KR20230072446A - Synergic combination of 2,3-dioxygenase inhibitor and immune checkpoint inhibitor for the treatment of cancer - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating a cancer, which comprises a combination of a first compartment comprising a derivative having a certain cyclohexyl-ethylene-amino-heteroaryl moiety or pharmaceutically acceptable salt thereof as an active ingredient and a second compartment comprising an immune checkpoint inhibitor as an active ingredient. In addition, the present invention provides a method for treating cancer, which comprises a step of administering a therapeutically effective amount of a derivative having a certain cyclohexyl-ethylene-amino-heteroaryl moiety or pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of an immune checkpoint inhibitor to a mammal.

Description

SYNERGIC COMBINATION OF 2,3-DIOXYGENASE INHIBITOR AND IMMUNE CHECKPOINT INHIBITOR FOR THE TREATMENT OF CANCER}

The present invention relates to synergistic combinations of indoleamine 2,3-dioxygenase inhibitors and immune checkpoint inhibitors useful in the treatment of cancer. More specifically, the present invention provides a selective inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1) and an immune checkpoint inhibitor, particularly a PD-1/PD-L1 signaling inhibitor, For example, it relates to synergic combinations of programmed death 1 protein (PD-1) antagonists for the treatment of cancer.

Some tumors, including non-small cell lung cancer (NSCLC), suppress immune function by altering the tumor microenvironment as a defense mechanism to evade attack by the immune system, or develop T-cell immunotolerance or immunity. Immune escape is attempted through immuno-editing, etc. Immune checkpoints are proteins that interfere with the destruction of cancer cells, and cancers become tumor resistant by activating immune suppressive molecules to evade T cell attack. Typically, when a specific cell surface protein called PD-L1 is expressed in cancer cells, it binds to PD-1 present in T cells to suppress T cell functions and evade immunity.

Immune checkpoint inhibitors block immune evasion signals by directly binding to immune checkpoint receptors (CTLA-4, PD-1) that control the activity of T cells that attack cancer, or by binding to PD-L1 on the surface of cancer cells. By doing so, the formation of immunological synapses is prevented, and thus T cells that are not interfered with immune evasion have a mechanism to destroy cancer cells. Representative immune checkpoint inhibitors include CTLA-4 monoclonal antibodies [eg, Ipilimumab (YERVOY ^TM ), etc.], PD-1 monoclonal antibodies [Nivolumab (Opdivo ^TM ), Pembrolizumab (Keytruda ^TM ), etc.], PD -L1 monoclonal antibodies [Atezolizumab (TECENTRIQ ^TM ), Durvalumab (IMFINZI ^TM ), etc.] are known. However, it is known that immune checkpoint inhibitors respond successfully to only a portion of the entire cancer patient population and overcome side effects such as autoimmune diseases.

Tryptophan is an essential amino acid for cell proliferation and survival. Indolamine 2,3-dioxygenase 1 (commonly referred to as 'IDO-1') is the first, rate-determining step in the degradation of L-tryptophan, an essential amino acid, into N-formyl-kynurenine. It is a heme-containing intracellular enzyme that catalyzes IDO-1 acts on the metabolism of L-tryptophan to break it down into N-formyl-kynurenine, and N-formyl-kynurenine is metabolized by various steps to produce nicotinamide adenine dinucleotide (NAD+). Tryptophan catabolites produced from N-formyl-kynurenine, such as kynurenine, are known to be cytotoxic to T-cells. The action of IDO depletes tryptophan and produces kynurenine, and these two cooperate with each other to inhibit the activity of immune cells including T-cells through various mechanisms (Mellor, A. L. & Munn, D. H. Nature Rev. Immunol. 8, 74-80 (2008), Fallarino, F., Gizzi, S., Mosci, P., Gronmann, U. & Puccetti, P. Curr. Drug Metab. 8, 209-216 (2007)). IDO-1 is distributed in dendritic cells and regulatory B cells as well as cancer cells, and acts on these cells to suppress the ability of the immune system to recognize and attack cancer cells. Therefore, overexpression of IDO-1 can lead to increased resistance in the tumor microenvironment, which acts to grow cancer tissue.

In the case of cancer patients, it has been reported that if IDO-1 is upregulated, the prognosis becomes poor (Uyttenhove, C. et al. Nature Med. 9, 1269-1274 (2003)). From tests in which the IDO-1 gene was removed from mice, it was confirmed that IDO-1 plays a key role in the process of immune tolerance and inflammatory carcinogenesis (Muller, A. J., Mandik-Nayak, L. & Prendergast, G. C. Immunotherapy 2, 293-297 (2010) Muller, A. J. et al. Proc. Natl Acad. Sci. USA 105, 17073-17078 (2008)). In particular, it has been reported that the use of IDO-1 inhibitors as adjuvant treatments can improve the effects of immunochemotherapy, radiotherapy, and anticancer vaccines (Muller, A. J., DuHadaway, J. B., Donover, P. S., Sutanto-Ward, E. & Prendergast, G. C. Nature Med. 11, 312-319 (2005)). In addition, it has been reported that the reason why the anticancer drug imatinib (Gleevec) shows a strong effect on solid gastrointestinal stromal tumors is because it inhibits IDO-1 (Balachandran, V. P. et al. Nature Med. 17, 1094 -1100 (2011)).

Therefore, IDO-1 inhibitors can effectively inhibit cancer metastasis and cancer growth, and can be usefully used for the treatment and prevention of viral infections and autoimmune diseases such as rheumatoid arthritis. In addition, IDO-1 inhibitors can be used to activate T cells during T cell suppression by pregnancy, malignant tumors or viruses. It is expected that IDO-1 inhibitors can also be used for the treatment of patients. The present inventors have found that a derivative having a specific cyclohexyl-ethylene-amino-heteroaryl moiety or a pharmaceutically acceptable salt thereof not only has an excellent inhibitory activity against IDO-1 but also exhibits remarkably high exposure to the body upon oral administration. (Korean Patent Application No. 10-2021-0037824, filed on March 24, 2021). The derivative or pharmaceutically acceptable salt thereof can be usefully used for the treatment and prevention of various diseases mediated by IDO-1, for example, proliferative disorders such as cancer, viral infections and/or autoimmune diseases. .

The present inventors have found that when a derivative having a specific cyclohexyl-ethylene-amino-heteroaryl moiety or a pharmaceutically acceptable salt thereof is administered in combination with an immune checkpoint inhibitor, the increase is higher than that when the immune checkpoint inhibitor is administered alone. It was found to exhibit significant tumor suppressive activity.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising a combination of a derivative having a specific cyclohexyl-ethylene-amino-heteroaryl moiety or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor. aims to

Another object of the present invention is to provide a method for treating cancer comprising administering a derivative having a specific cyclohexyl-ethylene-amino-heteroaryl moiety or a pharmaceutically acceptable salt thereof in combination with an immune checkpoint inhibitor. to be

According to one aspect of the present invention, the first compartment containing the compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient and the second compartment containing an immune checkpoint inhibitor as an active ingredient A pharmaceutical composition for preventing or treating cancer, including the combination, is provided.

<Formula 1>

during the ceremony,

R ₁ is a C ₁ -C ₆ alkyl group;

A is a heteroaryl group selected from the group consisting of benzo[d]oxazolyl and quinazolinyl, and the heteroaryl group is substituted with one or two substituents selected from the group consisting of halogen and C ₁ -C ₆ alkyl.

According to another aspect of the present invention, comprising administering to a mammal a therapeutically effective amount of the compound of Formula 1 or a pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of an immune checkpoint inhibitor, A method of treating cancer is provided.

According to another aspect of the present invention there is provided the use of a combination of a compound of Formula 1 or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor for use in the manufacture of a medicament for the treatment of cancer in a mammal. do.

When a derivative having a specific cyclohexyl-ethylene-amino-heteroaryl moiety (i.e., a compound of Formula 1) or a pharmaceutically acceptable salt thereof is administered in combination with an immune checkpoint inhibitor, the immune checkpoint inhibitor is administered alone. It was found by the present invention that it exhibits a synergistic tumor suppression activity than when administered as . Therefore, the combination of the compound of Formula 1 or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor can be usefully applied to suppress cancer metastasis and cancer recurrence.

