond. For example, there are methoxy, ethoxy, propoxy, n-butoxy, tert-butoxy, 1-methylpropoxy, etc.

[0055] The term "haloalkyl" represents an alkyl group in which one or more hydrogen atoms of the alkyl group are replaced by a halo group, and the alkyl and halo groups are as disclosed above.

[0056] The term "haloalkoxy" represents an alkoxy group in which one or more hydrogen atoms of the alkoxy group are replaced by a halo group, and halo and alkoxy are as disclosed above.

[0057] The term "alkoxyalkoxy" represents an alkoxy group in which one or more hydrogen atoms of the alkoxy group are further replaced by other alkoxy groups, and the alkoxy is as disclosed above.

[0058] The terms "cyclic" and "ring" refer to a substituted or unsubstituted and / or heteroatom-containing or non-containing substituted or aromatic group which may be monocyclic, bicyclic or polycyclic.

[0059] The term "cycloalkyl" means a cyclic single-bonded saturated hydrocarbon group. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[3.1.1]heptyl, spiro[4.5]decyl, spiro[5.5]undecyl, adamantyl and the like.

[0060] The term "heterocycloalkyl" refers to a cyclic single-bonded saturated hydrocarbon group containing one or more heteroatoms such as N, O, or S, and depending on the number and type of heteroatoms contained in the ring and the number of carbon atoms, includes aziridinyl, pyrrolidinyl, piperidinyl, oxopiperidinyl, morpholinyl, piperazinyl, oxopiperazinyl, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, oxazepanyl, thiazepanyl, dioxothiazepanyl, azocanyl, tetrahydroisoquinolinyl, phthalanyl, phthaloyl, tetrahydrofuranyl, tetrahydropyranyl, oxazolidinyl, dioxanyl, dioxolanyl and the like.

[0061] The term "aryl" means an aromatic substituent containing at least one ring having a shared pi electron system, and examples include phenyl, naphthyl, anthryl, non-phenyl, triphenyl and the like.

[0062] The term "heteroaryl" refers to an aromatic ring compound containing one or more heteroatoms such as N, O, or S, and depending on the number and type of heteroatoms contained in the ring and the number of carbon atoms, pyridyl, pyrrolyl, pyrrolidinyl, pyridinyl, furanyl, quinolidinyl, indolyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, pyranyl, thiophenyl, thiazolyl, dibenzothiophenyl, dibenzofuranyl, dibenzoselenophenyl, thiophene, benzofuran, benzothiophenyl, benzoselrenophenyl, carbazolyl, indolocarbazolyl, pyridylindolyl, pyrrolodipyridinyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, oxadiazolyl, oxatriazolyl, dioxazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrazinyl, triazinyl, oxazinyl, oxathiazinyl, oxadiazinyl, indolyl, benzimidazolyl, indazolyl, indoxazinyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, phthalazinyl, pteridinyl, xanthenyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, benzofuropyridinyl, furodipyridinyl, benzothienopyridinyl, thienodipyridinyl, benzoselenophenopyridinyl, and selenophenodipyridinyl, etc.

[0063] The term "cycloalkylalkyl" means a cycloalkyl group substituted with an alkyl group, and cycloalkyl and alkyl are as disclosed above.

[0064] The term "heterocycloalkylalkyl" means a heterocycloalkyl group substituted with an alkyl group, and heterocycloalkyl and alkyl are as disclosed above.

[0065] The term "arylalkyl" means an aryl group substituted with an alkyl group, and aryl and alkyl are as disclosed above.

[0066] The term "heteroarylalkyl" means a heteroaryl group substituted with an alkyl group, where heteroaryl and alkyl are as disclosed above.

[0067] Furthermore, the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All such compounds and diastereoisomers are included within the scope of the present invention.

[0068] In the present invention, the pharmaceutically acceptable salts mean salts or complexes of the compounds represented by Formula 1, 2, 3 or 4 having preferable biological activity. Examples of such salts include, but are not limited to, acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid. The above compounds may also be administered as pharmaceutically acceptable quaternary salts known to those skilled in the art, particularly including chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (e.g., benzoate, succinate, acetate, glycolate, maleate, malate, fumarate, citrate, tartrate, ascorbate, cinnamate, mandelate, and diphenylacetate). The compounds represented by Formula 1, 2, 3 or 4 of the present invention may include, in addition to pharmaceutically acceptable salts, all salts, hydrates, solvates and prodrugs that can be produced by ordinary methods.

[0069] The acid addition salts according to the present invention can be produced by ordinary methods. For example, derivatives of the compounds represented by Chemical Formula 1, 2, 3, or 4 are dissolved in an organic solvent such as methanol, ethanol, acetone, dichloromethane, acetonitrile, etc., an organic acid or an inorganic acid is added, and the resulting precipitate is filtered and dried to produce it, or the solvent and the excess acid are distilled under reduced pressure and then dried, and crystallized under an organic solvent to produce it.

[0070] Also, pharmaceutically acceptable metal salts can be made using a base. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in a solution of an excess of an alkali metal hydroxide or an alkaline earth metal hydroxide, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to produce sodium, potassium, or calcium salts as the metal salts. Also, the corresponding salts are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

[0071] On the other hand, the present invention relates to a pharmaceutical composition for preventing or treating cancer, viral infectious diseases, Parkinson's disease, non-alcoholic steatohepatitis, or tuberculosis, containing as an active ingredient the compound represented by the above Chemical Formula 1, 2, 3, or 4, its optical isomers, or its pharmaceutically acceptable salts, and the compound represented by the above Chemical Formula 1, 2, 3, or 4 has an activity of inhibiting both HPK1 and MLK3.

