Compound comprising 2, 4-diaminopyrimidine, method for preparing same, and pharmaceutical composition for preventing or treating cancer containing same as active ingredient

Abstract

The present invention relates to an isobenzofuran-1 (3H)-one derivative as a kinase inhibitor and a use thereof, and more specifically, to an isobenzofuran-1 (3H)-one derivative as a kinase inhibitor, 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 for preventing or treating cancer, viral infection diseases, Parkinson's disease, non-alcoholic steatohepatitis, or tuberculosis comprising the same. In addition, the compound can be effectively used as a composition for preventing or treating cancer, including administration in combination with an anti-cancer drug or in combination with a cell therapeutic agent.

Description

Technical Field

[0001] The present invention relates to an isobenzofuran-1(3H)-one derivative and uses thereof, and more specifically, to 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 infection diseases, Parkinson's disease, non-alcoholic fatty hepatitis or tuberculosis. Background Art

[0002] Protein kinase is a catalytic enzyme that catalyzes the transfer of the terminal phosphate group of adenosine triphosphate (ATP) to specific residues (tyrosine, serine and threonine) of proteins and participates in the signaling that regulates cell activity, growth and differentiation 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 phosphorylated (Hanks, SK et al., FASEB J., 9(8), 576-596, 1995). The serine / threonine kinase group includes protein kinase C isoforms (Newton, AC, J Biol Chem., 270(48), 28495-28498, 1995), cyclin-dependent kinase group and cdc2 (Pines, J., Trends Biochem Sci., 18(6), 197, 1993). The tyrosine kinase component is divided into: transmembrane 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, AC et al., Annu Rev Immunol., 12, 555-592, 1994).

[0004] Inappropriately high protein kinase activity is directly or indirectly associated with a variety of diseases that result from abnormal cellular action. For example, disease may arise due to failure of appropriate regulatory mechanisms of kinases associated with mutation, overexpression or inappropriate enzyme activity, or overproduction or underproduction of factors involved in signal transduction upstream or downstream of cytokines or kinases. Thus, selective inhibition of kinase activity may be a favorable target for the development of new drugs for the treatment of disease.

[0005] MLK3 is a member of the mixed lineage kinase (MLK) family and can regulate MAP kinases (especially JNK) that play an important role in the proliferation and survival of cancer cells by using a variety of signal transduction systems.

[0006] In addition, many studies are currently underway to develop compounds that inhibit MLK3 enzyme activity as therapeutic agents for cancer, viral infections, Parkinson's disease, non-alcoholic steatohepatitis, etc. (Chadee, DN, 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 Set al., J Med Chem., 56(20), 8032-8048, 2013; Jiang, JX et al., Livermore Int.,34(8),1131-1132,2014;Tomita,K.et al.,JCI Insight.,2(15),94488,2017;Ibrahim,SHetal.,LiverInt.,34(3),427-437,2014;Parkinson Study Group PRECEPT Investigators, Neurology, 69(15), 1480-1490, 2007; Kline, EM et al., Exp Neurol., 318, 157-164, 2019).

[0007] Hematopoietic progenitor kinase 1 (HPK1) cloned from primitive hematopoietic progenitor cells is a series of many MAP kinase kinase kinase kinases (MAP4Ks), including: MAP4K1 / HPK1, MAP4K2 / GCK, MAP4K3 / GLK, MAP4K4 / HGK, MAP4K5 / KHS and MAP4K6 / MINK (Hu, MC, et al., Genes Dev, 1996. 10 (18): p. 2251-64). Interestingly, HPK1 is mainly expressed in hematopoietic cells such as T cells, B cells, macrophages, dendritic cells, neutrophils and mast cells (Hu, MC, et al., Genes Dev, 1996. 10 (18): p. 2251-64; Kiefer, F., et al., EMBO J, 1996. 15 (24): p. 7013-25). It is proposed that HPK1 kinase activity is induced upon activation of 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 receptor (EP2 and EP4) (Ikegami, R., et al., J Immunol, 2001. 166(7): p.4689-96). As mentioned above, HPK1 regulates multiple functions of various immune cells.

[0008] HPK1 is very important in regulating the functions of various immune cells and is associated with autoimmune diseases and anti-tumor immunity (Shui, JW, 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, JW, et al., Nat Immunol, 2007.8(1): p.84-91). In humans, HPK1 is downregulated in peripheral blood mononuclear cells of patients with psoriatic arthritis and T cells of patients with systemic lupus erythematosus (SLE) (Batliwalla, FM, et al., Mol Med, 2005.11(1-12): p.21-9). These observations suggest that the reduction in HPK1 activity may be caused by the patient's autoimmunity. In addition, HPK1 can also control anti-tumor immunity through T cell-dependent mechanisms. In a PGE2-producing lung cancer tumor model, tumors developed more slowly in HPK1 knockout mice compared to wild-type mice (see US2007 / 0087988). In addition, adoptive transfer of HPK1-deficient T cells has been proposed to be 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 are more effective in increasing T cell responses to eradicate Lewis lung cancer compared to wild-type BMDCs (Alzabin, S., et al., J Immunol, 2009.182(10): p.6187-94). In addition, it has been reported that cancer patients with high HPK1 expression have a lower survival rate than cancer patients with low expression. However, according to recent reports, inhibiting HPK1 expression in cancer cells themselves can inhibit cancer growth. In addition, in various CAR-T cells such as CD19CAR-T, Her2 CAR-T, B cell maturation antigen (BCMA) CAR-T (chimeric antigen receptor T cell), etc., which are anti-cancer immune cells, if HPK1 expression is inhibited, the anti-cancer immune ability of CAR-T is increased, thereby effectively inhibiting the proliferation and growth of cancer cells (Si, J., et al., Cancer Cell, 2020, 38(4): p.551-66).These data, together with the restricted expression of HPK1 in hematopoietic cells and the lack of effect on the normal development of immune cells, suggest that HPK1 may be an excellent drug target for enhancing anti-tumor immunity. In addition, the most important immunotherapy using T cells is ex vivo culture, which is a technology for rapidly proliferating a large number of high-quality T cells in a culture container (Dudley, ME., et al., Nat. Rev. Cancer, 2003, 3(9): p.666-75). If combined with a technology that can regulate the differentiation and proliferation of hematopoietic cells in a culture container, new T cell culture technology for immunotherapy can be developed. Therefore, new compounds that regulate HPK1 activity are needed.