1a is a plasma concentration profile obtained after oral administration of the compound of Example 1 and a control substance (BMS-986205) to rats.
Figure 1b is a blood concentration profile obtained after orally administering the compound of Examples 2 to 5 to rats together with a virtual blood concentration profile calculated by orally administering the compound of Example 1 to rats.
Figure 2 shows the results obtained by evaluating the tumor growth inhibitory activity according to the combined administration of the compound of Example 1 and the anti-PD-1 antibody.
Figure 3 is a waterfall plot showing inhibition of tumor growth on day 11.
Figure 4 shows survival curves of each group of C57BL/6 mice bearing MC38 transplanted tumors.
5A-5C show tumor volume tracing curves of surviving individuals who showed complete cure.

The present invention includes a combination of a first compartment containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient and a second compartment containing an immune checkpoint inhibitor as an active ingredient, Provided is a pharmaceutical composition for preventing or treating cancer.

<Formula 1>

during the ceremony,

R ₁ is a C ₁ -C ₆ alkyl group;

A is a heteroaryl group selected from the group consisting of benzo[d]oxazolyl and quinazolinyl, and the heteroaryl group is substituted with one or two substituents selected from the group consisting of halogen and C ₁ -C ₆ alkyl.

Preferably, the compound represented by Formula 1 contained in the first compartment may be selected from the group consisting of the following compounds:

6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazol-2-amine ;

6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)butan-2-yl)benzo[d]oxazol-2-amine ;

6-fluoro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazol-2- amine;

6,7-difluoro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazole -2-amine; and

2-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)-7-methylquinazolin-4-amine .

More preferably, the compound of Formula 1 contained in the first compartment is 6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl) cyclohexyl)propan-2-yl)benzo[d]oxazol-2-amine.

The compound of Formula 1 may be in the form of a pharmaceutically acceptable salt. Such salts include conventional acid addition salts, for example salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid or nitric acid, and acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, citric acid, maleic acid, malic acid. salts derived from organic acids such as ronic acid, methanesulfonic acid, tartaric acid, malic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, p-toluenesulfonic acid, oxalic acid or trifluoroacetic acid. In addition, the salt includes a common metal salt form, for example, a salt derived from a metal such as lithium, sodium, potassium, magnesium, or calcium. The acid addition salt or metal salt may be prepared according to a conventional method.

The compound of Formula 1 or a pharmaceutically acceptable salt thereof may be prepared according to the prior application of the present applicant, that is, Korean Patent Application No. 10-2021-0037824 (filed on March 24, 2021). For example, the compound of Formula 1 or a pharmaceutically acceptable salt thereof may be obtained by reacting a compound of Formula 2 or a salt thereof with a compound of Formula 3 to obtain a compound of Formula 1; and optionally, converting the compound of Formula 1 into a pharmaceutically acceptable salt thereof.

<Formula 2>

<Formula 3>

X-A

In the formula, R ₁ and A are as defined above, and X is halogen.

Compounds of Formula 3 are commercially available. The reaction between the compound of Formula 2 or a salt thereof (eg, hydrochloride) and the compound of Formula 3 may be carried out in the presence of a base and a solvent. The base may be cesium carbonate, potassium carbonate, sodium carbonate, triethylamine, etc., and the solvent may be an organic solvent such as N,N-dimethylformamide, 1,4-dioxane, tetrahydrofuran, ethanol, isopropyl alcohol, etc. can In addition, the reaction may be carried out at room temperature to 100 ℃.

The compound of Formula 2 or a salt thereof may be prepared, for example, according to Reaction Scheme 1 below.

<Scheme 1>

In Reaction Scheme 1, R ₁ is the same as defined above.

The compound of Formula 5 may be prepared through a Suzuki reaction between a commercially available compound of Formula 4 and 4-chloro-6-fluoroquinoline. The reaction may be carried out using a palladium catalyst, wherein the palladium catalyst is palladium (II) acetate (Pd(OAc) ₂ ), tris (dibenzylideneacetone) dipalladium (tris (dibenzylideneacetone) dipalladium, Pd ₂ ( dba) ₃ ), tetrakis(triphenylphosphine)palladium(0)(Pd(PPh ₃ ) ₄ ) or [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)(Pd( dppf)Cl ₂ ) and the like. In addition to the palladium catalyst, the reaction can be carried out by adding a ligand and a base. The ligand is (S) -2,2-bis (diphenylphosphino) -1,1-binaphthyl (BINAP), 1,1'-bis (diphenylphosphino) ferrocene (dppf) or (tri- O-tolyl)phosphine (P(O-Tol) ₃ ); and the like. The base is cesium carbonate (Cs ₂ CO ₃ ), sodium carbonate (Na ₂ CO ₃ ), potassium carbonate (K ₂ CO ₃ ), potassium fluoride (KF), cesium fluoride (CsF), sodium hydroxide (NaOH ), potassium phosphate (K ₃ PO ₄ ), sodium tert-butoxide (tert-BuONa) or potassium tert-butoxide (tert-BuOK). The reaction is carried out in a non-polar organic solvent such as benzene or toluene or a polar organic solvent such as 1,4-dioxane, tetrahydrofuran, acetonitrile, 1,2-dimethoxyethane, or N,N-dimethylformamide, It may be performed at 50 °C to 150 °C, preferably at 80 °C to 110 °C. Other reaction conditions including reaction time can be performed according to a known method for Suzuki reaction (Barbara Czako and Laszlo Kurti, STRATEGIC APPLICATIONS of NAMED REACTIONS in ORGANIC SYNTHESIS, 2005).

Reduction of the compound of Formula 5 may be performed using palladium/carbon in an organic solvent such as ethyl acetate or methanol. The reduction reaction can typically be carried out at room temperature using hydrogen.

Reduction of the compound of Formula 6 may be performed using lithium aluminum hydride in an organic solvent such as tetrahydrofuran or dichloromethane. The reduction reaction may be typically carried out at -78°C to room temperature.

Oxidation of the compound of Formula 7 may be performed using an oxidizing agent in an organic solvent such as ethyl acetate or dichloromethane. The oxidation reaction may typically be carried out at 0°C to room temperature.

The compound of Formula 9 may be prepared by condensation of the compound of Formula 8 and (S)-(-)-2-methyl-2-propanesulfinamide. The condensation may be performed in an organic solvent such as ethyl acetate, dichloromethane, or tetrahydrofuran in the presence of a Lewis acid catalyst such as titanium (IV) isopropoxide or titanium (IV) epoxide. The reaction may be carried out at -78 ° C to room temperature.

The compound of Formula 10 may be prepared by reacting the compound of Formula 9 with an alkyl Grignard reagent. In addition, the compound of Formula 2 or a salt thereof (eg, a hydrochloride salt) may be prepared by deprotecting the compound of Formula 10. The deprotection reaction may be performed according to a known method (Theodora W. Greene and Peter G. M. Wuts, Protective groups in organic synthesis, 3rd Ed., 1999). For example, the deprotection reaction may be performed at room temperature using a solution of trifluoroacetic acid or hydrochloric acid in an organic solvent such as dichloromethane, 1,4-dioxane or ethyl acetate.

In the pharmaceutical composition of the present invention, the immune checkpoint inhibitor is, for example, the PD-1 / PD-L1 signaling pathway (Programmed Death Receptor-1 (PD-1) / Programmed Death Ligand-1 (PD-L1) signaling pathway , PD-1/PD-L1 signaling pathway) inhibitors. The immune checkpoint inhibitor is preferably an anti-CTLA-4 antibody (eg, Ipilimumab, etc.), an anti-PD-1 antibody (Nivolumab, Pembrolizumab, etc.) , or an anti-PD-L1 antibody [Atezolizumab, Durvalumab, etc.], more preferably an anti-PD-1 antibody [Nivolumab, Pembrolizumab ), etc.], but is not limited thereto. The antibody may be a monoclonal antibody or an antigen-binding fragment thereof. The antigen-binding fragment refers to a fragment that retains the function of binding to an antigen of a monoclonal antibody, and includes Fab, Fab', F(ab')2, scFv (scFv)2, scFv-Fc, and Fv Include etc.

In the pharmaceutical composition of the present invention, the cancer may be a solid tumor, for example, squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myelogenous leukemia , multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme , cervical cancer, brain cancer, bladder cancer, breast cancer, colon carcinoma, urothelial cancer, head and neck cancer, etc., but is not limited thereto.

