WO2025063645A1 - Novel compound having inhibitory activity against hpk1 and mlk3, and anticancer composition containing same - Google Patents

Abstract

The present invention relates to a novel compound inhibiting hematopoietic progenitor kinase 1 (HPK1) and mixed lineage kinase 3 (MLK3), and a composition containing same. The compound can be effectively used as a pharmaceutical composition for preventing or treating cancer, viral infectious diseases, Parkinson's disease, nonalcoholic steatohepatitis, or tuberculosis.

Description

Novel compound having HPK1 and MLK3 inhibitory activity and anticancer composition containing same

The present invention relates to a novel compound having HPK1 (Hematopoietic Progenitor Kinase 1) and MLK3 (Mixed lineage kinase 3) inhibitory activity and an anticancer composition containing the same.

Protein kinases transfer a phosphate group (γ-phosphate group) of a nucleoside triphosphate to the hydroxyl group of a serine, threonine, or tyrosine residue in a protein. In this process, signaling pathways that are important in various biological processes such as metabolism, transcription, cell cycle progression, cell movement, apoptosis, and differentiation, which are most of the cellular functions, are activated or inactivated, so it can be said that protein kinases play a central role in almost all aspects of cellular function.

In general, protein kinases are classified into the serine and/or threonine kinase group and the tyrosine kinase group according to the substrates they phosphorylate. The serine/threonine kinase group includes the protein kinase C isoform, the cyclin-dependent kinase group, and cdc2. The tyrosine kinase group is divided into the membrane-spanning growth factor receptors including the epidermal growth factor receptor, the cytoplasmic non-receptor kinases including p56tck, p59fYn, ZAP-70, and the C-terminal Src kinase. Inappropriately high protein kinase activity is directly or indirectly associated with a number of diseases resulting from abnormal cellular function. For example, diseases may be caused by failure of the proper regulatory mechanisms of the kinase, which are related to mutations, overexpression, or inappropriate enzymatic activity; or by excessive or deficient production of cytokines or factors involved in signal transduction upstream or downstream of the kinase. Therefore, selective inhibition of kinase activity may be a beneficial target for the development of novel drugs to treat diseases.

Mixed lineage kinase 3 (MLK3) is one of the MLK family, and can regulate MAP kinases (especially JNK), which play an important role in the proliferation and survival of cancer cells, by utilizing various signal transduction systems. In addition, many studies are being conducted to develop compounds that inhibit the enzymatic activity of MLK3 as treatments for cancer, viral infection diseases, Parkinson's disease, non-alcoholic steatohepatitis, etc. Hematopoietic Progenitor Kinase 1 (HPK1), originally cloned from hematopoietic progenitor cells, is a member of many MAP kinase kinase kinase kinase kinase (MAP4K) family members, including MAP4K1/HPK1, MAP4K2/GCK, MAP4K3/GLK, MAP4K4/HGK, MAP4K5/KHS, and MAP4K6/MINK.

HPK1 (Hematopoietic Progenitor Kinase 1) is of particular interest because it is mainly expressed in hematopoietic cells such as T cells, B cells, macrophages, dendritic cells, neutrophils, and mast cells. HPK1 kinase activity has been suggested to be induced upon activation of the T cell receptor (TCR), B cell receptor (BCR), transforming growth factor receptor (TGF-βR), or Gs-coupled PGE2 receptors (EP2 and EP4). As such, HPK1 regulates various functions of various immune cells. HPK1 is known to be important in regulating the functions of various immune cells and is related to autoimmune diseases and antitumor activity. In addition, it has been reported that cancer patients with high expression of HPK1 have a lower survival rate than cancer patients with low expression, and it has been said that suppression of HPK1 expression in cancer cells themselves inhibits cancer growth. Therefore, there is a need for novel compounds that regulate HPK1 activity.

[Prior art literature]

[Patent Document]

(Patent Document 1) Republic of Korea Registered Patent No. 10-0832602 (Title of invention: Novel multi-ring compound and its use, Applicant: Sepharon, Inc., Registration date: May 20, 2008)

(Patent Document 2) Republic of Korea Registered Patent No. 10-2358632 (Title of the invention: Pharmaceutical composition for preventing or treating colon cancer containing streptonigrin and anticancer agent, Applicant: MD Biopharm Co., Ltd., Registration date: January 27, 2022)

(Patent Document 3) Republic of Korea Registered Patent No. 10-2147721 (Title of the invention: Pharmaceutical composition for preventing or treating cancer, containing streptonigrin and rapamycin as active ingredients, Applicant: MD Biopharm Co., Ltd., Registration date: August 19, 2020)

(Patent Document 4) US Patent Publication No. 2007-0087988 (Title of the invention: Hematopoietic progenitor kinase 1 for modulation of an immune response, Applicant: New York University, Publication date: April 19, 2007)

(Patent Document 5) International Publication No. 2020-159203 (Title of the invention: Composition comprising streptonigrin and anticancer agent for preventing or treating brain tumor, Applicant: MD Biopharm Co., Ltd., Publication date: August 6, 2020)

The purpose of the present invention is to provide a novel compound for inhibiting the activity of HPK1 (Hematopoietic Progenitor Kinase 1) and MLK3 (Mixed lineage kinase 3) and a composition containing the same for preventing or treating cancer.