[0009] On the other hand, as an existing document related to isobenzofuran-1(3H)-one derivatives having HPK1 inhibitory activity and MLK3 inhibitory activity and pharmaceutical compositions containing the same for preventing or treating cancer, viral infection diseases, Parkinson's disease, non-alcoholic fatty hepatitis or tuberculosis, Korean Patent No. 10-0832602 discloses a novel polycyclic compound related to the activity of MLK3 enzyme and its use. However, there is no mention or report of isobenzofuran-1(3H)-one derivatives of Chemical Formula 1 and Chemical Formula 2 of the present invention and the HPK1 and MLK3 inhibitory activities of the derivatives, and the ability to treat cancer, viral infection diseases, Parkinson's disease, non-alcoholic fatty hepatitis or tuberculosis thereby.

[0010] Therefore, the present inventors confirmed the HPK1 inhibitory activity and the MLK3 inhibitory activity during the process of studying isobenzofuran-1(3H)-one derivatives, thereby completing the present invention. Summary of the invention

[0011] Problem that the invention aims to solve

[0012] The object of the present invention is to provide an isobenzofuran-1(3H)-one derivative and its use, more specifically, to provide 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 infection diseases, Parkinson's disease, non-alcoholic fatty hepatitis or tuberculosis.

[0013] Means used to solve problems

[0014] The present invention relates to a compound represented by the following Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 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 1]

[0017]

[0018] [Chemical formula 2]

[0019]

[0020] In the chemical formula 1 or chemical formula 2,

[0021] R1 is independently hydrogen, C1-C4 alkyl or C1-C4 alkoxy,

[0022] Alternatively, two R1s and the C adjacent to R1 together form a 3- to 7-membered saturated spiro ring,

[0023] R2 is hydrogen, C1-C4 alkyl or C1-C4 alkoxy,

[0024] R3 is hydrogen, C1-C4 alkyl, halogenated C1-C4 alkyl, hydroxy C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, C1-C4 alkoxy C1-C4 alkoxy, -COR4, -CONH2, -CONHR4, -CON(R4)2, -SO3H or -SO2R4,

[0025] R4 is substituted or unsubstituted 3-12 membered cycloalkyl, substituted or unsubstituted 3-12 membered heterocycloalkyl, substituted or unsubstituted 4-12 membered aryl, substituted or unsubstituted 4-12 membered heteroaryl, substituted or unsubstituted 3-12 membered cycloalkyl C1-C4 alkyl, substituted or unsubstituted 3-12 membered heterocycloalkyl C1-C4 alkyl, substituted or unsubstituted 4-12 membered aryl C1-C4 alkyl, substituted or unsubstituted 4-12 membered heteroaryl C1-C4 alkyl, substituted or unsubstituted C1-C4 alkyl, halo C1-C4 alkyl, hydroxy C1-C4 alkyl, C1-C4 alkoxy, halo C1-C4 alkoxy or C1-C4 alkoxy C1-C4 alkoxy,

[0026] wherein the substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl or alkyl is substituted at a position capable of hydrogen bonding by one or more substituents selected from the group consisting of OH, NO2, NH2, CN, halogen, C1-C4 alkyl, halo-C1-C4 alkyl, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, halo-C1-C4 alkoxy and C1-C4 alkoxy-C1-C4 alkoxy,

[0027] R5 is independently hydrogen, C1-C4 alkyl or C1-C4 alkoxy,

[0028] Alternatively, two R5 and the C adjacent to R5 together form a 3- to 7-membered saturated spiro ring,

[0029] A is O or NH,

[0030] B is O or S,

[0031] X is a halogen, and

[0032] n is an integer of 0-6.

[0033] In addition, the present invention 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.

[0034] [Chemical formula 3]

[0035]

[0036] [Chemical formula 4]

[0037]

[0038] In the chemical formula 3 or chemical formula 4,

[0039] R1 is independently hydrogen, C1-C4 alkyl or C1-C4 alkoxy,

[0040] Alternatively, two R1s and the C adjacent to R1 together form a 3- to 7-membered saturated spiro ring,

[0041] R2 is hydrogen, C1-C4 alkyl or C1-C4 alkoxy,

[0042] R3 is hydrogen, C1-C4 alkyl, halogenated C1-C4 alkyl, hydroxy C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, C1-C4 alkoxy C1-C4 alkoxy, -COR4, -CONH2, -CONHR4, -CON(R4)2, -SO3H or -SO2R4, R4 is substituted or unsubstituted 3-12 membered cycloalkyl, substituted or unsubstituted 3-12 membered heterocycloalkyl, substituted or unsubstituted 4-12 membered aryl, substituted or unsubstituted 4-12 membered heteroaryl, substituted or unsubstituted 3-12 membered cycloalkyl C1-C4 alkyl, substituted or unsubstituted 3-12 membered heterocycloalkyl C1-C4 alkyl, substituted or unsubstituted 4-12 membered aryl C1-C4 alkyl, substituted or unsubstituted 4-12 membered heteroaryl C1-C4 alkyl, substituted or unsubstituted C1-C4 alkyl, halogenated C1-C4 alkyl, hydroxy C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy or C1-C4 alkoxy C1-C4 alkoxy,

[0043] wherein the substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl or alkyl is substituted at a position capable of hydrogen bonding by one or more substituents selected from the group consisting of OH, NO2, NH2, CN, halogen, C1-C4 alkyl, halo-C1-C4 alkyl, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, halo-C1-C4 alkoxy and C1-C4 alkoxy-C1-C4 alkoxy,

[0044] R5 is independently hydrogen, C1-C4 alkyl or C1-C4 alkoxy,

[0045] Alternatively, two R5 and the C adjacent to R5 together form a 3- to 7-membered saturated spiro ring,

[0046] A is O or NH,

[0047] B is O or S,

[0048] X is a halogen, and

[0049] n is an integer of 0-6.

[0050] Specifically, the compound is a compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that:

[0051] Selected from 5-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one (Compound 1);

[0052] 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

[0053] The compound is selected from the group consisting of 5,5′-((5-chloropyrimidine-2,4-diyl)bis(imino))bis(3,3-dimethylisobenzofuran-1(3H)-one) (Compound 3).

[0054] Unless otherwise specified, the following terms in the present invention have the following meanings. Any undefined terms have the meanings understood in the technical field.

[0055] The terms "halo" and "halogen" are used in their conventional sense to refer to fluoro, chloro, bromo or iodo substituents.

[0056] The term "alkyl" refers to a single bond straight chain or branched hydrocarbon group. For example, the alkyl group includes methyl, ethyl, propyl, n-butyl, isobutyl, tert-butyl, 1-methylpropyl, etc.

[0057] The term "alkoxy" refers to an oxy group bonded to a single-bond straight-chain or branched hydrocarbon group. For example, the alkoxy group includes methoxy, ethoxy, propoxy, n-butoxy, tert-butoxy, 1-methylpropoxy, and the like.

[0058] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms in the alkyl group are substituted by a halogen group, wherein the alkyl group and the halogen group are as described above.