In the pharmaceutical composition of the present invention, the first compartment may be an oral administration compartment, and the second compartment may be an injection compartment. Therefore, the first compartment contains pharmaceutically acceptable carriers such as excipients (for example, lactose, corn starch, etc.), disintegrants, and lubricants (magnesium stearate, etc.) commonly used in the field of pharmaceuticals. and may be formulated into oral preparations such as tablets, capsules, powders, granules and suspensions, emulsions or syrups according to conventional methods. The first compartment of the oral dosage form may be, for example, a single-dose or multi-dose dosage form. The second compartment may be in the form of a preparation for parenteral administration such as an injection, for example, a form suitable for intramuscular, intraperitoneal, subcutaneous or intravenous administration, and may be a single or multiple dose dosage form. form) can be For the injection, a sterile solution of an immune checkpoint inhibitor is usually prepared, and the pH of the solution must be appropriately adjusted and buffered. For intravenous administration, the total concentration of solutes should be adjusted to impart isotonicity to the formulation. The second compartment may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier such as saline having a pH of 7.4.

In one embodiment of the pharmaceutical composition of the present invention, the first compartment is administered orally 1 to 4 times a day at a dose of 5 to 200 mg/kg of the compound of Formula 1 or a pharmaceutically acceptable salt thereof. It can be. In a preferred embodiment, the first compartment may be orally administered at a dose of 10 to 20 mg/kg of the compound of Formula 1 or a pharmaceutically acceptable salt thereof, 1 to 2 times a day. Of course, the dosage and frequency of administration may be appropriately adjusted according to the patient's condition, age, severity of cancer, and the like.

In another embodiment of the pharmaceutical composition of the present invention, the second compartment may be injected 1 to 5 times a week at a dose of 0.005 to 10 mg/kg of the immune checkpoint inhibitor. In a preferred embodiment, the second compartment is injected at a dose of 1 to 10 mg/kg of the anti-PD-1 antibody, 1 to 5 times per week. Of course, the dosage and frequency of administration may be appropriately adjusted according to the patient's condition, age, severity of cancer, and the like.

The present invention also relates to a method for treating cancer comprising administering to a mammal a therapeutically effective amount of a compound of Formula 1 below or a pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of an immune checkpoint inhibitor. provides

<Formula 1>

during the ceremony,

R ₁ is a C ₁ -C ₆ alkyl group;

A is a heteroaryl group selected from the group consisting of benzo[d]oxazolyl and quinazolinyl, and the heteroaryl group is substituted with one or two substituents selected from the group consisting of halogen and C ₁ -C ₆ alkyl.

Preferably, the compound of Formula 1 may be selected from the group consisting of the following compounds:

6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazol-2-amine ;

6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)butan-2-yl)benzo[d]oxazol-2-amine ;

6-fluoro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazol-2- amine;

6,7-difluoro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazole -2-amine; and

2-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)-7-methylquinazolin-4-amine .

More preferably, the compound of Formula 1 is 6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl ) benzo[d]oxazol-2-amine.

In the treatment method of the present invention, the compound of Formula 1 or a pharmaceutically acceptable salt thereof, an immune checkpoint inhibitor, and the type of cancer are the same as described in relation to the pharmaceutical composition of the present invention.

In one embodiment of the treatment method of the present invention, the compound of Formula 1 or a pharmaceutically acceptable salt thereof may be administered orally, and the immune checkpoint inhibitor may be injected. Therefore, the compound of Formula 1 or a pharmaceutically acceptable salt thereof may be administered in an oral dosage form, and the oral dosage form is the same as described in relation to the first compartment in the pharmaceutical composition of the present invention. . In addition, the immune checkpoint inhibitor may be administered in the form of an injection, and the form of the preparation for parenteral administration, such as the injection, is the same as that described in relation to the second compartment in the pharmaceutical composition of the present invention.

In one embodiment of the treatment method of the present invention, the compound of Formula 1 or a pharmaceutically acceptable salt thereof may be orally administered at a dose of 5 to 200 mg/kg, 1 to 4 times a day. In a preferred embodiment, the compound of Formula 1 or a pharmaceutically acceptable salt thereof may be orally administered at a dosage of 10 to 20 mg/kg, 1 to 2 times a day. Of course, the dosage and frequency of administration may be appropriately adjusted according to the patient's condition, age, severity of cancer, and the like.

In another embodiment of the treatment method of the present invention, the immune checkpoint inhibitor may be injected at a dose of 0.005 to 10 mg/kg, 1 to 5 times a week. In a preferred embodiment, the anti-PD-1 antibody is injected at a dose of 1 to 10 mg/kg, 1 to 5 times per week. Of course, the dosage and frequency of administration may be appropriately adjusted according to the patient's condition, age, severity of cancer, and the like.

The present invention also provides the use of a compound of Formula 1 or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor in combination for use in the manufacture of a medicament for the treatment of cancer in a mammal. In the use of the present invention, the compound of Formula 1 or a pharmaceutically acceptable salt thereof, an immune checkpoint inhibitor, and the type of cancer are the same as those described in relation to the pharmaceutical composition of the present invention. In addition, the compound of Formula 1 or a pharmaceutically acceptable salt thereof and the form, dosage, administration method, etc. of each containing the immune checkpoint inhibitor are the same as those described in relation to the treatment method of the present invention.

Hereinafter, the present invention will be described in more detail through examples and test examples. However, these Examples and Test Examples illustrate the present invention, and the present invention is not limited thereto.

Analysis of the compounds prepared in the following examples was performed as follows: nuclear magnetic resonance (NMR) spectral analysis was performed on a Bruker 400 MHz spectrometer, chemical shifts were analyzed in ppm, Column chromatography was performed on silica gel (Merck, 70-230 mesh) (W. C. Still, J. Org. Chem., 43, 2923, 1978). Starting materials were known compounds that were synthesized according to the literature or purchased from Sigma-Aldrich, etc.

Example 1. 6-Chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazole -2-amine

Step 1: Ethyl 2-(4-(6-fluoroquinolin-4-yl)cyclohex-3-en-1-yl)acetate

Ethyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-en-1-yl)acetate (5.83 g), 4 -Chloro-6-fluoroquinoline (3.00 g) and sodium carbonate (5.35 g) were dissolved in a mixed solvent of 1,4-dioxane (30 ml) and water (30 ml), [1,1'-bis( Diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl ₂ ) (675 mg) was added and stirred at 95°C overnight. After concentrating the reaction mixture, ethyl acetate was added, washed with distilled water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a yellow liquid residue. The residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate = 1/1, v/v) to obtain 4.40 g of the title compound as a white solid. (Yield: 82.4%)

¹ H-NMR (CDCl ₃ ) δ 8.79 (d, 1H), 8.10 (t, 1H), 7.61 (d, 1H), 7.51 (t, 1H). 7.18 (d, 1H), 5.81 (s, 1H), 4.18 (q, 2H), 2.51-2.28 (m, 7H), 2.02 (m, 2H), 1.58 (m, 1H), 1.28 (t, 3H)

Step 2: Ethyl 2-(4-(6-fluoroquinolin-4-yl)cyclohexyl)acetate

Ethyl 2-(4-(6-fluoroquinolin-4-yl)cyclohex-3-en-1yl)acetate (4.40 g) prepared in step 1 and 10% Pd/C (440 mg), acetic acid ( 0.16 ml) was dissolved in 30 ml of methanol and stirred for 12 hours under a hydrogen atmosphere. After drying and filtering the reaction mixture, the resulting residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate = 1/1, v/v) to obtain 4.17 g of the title compound as a white solid. (Yield: 94.2%)

¹ H-NMR (CDCl ₃ ) δ 8.80 (s, 1H), 8.11 (t, 1H), 7.65 (d, 1H), 7.46 (t, 1H), 7.30 (d, 1H), 4.16 (q, 2H) , 3.21-3.06 (m, 1H), 2.49 (s, 1H), 2.30 (d, 1H), 2.04-1.72 (m, 7H), 1.62 (q, 1H), 1.37-1.24 (m, 4H)

Step 3: 2-(4-(6-fluoroquinolin-4-yl)cyclohexyl)ethane-1-ol

Ethyl 2-(4-(6-fluoroquinolin-4-yl)cyclohexyl)acetate (2.27 g) prepared in step 2 was dissolved in tetrahydrofuran (24 ml), stirred at 0 ° C. for 10 minutes, and lithium Aluminum hydride (355 mg) was added slowly. The reaction mixture was stirred at room temperature for 6 hours. After stopping the reaction by adding water, extraction was performed with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and filtered. The obtained filtrate was concentrated under reduced pressure. Two stereoisomers confirmed by TLC were collected and purified by silica gel column chromatography (n-hexane/ethyl acetate/=1/2, v/v) to obtain 1.53 g of the title compound as a white solid. (Yield: 73.2%)