[0072] Also, the cancer may be selected from the group consisting of lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, blood cancer, colon cancer, non-small cell lung cancer, pancreatic cancer, skin cancer, head and neck cancer, small intestine cancer, rectal cancer, endometrial cancer, vaginal cancer, testicular cancer, esophageal cancer, biliary tract cancer, lymphoma, gallbladder cancer, endocrine adenocarcinoma, adrenal cancer, lymphoma, multiple myeloma, thymoma, mesothelioma, kidney cancer, brain cancer, central nervous system tumor, brainstem glioma, and pituitary adenoma, but is not particularly limited thereto.

[0073] The viral infectious disease may be a disease infected by one or more viruses selected from the group consisting of Zika virus, human immunodeficiency virus (HIV), influenza virus, Influenza A virus subtype H1N1, avian influenza virus, rhinovirus, adenovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, Herpesvirus (HSV), rotavirus, and hepatitis virus, but is not particularly limited thereto.

[0074] The present invention also relates to a method for suppressing kinase activity in a subject in need of suppressing kinase activity including HPK1, by administering an effective amount of a compound represented by Chemical Formula 1, 2, 3, or 4, or a pharmaceutically acceptable salt or stereoisomer thereof to the subject.

[0075] The present invention also relates to a pharmaceutical composition for preventing or treating cancer, characterized by including combined administration of a compound represented by Chemical Formula 1, 2, 3, or 4 and an anticancer drug.

[0076] The anticancer drug may be selected from taxane-based anticancer drugs, antitumor alkylating agents, antitumor antimetabolites, antitumor antibiotics, plant-derived antitumor agents, antitumor platinum complexes, antitumor camptothecin derivatives, antitumor kinase inhibitors, antitumor antibodies, hormonal antitumor agents, antitumor virus agents, and angiogenesis inhibitors.

[0077] The taxane-based anticancer drug is paclitaxel, docetaxel, or cabazitaxel,

[0078] The anti-tumor alkylating agent is nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, melphalan, busulfan, mitobronitol, carboquone, thiotepa, ranimustine, nimustine, temozolomide or carmustine,

[0079] The anti-tumor antimetabolite is methotrexate, 6-mercaptopurine riboside, mercaptopurine, 5-fluorouracil, tegafur, doxifluridine, camofur, cytarabine, cytarabine ocfosfate, enocitabine, S-1, gemcitabine, fludarabine or pemetrexed disodium,

[0080] The anti-tumor antibiotic is actinomycin D, doxorubicin, daunorubicin, neocarzinostatin, bleomycin, peplomycin, mitomycin C, aclarubicin, pirarubicin, epirubicin, dinostatin stimalamer, idarubicin, sirolimus, or valrubicin,

[0081] The anti-tumor agent derived from plants is vincristine, vinblastine, vindesine, etoposide, sobuzoxane, docetaxel, paclitaxel or vinorelbine,

[0082] The anti-tumor platinum complex is cisplatin, carboplatin, nedaplatin, or oxaliplatin,

[0083] The anti-tumor camptothecin derivative is irinotecan, topotecan, or camptothecin,

[0084] The anti-tumor kinase inhibitor is gefitinib, imatinib or erlotinib,

[0085] The anti-tumor antibody is cetuximab, bevacizumab, rituximab, bevacizumab, alemtuzumab, or trastuzumab,

[0086] The hormonal anti-tumor agent is goserelin, leuprolide or tamoxifen, and

[0087] the anti-tumor virus agent is Imlygic,

[0088] the angiogenesis inhibitor may be, but is not particularly limited to, Avastin, bevacizumab, ranibizumab, pegaptanib, aflibercept, erlotinib, cabozantinib, aflibercept, brivanib, tivozanib, ramucirumab, or motesanib.

[0089] The present invention also relates to a pharmaceutical composition for preventing or treating cancer, which comprises a combined administration of a compound represented by Chemical Formula 1, 2, 3 or 4 and a cell therapy agent.

[0090] The cell therapy agent may be selected from Lymphokine Activated Killer Cell (LAK), Dendritic cell (DC), Natural Killer (NK) cell, Tumor-Infiltrating Lymphocyte (TIL), Engineered T cell Receptor Therapy (TCR-T) cells for treatment, Chimeric Antigen Receptor-modified T cells (CAR-T), and Chimeric Antigen Receptor-modified NK cells (CAR-NK).

[0091] Furthermore, the present invention relates to a composition containing a compound represented by Chemical Formula 1, 2, 3, or 4 or a pharmaceutically acceptable salt thereof for the production of immunologically activated cells or the amplified production of immune cells by in vitro (ex vivo) culture of therapeutic cells used in the immunomodulatory cell therapy agent.

[0092] Furthermore, the present invention relates to a method for treating a subject having cancer, the method comprising administering to the subject an effective amount of a compound represented by Chemical Formula 1, 2, 3, or 4, or a pharmaceutically acceptable salt thereof, and an effective amount of the immunomodulatory cell therapy agent or immunomodulatory preparation, such as a checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-CTLA4 antibody, or an anti-PD-L1 antibody) or an inhibitor of tryptophan oxidation (e.g., an IDO1, IDO2, or TDO2 inhibitor).