[0059] The term "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms in the alkoxy group are substituted with a halogen group, wherein the halogen group and the alkoxy group are as described above.

[0060] The term "alkoxyalkoxy" refers to an alkoxy group in which one or more hydrogen atoms in the alkoxy group are replaced by another alkoxy group, wherein the alkoxy group is as described above.

[0061] The terms "cyclic group" and "ring" refer to a substituent group or an aromatic group which may be substituted or unsubstituted and / or may contain heteroatoms or may not contain heteroatoms and may be a monocyclic group, a bicyclic group or a polycyclic group.

[0062] The term "cycloalkyl" refers to a cyclic single-bond saturated hydrocarbon group. For example, the cycloalkyl group includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[3.1.1]heptyl, spiro[4.5]decyl, spiro[5.5]undecyl, adamantyl, etc.

[0063] The term "heterocycloalkyl" refers to a cyclic single-bond saturated hydrocarbon group containing one or more heteroatoms such as N, O or S, and may include aziridinyl, pyrrolidinyl, piperidinyl, oxopiperidinyl, morpholinyl, piperazinyl, oxopiperazinyl, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, oxazepane, thiazepane, dioxathiazepane, azocanyl, tetrahydroisoquinolinyl, phthaloyl, phthaloyl, tetrahydrofuranyl, tetrahydropyranyl, oxazolidinyl, dioxanyl, dioxolanyl, and the like, depending on the number and type of heteroatoms contained in the ring.

[0064] The term "aryl" refers to an aromatic substituent having at least one ring with a conjugated π electron system, including, for example, phenyl, naphthyl, anthracenyl, biphenyl and terphenyl.

[0065] The term "heteroaryl" refers to an aromatic ring compound containing one or more heteroatoms such as N, O or S, and may include pyridyl, pyrrolyl, pyrrolidinyl, pyridyl, furanyl, quinolyl, indolyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, pyranyl, thienyl, thiazolyl, dibenzothienyl, dibenzofuranyl, dibenzoselenophene, thiophene, benzofuran, benzothienyl, benzoselenophene, carbazolyl, indolocarbazolyl, pyridindolyl, pyrrolobipyridyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, oxazolyl, oxadiazolyl, oxatriazolyl, dioxazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrazinyl, triazinyl, oxazinyl, oxathiazinyl, indolyl, benzimidazolyl, indazolyl, indoxazinyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, quinolyl, isoquinolyl, sinolinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, phthalazinyl, pteridinyl, xanthenyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, benzofuranpyridyl, furandipyridyl, benzothienopyridyl, thienodipyridyl, benzoselenophenopyridyl and selenophenodipyridyl, etc.

[0066] The term "cycloalkylalkyl" refers to a cycloalkyl group substituted by an alkyl group, wherein the cycloalkyl group and the alkyl group are as described above.

[0067] The term "heterocycloalkylalkyl" refers to a heterocycloalkyl group substituted by an alkyl group, wherein the heterocycloalkyl group and the alkyl group are as described above.

[0068] The term "arylalkyl" refers to an aryl group substituted by an alkyl group, wherein the aryl group and the alkyl group are as described above.

[0069] The term "heteroarylalkyl" refers to a heteroaryl group substituted by an alkyl group, wherein the heteroaryl group and the alkyl group are as described above.

[0070] Furthermore, the compounds of the present invention may contain more than one asymmetric carbon atom and may exist in racemic and optically active forms. All such compounds and non-stereoisomers are included within the scope of the present invention.

[0071] In the present invention, the pharmaceutically acceptable salt refers to a salt or complex of a compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 having preferred biological activity. Examples of such salts include: acid addition salts formed by inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid); salts formed by 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, naphthalene disulfonic acid and polygalacturonic acid), but are not limited thereto. The compound can also be administered in the form of a pharmaceutically acceptable quaternary salt known to those of ordinary skill in the art, particularly including chloride, bromide, iodide, -O-alkyl, toluenesulfonate, mesylate, sulfonate, phosphate or carboxylate (e.g., benzoate, succinate, acetate, glycolate, maleate, malate, fumarate, citrate, tartrate, ascorbate, cinnamate, mandelate and diphenylacetate). The compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 of the present invention can include not only pharmaceutically acceptable salts, but also all salts, hydrates, solvates and prodrugs that can be prepared by conventional methods.

[0072] The acid addition salt of the present invention can be prepared by conventional methods. For example, the derivative of the compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 can be dissolved in an organic solvent such as methanol, ethanol, acetone, dichloromethane and acetonitrile, and an organic acid or an inorganic acid is added. The resulting precipitate is filtered and dried to prepare the acid addition salt. Alternatively, the solvent and excess acid are subjected to reduced pressure distillation and then dried, and then crystallized under organic solvent conditions to prepare the acid addition salt.

[0073] In addition, a base can be used to prepare a pharmaceutically acceptable metal salt. For example, an alkali metal or alkaline earth metal salt can be obtained by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. In this case, from a pharmaceutical perspective, a sodium salt, a potassium salt or a calcium salt is suitable for preparing the metal salt. In addition, a corresponding salt can be obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (e.g., silver nitrate).

[0074] On the other hand, the present invention relates to a pharmaceutical composition for preventing or treating cancer, viral infection, Parkinson's disease, non-alcoholic fatty hepatitis or tuberculosis, comprising a compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 has an activity of inhibiting both HPK1 and MLK3.

[0075] In addition, the cancer can 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, bile duct cancer, lymph node cancer, gallbladder cancer, endocrine cancer, adrenal cancer, lymphoma, multiple myeloma, thymoma, mesothelioma, kidney cancer, brain cancer, central nervous system tumors, brain stem glioma and pituitary adenoma, but is not limited to this.

[0076] The viral infection disease can be selected from one or more viral infection diseases in 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, herpes virus (HSV), rotavirus and hepatitis virus, but is not limited to this.

[0077] In addition, the present invention relates to a method for inhibiting kinase activity in a subject, the method comprising the following steps: administering an effective amount of a compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4, or a pharmaceutically acceptable salt or stereoisomer thereof to a subject in need of inhibition of kinase activity such as HPK1.

[0078] In addition, the present invention relates to a pharmaceutical composition for preventing or treating cancer, characterized in that it comprises a compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 and an anticancer drug for combined administration.

[0079] The anticancer agent can be selected from taxane anticancer agents, 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 viral agents and angiogenesis inhibitors.