According to one aspect of the present invention, a compound represented by the following chemical formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof is provided.

[Chemical Formula 1]

In the above chemical formula 1

A or B is selected from the group consisting of carbon, which is substituted or unsubstituted with R ₅ and R ₆ , nitrogen, oxygen and carbonyl, which is substituted or unsubstituted with R ₇ (provided that when A and B are nitrogen, B is bonded as imine),

R ₁ , R ₂ , R ₅ , R ₆ and R ₇ are 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 or hydroxy C ₁ -C ₄ alkyl;

R ₄ is a 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, a substituted or unsubstituted C ₅ -C ₁₂ cycloalkyl, a substituted or unsubstituted C ₅ -C ₁₂ heterocycloalkyl, a substituted or unsubstituted C ₅ -C ₁₂ aryl, a substituted or unsubstituted C ₅ -C ₁₂ heteroaryl group,

X is selected from a halogen group, a cyano group, a nitro group, an amide group, a thiol group, a sulfone group, a phosphoric acid group, and an amine group.

The compound represented by the chemical formula 1 of the present invention may be selected from at least one of the following 15 compounds.

The compound represented by the above chemical formula 1 is characterized by having an inhibitory effect on HPK1 (Hematopoietic Progenitor Kinase 1) and MLK3 (Mixed lineage kinase 3) activity.

The compound represented by the chemical formula 1 according to the present invention may contain one or more asymmetric carbon atoms and may exist in racemic form and optically active form. All such compounds and diastereomers are included in the scope of the present invention.

According to another aspect of the present invention, an inhibitor of HPK1 (Hematopoietic Progenitor Kinase 1) and MLK3 (Mixed lineage kinase 3) activity is provided, comprising a compound represented by the above-mentioned chemical formula 1 or a pharmaceutically acceptable salt thereof.

The above "pharmaceutically acceptable 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, naphthalene sulfonic acid, naphthalene disulfonic acid, and poly-galacturonic acid. The above compounds may also be administered as pharmaceutically acceptable quaternary salts known to those skilled in the art, particularly chlorides, bromides, iodides, -O-alkyls, toluenesulfonates, methylsulfonates, sulfonates, phosphates, or carboxylates (e.g., benzoates, succinates, acetates, glycolates, maleates, malates, fumarates, citrates, tartrate, ascorbates, cinnamoates, mandeloates and diphenylacetates).

The compound represented by chemical formula 1 of the present invention may include not only pharmaceutically acceptable salts, but also all salts, hydrates, solvates, and prodrugs that can be prepared by conventional methods.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound represented by the chemical formula 1 in an organic solvent such as methanol, ethanol, acetone, dichloromethane, acetonitrile, etc., adding an organic acid or inorganic acid, filtering and drying the resulting precipitate, or by distilling the solvent and an excess acid under reduced pressure, drying, and crystallizing in the presence of an organic solvent.

In the present invention, a pharmaceutically acceptable metal salt can also be prepared using a base. An alkali metal or alkaline earth metal salt is obtained, for example, by dissolving a compound in an excess of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. In addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

On the other hand, the compound represented by the chemical formula 1 has the effect of preventing or treating a disease caused by increased activity of HPK1 (Hematopoietic Progenitor Kinase 1) and MLK3 (Mixed lineage kinase 3).

The above "disease caused by increased HPK1 and MLK3 activity" may be cancer, viral infection disease, Parkinson's disease, non-alcoholic steatohepatitis, or tuberculosis.

Accordingly, the present invention relates to a pharmaceutical composition for preventing or treating cancer, viral infectious diseases, Parkinson's disease, non-alcoholic fatty liver disease or tuberculosis, comprising a compound represented by the chemical formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the cancer may be selected from the group consisting of lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, leukemia, 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 cancer, adrenal cancer, lymphoma, multiple myeloma, thymoma, mesothelioma, kidney cancer, brain tumor, central nervous system tumor, brainstem glioma, and pituitary adenoma, but is not particularly limited thereto. The above renal cancer is characterized by being selected from the group consisting of, but not limited to, clear cell RCC, papillary RCC, chromophobe RCC, collecting tubular RCC, and urothelial cancer (transitional cell carcinoma, TCC).

The above viral infectious disease may be a disease caused 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.

In addition, the present invention provides a method for inhibiting HPK1 (Hematopoietic Progenitor Kinase 1) and MLK3 (Mixed lineage kinase 3) activity in a subject by administering to the subject a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof. The subject may include any animal or cell. The animal may include any individual that is a test subject. It may include humans, mammals other than humans, and rodents (rats, hamsters, etc.), and as mammals, it may include various all animals, beasts, livestock, etc., such as dogs, primates, pigs, cats, rabbits, chickens, and sheep.

Through the above description, the present invention can also provide a pharmaceutical composition for preventing or treating cancer, characterized by including combined administration of a compound represented by Chemical Formula 1 and an anticancer drug. The anticancer drug can be selected from taxane-based 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.