[0093] The pharmaceutical composition according to the present invention can be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not generally cause allergic reactions or similar reactions such as gastrointestinal disorders, dizziness, etc. when administered to humans. Also, each of the compositions may be formulated and used in the form of an oral dosage form such as a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, etc., an external preparation, a suppository, and a sterile injection solution by a conventional method.

[0094] Carriers, excipients, and diluents that may be included in the composition may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil. When formulating, it is generally prepared using diluents or excipients such as fillers, stabilizers, binders, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations are prepared by mixing the compound of the present invention with at least one excipient, such as starch, microcrystalline cellulose, sucrose, or lactose, low-substituted hydroxypropyl cellulose, hypromellose, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. For non-aqueous solvents and suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. For the base of suppositories, witepsol, macrogol, tween 61, cocoa butter, laurin fat, glycerol, gelatin, etc. may be used. To formulate into a parenteral dosage form, the isobenzofuran-1(3H)-one derivative compound represented by the above chemical formula 1, 2, 3, or 4, or a pharmaceutically acceptable salt thereof, is mixed with water together with sterilizing agents, or auxiliary agents such as preservatives, stabilizers, hydrating agents or emulsification promoters, salts or buffers for osmotic pressure adjustment, and other therapeutically useful substances to produce a solution or suspension, which can be manufactured into ampoule or vial unit dosage forms.

[0095] The pharmaceutical composition containing, as an active ingredient, a compound represented by Chemical Formula 1, 2, 3 or 4 disclosed in the present invention may be administered to mammals such as mice, livestock, and humans by various routes.

[0096] All modes of administration may be contemplated. For example, it may be administered orally, rectally or by intravenous, intramuscular, subcutaneous, intrauterine epidural or intracerebrovascular injection. The dosage may vary depending on the age, sex, weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the administration time, the administration route, the absorption, distribution and excretion rates of the drug, the type of other drugs used, and the judgment of the prescriber. Determination of the dosage based on such factors is within the level of those skilled in the art. Generally, the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferred dosage is from 1 mg / kg / day to 500 mg / kg / day. Administration may be carried out once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way.

[0097] In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, chemotherapy and biological response modifiers for the prevention or treatment of cancer, viral infectious diseases, Parkinson's disease, non-alcoholic steatohepatitis, or tuberculosis.

Advantages of the Invention

[0098] The present invention relates to an isobenzofuran-1(3H)-one derivative and its use. The isobenzofuran-1(3H)-one derivative is an isobenzofuran-1(3H)-one derivative having HPK1 inhibitory activity and MLK3 inhibitory activity and can be usefully used as a pharmaceutical composition for the prevention or treatment of cancer, viral infectious diseases, Parkinson's disease, non-alcoholic steatohepatitis or tuberculosis containing the same. Further, the isobenzofuran-1(3H)-one derivative of the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, which is characterized by having an excellent anti-cancer effect when administered in combination with an anti-cancer drug or a cell therapy agent.

Brief Description of the Drawings

[0099]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0100] Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the said embodiments are provided so that the content introduced herein becomes thorough and complete, and fully conveys the idea of the present invention to those skilled in the art.

[0101] <Example 1. Synthesis of Isobenzofuran-1(3H)-one Derivatives and Confirmation of Physicochemical Properties>

[0102] The synthesis processes and physicochemical properties of Compounds 1 to 3 of the present invention are as follows.

[0103] Compound 1. 5-((5-Chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one

[0104]

Chemical Formula

[0105] Step 1: Synthesis of 2-(5-bromo-2-(hydroxymethyl)phenyl)propan-2-ol

[0106]

Chemical Formula

[0107] Et 2 After filling nitrogen into a solution of Compound 1-1 (6-bromoisobenzofuran-1(3H)-one; 1.00 g, 4.69 mmol) in Et 2 O (30 ml) for 5 minutes, 3M CH 3 MgBr in Et 4 O (4.69 ml, 14.1 mmol) was slowly added dropwise at 0 °C for 20 minutes. The reaction mixture was left at room temperature for 3 hours. After 3 hours, the reaction mixture was cooled to 0 °C and then quenched with saturated NH 4 Cl (10 mL). The crude mixture was extracted with MC, washed with brine, dried over MgSO

[0108] Step 2: Synthesis of 5-bromo-3,3-dimethylisobenzofuran-1(3H)-one

[0109]

Chemical formula

[0110] To a solution of Compound 1-2 (1.15 g, 4.69 mmol) in THF (10 mL), 85% activated MnO 2 (4.08 g, 46.9 mmol) was added, and then the mixture was stirred at 70 °C for 16 hours. After 7 hours, the reaction mixture was filtered through Celite, the residue was concentrated, and softening treatment was carried out using EA and Hex to obtain Compound 1-3 (926 mg, 3.84 mmol) as a white solid.

[0111] Step 3: Synthesis of tert-butyl(3,3-dimethyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)carbamate

[0112]

Chemical formula

[0113] Pd 2 (dba)3 (57.0 mg, 0.0620 mmol) and Xantphos (36.0 mg, 0.0207 mmol) were added to an oven-dried tube, which was then refilled with nitrogen under vacuum. Anhydrous 1,4-dioxane (2 mL), compound 1-3 (500 mg, 2.07 mmol), and tert-butyl carbamate (364 mg, 3.11 mmol) were added to the reaction tube. Cs 2 CO 3 (1.35 g, 4.15 mmol) was added. The tube was sealed, and the mixture was stirred at 100 °C for 2 h. After completion of the reaction, the reaction mixture was quenched with water, extracted with DCM, and concentrated under vacuum. The crude product was purified by column chromatography on silica gel (eluent: 30% EA in Hex), and compound 1-4 (250 mg, 0.901 mmol, 44%) was obtained as a white solid.