[0080] The taxane anticancer agent may be paclitaxel, docetaxel and cabazitaxel, the antitumor alkylating agent may be nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, melphalan, busulfan, dibromomannitol, carboquinone, thiotepa, ranimustine, nimustine, temozolomide or carmustine,

[0081] The anti-tumor antimetabolite may be methotrexate, 6-mercaptopurine nucleoside, mercaptopurine, 5-fluorouracil, tegafur, doceflurane, carmofur, cytarabine, cytarabine alkylphosphite, enocitabine, S-1, gemcitabine, fludarabine or pemetrexed disodium,

[0082] The antitumor antibiotic may be actinomycin D, doxorubicin, daunorubicin, neocarcinostatis, bleomycin, pelocycin, mitomycin C, aclarubicin, pirarubicin, epirubicin, statins, idarubicin, sirolimus or valrubicin,

[0083] The plant-derived antitumor agent may be vincristine, vinblastine, vindesine, etoposide, sobuzoxane, docetaxel, paclitaxel or vinorelbine.

[0084] The anti-tumor platinum complex is cisplatin, carboplatin, nedaplatin or oxaliplatin,

[0085] The anti-tumor camptothecin derivative may be irinotecan, topotecan or camptothecin,

[0086] The anti-tumor kinase inhibitor may be gefitinib, imatinib or erlotinib,

[0087] The anti-tumor antibody may be cetuximab, bevacizumab, rituximab, bevacizumab, alemtuzumab or trastuzumab,

[0088] The hormonal antitumor agent may be goserelin, leuprolide or tamoxifen.

[0089] The anti-tumor virus agent may be Imlygic,

[0090] The angiogenesis inhibitor may be Avastin, bevacizumab, ranibizumab, pegaptanib, aflibercept, axitinib, cabozantinib, aflibercept, brivanib, tivozanib, ramucirumab or motesanib, but is not limited thereto.

[0091] In addition, the present invention relates to a pharmaceutical composition for preventing or treating cancer, characterized in that it comprises a compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 and a cell therapy agent for combined administration.

[0092] The cell therapeutic agent can be selected from lymphokine activated killer cells (Lymphokine Activated Killer Cell, LAK), dendritic cells (Dendritic cell, DC), natural killer (Natural Killer, NK) cells, tumor infiltrating lymphocytes (Tumor-Infiltrating Lymphocyte, TIL), engineered T cell receptor therapy cells (Engineered T cell Receptor Therapy, TCR-T), chimeric antigen receptor T cells (Chimeric Antigen Receptor-modified T cell, CAR-T), chimeric antigen receptor NK cells (Chimeric Antigen Receptor modified NK cell, CAR-NK).

[0093] In addition, the present invention relates to a composition comprising a compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 or a pharmaceutically acceptable salt thereof, which is used for in vitro (ex vivo) culturing therapeutic cells used in the immunomodulatory cell therapeutic agent to achieve the preparation of immune active amplified cells or the amplified production of immune cells.

[0094] In addition, the present invention relates to a method for treating a subject suffering from cancer, the method comprising the following steps: administering to the subject an effective amount of a compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4, or a pharmaceutically acceptable salt thereof, and an effective amount of the immunomodulatory cell therapy agent or immunomodulatory preparation, for example, an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-CTLA4 antibody or an anti-PD-L1 antibody) or a tryptophan oxidation inhibitor (e.g., an IDO1, IDO2 or TDO2 inhibitor).

[0095] The pharmaceutical composition of the present invention can be formulated into a suitable dosage form together with a commonly used pharmaceutically acceptable carrier. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions or similar reactions such as gastrointestinal discomfort, dizziness, etc. when administered to humans. In addition, the composition can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols; external preparations; suppositories and sterile injections for use according to conventional methods.

[0096] The carrier, excipient and diluent that can be included in the composition, for example, can be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylparaben, propylparaben, talcum powder, magnesium stearate and mineral oil, but not limited to this. When formulation, diluents or excipients such as conventional fillers, stabilizers, adhesives, disintegrants and surfactants are used to prepare. Solid preparations for oral administration include tablets, pills, powders, granules and capsules, etc., and this solid preparation is prepared by mixing at least one excipient in the compound of the present invention, and the excipient can be, for example, starch, microcrystalline cellulose, sucrose or lactose, low-substituted hydroxypropyl cellulose and hydroxypropyl methylcellulose, etc. In addition, in addition to simple excipients, slip agents such as magnesium stearate and talcum powder are also used. Liquid preparations for oral administration include suspensions, oral liquids, emulsions and syrups, etc. For liquid preparations, in addition to water and liquid paraffin as common simple diluents, various excipients may be included, such as wetting agents, sweeteners, aromatics and preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions can use vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate, etc. The matrix of the suppository can use semi-synthetic fatty acid esters (witepsol), polyethylene glycol, tween 61, cocoa butter, laurel oil, glycerol and gelatin, etc. In order to formulate into a dosage form for non-oral administration, the isobenzofuran-1(3H)-one derivative compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 or a salt thereof acceptable to students can be sterilized, or mixed with a preservative, a stabilizer, a hydrating agent or an emulsifying promoter, auxiliary agents such as salts or buffers for adjusting osmotic pressure, and other therapeutically useful substances in water to prepare a solution or suspension, and then prepared into an ampoule or vial unit dosage form.

[0097] The pharmaceutical composition comprising the compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 disclosed in the present invention as an active ingredient can be administered to mammals such as mice, livestock and humans through various routes.

[0098] All modes of administration can be expected, for example, oral administration, rectal administration or administration by intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebral injection. The dosage can be different according to the age, sex, body weight, specific disease to be treated or pathological condition, severity of disease or pathological condition, administration time, route of administration, absorption, distribution and excretion rate of drug, the type of other drugs used and the judgment of the prescriber etc. of the treatment object. Those of ordinary skill in the art can easily realize the determination of dosage based on these factors, and usually dosage is in the range of 0.01mg / Kg / day to about 2000mg / Kg / day. The preferred dosage is 1mg / Kg / day to 500mg / Kg / day. Administration can be administered once a day, or it can be administered several times. The dosage does not limit the scope of the present invention in any way.

[0099] In addition, in order to prevent or treat cancer, viral infection, Parkinson's disease, non-alcoholic fatty hepatitis or tuberculosis, the pharmaceutical composition of the present invention can be used alone or in combination with surgery, hormone therapy, chemotherapy and the use of biological response modifiers.

[0100] Effects of the Invention

[0101] The present invention relates to an isobenzofuran-1(3H)-one derivative and its use, and to a pharmaceutical composition for preventing or treating cancer, characterized in that the isobenzofuran-1(3H)-one derivative can be effectively used as an isobenzofuran-1(3H)-one derivative having HPK1 inhibitory activity and MLK3 inhibitory activity, and a pharmaceutical composition containing the isobenzofuran-1(3H)-one derivative for preventing or treating cancer, viral infection diseases, Parkinson's disease, non-alcoholic fatty hepatitis or tuberculosis. In addition, the isobenzofuran-1(3H)-one derivative of the present invention has an excellent anti-cancer effect by being administered in combination with an anti-cancer drug or a cell therapy agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 The results show the anticancer activities of the compounds of the present invention on A2780 cell line (human ovarian cancer cell line) and 2780 / cis cell line (cisplatin-resistant cell line).