The above taxane anticancer agent is paclitaxel, docetaxel or cabazitaxel, the above antitumor alkylating agent is nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, melphalan, busulan, mitobronitol, carboquone, thiotepa, ranimustine, nimustine, temozolomide or carmustine, the above antitumor antimetabolite is methotrexate, 6-mercaptopurine riboside, mercaptopurine, 5-fluorouracil, tegafur, doxifluridine, camofur, cytarabine, cytarabine ocphosphate, enocitabine, S-1, gemcitabine, fludarabine or pemetrexed disodium, and the above antitumor antibiotic is actinomycin D, doxorubicin, daunorubicin, neocasinostatin, bleomycin, peplomycin, mitomycin C, aclarubicin, pirarubicin, epirubicin, zinostatin stimalamer, idarubicin, sirolimus or valrubicin, wherein the plant-derived antitumor agent is vincristine, vinblastine, vindesine, etoposide, sobuzoxane, docetaxol, paclitaxel or vinorelbine, wherein the antitumor platinum complex is cisplatin, carboplatin, nedaplatin or oxaliplatin, wherein the antitumor camptothecin derivative is irinotecan, topotecan or camptothecin, wherein the antitumor kinase inhibitor is gefitinib, imatinib or erlotinib, wherein the antitumor antibody is cetuximab, bevacizumab, rituximab, bevacizumab, alemtuzumab or trastuzumab, wherein The hormonal antineoplastic agent may be goseserelin, leuprolide or tamoxifen, the antineoplastic virus agent may be Imlygic, and the angiogenesis inhibitor may be, but is not particularly limited to, avastin, bevacizumab, ranibizumab, pegaptanib, aflibercept, axitinib, cabozantinib, aflibercept, brivanib, tivozanib, ramucirumab or motesanib.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating cancer, characterized by including co-administration of a compound represented by Chemical Formula 1 and a cell therapeutic agent.

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

The above cell therapy agent can be cultured through ex vivo culture and used for treatment together with a compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof.

In addition, the present invention can be utilized in a method for treating a subject having cancer by administering to the subject the cell therapy agent, immune checkpoint inhibitor, or tryptophan oxidation inhibitor together with the compound represented by the chemical formula 1, or a pharmaceutically acceptable salt thereof.

The above immunomodulatory agent, for example, a checkpoint inhibitor, may be an anti-PD-1 antibody (anti-Programmed cell death protein 1 antibody), an anti-CTLA4 antibody (anti-Cytotoxic T-Lymphocyte associated protein 4 antibody), or an anti-PDL1 antibody (anti-Programmed death ligand 1 antibody).

The inhibitor of the above tryptophan oxidation may be an IDO1 (Indoleamine 2,3-dioxygenase 1), IDO2 (Indoleamine 2,3-dioxygenase 2) or TDO2 (Tryptophan 2,3-dioxygenase) inhibitor.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier in addition to the compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof. The pharmaceutically acceptable carrier may be one commonly used in the pharmaceutical field, and may be an excipient (e.g., starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, etc.) or a diluent (e.g., saline solution, purified water, etc.).

In addition, if necessary, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive other than the pharmaceutically acceptable carrier, for example, a binder, a disintegrant, a glidant, a coating agent, a film coating agent, an enteric film coating agent, a soft capsule agent, a solubilizing agent, an emulsifier, a suspending agent, a stabilizer, a buffer, an antioxidant, a surfactant, a sweetener, a flavoring agent, a preservative, a thickener, a fragrance, or a colorant.

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. In the case of oral administration, the composition of the present invention can be formulated in the form of a solid or liquid dosage form. For example, the solid dosage form can be a tablet, a capsule (soft & hard capsule), a powder, a granule, a pill, a troche, etc., and the liquid dosage form can be in the form of an elixir, a suspension, an emulsion, a solution, a syrup, a limonade, etc.

The dosage of the pharmaceutical composition of the present invention can be determined by considering the administration method, the age and sex of the recipient, the severity and condition of the patient, the inactivation rate, and the concomitantly administered drug, and can be administered once or in several divided doses.

A pharmaceutical composition containing a compound represented by the chemical formula 1 disclosed in the present invention as an active ingredient can be administered to mammals such as mice, livestock, and humans via various routes.

Any mode of administration may be envisaged, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine, or intracerebrovascular injection. The dosage will vary depending on the age, sex, and body 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 time of administration, the route of administration, the absorption, distribution and excretion rate of the drug, the types of other drugs used, and the judgment of the prescriber. Determination of the dosage based on these factors is within the level of those skilled in the art, and generally the dosage ranges from 0.01 mg/kg/day to about 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. The administration may be administered once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way.

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

The present invention relates to an anticancer composition having HPK1 (Hematopoietic Progenitor Kinase 1) and MLK3 (Mixed lineage kinase 3) inhibitory activity, and a compound corresponding to the anticancer composition can be usefully used as a pharmaceutical composition for preventing or treating cancer, viral infection disease, Parkinson's disease, non-alcoholic steatohepatitis, or tuberculosis.

Figure 1 shows a representative reaction scheme for synthesizing the compound of the present invention.

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 contents introduced herein are provided to sufficiently convey the idea of the present invention to those skilled in the art so that they are thorough and complete.