[0114] Step 4: Synthesis of 5-amino-3,3-dimethylisobenzofuran-1(3H)-one

[0115]

Chemical Structure

[0116] To a solution of compound 1-4 (250 mg, 0.901 mmol) in DCM (1.5 mL), 40% TFA was added, and the mixture was stirred for 1 h. The reaction mixture was quenched with water, basified with saturated NaHCO 3 (aq.), and extracted with DCM (30 mL × 2). The crude mixture was purified using MPLC (eluent: 15% EA in Hex), and compound 1-5 (130 mg, 0.901 mmol, 81%) was obtained as a white solid.

[0117] Step 5: Synthesis of 5-((2,5-dichloropyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one

[0118]

Chemical Structure

[0119] To a solution of 2,4,5-trichloropyrimidine (100 mg, 0.545 mmol) in IPA (1.5 mL) was added DIPEA (114 μL, 0.654 mmol), and the mixture was stirred. After adding compound 1-5 (116 mg, 0.654 mmol) to the reaction mixture, the mixture was stirred at 90 °C for 4 h. The reaction mixture was cooled, dissolved in EA, and washed with water. The organic layer was dried over MgSO 4 and concentrated using a rotary evaporator. The reaction mixture was purified using MPLC (20% EA / Hex) to give compound 1-6 (46.0 mg, 0.142 mmol, 26%) as a white solid.

[0120] Step 6: Synthesis of 5-((5-chloro-2-((2-(2,2,2-trifluoroacetyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one

[0121]

Chemical Structure

[0122] To a solution of compound 1-6 (43.0 mg, 0.133 mmol) in IPA (1 mL) were added compound 1-7 (35.6 mg, 0.146 mmol) and PTSA (22.8 mg, 0.123 mmol), and the mixture was stirred at 90 °C for 16 h. The reaction mixture was quenched with water and then extracted with EA (30 mL × 2). The organic layer was dried over MgSO 4 and concentrated under vacuum. The reaction mixture was purified using MPLC (30% EA / Hex) to give compound 1-8 (34.0 mg, 0.0064 mmol, 48%) as a white solid.

[0123] Step 7: Synthesis of 5-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one (Compound 1)

[0124]

Chem.

[0125] To a solution of Compound 1-8 (30.0 mg, 0.0560 mmol) in THF / MeOH / H2O (1 mL) was added LiOH·H 2 O (2.37 mg, 0.0560 mmol), and the mixture was stirred for 2 hours. The reaction mixture was purified using 1N HCl, NaHCO 3 (acid / base workup) to obtain Compound 1 (19.0 mg, 0.0440 mmol) as a white solid.

[0126] 1 1H NMR (400 MHz, DMSO-d 6 ) δ 9.27 (s, 1H), 9.19 (s, 1H), 8.23 (s, 1H), 7.98 (dd, J = 8.4, 1.8 Hz, 1H), 7.92 (s, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.30 (dd, J = 8.3, 2.2 Hz, 1H), 7.25 (s, 1H), 6.86 (d, J = 8.3 Hz, 1H), 3.76 (s, 2H), 2.88 (t, J = 5.8 Hz, 2H), 2.53 - 2.51 (m, 2H), 1.57 (s, 6H).

[0127] LC / MS : 436.39 [M+H] +

[0128] Compound 2. 6-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one

[0129]

Chem.

[0130] Step 1: Synthesis of 3,3-dimethylisobenzofuran-1(3H)-one

[0131]

Chem.

[0132] To a stirred solution of compound 2-1 (isobenzofuran-1,3-dione; 1.00 g, 6.75 mmol) in THF (30 mL) in a one-neck round-bottom flask under nitrogen, MeMgBr (3 M in Et 2 O, 4.97 mL, 14.9 mmol) was added dropwise at 0 °C in an ice bath. The resulting solution was stirred at room temperature for 4 h. 150 mL of HCl (10 wt%) was added to quench the reaction. The resulting solution was extracted with 2 × 100 mL of EtOAc, and the combined organic phases were dried over anhydrous sodium sulfate. The solid was filtered off, and the resulting mixture was concentrated under vacuum. The residue was eluted from silica gel with EtOAc / Hex (1:9) to give compound 2-2 (439 mg, 2.71 mmol) as a white solid.

[0133] Step 2: Synthesis of 3,3-dimethyl-6-nitroisobenzofuran-1(3H)-one

[0134]

Chem.

[0135] H 2 SO 4To a solution of Compound 2-2 (3,3-dimethylisobenzofuran-1(3H)-one; 33.0 mg, 0.206 mmol) in (1 mL), nitric acid (19.2 mg, 0.305 mmol) was added, and then the mixture was stirred at 0 °C for 1 hour. After adding water to the reaction mixture to quench the reaction, it was extracted with EA (20 mL × 2). The organic layer was dried over MgSO 4 and then evaporated using a rotary evaporator to obtain Compound 2-3 (41.2 mg, 0.203 mmol) as a white solid.