[0103] Figure 2 These are results showing the T cell activity increasing effect of the compound of the present invention. DETAILED DESCRIPTION

[0104] The preferred embodiments of the present invention are described in detail below. However, the present invention is not limited to the embodiments described herein, and may also be embodied in other forms. Instead, the present invention is provided to make the contents described herein thorough and complete, and to fully convey the ideas of the present invention to those skilled in the art.

[0105] <Example 1. Synthesis of Isobenzofuran-1(3H)-one Derivatives and Confirmation of Physical and Chemical Properties>

[0106] The synthesis processes and physicochemical properties of compounds 1 to 3 of the present invention are as follows.

[0107] Compound 1. 5-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one

[0108]

[0109] Step 1: Synthesis of 2-(5-bromo-2-(hydroxymethyl)phenyl)propan-2-ol

[0110]

[0111] After nitrogen was filled into a solution of compound 1-1 (6-bromoisobenzofuran-1(3H)-one; 1.00 g, 4.69 mmol) in Et2O (30 ml) for 5 minutes, 3M CH3MgBr in Et2O (4.69 ml, 14.1 mmol) was slowly added dropwise for 20 minutes at 0°C. The reaction mixture was left at room temperature for 3 hours. After 3 hours, the reaction mixture was cooled to 0°C and quenched with saturated NH4Cl (10 mL). The crude mixture was extracted with MC, washed with brine, and dried over MgSO4 to obtain compound 1-2 (1.15 g, 4.69 mmol) as a colorless oil.

[0112] Step 2: Synthesis of 5-bromo-3,3-dimethylisobenzofuran-1(3H)-one

[0113]

[0114] After adding 85% activated MnO2 (4.08 g, 46.9 mmol) to a solution of compound 1-2 (1.15 g, 4.69 mmol) in THF (10 mL), the mixture was stirred for 16 hours at 70° C. After 7 hours, the reaction mixture was filtered through celite and the residue was concentrated and softened with EA and Hex to obtain compound 1-3 (926 mg, 3.84 mmol) as a white solid.

[0115] Step 3: Synthesis of tert-butyl(3,3-dimethyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)carbamate

[0116]

[0117] Pd2(dba)3 (57.0 mg, 0.0620 mmol) and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (Xantphos) (36.0 mg, 0.0207 mmol) were added to an oven-dried tube, which was then placed under vacuum and flushed with nitrogen again, and 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. After adding Cs2CO3 (1.35 g, 4.15 mmol), the tube was sealed and the mixture was stirred at 100 ° C for 2 hours. After the reaction was completed, the reaction mixture was quenched with water and extracted with DCM and concentrated under vacuum. The crude product was purified by silica gel chromatography (eluent: 30% EA in Hex) to obtain compound 1-4 (250 mg, 0.901 mmol, 44%) as a white solid.

[0118] Step 4: Synthesis of 5-amino-3,3-dimethylisobenzofuran-1(3H)-one

[0119]

[0120] In the solution of DCM (1.5mL) containing compound 1-4 (250mg, 0.901mmol), 40% TFA was added, and then stirred for 1 hour. The reaction mixture was quenched with water, alkalized with saturated NaHCO (aq.) and extracted with DCM (30ml×2). The crude mixture was purified using MPLC (eluent: Hex containing 15% EA) to obtain compound 1-5 (130mg, 0.901mmol, 81%) as a white solid.

[0121] Step 5: Synthesis of 5-((2,5-dichloropyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one

[0122]

[0123] DIPEA (114uL, 0.654mmol) was added to a solution of IPA (1.5mL) containing 2,4,5-trichloropyrimidine (100mg, 0.545mmol) and stirred. After adding compound 1-5 (116mg, 0.654mmol) to the reaction mixture, it was stirred for 4 hours at a temperature of 90°C. The reaction mixture was cooled and dissolved in EA and washed with water. The organic layer was dried over MgSO4 and concentrated using a rotary evaporator. The reaction mixture was purified using MPLC (20% EA / Hex) to obtain compound 1-6 (46.0mg, 0.142mmol, 26%) as a white solid.

[0124] 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

[0125]

[0126] Compound 1-7 (35.6 mg, 0.146 mmol) and PTSA (22.8 mg, 0.123 mmol) were added to a solution of IPA (1 mL) containing compound 1-6 (43.0 mg, 0.133 mmol), and stirred at 90° C. for 16 hours. The reaction mixture was quenched with water and extracted with EA (30 mL×2). The organic layer was dried over MgSO4 and concentrated under vacuum. The reaction mixture was purified using MPLC (30% EA / Hex) to obtain compound 1-8 (34.0 mg, 0.0064 mmol, 48%) as a white solid.

[0127] 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)

[0128]

[0129] After adding LiOH·H2O (2.37 mg, 0.0560 mmol) to a solution of compound 1-8 (30.0 mg, 0.0560 mmol) in THF / MeOH / H2O (1 mL), the mixture was stirred for 2 hours. The reaction mixture was purified using 1N HCl and NaHCO3 (acid / base post-treatment) to obtain compound 1 (19.0 mg, 0.0440 mmol) as a white solid.

[0130] 1 H NMR(400MHz,DMSO-d 6) δ9.27(s,1H),9.19(s,1H),8.23(s,1H),7.98(dd,J=8.4,1.8Hz,1H),7.92(s,1H),7.71(d,J=8.4Hz,1H),7.30(dd,J= 8.3, 2.2Hz, 1H), 7.25 (s, 1H), 6.86 (d, J = 8.3Hz, 1H), 3.76 (s, 2H), 2.88 (t, J = 5.8Hz, 2H), 2.53-2.51 (m, 2H), 1.57 (s, 6H).

[0131] LC / MS: 436.39 [M+H] +

[0132] Compound 2. 6-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one

[0133]

[0134] Step 1: Synthesis of 3,3-dimethylisobenzofuran-1(3H)-one

[0135]

[0136] Under nitrogen, in a round-bottom flask, in a stirred solution of THF (30 mL) containing compound 2-1 (isobenzofuran-1,3-dione; 1.00 g, 6.75 mmol), MeMgBr (3M, 4.97 mL, 14.9 mmol) in Et2O was added dropwise under ice-water bath conditions at 0°C. The resulting solution was stirred at room temperature for 4 hours. The reaction was quenched by adding 150 mL of HCl (10 wt%). 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 and the resulting mixture was concentrated under vacuum. The residue was eluted from silica gel using EtOAc / Hex (1:9) to give compound 2-2 (439 mg, 2.71 mmol) as a white solid.