[Reaction Formula 1]

Intermediate production 1

Pyrimidine (1 mmol, 1 eq), DIEA (Diisopropylamine, 2 eq) and DMF (Dimethylformamide, 2 mL) were mixed, and then a solution of R ² NH ₂ (1 eq) dissolved in DMF (1 mL) was slowly added at 0 °C and stirred for 1 hour. After 1 hour, the reaction mixture was stirred at room temperature for 5 hours. Water was added to the reaction mixture, and the resulting solid was filtered and washed with water to obtain the crude product compound 2 (yield: 50-94%).

Intermediate production 2

The obtained compound 2 (2 mmol, 1 eq) and 1-(6-amino-3,4-dihydroisoquinolin-2(1H)-yl)-2,2,2-trifluoroethanone (1.2 eq) and PTSA (p-Toluene sulfonic acid, 0.9 eq) were mixed in IPA (1 mL) and DMF (1 mL), and stirred at 100 ℃. Then, the mixture was diluted with EtOAc (ethyl ethanoate), washed with water and brine, dried over Na ₂ SO ₄ , and concentrated. The crude product was purified by column chromatography to obtain compound 3 (yield: 48-83%).

Obtain the final product

The obtained compound 3 (0.04 mmol, 1 eq) was dissolved in MeOH (1.3 mL) and DMF (0.65 mL), 10% K ₂ CO ₃ (0.5 mL) was slowly added at room temperature, and the mixture was stirred at room temperature for 1 h. The organic solvent of the reaction mixture was concentrated under reduced pressure, 1 M HCl was added, and the mixture was washed several times with EtOAc. Saturated (Sat) NaHCO ₃ was slowly added dropwise, and the resulting solid was filtered to obtain the final product compound 4 (yield: 50-96%).

The specific structures and physicochemical information for each example of compound 4 synthesized through the above reaction are as follows: Synthetic Examples 1 to 16.

Synthetic Example 1. 5-chloro-N4-(1H-indazol-5-yl)-N2-(1,2,3,4-tetrahydroisoquinolin-6-yl)pyrimidine-2,4-diamine (Compound 3703)

MS (ESI): m/z = 391.28 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 13.06 (s, 1H), 9.09 (s, 1H), 8.87 (s, 1H), 8.09 (s, 1H), 8.02 (s, 1H), 7.91 (s, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.23 (s, 1H), 7.19 (d, J = 8.0 Hz, 1H), 6.68 (d, J = 8.0 Hz, 1H), 3.69 (s, 2H), 2.80-2.77 (m, 2H), 2.18 (s, 2H).

Synthetic Example 2. 5-chloro-N4-(1-methyl-1H-indazol-6-yl)-N2-(1,2,3,4-tetrahydroisoquinolin-6-yl)pyrimidine-2,4-diamine (Compound 3704)

MS (ESI): m/z = 405.0 5 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 9.11 (s, 1H), 8.90 (s, 1H), 8.08 (s, 1H), 7.99 (s, 1H), 7.90 (s, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.23 (s, 1H), 7.16 (d, J = 7.3 Hz,1H), 6.70 (d, J = 7.9 Hz, 1H), 4.06 (s, 4H), 3.70 (s, 2H), 2.79 (s, 2H), 2.13 (s, 2H).

Synthetic Example 3. 5-chloro-N4-(1H-indazol-6-yl)-N2-(1,2,3,4-tetrahydroisoquinolin-6-yl)pyrimidine-2,4-diamine (Compound 3705)

MS (ESI): m/z = 391.18 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.98 (s, 1H), 9.18 (s, 1H), 8.93 (s, 1H), 8.13 (s, 1H), 8.03 (m, 1H), 7.72-7.70 (m, 2H), 7.35-7.33 (m, 2H), 7.25-7.23 (m, 1H), 6.72 (d, J = 8.4 Hz, 2H), 3.70 (s, 2H), 2.80-2.77 (t, J = 5.8 Hz, 2H), 2.23 (s, 2H).

Synthetic Example 4. 5-chloro-N4-(1-methyl-1H-indazol-6-yl)-N2-(1,2,3,4-tetrahydroisoquinolin-6-yl)pyrimidine-2,4-diamine (Compound 3706)

MS (ESI): m/z = 405.03 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 9.22 (s, 1H), 8.96 (s, 1H), 8.15 (s, 1H), 7.99 (s, 1H), 7.93 (s, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.36-7.33 (m, 2H), 7.24 (d, J = 8 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 3.89 (s, 4H), 3.71 (s, 2H), 2.79-2.76 (t, J = 5.3 Hz, 2H), 2.21 (s, 2H).

Synthetic Example 5. 5-chloro-N4-(1,3-dimethyl-1H-indazol-5-yl)-N2-(1,2,3,4-tetrahydroisoquinolin-6-yl)pyrimidine-2,4-diamine (Compound 3707)

MS (ESI): m/z = 419.32 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 9.10 (s, 1H), 8.90 (s, 1H), 8.08 (s, 1H), 7.78 (s, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.49-7.46 (dd, J = 8.9 Hz, 1.6 Hz, 1H), 7.22 (s, 1H), 7.15 (d, J = 8 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 3.97 (s, 3H), 3.68 (s, 2H), 2.79-2.73 (m, 2H), 2.40 (s, 3H), 2.09 (s, 2H).