[0136] Step 3: Synthesis of 6-amino-3,3-dimethylisobenzofuran-1(3H)-one

[0137]

Chemical Structure

[0138] To a solution of Compound 2-3 (3,3-dimethyl-5-nitroisobenzofuran-1(3H)-one; 33.0 mg, 0.159 mmol) in EA (5 mL), 10% Pd / C (5 mg) was added, and then the mixture was stirred for 2 hours. After filtering the reaction mixture through a Celite filter, it was evaporated using an evaporator to obtain Compound 2-4 (28.2 mg, 0.159 mmol) as a white solid.

[0139] Step 4: Synthesis of 6-((2,5-dichloropyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one

[0140]

Chemical Structure

[0141] To a solution of 2,4,5-trichloropyrimidine (20.0 mg, 0.109 mmol) in IPA (1.5 mL), DIPEA (23.0 uL, 0.131 mmol) was added and stirred. After adding compound 2-4 (6-amino-3,3-dimethylisobenzofuran-1(3H)-one; 20.3 mg, 0.114 mmol) to the reaction mixture, it was stirred at 90 °C for 2 hours. The reaction mixture was cooled, dissolved in EA, and washed with water. The organic layer was dried over MgSO 4 and concentrated using a rotary evaporator. The reaction mixture was purified using MPLC (20% EA / Hex) to obtain compound 2-5 (33.9 mg, 0.109 mmol) as a white solid.

[0142] Step 5: Synthesis of 6-((5-chloro-2-((2-(2,2,2-trifluoroacetyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one

[0143]

Chemical Structure

[0144] To a solution of compound 2-5 (20.0 mg, 0.0620 mmol) in IPA (1 mL), 1-(6-amino-3,4-dihydroisoquinolin-2(1H)-yl)-2,2,2-trifluoroethan-1-one (16.6 mg, 0.0680 mmol) and PTSA (10.6 mg, 0.0620 mmol) were added, and then it was stirred at 90 °C for 4 hours. After quenching the reaction mixture with water, it was extracted with EA (30 mL × 2). The organic layer was dried over MgSO 4 and concentrated under vacuum. The reaction mixture was purified using MPLC (30% EA / Hex) to obtain compound 2-6 (20.6 mg, 0.0390 mmol, 63%) as a white solid.

[0145] Step 6: Synthesis of 6-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one (Compound 2)

[0146]

Chem.

[0147] THF / MeOH / H 2 To a solution of Compound 2-6 (20.0 mg, 0.038 mmol) in O (1 mL), LiOH·H 2 O (4.74 mg, 0.113 mmol) was added, and then the mixture was stirred at room temperature for 1 hour. The acid / base reaction was carried out to obtain Compound 2 (9.5 mg, 0.0220 mmol) as a white solid.

[0148] 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.26 (s, 1H), 9.13 (s, 1H), 8.18 (s, 1H), 8.05 (dd, J = 8.2, 2.0 Hz, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.36 (d, J = 2.2 Hz, 1H), 7.28 - 7.13 (m, 1H), 6.80 (d, J = 8.3 Hz, 1H), 3.74 (s, 2H), 3.51 (s, 1H), 2.86 (t, J = 5.9 Hz, 2H), 2.45 - 2.40 (m, 2H), 1.65 (s, 6H).

[0149] LC / MS: 434.43 [M-H] +

[0150] Compound 3.5,5’-((5-chloropyrimidine-2,4-diyl)bis(azanediyl))bis(3,3-dimethylisobenzofuran-1(3H)-one)

[0151]

Chem.

[0152] To a solution of 2,4,5-trichloropyrimidine (100 mg, 0.545 mmol) in IPA (1.5 mL) was added DIPEA (114 μL, 0.654 mmol), and the mixture was stirred. After adding compound 1-5 (116 mg, 0.654 mmol) to the reaction mixture, it was stirred at 90 °C for 4 h. The reaction mixture was cooled, dissolved in EA, and washed with water. The organic layer was dried over MgSO 4 4 and concentrated using a rotary evaporator. The reaction mixture was purified using MPLC (20% EA / Hex) to obtain compound 3 (34.0 mg, 0.073 mmol, 13%) as a white solid.

[0153] 1 H NMR (400 MHz, CDCl 3 ) δ 8.28 (s, 1H), 7.97 - 7.88 (m, 2H), 7.83 - 7.75 (m, 2H), 7.61 (dd, J = 8.4, 1.8 Hz, 1H), 7.54 (d, J = 1.7 Hz, 1H), 7.42 (s, 1H), 7.32 (s, 1H), 1.70 (s, 6H), 1.66 (s, 6H).

[0154] <Experimental Example 1. Evaluation of the kinase activity of the compounds of the present invention against MLK3 or HPK1>

[0155] 1-1. Evaluation of kinase activity inhibition against MLK3

[0156] The inhibition of kinase activity against MLK3 is calculated as follows. Recombinant human MKK3 protein is mixed with a compound in a reaction solution (8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.33 mg / mL myelin basic protein, 5 mM DTT, 10 mM magnesium acetate, 45 μM [γ-33P-ATP]), and then reacted at room temperature for 40 minutes. Then, phosphoric acid is added to a concentration of 0.5% to terminate the reaction, and 10 μL of the reaction solution is dropped onto a P30 filter. The dropped filter is washed with a 0.425% phosphoric acid solution for 4 minutes, and this process is repeated 4 times, then further washed with methanol and dried, and the activity of MLK3 is measured by scintillation counting. The activity value obtained from the group without the compound is determined as 100% of the control group (100% of control), and the degree of inhibition of MLK3 by the compound is converted. IC 50 To calculate the value, the compound is diluted stepwise by one-third from a maximum concentration of 1 μM and treated at a total of 9 concentrations, and the degree of inhibition is evaluated to define the concentration at which 50% inhibition occurs as the IC 50 value.