¹ H-NMR (DMSO-d ₆ ) δ 8.77 (d, 1H), 8.06 (t, 1H), 7.89 (d, 1H), 7.62 (t, 1H), 7.35 (d, 1H), 4.63 (t, 1/2 H), 4.39 (t, 1/2 H), 3.47 (q, 2H), 3.20 (t, 1/2 H), 3.12 (t, 1/2 H), 1.87-1.72 (m, 4H) ), 1.50-1.36 (m, 4H), 1.21-1.14 (q, 2H)

Step 4: 2-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)acetaldehyde

2- (4- (6-fluoroquinolin-4-yl) cyclohexyl) ethane-1-ol (15.14 g) prepared in step 3 was dissolved in dichloromethane (185 ml) and stirred at 0 ° C for 30 minutes After that, Dess-Martin oxidizer (35.2 g) was added slowly. The reaction mixture was stirred at room temperature for 5 hours. After stopping the reaction by adding water, extraction was performed with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and filtered. The obtained filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (n-hexane/ethyl acetate/=3/1, v/v) to obtain 8.72 g of the title compound as a white solid among two stereoisomers confirmed by TLC. did (Yield: 57.9%)

¹ H-NMR (CDCl ₃ ) δ 9.82 (s, 1H), 8.81 (d, 1H), 8.12 (t, 1H), 7.64 (d, 1H), 7.48 (t, 1H), 7.31 (d, 1H) , 3.22 (t, 1H), 2.61 (s, 3H), 1.93-1.67 (m, 8H)

Step 5: (S)-N-(2-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethylidene)-2-methylpropane-2-sulfinamide

2-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)acetaldehyde prepared in step 4 (4.50 g) and (S)-(-)-2-methyl-2-propane Sulfinamide (4.02 g) was dissolved in dichloromethane (55 ml), stirred at 0° C. for 10 minutes, and then titanium (IV) isopropoxide (9.82 ml) was slowly added. After stirring the reaction mixture at room temperature for 8 hours, water was added to stop the reaction, and then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and filtered. The obtained filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (n-hexane/ethyl acetate/=1/1, v/v) to obtain 5.22 g of the title compound as a white solid. (Yield: 84.0%)

¹ H-NMR (CDCl ₃ ) δ 8.82 (1H, d), 8.14-8.10 (m, 2H), 7.64 (d, 1H), 7.46 (t, 1H), 7.33 (d, 1H), 3.22 (t, 1H), 2.72 (t, 2H), 2.04 (s, 1H), 1.95-1.68 (m, 8H), 1.21 (s, 9H)

Step 6: (R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-amine hydrochloride

(S)-N-(2-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethylidene)-2-methylpropane-2-sulfinamide (2.54 g) was dissolved in dichloromethane (23 ml), stirred at 0° C. for 10 minutes, and then 3.0 M methylmagnesium bromide (diethyl ether solution) (4.6 ml) was slowly added. After stirring the reaction mixture at room temperature for 2 hours, the reaction was stopped by adding saturated ammonium chloride aqueous solution, and then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and filtered. The obtained filtrate was concentrated under reduced pressure. The obtained material was dissolved in ethyl acetate (25 ml), 4N hydrochloric acid and 1,4-dioxane solution (2 ml) was added dropwise, and the mixture was stirred at room temperature for 8 hours. The obtained solid was filtered under reduced pressure and washed with ethyl acetate to obtain 1.67 g of the title compound as a white solid. (Yield: 86.0%)

¹ H-NMR (DMSO-d ₆ ) δ 9.23 (d, 1H), 8.56 (t, 1H), 8.42 (d, 1H), 8.27 (s, 2H), 8.08 (t, 2H), 3.63 (s, 1H), 3.19 (s, 1H), 2.11 (m, 11H), 1.26 (d, 3H)

Step 7: 6-Chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazol- 2-amine

(R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-amine hydrochloride prepared in step 6 (22 mg) and 2,6-dichlorobenzo Oxazole (15 mg) was dissolved in 1,4-dioxane (1.0 ml), and N,N-diisopropylethylamine (40 uL) was slowly added. The reaction mixture was stirred at 80°C for 12 hours. The reaction mixture was cooled to room temperature, water was added to stop the reaction, and then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and filtered. The obtained filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (n-hexane/ethyl acetate/=1/2, v/v) to obtain 14.7 mg of the title compound. (Yield: 43.7%)

¹ H-NMR (CDCl ₃ ) δ 8.79 (d, 1H), 8.13-8.10 (m, 1H), 7.67-7.64 (d, 1H), 7.49-7.44 (m, 1H), 7.31 (d, 1H), 7.26-7.24 (m, 1H), 7.15 (d, 1H), 4.92 (d, 1H), 4.06-4.02 (m, 1H), 3.21-3.19 (m, 1H), 2.04-2.00 (m, 1H), 1.88-1.64 (m, 11H), 1.36 (d, 3H)

Example 2. 6-Chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)butan-2-yl)benzo[d]oxazole -2-amine

Step 1: (R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)butan-2-amine hydrochloride

(S)-N-(2-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethylidene)-2-methylpropane-2- prepared in step 5 of Example 1 Sulfinamide (3.0 g) was dissolved in dichloromethane (30 ml), stirred at -20 °C for 10 minutes, and then 1.0 M ethyl magnesium bromide (THF solution) (5.34 mL) was slowly added. The reaction mixture was stirred at -20 °C for 6 hours and then warmed to room temperature. After quenching the reaction by adding a saturated aqueous ammonium chloride solution, extraction was performed with ethyl acetate. The extracted organic layer was washed with brine and dried over anhydrous magnesium sulfate. filtered. The obtained filtrate was concentrated under reduced pressure. The obtained material was dissolved in ethyl acetate (30 ml), 4N hydrochloric acid and 1,4-dioxane solution (8 ml) was added dropwise, and the mixture was stirred at room temperature for 8 hours. The obtained solid was filtered under reduced pressure and washed with ethyl acetate to obtain 2.33 g of the title compound as a white solid. (Yield: 86.0%)

¹ H-NMR (DMSO-d ₆ ) δ 9.21 (d, 1H), 8.57 (q, 1H), 8.40 (d, 1H), 8.26 (s, 2H), 8.08 (q, 2H), 3.61 (s, 1H), 3.01 (s, 1H), 2.00-1.62 (m, 13H), 0.95 (t, 3H)

Step 2: 6-Chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)butan-2-yl)benzo[d]oxazol- 2-amine

(R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)butan-2-amine hydrochloride prepared in step 1 (70 mg), 2,6-dichlorobenzo Oxazole (32 mg), and potassium carbonate (57 mg) were dissolved in N,N-dimethylformamide (1.0 ml), and triethylamine (30 uL) was slowly added. The reaction mixture was stirred at 80°C for 12 hours. The reaction mixture was cooled to room temperature, water was added to stop the reaction, and then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and filtered. The obtained filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (n-hexane/ethyl acetate/=1/2, v/v) to obtain 42 mg of the title compound. (Yield: 44.7%)

¹ H-NMR (CDCl ₃ ) δ 8.79 (d, 1H), 8.13-8.09 (m, 1H), 7.64 (d, 1H), 7.45 (t, 1H), 7.26-7.21 (m, 3H), 7.14 ( d, 1H), 5.60 (s, 1H), 3.88 (s, 1H), 3.19 (s, 1H), 2.04 (s, 1H), 1.82-1.61 (m, 12H), 1.01 (t, 3H)

Example 3. 6-Fluoro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxa sol-2-amine

(R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-amine hydrochloride prepared in step 6 of Example 1 (30 mg) and 2-chloro -6-Fluoro-1,3-benzooxazole (16 mg) was dissolved in 1,4-dioxane (1.0 ml), and N,N-diisopropylethylamine (50 uL) was slowly added. The reaction mixture was stirred at 100°C for 12 hours. The reaction mixture was cooled to room temperature, water was added to stop the reaction, and then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and filtered. The obtained filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (n-hexane/ethyl acetate/=1/2, v/v) to obtain 3.9 mg of the title compound. (Yield: 9.5%)

¹ H-NMR (CDCl ₃ ) δ 8.80 (d, 1H), 8.13-8.10 (m, 1H), 7.64 (d, 1H), 7.49-7.47 (m, 1H), 7.31 (d, 1H), 7.26- 7.24 (m, 1H), 7.01 (d, 1H), 6.93-6.88 (m, 1H), 4.74 (m, 1H), 4.05-4.01 (m, 1H), 3.21-3.19 (m, 1H), 2.04- 2.00 (m, 1H), 1.88-1.64 (m, 11H), 1.36 (d, 3H)