Synthesis Example 6. 6-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)isoindolin-1-one (Compound 3708)

MS (ESI): m/z = 406.22 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 9.18 (s, 1H), 9.01 (s, 1H), 8.58 (s, 1H), 8.14 (s, 1H), 7.85 (s, 1H), 7.77 (s, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.28 (s, 1H), 7.23 (d, J = 8.1 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H), 4.37 (s, 2H), 3.73 (s, 2H), 2.89-2.85 (m, 2H), 2.39 (s, 2H).

Synthetic Example 7. 5-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)isoindolin-1-one (Compound 3710)

MS (ESI): m/z = 406.24 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 9.45 (s, 1H), 9.26 (s, 1H), 9.10 (s, 1H), 8.44 (s, 1H), 8.22-8.20 (m, 1H), 7.95-7.92 (m, 1H), 7.80-7.77 (m, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.48-7.44 (m, 2H), 7.04 (d, J = 8.4 Hz, 1H), 4.35 (s, 2H), 4.14 (s, 2H), 3.29-3.26 (m, 2H), 2.79-2.77 (m, 2H).

Synthetic Example 8. 5-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-2-methylisoindolin-1-one (Compound 3711)

MS (ESI): m/z = 420.10 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 9.46 (s, 1H), 9.11 (s, 2H), 8.22 (s, 1H), 7.92 (s, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.49-7.45 (m, 2H), 7.04 (d, J = 8.3 Hz, 1H), 4.43 (s, 2H), 4.16 (s, 2H), 3.30-3.28 (m, 2H), 3.09 (s, 3H), 2.76 (s, 2H).

Synthetic Example 9. 5-((5-chloro-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-1-methylindolin-2-one (Compound 3713)

MS (ESI): m/z = 420.28 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 9.17 (s, 1H), 8.80 (s, 1H), 8.08 (s, 1H), 7.46-7.43 (m, 2H), 7.33 (s, 1H), 7.26 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 3.87 (s, 2H), 3.54 (s, 2H), 3.15 (s, 3H), 3.01-2.97 (m, 2H), 2.50-2.45 (m, 2H).

Synthesis Example 10. 6-((5-amino-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)amino)-3,3-dimethylisobenzofuran-1(3H)-one (Compound 3716)

MS (ESI): m/z = 416.17 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 11.76 (s, 1H), 9.68 (s, 1H), 8.77 (s, 1H), 7.99-7.94 (m, 3H), 7.72-7.70 (m, 2H), 7.51-7.49 (m, 2H), 7.35 (d, J = 7.9 Hz, 1H), 7.36-6.90 (m, 1H), 3.90 (s, 1H), 3.82 (s, 1H), 3.61-3.57 (m, 1H), 2.93 (s, 1H), 2.81 (s, 1H), 2.69 (s, 1H), 1.70 (s, 6H).

Synthetic Example 11. 4-((1H-indazol-6-yl)amino)-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidine-5-carboxamide (Compound 3720)

MS (ESI): m/z = 400.21 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.92 (s, 1H), 11.66 (s, 1H), 9.57 (s, 1H), 8.72 (s, 1H), 8.01 (s, 2H), 7.95 (s, 1H), 7.86 (s, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.46 (s, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.5 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 3.77 (s, 2H), 2.89-2.84 (m, 3H), 2.73 (s, 1H), 2.42 (s, 2H).

Synthetic Example 12. 4-((1-methyl-2-oxoindolin-5-yl)amino)-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidine-5-carboxamide (Compound 3721)

MS (ESI): m/z = 429.13 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 11.38 (s, 1H), 9.46 (s, 1H), 8.67 (s, 1H), 7.95 (s, 1H), 7.59 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.34 (s, 3H), 6.93 (d, J = 8.4 Hz, 2H), 4.4 (s, 1H), 3.77 (s, 1H), 3.51-3.48 (m, 3H), 3.27 (s, 1H), 3.13 (s, 1H), 2.89 (s, 1H).

Synthetic Example 13. 4-((1-methyl-1H-indazol-5-yl)amino)-2-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidine-5-carboxamide (Compound 3722)

MS (ESI): m/z = 414.23 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 11.46 (s, 1H), 9.51 (s, 1H), 8.69 (s, 1H), 8.15 (s, 1H), 7.97-7.93 (m, 2H), 7.63 (d, J = 8.9 Hz, 1H), 7.43-7.41 (m, 3H), 7.32 (d, J = 7.8 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 4.05 (s, 3H), 3.84 (s, 2H), 2.94-2.91 (m, 2H), 2.39 (s, 2H).

In addition, two compounds were synthesized through the following reaction scheme 2 as in reaction scheme 1, and the physicochemical characteristics of each compound are shown in synthesis examples 14 and 15.

[Reaction Formula 2]

Synthetic Example 14. 5-chloro-N4-(1H-indazol-5-yl)-N2-(1,2,3,4-tetrahydroisoquinolin-7-yl)pyrimidine-2,4-diamine (Compound 3724)

MS (ESI): m/z = 391.18 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 13.10 (s, 1H), 9.09 (s, 1H), 8.86 (s, 1H), 8.08 (s, 1H), 8.01 (s, 1H), 7.93 (s, 1H), 7.53 (s, 1H), 7.49 (dd, J = 8.3 Hz, 1H), 7.22-7.18 (m, 2H), 6.75 (s, 1H), 3.41 (s, 2H), 2.89-2.85 (m, 2H), 2.53-2.51 (m, 2H).