[0157] 1-2. Evaluation of kinase activity inhibition against HPK1

[0158] The inhibition of kinase activity against HPK1 is calculated as follows. Recombinant human HPK1 protein is mixed with a compound in a reaction solution (8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.33 mg / mL myelin basic protein, 10 mM magnesium acetate, 15 μM [γ-33P-ATP]), and then reacted at room temperature for 40 minutes. Then, phosphoric acid is added to a concentration of 0.5% to terminate the reaction, and 10 μL of the reaction solution is dropped onto a P30 filter. The dropped filter is washed with a 0.425% phosphoric acid solution for 4 minutes, and this process is repeated 4 times, then further washed with methanol and dried, and the activity of HPK1 is measured by scintillation counting. The activity value obtained from the group without the compound is determined as 100% of the control group (100% of control), and the degree of inhibition of HPK1 by the compound is converted. IC 50To calculate the value, the compound was diluted stepwise by one-third from the highest concentration of 1 μM and treated at a total of nine concentrations, and the degree of inhibition was evaluated to define the concentration at which 50% inhibition occurred as the IC 50 value.

[0159] 1-3. Results of kinase activity inhibition against MLK3 or HPK1

[0160] Using the compounds 1 to 3 of the present invention synthesized in Example 1 above, the inhibition of kinase activity against MLK3 or HPK1 was confirmed, and the results are shown in Table 1.

[0161]

Table 1

[0162] Looking at Table 1 showing the results, the 50% inhibitory concentration (IC 50 ) of HPK1 treated with Compound 1 was approximately 30 nM, the IC 50 at the time of treatment with Compound 2 was approximately 50 nM, and the IC 50 at the time of treatment with Compound 3 was approximately 900 nM. Also, the 50% inhibitory concentration (IC 50 ) of MLK3 treated with Compound 1 was approximately 140 nM, and the IC 50 at the time of treatment with Compound 2 was 200 nM. The isobenzofuran-1(3H)-one derivative compound of the present invention simultaneously exhibited high inhibitory activity against HPK1 and MLK3 at low concentrations (nM).

[0163] <Experimental Example 2. Confirmation of the anti-cancer efficacy of the compound of the present invention>

[0164] The anti-cancer activities of the MLK3 and HPK1 kinase inhibitory compounds were evaluated as follows using the human ovarian cancer cell line A2780 cell line and the cisplatin-resistant cell line A2780 / cis. A2780 and A2780 / cis cells were cultured in a 96-well plate at 4000 cells per well and stabilized for 1 day, and the compounds 1 to 3 of the present invention synthesized in Example 1 above were serially diluted from 10 μM and treated with the cells. The cells treated with the compound were cultured for 3 days, treated with CCK8 reagent to measure the cell viability, and the concentration at which 50% of the cancer cells died was expressed as GI 50 .

[0165] Looking at Figure 1 showing the results, the 50% growth inhibitory concentration (GI 50 ) of the A2780 cell line treated with Compound 1 was approximately 200 nM, and the GI 50 when treated with Compound 2 was approximately 210 nM. Also, the 50% growth inhibitory concentration (GI 50 ) of the A2780 / cis cell line treated with Compound 1 was approximately 400 nM, and the GI 50 when treated with Compound 2 was 310 nM. It was confirmed that the isobenzofuran-1(3H)-one derivative compounds 1 to 3 of the present invention have anti-cancer effects in both the cancer cell line A2780 and the resistant cancer cell line A2780 / cis.

[0166] In addition, the anti-cancer activities in cancer cell lines other than ovarian cancer (blood cancer, breast cancer, ovarian cancer, lung cancer, brain cancer) and anti-cancer agent-resistant cancer cell lines (doxorubicin-resistant blood cancer, K562 / ADM; paclitaxel-resistant breast cancer, MDA-MB-231 / PTX) were observed. The anti-cancer activity effects of the compounds of the present invention are shown in Table 2.

[0167]

Table 2

[0168] Looking at Table 2 showing the results, it was confirmed that treatment with Compound 1, 2, or 3 has anti-cancer activity in various cancer cells and anti-cancer agent-resistant cancer cells.

[0169] <Experimental Example 3. Measurement of IL-2 for Evaluation of Increased T Cell Activity of the Compounds of the Present Invention>

[0170] That HPK1 can be inhibited to increase the activity of T cells was observed from the following experiments. Human PBMC cells were seeded at 2×10 per well in a 96-well plate. 5They were diluted serially from 3 μM of the compounds 1 to 3 of the present invention synthesized in Example 1 and pretreated for 1 hour. Then, CD3 / CD28 dynabeads were treated at a ratio of 1:1 with the cell number and cultured for 6 days. After culturing, the cells and the medium were measured with an iQue device using a human T cell activation cell and cytokine profiling kit manufactured by Zaltron. IL-2, which is utilized as a biomarker for increased T cell activity, was measured, and the result value was calculated with the positive control group treated with dynabeads only set as 100%.

[0171] Looking at FIG. 2 showing the results, since Compound 1 or 2 increases IL-2, it can be seen that the compound has an activity of increasing T cell activity.