Example 4. 6,7-difluoro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[ d] oxazol-2-amine

(R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-amine hydrochloride prepared in step 6 of Example 1 (50 mg) and 2-chloro -6,7-difluoro-1,3-benzooxazole (33 mg) was dissolved in tetrahydrofuran (1.0 ml). The obtained solution was placed in a sealed tube, triethylamine (110 μL) was added dropwise, and then sealed. The reaction mixture was stirred at 120°C for 12 hours, cooled to room temperature, and water was added to stop the reaction, followed by extraction with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and filtered. The obtained filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (n-hexane/ethyl acetate/=1/2) to obtain 51.5 mg of the title compound. (Yield: 75.6%)

¹ H-NMR (CDCl ₃ ) δ 8.79 (d, 1H), 8.12 (dd, 1H), 7.64 (dd, 1H), 7.48 (td, 1H), 7.32 (d, 1H), 7.02-6.94 (m, 2H), 5.38(d, 1H), 4.04(m, 1H), 3.21(m, 1H), 2.08(br, 1H), 1.81-1.71(m, 10H), 1.38(d, 3H)

Example 5. 2-Chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)-7-methylquinazoline -4-amine

(R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-amine hydrochloride prepared in step 6 of Example 1 (50 mg) and 2,4 -Dichloro-7-methylquinazoline (37 mg) was dissolved in tetrahydrofuran (1.0 ml). The obtained solution was placed in a sealed tube, triethylamine (110 μL) was added dropwise, and sealed. The reaction mixture was stirred at 80°C for 12 hours, cooled to room temperature, and water was added to stop the reaction, followed by extraction with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and filtered. The obtained filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (n-hexane/ethyl acetate/=1/2) to obtain 49.5 mg of the title compound in the form of a pale yellow liquid. (Yield: 69.3%)

¹ H-NMR (CDCl ₃ ) δ 8.81 (d, 1H), 8.12 (t, 1H), 7.67 (dd, 1H), 7.59-7.56 (m, 2H), 7.47 (td, 1H), 7.36 (d, 1H), 7.28(m, 1H), 5.67(d, 1H), 4.58(m, 1H), 3.21(m, 1H), 2.51(s, 3H), 1.99-1.62(m, 11H), 1.39(d) , 3H)

Test Example 1. IDO1 enzyme activity assay (IDO1 enzyme assay)

Using the IDO1 fluorogenic inhibitor screening assay kit catalog #72037 BPS Bioscience, IDO enzyme activity analysis of the compounds prepared in Examples was performed. In a black 384 well plate, 5 μl of the test substance solution (prepared by dissolving the compounds of the examples in 10 μl of DMSO and then mixing with 90 μl of phosphate buffered saline (PBS)) was added, and 5 μl of IDO1 After adding His-tag enzyme (40 ng/μl in IDO1 assay buffer), pre-incubation was performed at 37°C for 2 hours. 90 μl of IDO1 fluorogenic reaction solution per well was added and reacted at room temperature for 1 hour. After adding 20 μl of fluorescence solution per well and reacting at 37° C. for 4 hours, the plate was cooled at room temperature for 10 minutes. Fluorescence was measured in a fluorescence meter at 400 nm for excitation and 510 nm for emission. For the analysis of the results, the fluorescence (Ft) of wells containing IDO protein and not treated with the test substance was set to 100%, and the fluorescence (Fb) of wells without IDO protein and not treated with the test substance was set to 0%. , and the % fluorescence was calculated.

% Fluorescence = (F-Fb)/(Ft-Fb)

(F = fluorescence of wells treated with test substance)

The results obtained by calculating the 50% inhibitory concentration (IC ₅₀ ) of the test substance obtained as above are shown in Table 1 below.

Test Example 2. IDO1 HeLa cell activity assay (IDO1 HeLa cell assay)

20000 cells/well of Hela cells were added to 100 μl of culture medium (EMEM containing 10% FBS, penicillin 100 U/ml, and streptomycin 100 μg/ml) in a tissue culture-treated 96 well plate. Inoculated. After culturing for 24 hours in a 5% carbon dioxide incubator, recombinant human interferon gamma was treated at a concentration of 50 ng/ml, and cultured in a 5% carbon dioxide incubator for 48 hours to induce IDO expression. made it

Activity was measured using an IDO1 cellular activity quickDetect supplements catalog: #62000-2, BPS Bioscience. Specifically, an assay medium was prepared by diluting IDO1 assay medium substrate 1 (IDO1 assay medium supplement 1) and IDO1 assay medium supplement 2 (IDO1 assay medium supplement 2) in a culture medium at a ratio of 1:100, respectively. Dilute the test substance (compound of each example) in the assay medium by 1/3 from 1 uM to make 10 concentrations, remove the culture medium using a multi-pipet, and then add the assay medium 200 containing the test substance to each well. [mu]l was added and incubated for 24 hours in a 5% carbon dioxide incubator. The next day, 140 μl of the culture medium from each well was transferred to a new 96-well plate, and 10 μl of 6.1N trichloroacetic acid was dispensed into each well, followed by incubation in a 50° C. incubator for 30 minutes. The plate was centrifuged at 2500 rpm for 10 minutes to settle all precipitates. A detection reagent solution (component D kit, catalog: #62000-2, BPS Bioscience) was diluted 50 times in acetic acid to prepare a detection reagent solution. From the centrifuged plate, 100 μl of the supernatant per well was transferred to a new transparent 96-well plate, 100 μl of the assay reagent solution was added to each well, reacted at room temperature for 10 minutes, and absorbance was measured at a wavelength of 480 nm. In the analysis of the results, the absorbance (At) of wells containing Hela cells in which IDO protein expression was induced without treatment with the inhibitor was set to 100%, and the absorbance (Ab) of wells containing Hela cells in which IDO protein expression was not induced was set to 0. The % absorbance was calculated as %. (% Absorbance = (A-Ab)/(At-Ab), A = Absorbance of wells treated with test substance)

The results obtained by calculating the 50% inhibitory concentration (IC ₅₀ ) of the test substance obtained as above are shown in Table 1 below.

Test Example 3. IDO1 HEK293 cell activity assay

30000 cells/well of HEK293 cells in 100 μl of culture medium (DMEM containing 10% FBS, penicillin 100 U/ml, and streptomycin 100 μg/ml) in a tissue culture-treated 96 well plate. Inoculated. After culturing for 24 hours in a 5% carbon dioxide incubator, the IDO1 expression vector (component A in IDO1 cell-based assay kit catalog#72031 BPS Bioscience) was transfected using Lipofectamine 2000 life technologies #11668027 ( transfection), and cultured for 24 hours in a 5% carbon dioxide incubator to express IDO1.

Activity was measured using the IDO1 cell-based assay kit catalog #72031 BPS Bioscience. Specifically, an assay medium was prepared by diluting IDO1 assay medium substrate 1 (IDO1 assay medium supplement 1) and IDO1 assay medium supplement 2 (IDO1 assay medium supplement 2) in a culture medium at a ratio of 1:100, respectively. Dilute the test substance (compound of each example) in the assay medium by 1/3 from 1 uM to make 10 concentrations, remove the culture medium using a multi-pipet, and then add 200 assay medium containing the test substance to each well. [mu]l was added and incubated for 24 hours in a 5% carbon dioxide incubator. The next day, 140 μl of the culture medium from each well was transferred to a new 96-well plate, and 10 μl of 6.1N trichloroacetic acid was dispensed into each well, followed by incubation in a 50° C. incubator for 30 minutes. The plate was centrifuged at 2500 rpm for 10 minutes to settle all precipitates. A detection reagent solution (component D in IDO1 cell-based assay kit catalog # 72031 BPS Bioscience) was diluted 50 times in acetic acid to prepare a detection reagent solution. From the centrifuged plate, 100 μl of the supernatant per well was transferred to a new transparent 96-well plate, 100 μl of the assay reagent solution was added to each well, reacted at room temperature for 10 minutes, and absorbance was measured at a wavelength of 480 nm. For the analysis of the results, the absorbance (At) of the well containing HEK293 cells expressing IDO protein without treatment with the inhibitor was set to 100%, and the absorbance (Ab) of the well containing HEK293 cells without IDO protein expression was set to 0%. Absorbance was calculated. (% Absorbance = (A-Ab)/(At-Ab), A = Absorbance of wells treated with test substance)

The results obtained by calculating the 50% inhibitory concentration (IC ₅₀ ) of the test substance obtained as above are shown in Table 1 below.