Synthetic Example 15. 5-chloro-N4-(1H-indazol-5-yl)-N2-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)pyrimidine-2,4-diamine (Compound 3726)

MS (ESI): m/z = 405.17 (M ⁺ +1)

^1H NMR (400 MHz, DMSO-d ₆ ) δ 13.08 (s, 1H), 9.13 (s, 1H), 8.89 (s, 1H), 8.09-8.06 (m, 1H), 8.05 (s, 2H), 7.86 (s, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.42 (dd, J = 8.9, 1.8 Hz, 1H), 7.27 (s, 1H), 7.10 (d, J = 7.9 Hz, 1H), 6.78 (d, J = 8.3 Hz, 1H), 2.78 (s, 2H), 2.65-2.63 (m, 2H), 2.47 (s, 2H), 2.19 (s, 3H).

<Experimental Example 1. Evaluation of kinase activity of the compound of the present invention against MLK3 or HPK1>

Experimental Example 1-1. Evaluation of kinase activity inhibition for MLK3

Kinase activity inhibition for MLK3 is calculated as follows. Recombinant human MKK3 protein is mixed with the 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 [gamma-33PATP]) and reacted at room temperature for 40 minutes. After that, phosphoric acid is added to 0.5% to terminate the reaction, and 10 μl of the reaction solution is spotted onto a P30 filter. The spotted filter is washed four times in a 0.425% phosphoric acid solution for 4 minutes, then washed once more in methanol, and dried to measure the activity of MLK3 by scintillation counting. The activity value from the group in which the compound was not added is set as 100% of the control, and the degree of MLK3 inhibition by the compound is converted. To calculate the IC ₅₀ value, the compound is diluted by 1/3 from the highest concentration of 1 μM and treated at a total of 9 concentrations to evaluate the degree of inhibition, and the concentration that causes 50% inhibition is defined as the IC ₅₀ value.

Experimental Example 1-2. Evaluation of Kinase Activity Inhibition for HPK1

Kinase activity inhibition for HPK1 was calculated as follows. Recombinant human HPK1 protein was mixed with the 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 [gamma-33P-ATP]) and reacted at room temperature for 40 minutes. After that, phosphoric acid was added to 0.5% to terminate the reaction, and 10 μl of the reaction solution was spotted onto a P30 filter. The spotted filter was washed four times in a 0.425% phosphoric acid solution for 4 minutes, then washed once more in methanol, and dried to measure the activity of HPK1 by scintillation counting. The activity value from the group in which the compound was not added was set as 100% of the control, and the degree of HPK1 inhibition by the compound was calculated. To calculate the IC ₅₀ value, the compound is diluted by 1/3 from the highest concentration of 1 μM and treated at a total of 9 concentrations to evaluate the degree of inhibition, and the concentration that causes 50% inhibition is defined as the IC ₅₀ value.

Experimental Example 1-3. Results of kinase activity inhibition for MLK3 or HPK1

Using the compounds synthesized in the present invention, inhibition of kinase activity against MLK3 or HPK1 was confirmed, and the results are shown in Table 1.

compound HPK1 activity
IC ₅₀ (nM) MLK3 activity
IC ₅₀ (nM) 3703 5.0 3.6 3704 12.2 6.3 3705 6.2 1.3 3706 25.8 21.8 3707 27.8 35.7 3708 126.0 146.2 3710 21.9 5.7 3711 14.4 6.2 3713 24.1 8.2 3720 2.1 1.4 3721 5.6 9.5 3722 3.2 4.8 3724 3.5 5.8 3726 1.0 9.2

Referring to Table 1 above, it was confirmed that the compounds synthesized in the present invention simultaneously exhibit excellent inhibitory activity against HPK1 and MLK3.

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

The anticancer activity of MLK3 and HPK1 kinase inhibitors was evaluated in renal cell carcinoma ACHN (RCC), gastric cancer cells with naturally regulated MLK3 expression in patients with gastric cancer, YCC-18 (GC, MLK3 Mutation), YCC-30 (MLK3 Amplification), SNU-668 (MLK3 Low expression) cells, and other various solid tumors as follows.

Each cell was cultured in a 96-well plate at 10,000 cells per well, stabilized for one day, and the compounds synthesized in the present invention were serially diluted from 10 μM and treated to the cells. The cells treated with the compounds were cultured for 3 days, and the cell viability was measured by treating with WST reagent. The concentration at which 50% of the cancer cells were killed was expressed as IC ₅₀ in Tables 2 to 4 below. Meanwhile, in Table 2 and the results below, '-' means no activity.

compound Cell viability IC ₅₀ (nM) ACHN (RCC) YCC-18
(GC, MLK3 Mutation) YCC-30 (MLK3 Amplification) SNU-668 (MLK3 Low expression) 3703 344.1 5278.0 126.6 1953.0 3704 71.5 709.2 141.8 388.6 3705 72.0 628.8 104.5 411.5 3706 407.5 1698.0 71.7 1330.0 3707 283.1 824.8 210.7 892.0 3708 290.2 2393.0 137.8 3386.0 3710 279.5 - 923.6 1846.0 3711 241.2 2980.0 208.1 1470.0 3713 89.1 1537.0 305.7 1233.0 3716 - - 579.5 - 3720 - - 899.2 - 3722 - - 789.8 - 3724 1114.0 2997.0 79.87 2868.0 2726 1326.0 1597.0 70.58 2284.0