[0172] In addition, the characteristic HPK1 and MLK3 inhibitory activities of the isobenzofuran-1(3H)-one derivatives of the present invention were confirmed to be produced as immune activity-amplifying cells by co-administration during ex vivo culture of therapeutic cells used in combination with conventional anticancer drugs or immunomodulatory cell therapy agents, or that immune cells can be amplified and produced. From this, a synergistic anticancer effect was confirmed.

[0173] <Formulation Example 1. Production of Tablets>

[0174] 100 mg of Compound 1 of the present invention, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropyl cellulose, and 2 mg of magnesium stearate were mixed and then tabletted by a usual tablet manufacturing method to produce tablets.

[0175] <Formulation Example 2. Production of Capsules>

[0176] 100 mg of the compound of the present invention, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropyl cellulose, and 2 mg of magnesium stearate were mixed, and then the above components were mixed by a conventional method for producing capsules and filled into gelatin capsules to produce capsules.

[0177] <Formulation Example 3. Production of Injectable Solution>

[0178] 10 mg of the compound of the present invention, an appropriate amount of sterile distilled water for injection, and an appropriate amount of pH adjuster were mixed, and then the components were produced in an amount per ampoule (2 ml) by a conventional method for producing injectable solutions.

Claims

1. The following chemical formula 1 or chemical formula 2: 【Chemical 1】 [Chemical Formula 2] (In the above chemical formula 1 or chemical formula 2, R １ is independently hydrogen, C １ -C ４ -alkyl or C １ -C ４ -alkoxy, or Alternatively, two Rs １ and R １ together with adjacent C form a 3- to 7-membered saturated spiro ring; R ２ is hydrogen, C １ -C ４ -alkyl or C １ -C ４ -alkoxy; R ３ is hydrogen, C １ -C ４ -alkyl, halo C １ -C ４ -alkyl, hydroxy C １ -C ４ -alkyl, C １ -C ４ -alkoxy, halo C １ -C ４ -alkoxy, C １ -C ４ -alkoxy C １ -C ４ -alkoxy, -COR ４ , -CONH ２ , -CONHR ４ , -CON(R ４ ) ２ , -SO ３ H, or -SO ２ R ４ ; R ４ is a substituted or unsubstituted 3- to 12-membered cycloalkyl, a substituted or unsubstituted 3- to 12-membered heterocycloalkyl, a substituted or unsubstituted 4- to 12-membered aryl, a substituted or unsubstituted 4- to 12-membered heteroaryl, a substituted or unsubstituted 3- to 12-membered cycloalkylC １ -C ４ alkyl, a substituted or unsubstituted 3- to 12-membered heterocycloalkylC １ -C ４ alkyl, a substituted or unsubstituted 4- to 12-membered arylC １ -C ４ alkyl, a substituted or unsubstituted 4- to 12-membered heteroarylC １ -C ４ alkyl, a substituted or unsubstituted C １ -C ４ alkyl, haloC １ -C ４ alkyl, hydroxyC １ -C ４ alkyl, C １ -C ４ alkoxy, haloC １ -C ４ alkoxy, or C １ -C ４ alkoxyC １ -C ４ alkoxy, and Furthermore, here, the substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl or alkyl is OH, NO ２ , NH ２ , CN, halogen, C １ -C ４ alkyl, halo C １ -C ４ alkyl, hydroxy C １ -C ４ alkyl, C １ -C ４ alkoxy, halo C １ -C ４ alkoxy and C １ -C ４ alkoxy C １ -C ４ substituted with one or more substituents selected from the group consisting of alkoxy; R ５ is independently hydrogen, C １ -C ４ -alkyl or C １ -C ４ -alkoxy, or Alternatively, two Rs ５ and R ５ together with the adjacent C form a 3- to 7-membered saturated spiro ring; A is O or NH; B is O or S; X is a halogen; and n is an integer from 0 to 6;) A compound represented by characterized by being a compound, its optical isomer, or its pharmaceutically acceptable salt.

2. The following chemical formula 3 or chemical formula 4: 【Chemical Formula 3】 【Chemical Formula 4】 (In the above chemical formula 3 or chemical formula 4, R １ is each independently hydrogen, C １ -C ４ -alkyl or C １ -C ４ -alkoxy, or Alternatively, two Rs １ and R １ together with the adjacent C form a 3- to 7-membered saturated spiro ring; R ２ is hydrogen, C １ -C ４ -alkyl or C １ -C ４ -alkoxy; R ３ is hydrogen, C １ -C ４ -alkyl, halo C １ -C ４ -alkyl, hydroxy C １ -C ４ -alkyl, C １ -C ４ -alkoxy, halo C １ -C ４ -alkoxy, C １ -C ４ -alkoxy C １ -C ４ -alkoxy, -COR ４ , -CONH ２ , -CONHR ４ , -CON(R ４ ) ２ , -SO ３ H, or -SO ２ R ４ ; R ４ is a substituted or unsubstituted 3- to 12-membered cycloalkyl, a substituted or unsubstituted 3- to 12-membered heterocycloalkyl, a substituted or unsubstituted 4- to 12-membered aryl, a substituted or unsubstituted 4- to 12-membered heteroaryl, a substituted or unsubstituted 3- to 12-membered cycloalkylC １ -C ４ -alkyl, a substituted or unsubstituted 3- to 12-membered heterocycloalkylC １ -C ４ -alkyl, a substituted or unsubstituted 4- to 12-membered arylC １ -C ４ -alkyl, a substituted or unsubstituted 4- to 12-membered heteroarylC １ -C ４ -alkyl, a substituted or unsubstituted C １ -C ４ -alkyl, haloC １ -C ４ -alkyl, hydroxyC １ -C ４ -alkyl, C １ -C ４ -alkoxy, haloC １ -C ４ -alkoxy, or C １ -C ４ -alkoxyC １ -C ４ -alkoxy, and Furthermore, here, the substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl or alkyl is OH, NO ２ , NH ２ , CN, halogen, C １ -C ４ alkyl, halo C １ -C ４ alkyl, hydroxy C １ -C ４ alkyl, C １ -C ４ alkoxy, halo C １ -C ４ alkoxy and C １ -C ４ alkoxy C １ -C ４ substituted with one or more substituents selected from the group consisting of alkoxy; R ５ is independently hydrogen, C １ -C ４ -alkyl or C １ -C ４ -alkoxy, or Alternatively, two Rs ５ and R ５ together with the adjacent C form a 3- to 7-membered saturated spiro ring; A is O or NH; B is O or S; X is a halogen; and n is an integer from 0 to 6;) A compound represented by the compound, its optical isomer, or its pharmaceutically acceptable salt according to Claim 1.