Inhibitory activity (IC ₅₀ , nM) IDO1 enzyme activity
(Test Example 1) IDO1 Hela cell activity
(Test Example 2) IDO1 Hek293 cell activity
(Test Example 3) Example 1 42.1 4.4 5.2 Example 2 44.8 23.1 8.4 Example 3 98.4 9.8 9.9 Example 4 47.3 - 5.0 Example 5 29.6 - 9.9

From the results of Table 1, it can be seen that the compounds of Examples 1 to 5 exhibit excellent inhibitory activity against indoleamine 2,3-dioxygenase 1.

Test Example 4-1. Comparative test of pharmacokinetics through oral administration in normal rats

The pharmacokinetics of the compound of Example 1 and BMS-986205 (control substance) were measured in rats, respectively. The compound of Example 1 and BMS-986205 (control substance) were prepared by suspending them in 0.5% methylcellulose to which 0.2% of Tween 80 was added, and then orally administered to rats at a dose of 10 mg/kg/5 mL. Blood samples were taken from rats at predetermined times, and the concentrations of compounds in each sample were analyzed to obtain plasma concentration profiles (Fig. 1a), and the pharmacokinetic parameters obtained therefrom are shown in Table 2 below.

highest blood level
(ng/mL) Time to reach peak blood concentration (hr) area under the curve
(ng hr/mL) bioavailability
(%) BMS-986205 656.8 One 3976.7 39.5 Example 1 2170.3 2 16018 62.8

As can be seen from the results of Table 2, the compound of Example 1 had a peak blood concentration and an area under the blood concentration curve that were 3.3 and 4.0 times higher, respectively, in normal rats than the control substance (BMS-986205), This shows that the compound of Example 1 exhibits a remarkably high in vivo exposure. Therefore, the compound of Example 1 is expected to exhibit excellent pharmacological efficacy by exhibiting significantly higher in vivo exposure at the same dose than BMS-986205. In addition, the compound of Example 1 is expected to exhibit excellent safety because it can obtain a similar in vivo exposure to BMS-986205 even when administered at a low dose.

Test Example 4-2. Comparative test of pharmacokinetics through oral administration in normal rats

The pharmacokinetics of the compounds of Examples 2 to 5 were measured in rats, respectively. The compounds of Examples 2 to 5 were prepared by suspending them in 0.5% methylcellulose to which 0.2% of Tween 80 was added, and then orally administered to rats at a dose of 3 mg/kg/5 mL. Blood samples were taken from rats at predetermined times, and the concentrations of compounds in each sample were analyzed to obtain plasma concentration profiles (Fig. 1b), and the pharmacokinetic parameters obtained therefrom are shown in Table 3 below.

highest blood level
(ng/mL) Time to reach peak blood concentration (hr) area under the curve
(ng hr/mL) bioavailability
(%) BMS-986205
(Value converted to 3mg/kg dose) 197.1 One 1193 39.5 Example 1
(Value converted to 3mg/kg dose) 651.2 2 4806 62.8 Example 2 417.0 2 2763 38.9 Example 3 548.3 0.5 3284 75.2 Example 4 198.0 2 2097 55.2 Example 5 641.7 One 2835 46.8

As can be seen from the results in Table 3, the areas under the blood concentration curves of the compounds of Examples 2 to 5 were 1.8 to 2.8 times higher than those of the control substance (BMS-986205) in normal rats, respectively. It is shown that the compounds of Examples 2 to 5 exhibit high in vivo exposure. Therefore, the compounds of Examples 2 to 5 are expected to exhibit excellent pharmacological efficacy by showing significantly higher in vivo exposure at the same dose than BMS-986205. In addition, the compounds of Examples 2 to 5 are expected to exhibit excellent safety because they can obtain in vivo exposure similar to that of BMS-986205 even when administered at a low dose.

Test Example 5-1. Biomarker change and pharmacokinetic study according to administration of the compound of Example 1 in mouse subcutaneous MC38 tumor model

In a C57BL/6 mouse model subcutaneously implanted with MC38 colorectal cells, changes in biomarkers and pharmacokinetics according to administration of the compound of Example 1 were studied. As a control drug, BMS-986205 was also evaluated.

(1) Cell culture

MC38 tumor cells were monolayer cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 1% antibiotics-antimycotics at 37° C. in 5% CO ₂ conditions. Tumor cells were subcultured twice a week by trypsin-EDTA treatment. Subculture was performed while maintaining about 70-80% confluency, and after counting the cells using Adam, an automatic cell counting equipment, 1 x 10 ⁶ cells were transferred to a T-75 flask for maintenance and culture did Finally, growing cells in exponential growth phase were harvested and counted for tumor implantation.

(2) Tumor implantation and animal grouping

Each mouse was implanted subcutaneously in the right upper flank with MC38 cells (5 x 10 ⁶ cells) in 0.1 mL of PBS for tumor development. When the average tumor volume reached about 114 mm ³ at about 2 weeks after tumor transplantation, group separation was performed so that the average tumor volume in each group was similar, and the test substance was tested according to the test design in Table 4 below. After repeated administration for 3 days, blood at 0, 2, 12, 14 and 24 hours and tumor tissue at 24 hours were collected to measure drug, tryptophan, and kynurenine concentrations.

army process dose administration route of administration sex N G1 Vehicle - qd p.o. F 6 G2 BMS-986205 100 mg/kg qd x 3 days p.o. F 6 G3 Compound of Example 1 50 mg/kg qd x 1 day p.o. F 6 G4 Compound of Example 1 100 mg/kg qd x 1 day p.o. F 6 G5 Compound of Example 1 100 mg/kg bid x 1 days p.o. F 6 G6 Compound of Example 1 50 mg/kg qd x 3 days p.o. F 6 G7 Compound of Example 1 100 mg/kg qd x 3 days p.o. F 6 G8 Compound of Example 1 100 mg/kg bid x 3 days p.o. F 6

After repeated administration for 1 day and 3 days as described above, the results of analyzing the concentrations of tryptophan and kynurenine in tumor tissue are shown in Table 5 below, and about the compound of Example 1 obtained from plasma concentration profiles Table 6 shows the kinetic parameters and the resulting dose proportionality and accumulation in the body calculated therefrom.

process intratumoral
tryptophan
(μg/g) intratumoral
kynurenine
(μg/g) K/T ratio % inhibition of kynurenine average SD average SD average SD Vehicle (QD, 1 day) 37.62 5.19 1.65 1.39 0.043 0.035 - BMS-986205 (100 mg/kg, QD, 3 days) 38.92 7.82 1.63 1.94 0.045 0.058 1.1 Compound of Example 1 (50 mg/kg, QD, 1 day) 38.35 2.48 0.75 0.62 0.019 0.016 54.4 Compound of Example 1 (100 mg/kg, QD, 1 day) 38.25 6.84 0.51 0.36 0.013 0.009 69.4 Compound of Example 1 (100 mg/kg, BID, 1 day) 47.75 5.48 1.04 1.06 0.023 0.020 37.2 Compound of Example 1 (50 mg/kg, QD, 3 days) 39.52 6.27 0.17 0.12 0.004 0.003 89.6 Compound of Example 1 (100 mg/kg, QD, 3 days) 40.63 2.60 0.09 0.04 0.002 0.001 94.3 Compound of Example 1 (100 mg/kg, BID, 3 days) 40.43 4.30 0.01 0.00 0.000 0.000 99.4

Dose (mg/kg) 1 day 3 days 50 (QD) 100 (QD) 100 (BID) 50 (QD) 100 (QD) 100 (BID) C _max (μg/ml) 9.5 12.9 12.7 8.3 12.4 19.1 C _trough (μg/ml) 2.2 4.1 7.4 2.6 4.4 12.9 ^* T _max (hr) 2.0 2.0 14.0 2.0 2.0 14.0 AUC _last (ng hr/mL) 98.9 162.4 204.5 108.9 170.7 352.0 Accumulation (ratio) 3 days / 1 day (C _max ) - - - 0.9 1.0 1.5 3 days/1 day (AUC _last ) - - - 1.1 1.1 1.7 Dose proportionality (Fold increase) Volume 1.0 2.0 4.0 ^** 1.0 2.0 4.0 ^** C _max 1.0 1.4 1.3 1.0 1.5 2.3 AUC _last 1.0 1.6 2.1 1.0 1.6 3.2

* mean, ** BID

As can be seen from the results in Table 5, the compound of Example 1 exhibited a significantly reduced kynurenine regardless of the dose concentration when administered on the 3rd day compared to the 1-day administration, and significantly decreased in tumor tissues when administered repeatedly on the 3rd day. kynurenine concentration. In particular, when 100 mg/kg BID of the compound of Example 1 was repeatedly administered for 3 days, the concentration of kynurenine in tumor tissues was almost completely reduced. In addition, the pharmacokinetic parameters obtained from the blood concentration profile obtained upon administration of the compound of Example 1 are shown in Table 6, and showed a low systemic exposure compared to the dose proportionality. At the time of administration of 50 mg / kg QD and 100 mg / kg QD, accumulation by repeated administration was hardly observed, but at the time of administration of 100 mg / kg BID, accumulation (1.7 times) was observed (Table 6).