Compound Cell viability IC ₅₀ (nM) Caki-1
(RCC) A498
(RCC) Hep3B
(Liver) SNU-398
(Liver) A2780
(Ovarian) SK-OV-3
(Ovarian) 3703 1385.0 2191.0 1181 796.2 494.1 - 3704 1109.0 3070.0 462.1 692.2 370.2 2270.0 3705 764.8 1678.0 323.9 491.5 166.0 1304.0 3706 1286.0 2505.0 758.7 1529.0 229.9 - 3707 1634.0 3493.0 950.1 1024.0 231.0 1656.0 3708 - - - 1797.0 602.2 - 3710 - - 2001 2725.0 1208.0 - 3711 - - 879.5 840.0 457.6 - 3713 751.2 - 1664 1138.0 298.4 - 3716 - - - - 4822.0 - 3719 - - - - 4391.0 - 3720 - - - - 5915.0 - 3722 - - - - 4220.0 - 3723 - - 4683.0 - 3582.0 1527.0 3724 1564.0 3561.0 1407.0 1409.0 812.2 999.8 3725 - - - - - - 3726 - 3519.0 1218.0 1507.0 778.7 -

Compound Cell viability IC ₅₀ (nM) MDA-MB-231
(Breast, TNBC) MCF-7
(Breast) HT-29
(Colon) SW-480
(Colon) A-549
(Lung) U-87MG
(Brain) 3703 878.3 1258.0 1359.0 962.3 180.7 1885.0 3704 515.8 626.9 816.0 460.3 757.3 1347.0 3705 361.8 823.7 523.1 185.1 650.4 730.8 3706 1250.0 1433.0 948.3 525.2 883.3 1741.0 3707 631.2 901.9 1550.0 1025.0 2186.0 1191.0 3708 1807.0 3182.0 1075.0 810.9 - - 3710 5311.0 3951.0 5591.0 1521.0 888.6 2175.0 3711 1387.0 1376.0 1796.0 499.6 - 990.8 3713 1065.0 1283.0 1333.0 551.1 - 1508.0 3716 - - - - 5780.0 - 3719 - - - - - - 3720 - - - 1211.0 - - 3723 2493.0 - 4987.0 - 4565.0 - 3724 1495.0 2033.0 1953.0 1079.0 2736.0 2513.0 3726 1424.0 1835.0 1510.0 1146.0 1235.0 2099.0

Referring to Tables 2 to 4 above, it can be confirmed that the compounds synthesized in the present invention have anticancer activity in various cancer cells. In particular, in ACHN cells, it can be seen that 9 compounds among the synthesized compounds have an IC ₅₀ of 1 μM or less, indicating excellent activity.

Meanwhile, as a result of evaluating the activity of substances according to the expression of MLK3, only compounds 3704, 3705, and 3707 were confirmed to have IC ₅₀ values of 1 μM or less in MLK3 protein mutant cells YCC-18, but most synthetic compounds had IC ₅₀ values of 1 μM or less in MLK3 protein overexpressing cells YCC-30, confirming excellent cancer cell inhibition ability.

Therefore, it can be seen that the compounds of the present invention have excellent cancer cell inhibition ability according to the expression of MLK3.

<Experimental Example 3. Measurement of immune factors and cytokines for evaluating the enhancement of T cell activity of the compound of the present invention>

The ability to increase T cell activity by inhibiting HPK1 was observed through the following experiment. Human PBMC cells were seeded at 8 x 10 ⁴ per well in a 96-well plate and pretreated with 100 nM of the compounds of the present invention for 2 hours. After that, CD3/CD28 Dynabeads were treated at a ratio of 1:1 with the number of cells and cultured for 6 days. After culture, the medium was measured using a BD Flow cytometer instrument using a Human Th1/Th2/Th17 CBA kit from BD Corporation to analyze the secretion of Cytokine human IL-2. After culture, the cells were fixed and reacted with Anti-p-SLP-76 antibody attached with a fluorescent antibody to measure the change in the expression of p-SLP76, which is a phosphorylated SLP76 (SH2-domain-containing leukocyte protein of 76 kDa) utilized as a T cell biomarker. After one day of reaction with the antibody, measurement was performed using a BD Flow cytometer instrument. The measured results are calculated by setting the positive control group treated only with CD3/CD28 Dynabeads as 100%.

compound p-SLP-76 Inhibiton (%) IL-2 Stimulation (%) 3703 45.8 228.6 3704 37.4 229.4 3705 52.6 191.6 3706 55.2 105.3 3707 68.4 117.9 3708 51.0 - 3710 61.0 - 3711 53.9 121.7 3713 64.8 109.3 3716 55.5 190.1 3720 75.8 - 3721 67.2 - 3722 72.6 - 3724 10.3 106.0 3726 12.4 121.9

The results are shown in Table 5, and it was confirmed that most of the compounds synthesized in the present invention inhibit the expression of p-SLP76, a representative T cell biomarker activated by HPK1, very well. In the case of cytokine Human IL-2, a representative biomarker that acts on the immune response of cancer cells by T cells, it was confirmed that compounds 3703, 3704, and 3709 were activated more than twice, and it was found that other compounds also showed some activity.