3. 5-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one (Compound 1); 6-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one (Compound 2); and 5,5'-((5-chloropyrimidine-2,4-diyl)bis(azanediyl))bis(3,3-dimethylisobenzofuran-1(3H)-one) (Compound 3); Selected from the group consisting of the compound, its optical isomer, or its pharmaceutically acceptable salt represented by chemical formula 1, 2, 3 or 4 according to Claim 1 or 2.

4. A pharmaceutical composition for preventing or treating cancer, viral infectious diseases, Parkinson's disease, non-alcoholic steatohepatitis or tuberculosis, comprising as an active ingredient the compound, its optical isomer, or its pharmaceutically acceptable salt represented by chemical formula 1, 2, 3 or 4 according to Claim 1 or 2.

5. The cancer is selected from the group consisting of lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, blood cancer, colon cancer, non-small cell lung cancer, pancreatic cancer, skin cancer, head and neck cancer, small intestine cancer, rectal cancer, endometrial cancer, vaginal cancer, testicular cancer, esophageal cancer, biliary tract cancer, lymphoma, gallbladder cancer, endocrine adenocarcinoma, adrenal cancer, lymphoma, multiple myeloma, thymoma, mesothelioma, kidney cancer, brain cancer, central nervous system tumor, brainstem glioma and pituitary adenoma The pharmaceutical composition for preventing or treating cancer, viral infectious diseases, Parkinson's disease, non-alcoholic steatohepatitis or tuberculosis according to Claim 4.

6. The viral infectious disease is selected from the group consisting of Zika virus, human immunodeficiency virus (HIV), influenza virus, novel influenza A virus (Influenza A virus subtype H1N1), avian influenza virus, rhinovirus, adenovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, Herpesvirus (HSV), rotavirus, and hepatitis virus A pharmaceutical composition for preventing or treating the cancer, viral infectious disease, Parkinson's disease, non-alcoholic steatohepatitis or tuberculosis according to claim 4

7. administering an effective amount of the compound represented by Chemical Formula 1, 2, 3 or 4 according to claim 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof, to a subject in need of suppressing kinase activity including HPK1 A method for suppressing kinase activity in the subject, characterized by the above

8. A pharmaceutical composition for preventing or treating cancer, characterized by including combined administration of the compound represented by Chemical Formula 1, 2, 3 or 4 according to claim 1 or 2 and an anticancer drug A pharmaceutical composition for preventing or treating cancer, characterized by the above

9. The anticancer drug is selected from taxane anticancer drugs, antitumor alkylating agents, antitumor antimetabolites, antitumor antibiotics, plant-derived antitumor agents, antitumor platinum complexes, antitumor camptothecin derivatives, antitumor kinase inhibitors, antitumor antibodies, hormonal antitumor agents, antitumor virus agents, and angiogenesis inhibitors A pharmaceutical composition for preventing or treating cancer according to claim 8

10. A pharmaceutical composition for preventing or treating cancer, characterized by including combined administration of the compound represented by Chemical Formula 1, 2, 3 or 4 according to claim 1 or 2 and a cell therapy agent A pharmaceutical composition for preventing or treating cancer, characterized by the above

11. The cell therapy agent is selected from lymphokine-activated killer cells (LAK), dendritic cells (DC), natural killer (NK) cells, tumor-infiltrating lymphocytes (TIL), engineered T cell receptor therapy cells (TCR-T), chimeric antigen receptor T cells (CAR-T), and chimeric antigen receptor NK cells (CAR-NK). The pharmaceutical composition for preventing or treating cancer according to claim 10.

12. The compound represented by Chemical Formula 1, 2, 3, or 4 according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for the production of immunologically activated cells or the amplified production of immune cells by in vitro (ex vivo) culture of the therapeutic cells used in the immunomodulatory cell therapy agent of claim 11. A composition characterized by the above.

13. A method for treating a subject with cancer, comprising administering to the subject an effective amount of the compound represented by Chemical Formula 1, 2, 3, or 4 according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, and an effective amount of the immunomodulatory cell therapy agent or immunomodulatory preparation of claim 11, such as an immune checkpoint inhibitor (e.g., anti-PD-1 antibody, anti-CTLA4 antibody, or anti-PD-L1 antibody) or an inhibitor of tryptophan oxidation (e.g., IDO1, IDO2, or TDO2 inhibitor). A method characterized by the above.

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