Test Example 5-2. Biomarker change and pharmacokinetic study according to administration of the compounds of Examples 1 to 5 in mouse MC38 tumor subcutaneous transplantation model

Changes in biomarkers and pharmacokinetics according to administration of the compounds of Examples 1 to 5 were performed in a C57BL/6 mouse model subcutaneously implanted with MC38 colorectal cells.

(1) Cell culture

MC38 tumor cells were cultured as a monolayer in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 1% antibiotics-antimycotics at 37° C. under 5% CO ₂ conditions. Tumor cells were subcultured twice weekly by trypsin-EDTA treatment. Subculture was performed while maintaining about 70-80% confluency, and after counting the cells using Adam, an automatic cell counting equipment, 1 x 10 ⁶ cells were transferred to a T-75 flask for maintenance and culture did Finally, growing cells in exponential growth phase were harvested and counted for tumor implantation.

(2) Tumor transplantation and test group separation

Each mouse was implanted subcutaneously in the right upper flank with MC38 cells (5 x 10 ⁶ cells) in 0.1 mL of PBS for tumor development. When the average tumor volume reached about 171 mm ³ at about 2 weeks after tumor transplantation, group separation was performed so that the average tumor volume in each group was similar, and the test substance was repeated 3 days according to the test design in Table 7 below. Blood at 0, 2, 4, 8, 14, and 24 hours after administration and tumor tissue at 24 hours were collected to measure drug, tryptophan, and kynurenine concentrations.

army process dose administration route of administration sex N G1 Vehicle - qd p.o. F 6 G2 Compound of Example 1 100 mg/kg qd x 3 days p.o. F 6 G3 Compound of Example 2 100 mg/kg qd x 3 days p.o. F 6 G4 Compound of Example 3 100 mg/kg qd x 3 days p.o. F 6 G5 Compound of Example 4 100 mg/kg qd x 3 days p.o. F 6 G6 Compound of Example 5 100 mg/kg qd x 3 days p.o. F 6

After repeated administration for 3 days as described above, the results of analyzing the concentrations of tryptophan and kynurenine in blood and tumor tissue are shown in Tables 8 and 9 below, respectively, and examples 1 to 10 obtained from plasma concentration profiles The pharmacokinetic parameters of the compound of 5 are shown in Table 10.

process blood
tryptophan
(μg hr/mL) blood
kynurenine
(μg hr/mL) K/T ratio % inhibition of kynurenine average SD average SD average SD Vehicle (QD) 428.283 39.229 7.722 1.158 0.437 0.053 - Compound of Example 1 (100 mg/kg, QD, 3 days) 553.583 54.224 1.457 0.186 0.065 0.014 81.1 Compound of Example 2 (100 mg/kg, QD, 3 days) 449.783 14.277 3.182 0.503 0.173 0.031 58.8 Compound of Example 3 (100 mg/kg, QD, 3 days) 476.683 53.145 2.721 0.375 0.142 0.027 64.8 Compound of Example 4 (100 mg/kg, QD, 3 days) 570.100 52.105 2.431 0.218 0.106 0.014 68.5 Compound of Example 5 (100 mg/kg, QD, 3 days) 569.417 77.972 3.044 0.544 0.136 0.019 60.6

process intratumoral
tryptophan
(μg/g) intratumoral
kynurenine
(μg/g) K/T ratio % inhibition of kynurenine average SD average SD average SD Vehicle (QD) 2.832 0.649 0.334 0.146 0.126 0.067 - Compound of Example 1 (100 mg/kg, QD, 3 days) 3.957 0.776 0.012 0.004 0.003 0.001 96.5 Compound of Example 2 (100 mg/kg, QD, 3 days) 3.480 0.672 0.032 0.008 0.009 0.002 90.5 Compound of Example 3 (100 mg/kg, QD, 3 days) 3.032 0.356 0.024 0.006 0.008 0.003 92.7 Compound of Example 4 (100 mg/kg, QD, 3 days) 3.848 0.543 0.029 0.007 0.008 0.001 91.3 Compound of Example 5 (100 mg/kg, QD, 3 days) 4.442 0.776 0.036 0.007 0.008 0.002 89.1

Dose (mg/kg) Example 1 Example 2 Example 3 Example 4 Example 5 100 (QD) 100 (QD) 100 (QD) 100 (QD) 100 (QD) C _max (μg/ml) 17.10 10.96 6.17 5.05 16.48 ^* _Tmax (hr) 2.00 2.00 2.00 2.00 2.00 AUC _last (ng hr/mL) 291.59 121.50 68.16 52.03 275.20 Blood concentration (μg/ml, at 24 hr) 8.87 3.10 0.58 1.10 9.01 Intratumoral concentration (μg/g, at 24 hr) 17.13 7.83 1.28 2.89 10.36 T/P Ratio 1.93 2.52 2.29 2.58 1.17

* average

As can be seen from the results of Tables 8 and 9, the compounds of Examples 1 to 5 showed a similar degree of kynurenine reduction in blood and tumor tissues when administered for 3 days, and showed excellent T / P ratios. (Table 10).

Test Example 6. In vivo evaluation of anti-tumor efficacy according to combined administration in mouse subcutaneous MC38 tumor model

According to the combined administration of the compound of Example 1 and an anti-PD-1 antibody (Anti-PD-1 antibody, clone RMP1-14 (BE0146), BioXcell) in a subcutaneous MC38 colorectal model in C57BL/6 mice In vivo antitumor efficacy was evaluated. As a control drug, BMS-986205 was also evaluated. MC38 tumor cells were cultured in the same manner as in Test Example 5 (1).

(1) Tumor implantation and animal grouping

Each mouse was implanted subcutaneously in the right upper flank with MC38 cells (1 x 10 ⁶ cells) in 0.1 mL of PBS for tumor development. Test substance administration was started when the average tumor volume reached approximately 50 mm ³ . Since the rate of tumor development in each mouse is different, the test substance was administered individually when the tumor volume was measured every day and reached 50 mm ³ . Since each mouse has a different drug administration time point, the first administration date was set as the first day. The number of mice in the test design is 20 for each group. However, internal sacrifice for the immune activation test (ex vivo) was performed for each of 5 rats, and tumor growth inhibition and survival rate tests were performed with the remaining 15 rats. The test was performed except when the tumor did not grow or the mouse died due to cannibalism. The test design for in vivo efficacy evaluation according to the concomitant administration of the test substance is shown in Table 11 below.

Trial design for co-administration army number of mice process Volume route of administration schedule One 13 Vehicle - p.o. QD 2 14 Anti-PD-1 antibody 10 mg/kg i.p. BIW (1,4,8,11) 3 14 BMS-986205 125 mg/kg p.o. QD 4 13 BMS-986205 125 mg/kg p.o. QD Anti-PD-1 antibody 10 mg/kg i.p. BIW (1,4,8,11) 5 15 Compound of Example 1 50 mg/kg p.o. BID 6 14 Compound of Example 1 50 mg/kg p.o. BID Anti-PD-1 antibody 10 mg/kg i.p. BIW (1,4,8,11) 7 13 Compound of Example 1 100 mg/kg p.o. BID 8 15 Compound of Example 1 100 mg/kg p.o. BID Anti-PD-1 antibody 10 mg/kg i.p. BIW (1,4,8,11)

(2) Tumor measurement and endpoints

Tumor volume was measured daily in two dimensions using vernier calipers, and volume was calculated in mm ³ using the formula:

V (volume) = 0.52axb ²

In the above formula, a and b are the major and minor diameters of the tumor, respectively.