[0137] Step 2: Synthesis of 3,3-dimethyl-6-nitroisobenzofuran-1(3H)-one

[0138]

[0139] Nitric acid (19.2 mg, 0.305 mmol) was added to a solution of H2SO4 (1 mL) containing compound 2-2 (3,3-dimethylisobenzofuran-1(3H)-one; 33.0 mg, 0.206 mmol), and the mixture was stirred at 0°C for 1 hour. The reaction was quenched by adding water to the reaction mixture, and then extracted with EA (20 mL×2). The organic layer was dried over MgSO4 and evaporated using a rotary evaporator to obtain compound 2-3 (41.2 mg, 0.203 mmol) as a white solid.

[0140] Step 3: Synthesis of 6-amino-3,3-dimethylisobenzofuran-1(3H)-one

[0141]

[0142] After adding 10% Pd / C (5 mg) 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), the mixture was stirred for 2 hours. The reaction mixture was filtered through a diatomaceous earth filter and evaporated using an evaporator to obtain compound 2-4 (28.2 mg, 0.159 mmol) as a white solid.

[0143] Step 4: Synthesis of 6-((2,5-dichloropyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one

[0144]

[0145] DIPEA (23.0uL, 0.131mmol) was added to a solution of IPA (1.5mL) containing 2,4,5-trichloropyrimidine (20.0mg, 0.109mmol) and stirred. Compound 2-4 (6-amino-3,3-dimethylisobenzofuran-1(3H)-one; 20.3mg, 0.114mmol) was added to the reaction mixture and stirred at 90°C for 2 hours. The reaction mixture was cooled and dissolved in EA and washed with water. The organic layer was dried over MgSO4 and concentrated using a rotary evaporator. The reaction mixture was purified using MPLC (20% EA / Hex) to obtain compound 2-5 (33.9mg, 0.109mmol) as a white solid.

[0146] 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

[0147]

[0148] After adding 1-(6-amino-3,4-dihydroisoquinolin-2(1H)-yl)-2,2,2-trifluoroethane-1-one (16.6 mg, 0.0680 mmol) and PTSA (10.6 mg, 0.0620 mmol) to a solution of IPA (1 mL) containing compound 2-5 (20.0 mg, 0.0620 mmol), the mixture was stirred at 90° C. for 4 hours. The reaction mixture was quenched with water and extracted with EA (30 mL×2). The organic layer was dried over MgSO4 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.

[0149] 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)

[0150]

[0151] After adding LiOH·H2O (4.74 mg, 0.113 mmol) to a solution of compound 2-6 (20.0 mg, 0.038 mmol) in THF / MeOH / H2O (1 mL), the mixture was stirred at room temperature for 1 hour to react with an acid / base to obtain compound 2 (9.5 mg, 0.0220 mmol) as a white solid.

[0152] 1 H NMR(400MHz,DMSO-d 6) δ9.26(s,1H),9.13(s,1H),8.18(s,1H),8.05(dd,J=8.2,2.0Hz,1H),7.91(d,J=2.0Hz,1H),7.70(d,J=8.2Hz,1H),7.36(d,J=2.2 Hz,1H),7.28-7.13(m,1H),6.80(d,J=8.3Hz,1H),3.74(s,2H),3.51(s,1H),2.86(t,J=5.9Hz,2H),2.45-2.40(m,2H),1.65(s,6H).

[0153] LC / MS: 434.43 [MH]+

[0154] Compound 3.5,5'-((5-chloropyrimidine-2,4-diyl)bis(imino))bis(3,3-dimethylisobenzofuran-1(3H)-one)

[0155]

[0156] DIPEA (114uL, 0.654mmol) was added to a solution of IPA (1.5mL) containing 2,4,5-trichloropyrimidine (100mg, 0.545mmol) and stirred. After adding compound 1-5 (116mg, 0.654mmol) to the reaction mixture, it was stirred for 4 hours at a temperature of 90°C. The reaction mixture was cooled and dissolved in EA and washed with water. The organic layer was dried over MgSO4 and concentrated using a rotary evaporator. The reaction mixture was purified using MPLC (20% EA / Hex) to obtain compound 3 (34.0mg, 0.073mmol, 13%) as a white solid.

[0157] 1 H NMR (400MHz, CDCl3) δ8.28 (s, 1H), 7.97-7.88 (m, 2H), 7.83-7.75 (m, 2H), 7.61 (dd, J = 8.4 ,1.8Hz,1H),7.54(d,J=1.7Hz,1H),7.42(s,1H),7.32(s,1H),1.70(s,6H),1.66(s,6H).

[0158] <Experimental Example 1. Evaluation of MLK3 or HPK1 Kinase Activity of the Compounds of the Present Invention>

[0159] 1-1. Evaluation of MLK3 kinase activity inhibition

[0160] The inhibition of MLK3 kinase activity was calculated by the following method. Recombinant human MKK3 protein was mixed with the compound in a reaction solution (8mM MOPS pH 7.0, 0.2mM EDTA, 0.33mg / mL myelin basic protein, 5mM DTT, 10mM magnesium acetate, 45uM [gamma-33P-ATP]) and reacted at room temperature for 40 minutes. Then, phosphoric acid was added to reach 0.5% to terminate the reaction, and 10μl of the reaction solution was added dropwise to a P30 filter. The added filter was washed with a 0.425% phosphoric acid solution for 4 minutes, and after repeating this washing process 4 times, it was washed again in methanol, then dried, and the activity of MLK3 was determined by scintillation counting. The activity value of the group without compound was set as 100% of control and converted to the degree of MLK3 inhibition based on the compound. 50 The compound was treated with 9 concentrations, the highest concentration was 1 μM, and then diluted 1 / 3 to evaluate the degree of inhibition, and the concentration of 50% inhibition was defined as IC 50 value.

[0161] 1-2. Evaluation of HPK1 kinase activity inhibition

[0162] The inhibition of HPK1 kinase activity was calculated by the following method. Recombinant human HPK1 protein was mixed with the compound in a reaction solution (8mM MOPS pH 7.0, 0.2mM EDTA, 0.33mg / mL myelin basic protein, 10mM magnesium acetate, and 15uM [gamma-33P-ATP]) and reacted at room temperature for 40 minutes. Then, phosphoric acid was added to 0.5% to terminate the reaction, and 10μl of the reaction solution was dripped onto a P30 filter. The dripped filter was washed with 0.425% phosphoric acid solution for 4 minutes, and this washing process was repeated 4 times, and then washed again in methanol, dried, and the activity of HPK1 was measured by scintillation counting. The activity value of the group without the compound was set as 100% of the control group, and the degree of HPK1 inhibition based on the compound was converted. To calculate IC 50The compound was treated with 9 concentrations, the highest concentration was 1 μM, and then diluted 1 / 3 to evaluate the degree of inhibition, and the concentration of 50% inhibition was defined as IC 50 value.