Therefore, since the compounds synthesized in the present invention suppress or activate biomarkers according to the inhibition of HPK1, it can be seen that the compounds of the present invention have excellent T cell activity enhancing ability.

<Example 1. Preparation of tablets>

100 mg of the compound 3706 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 compressed into tablets according to a conventional tablet manufacturing method.

<Preparation Example 2. Preparation of capsules>

After mixing 100 mg of the compound 3706 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, the above ingredients were mixed according to a conventional capsule manufacturing method and filled into a gelatin capsule to manufacture a capsule.

<Example 3. Preparation of injection>

10 mg of the compound 3706 of the present invention, an appropriate amount of sterile distilled water for injection, and an appropriate amount of a pH regulator were mixed, and then prepared with the above ingredient contents per 1 ampoule (2 ml) according to a conventional method for preparing injections.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (13)

A compound represented by the following chemical formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 1]

Among the above chemical formula 1 A or B is selected from the group consisting of carbon, which is substituted or unsubstituted with R ₅ and R ₆ , nitrogen, oxygen and carbonyl, which is substituted or unsubstituted with R ₇ (provided that when A and B are nitrogen, B is bonded as imine), R ₁ , R ₂ , R ₅ , R ₆ and R ₇ are 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 or hydroxy C ₁ -C ₄ alkyl; R ₄ is a 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, a substituted or unsubstituted C ₅ -C ₁₂ cycloalkyl, a substituted or unsubstituted C ₅ -C ₁₂ heterocycloalkyl, a substituted or unsubstituted C ₅ -C ₁₂ aryl, a substituted or unsubstituted C ₅ -C ₁₂ heteroaryl group, X is selected from a halogen group, a cyano group, a nitro group, an amide group, a thiol group, a sulfone group, a phosphoric acid group, and an amine group. In the first paragraph, The compound represented by the above chemical formula 1 is a compound characterized by having HPK1 (Hematopoietic Progenitor Kinase 1) or MLK3 (Mixed lineage kinase 3) inhibitory activity, an optical isomer thereof, or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for the prevention or treatment of cancer, viral infectious disease, Parkinson's disease, non-alcoholic fatty liver disease or tuberculosis, comprising a compound represented by the chemical formula 1 of claim 1, an optical isomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient. A method for inhibiting HPK1 (Hematopoietic Progenitor Kinase 1) and MLK3 (Mixed lineage kinase 3) activity in a subject by administering to the subject a compound represented by the chemical formula 1 of claim 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for preventing or treating cancer, characterized by comprising a compound represented by the chemical formula 1 of claim 1, an optical isomer thereof or a pharmaceutically acceptable salt thereof; and an anticancer drug for combination administration. In paragraph 5, A pharmaceutical composition for preventing or treating cancer, characterized in that the anticancer agent is selected from taxane-based 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. A pharmaceutical composition for preventing or treating cancer, characterized by comprising a compound represented by the chemical formula 1 of claim 1, an optical isomer thereof or a pharmaceutically acceptable salt thereof; and a cell therapeutic agent. In Article 7, A pharmaceutical composition for preventing or treating cancer, characterized in that the cell therapy agent is selected from among lymphokine activated killer cells (LAK), dendritic cells (DC), natural killer (NK) cells, tumor-infiltrating lymphocytes (TIL), engineered T cell receptor therapy (TCR-T), chimeric antigen receptor-modified T cells (CAR-T), and chimeric antigen receptor-modified NK cells (CAR-NK). A method for treating a subject having cancer, characterized by administering to the subject a compound represented by the chemical formula 1 of claim 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof. A compound represented by the chemical formula 1 of claim 1, or a pharmaceutically acceptable salt thereof; and A method of treating a subject having cancer, characterized by co-administering to the subject a cell therapy agent, an immune checkpoint inhibitor, or a tryptophan oxidation inhibitor. In Article 10, A method for treating a subject having cancer, characterized in that the cell therapy agent is selected from among lymphokine activated killer cells (LAK), dendritic cells (DC), natural killer (NK) cells, tumor-infiltrating lymphocytes (TIL), engineered T cell receptor therapy (TCR-T), chimeric antigen receptor-modified T cells (CAR-T), and chimeric antigen receptor-modified NK cells (CAR-NK). In Article 10, A method for treating a subject having cancer, wherein the immune checkpoint inhibitor is selected from an anti-PD-1 antibody (anti-Programmed cell death protein 1 antibody), an anti-CTLA4 antibody (anti-Cytotoxic T-Lymphocyte associated protein 4 antibody), or an anti-PDL1 antibody (anti-Programmed death ligand 1 antibody). In Article 10, A method for treating a subject having cancer, wherein the tryptophan oxidation inhibitor is selected from an IDO1 (Indoleamine 2,3-dioxygenase 1), IDO2 (Indoleamine 2,3-dioxygenase 2) or TDO2 (Tryptophan 2,3-dioxygenase) inhibitor.

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