[0163] 1-3. Results of inhibition of kinase activity of MLK3 or HPK1

[0164] The inhibition of the kinase activity of MLK3 or HPK1 was confirmed using Compounds 1 to 3 of the present invention synthesized in Example 1. The results are shown in Table 1.

[0165] Table 1

[0166]

[0167] +:IC 50 >1000nM,

[0168] ++: 500 <IC 50 <1000nM,

[0169] +++: 100 < IC 50 <500nM,

[0170] ++++: 1<IC 50 <100nM IC 50 values

[0171] Referring to Table 1 showing the results, the 50% inhibitory concentration (IC 50 ) was about 30 nM, and the IC 50 About 50nM, IC when treated with compound 3 50 In addition, the 50% inhibitory concentration (IC 50 ) was about 140 nM, and the IC 50 The isobenzofuran-1(3H)-one derivative compound of the present invention exhibits high inhibitory activity against both HPK1 and MLK3 at low concentration (nM).

[0172] <Experimental Example 2. Confirmation of the anticancer efficacy of the compound of the present invention>

[0173] The anticancer activity of MLK3 and HPK1 kinase inhibitory compounds was evaluated as follows using A2780 cell lines (human ovarian cancer cell lines) and A2780 / cis (cisplatin-resistant cell lines). A2780 and A2780 / cis cells were cultured in 96-well plates at 4,000 cells per well and stabilized for one day. Compounds 1 to 3 of the present invention synthesized in Example 1 were serially diluted from 10 μM and treated to the cells. The cells treated with the compounds were cultured for 3 days and treated with CCK8 reagent to determine the cell survival rate. The concentration at which 50% of the cancer cells apoptosis occurs is expressed as GI. 50 .

[0174] Refer to the results shown Figure 1 , the 50% growth inhibition concentration (GI) of A2780 cells treated with compound 1 50 ) is about 200 nM, and the GI of compound 2 is 50 In addition, the 50% growth inhibition concentration (GI) of A2780 / cis cells treated with compound 1 was about 210 nM. 50 ) is about 400 nM, and the GI of compound 2 is 50 It was confirmed that the isobenzofuran-1(3H)-one derivatives Compounds 1 to 3 of the present invention had anticancer effects in both A2780 (cancer cell line) and A2780 / cis (drug-resistant cancer cell line).

[0175] In addition, the anticancer activity of the compound of the present invention was observed in other cancer cell lines (leukemia, breast cancer, ovarian cancer, lung cancer and brain cancer) and anticancer drug-resistant cancer cell lines (doxorubicin-resistant leukemia, K562 / ADM; paclitaxel-resistant breast cancer, MDA-MB-231 / PTX) other than ovarian cancer. The anticancer activity effect of the compound of the present invention is shown in Table 2.

[0176] Table 2

[0177] <![CDATA[GI 50 (pM)]]> Compound 1 Compound 2 Compound 3 K582 0.43 1.07 >10μM K582 / ADM 3.74 2.28 >10μM U937 0.29 0.44 >10μM MDA-MB-231 1.25 1.03 1.35 MDA-MB-231 / PTX 0.99 0.77 1.11 MDA-MB-453 1.02 2.90 1.07 SKOV3 1.704 1.50 >10μM A549 1.61 1.15 >10μM SH-SY5Y 0.39 0.28 0.55

[0178] Referring to Table 2 showing the results, it was confirmed that when the compound 1, the compound 2 or the compound 3 was treated, the compound 1, the compound 2 or the compound 3 also had anticancer activity in various cancer cells and anticancer drug-resistant cancer cells.

[0179] <Experimental Example 3. IL-2 Assay for Evaluating T Cell Activity Enhancement of the Compounds of the Present Invention>

[0180] The following experiment observed that T cell activity can be increased by inhibiting HPK1. Human PBMC cells were plated at 2 × 10 5Inoculate into a 96-well plate, and dilute the compounds 1 to 3 of the present invention synthesized in Example 1 serially from 3 μM and pre-treat for one hour. Then, CD3 / CD28 magnetic beads (dynabead) were treated with the cell solution at a ratio of 1:1 and cultured for 6 days. The cultured cells and culture medium were measured in the iQue device using the human T cell activation cell and cytokine profiling kit from Sartorius. IL-2, a biomarker for increased T cell activity, was measured, and the positive control group treated only with magnetic beads (dynabead) was set to 100% to calculate its result value.

[0181] Refer to the results shown Figure 2 When compound 1 or compound 2 increases IL-2, it can be known that the compound has T cell activity enhancing activity.

[0182] In addition, when used in combination with existing anticancer drugs or when culturing therapeutic cells used in ex vivo immunomodulatory cell therapy agents, it was confirmed that the characteristic HPK1 and MLK3 inhibitory activity of the isobenzofuran-1(3H)-one derivatives of the present invention can be used to prepare immune-active amplified cells or to perform immune cell amplification production through combined administration, and thereby a synergistic anticancer effect was confirmed.

[0183] <Preparation Example 1. Preparation of tablets>

[0184] 100 mg of the 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 tablets were prepared by tableting according to a conventional tablet preparation method.

[0185] <Preparation Example 2. Preparation of capsules>

[0186] After mixing 100 mg of the present invention compound 1, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropyl cellulose and 2 mg of magnesium stearate, the ingredients are mixed according to a conventional capsule preparation method and filled into a gelatin capsule to prepare a capsule.

[0187] <Preparation Example 3. Preparation of injection>

[0188] After mixing 10 mg of the compound 1 of the present invention, an appropriate amount of sterile distilled water for injection and an appropriate amount of a pH adjuster, each ampoule (2 mL) was prepared according to the content of the above ingredients according to a conventional injection preparation method.

Claims

1. A compound, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: It is represented by the following chemical formula 1 or chemical formula 2, [Chemical formula 1] [Chemical formula 2] In the chemical formula 1 or chemical formula 2, R1 is independently hydrogen, C1-C4 alkyl or C1-C4 alkoxy, Alternatively, two R1s and the C adjacent to R1 together form a 3- to 7-membered saturated spiro ring, R2 is hydrogen, C1-C4 alkyl or C1-C4 alkoxy, R3 is hydrogen, C1-C4 alkyl, halogenated C1-C4 alkyl, hydroxy C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, C1-C4 alkoxy C1-C4 alkoxy, -COR4, -CONH2, -CONHR4, -CON(R4)2, -SO3H or -SO2R4, R4 is substituted or unsubstituted 3-12 membered cycloalkyl, substituted or unsubstituted 3-12 membered heterocycloalkyl, substituted or unsubstituted 4-12 membered aryl, substituted or unsubstituted 4-12 membered heteroaryl, substituted or unsubstituted 3-12 membered cycloalkyl C1-C4 alkyl, substituted or unsubstituted 3-12 membered heterocycloalkyl C1-C4 alkyl, substituted or unsubstituted 4-12 membered aryl C1-C4 alkyl, substituted or unsubstituted 4-12 membered heteroaryl C1-C4 alkyl, substituted or unsubstituted C1-C4 alkyl, halo C1-C4 alkyl, hydroxy C1-C4 alkyl, C1-C4 alkoxy, halo C1-C4 alkoxy or C1-C4 alkoxy C1-C4 alkoxy, wherein the substituted cycloalkyl, the substituted heterocycloalkyl, the substituted aryl, the substituted heteroaryl, the substituted cycloalkylalkyl, the substituted heterocycloalkylalkyl, the substituted arylalkyl, the substituted heteroarylalkyl or the substituted alkyl is substituted at a position capable of hydrogen bonding by one or more substituents selected from the group consisting of OH, NO2, NH2, CN, halogen, C1-C4 alkyl, halogenated C1-C4 alkyl, hydroxy C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy and C1-C4 alkoxy C1-C4 alkoxy, R5 is independently hydrogen, C1-C4 alkyl or C1-C4 alkoxy, Alternatively, two R5 and R5 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 of 0-6.

2. The compound according to claim 1, its optical isomer, or its pharmaceutically acceptable salt, characterized in that: The compound is represented by the following Chemical Formula 3 or Chemical Formula 4: [Chemical formula 3] [Chemical formula 4] In the chemical formula 3 or chemical formula 4, R1 is independently hydrogen, C1-C4 alkyl or C1-C4 alkoxy, Alternatively, two R1 and R1 together with the adjacent C form a 3- to 7-membered saturated spiro ring, R2 is hydrogen, C1-C4 alkyl or C1-C4 alkoxy, R3 is hydrogen, C1-C4 alkyl, halogenated C1-C4 alkyl, hydroxy C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, C1-C4 alkoxy C1-C4 alkoxy, -COR4, -CONH2, -CONHR4, -CON(R4)2, -SO3H or -SO2R4, R4 is substituted or unsubstituted 3-12 membered cycloalkyl, substituted or unsubstituted 3-12 membered heterocycloalkyl, substituted or unsubstituted 4-12 membered aryl, substituted or unsubstituted 4-12 membered heteroaryl, substituted or unsubstituted 3-12 membered cycloalkyl C1-C4 alkyl, substituted or unsubstituted 3-12 membered heterocycloalkyl C1-C4 alkyl, substituted or unsubstituted 4-12 membered aryl C1-C4 alkyl, substituted or unsubstituted 4-12 membered heteroaryl C1-C4 alkyl, substituted or unsubstituted C1-C4 alkyl, halo C1-C4 alkyl, hydroxy C1-C4 alkyl, C1-C4 alkoxy, halo C1-C4 alkoxy or C1-C4 alkoxy C1-C4 alkoxy, wherein the substituted cycloalkyl, the substituted heterocycloalkyl, the substituted aryl, the substituted heteroaryl, the substituted cycloalkylalkyl, the substituted heterocycloalkylalkyl, the substituted arylalkyl, the substituted heteroarylalkyl or the substituted alkyl is substituted at a position capable of hydrogen bonding by one or more substituents selected from the group consisting of OH, NO2, NH2, CN, halogen, C1-C4 alkyl, halogenated C1-C4 alkyl, hydroxy C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy and C1-C4 alkoxy C1-C4 alkoxy, R5 is independently hydrogen, C1-C4 alkyl or C1-C4 alkoxy, Alternatively, two R5 and R5 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 of 0-6.

3. The compound according to claim 1 or 2, its optical isomer, or its pharmaceutically acceptable salt, characterized in that: The compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 is selected from the group consisting of the following compounds: Compound 1: 5-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one; Compound 2: 6-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one; and Compound 3: 5,5′-((5-chloropyrimidine-2,4-diyl)bis(imino))bis(3,3-dimethylisobenzofuran-1(3H)-one).

4. A pharmaceutical composition for preventing or treating cancer, viral infection, Parkinson's disease, non-alcoholic fatty hepatitis or tuberculosis, characterized in that: The pharmaceutical composition comprises, as an active ingredient, the compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 according to claim 1 or 2, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition for preventing or treating cancer, viral infection, Parkinson's disease, non-alcoholic fatty hepatitis or tuberculosis according to claim 4, characterized in that: The cancer is selected from the group consisting of lung cancer, liver cancer, stomach 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, lymph node cancer, gallbladder cancer, endocrine cancer, adrenal cancer, lymphoma, multiple myeloma, thymoma, mesothelioma, kidney cancer, brain cancer, central nervous system tumors, brain stem glioma and pituitary adenoma.

6. The pharmaceutical composition for preventing or treating cancer, viral infection, Parkinson's disease, non-alcoholic fatty hepatitis or tuberculosis according to claim 4, characterized in that: The viral infection disease is selected from the group consisting of Zika virus, human immunodeficiency virus, influenza virus, influenza A virus H1N1 subtype, avian influenza virus, rhinovirus, adenovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, herpes virus, rotavirus and hepatitis virus.

7. A method for inhibiting kinase activity in a subject, characterized in that: The method comprises the following steps: An effective amount of the compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 according to claim 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof is administered to a subject in need of inhibition of kinase activity including HPK1.

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

9. The pharmaceutical composition for preventing or treating cancer according to claim 8, characterized in that: The anticancer agent is selected from the group consisting of taxane anticancer agents, 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 viral agents and angiogenesis inhibitors.

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

11. The pharmaceutical composition for preventing or treating cancer according to claim 10, characterized in that: The cell therapy agent is selected from lymphokine-activated killer cells, dendritic cells, natural killer cells, tumor-infiltrating lymphocytes, engineered T cell receptor therapeutic cells, chimeric antigen receptor T cells, and chimeric antigen receptor NK cells.

12. A composition comprising the compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: Used for in vitro culturing of therapeutic cells used in the immunoregulatory cell therapeutic agent according to claim 11 to achieve the preparation of immune active amplified cells or the amplified production of immune cells.

13. A method for treating a subject suffering from cancer, characterized in that: The steps include: An effective amount of the compound represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 as described in 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, for example, an immune checkpoint inhibitor (for example, an anti-PD-1 antibody, an anti-CTLA4 antibody or an anti-PD-L1 antibody) or a tryptophan oxidation inhibitor (for example, an IDO1, IDO2 or TDO2 inhibitor) are administered to the subject.

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