According to the test plan, the tumor volume of each mouse was input into Prism, and the average value was calculated. At the end of the tumor growth inhibition test, on day 11, TGI (tumor growth inhibition) was calculated for each group using the following formula:

TGI (%) = (Tn-Ti)/Ti x 100

In the above formula, Ti is the average tumor volume of mice of the control group (vehicle-administered group) on day 11, and Tn is the average tumor volume of mice of each administration group on day 11. Test animals were sacrificed when they reached a tumor volume of 2,500 mm ³ , and the time to reach this endpoint was used for Kaplan-Meier survival analysis.

(3) Statistical analysis

Statistical analysis of differences in tumor volume between groups was performed on data obtained at the time point 11 days after start of dosing. Two-way ANOVA was performed, and when a significant F-statistic (ratio of treatment variance to error variance) was obtained, comparisons between groups were performed using Tukey's multiple comparisons test. All data were analyzed using Prism 9.0. Statistically significant was considered from p < 0.05: *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001.

Statistical analysis of differences in survival rates between groups was performed on survival data up to day 50 after start of dosing. Kaplan-Meier tests were performed, all data were analyzed using GraphPad Prism 9.0, and between-group comparisons were performed with Log-rank tests, and statistical significance was considered from p < 0.05: * p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001.

(4) Results

The tumor growth curves of each group of C57BL/6 mice transplanted with MC38 tumor cells are shown in FIG. 2 . Figure 3 is a waterfall plot showing inhibition of tumor growth on day 11. Tumor growth inhibition (TGI) is a value calculated as a percentage of the difference in average tumor volume of mice in each administration group when the average tumor volume of mice in the control group (vehicle administration group) is 100%. In addition, the results of tumor volume and statistical analysis on day 11 are shown in Table 12 below.

treatment group Tumor volume at day 11 ± SEM (mm ³ ) T/C (%) TGI% *p value Control group (vehicle administration group) 2255.7 ± 104.8 - - - Anti-PD-1 antibody (10 mpk, BIW) 1784.5 ± 131.2 79.1 21.3% 0.0003 BMS-986205 (125 mpk, QD) 1584.7 ± 147.8 70.3 30.3% <0.0001 BMS-986205 (125 mpk, QD)
+ Anti-PD-1 antibody (10 mpk, BIW) 1379.6 ± 165.6 61.2 39.5% <0.0001 Compound of Example 1 (50 mpk, BID) 1429.8 ± 151.3 63.4 37.4% <0.0001 Compound of Example 1 (50 mpk, BID)
+ Anti-PD-1 antibody (10 mpk, BIW) 1240.2 ± 203.2 55.0 46.0% <0.0001 Compound of Example 1 (100 mpk, BID) 991.6 ± 179.6 44.0 57.2% <0.0001 Compound of Example 1 (100 mpk, BID)
+ Anti-PD-1 antibody (10 mpk, BIW) 339.4 ± 22.4 15.0 86.7% <0.0001

As can be seen from the above results, although the degree of TGI was different in all administration groups compared to the control group (vehicle administration group), tumor growth was significantly inhibited. The group administered with the IDO inhibitor and anti-PD-1 antibody showed statistically significant inhibition of tumor growth (p<0.0001 for each group). In particular, the combination administration group of the compound of Example 1 (100 mpk, BID) and the anti-PD-1 antibody showed a remarkably high (i.e., synergistic) tumor growth inhibitory activity of 86.7%, significantly compared to the other groups. showed reduced tumor volume (**** P < 0.0001).

Figure 4 shows the survival curves of each treatment group of C57BL/6 mice bearing MC38 tumors. Animals were sacrificed when tumor volumes reached greater than 2,500 mm ³ . From the results of FIG. 4, the statistical analysis results of the survival curves of each treatment group relative to the control group (vehicle administration group) are shown in Table 13 below. Comparison of each treatment group with respect to the control group (vehicle administration group) was performed using a log-rank test.

treatment group median survival
(number of days) P value for Log-rank test Tumor Free Mice at Day 50 Control group (vehicle administration group) 13 - 0/13 Anti-PD-1 antibody (10 mpk, BIW) 19 <0.0001 0/14 BMS-986205 (125 mpk, QD) 15 <0.0001 0/14 BMS-986205 (125 mpk, QD)
+ Anti-PD-1 antibody (10 mpk, BIW) 19 <0.0001 0/13 Compound of Example 1 (50 mpk, BID) 25 <0.0001 0/15 Compound of Example 1 (50 mpk, BID)
+ Anti-PD-1 antibody (10 mpk, BIW) 34.5 <0.0001 2/14 Compound of Example 1 (100 mpk, BID) 30 <0.0001 1/13 Compound of Example 1 (100 mpk, BID)
+ Anti-PD-1 antibody (10 mpk, BIW) not reaching <0.0001 5/15

As can be seen from the results of Table 13, compared to the control group (vehicle administration group), the anti-PD-1 antibody alone administration group, the BMS-986205 (125 mpk, QD) alone administration group and the anti-PD-1 antibody combination administration group, Both the group administered with the compound of Example 1 (50 mpk, BID / 100 mpk, BID) alone and the group administered with the anti-PD-1 antibody significantly extended the survival rate of the test animals (p<0.0001).

In addition, the statistical analysis results of the survival curves of each group for the anti-PD-1 antibody administration group using the Log-rank test are shown in Table 14 below.

treatment group median survival
(number of days) P value for Log-rank test Tumor Free Mice at Day 50 Anti-PD-1 antibody (10 mpk, BIW) 19 - 0/14 BMS-986205 (125 mpk, QD)
+ anti-PD-1 antibody (10 mpk, BIW) 19 0.2813 0/13 Compound of Example 1 (50 mpk, BID)
+ anti-PD-1 antibody (10 mpk, BIW) 34.5 0.0008 2/14 Compound of Example 1 (100 mpk, BID)
+ anti-PD-1 antibody (10 mpk, BIW) not reached <0.0001 5/15

As can be seen from the results in Table 14, the combined administration of BMS-986205 (125 mpk, QD) and anti-PD-1 antibody did not prolong the survival rate of the test animals (p=0.2813). In contrast, the combined administration of the compound of Example 1 (50 mpk, BID / 100 mpk, BID) and the anti-PD-1 antibody significantly prolonged the survival rate of the test animals (each p = 0.0008, p <0.0001). In addition, in the tumor-free mice on the 50th day of administration of the test substance, in the group administered with the compound of Example 1 (50 mpk, BID) and the anti-PD-1 antibody in combination, 2 out of 14 subjects had complete response (Complete Response, CR). ), and in the group administered with the compound of Example 1 (100 mpk, BID) and the anti-PD-1 antibody in combination, 5 out of 15 subjects showed complete remission (CR).

In addition, using the Log-rank test, the combination of the compound of Example 1 (100 mpk, BID) and the anti-PD-1 antibody for the compound of Example 1 (100 mpk, BID) alone administration group Statistical analysis results of survival curves for the administration groups are shown in Table 15 below.

treatment group median survival
(number of days) P value for Log-rank test Tumor Free Mice at Day 50 Compound of Example 1 (100 mpk, BID) 30 - 1/13 Compound of Example 1 (100 mpk, BID)
+ anti-PD-1 antibody (10 mpk, BIW) not reaching <0.0001 5/15

As can be seen from the results of Table 15, compared to the group administered with the compound of Example 1 (100 mpk, BID) alone, the combination of the compound of Example 1 (100 mpk, BID) and the anti-PD-1 antibody When administered, it showed a significantly high anti-tumor effect and significantly extended the survival rate of test animals (p<0.0001). In tumor-free mice on the 50th day of administration of the test substance, in the group administered with the compound of Example 1 (100 mpk, BID) alone, 1 out of 13 subjects showed complete remission (CR), whereas the compound of Example 1 showed complete remission (CR). (100 mpk, BID) and the anti-PD-1 antibody combined administration group, 5 out of 15 subjects showed complete remission (CR).

5A to 5C show tumor volume tracing curves of individuals who showed complete remission among surviving individuals. As can be seen from the results of FIGS. 5A to 5C , tumor recurrence did not occur even after drug administration was stopped.

From the above results, it can be confirmed that the combined administration of the compound of Example 1 and the anti-PD-1 antibody showed a clear synergistic antitumor activity in the MC38 colorectal cancer animal model and significantly extended the survival rate of the animal.

Claims (16)

Prevention of cancer comprising a combination of a first compartment containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient and a second compartment containing an immune checkpoint inhibitor as an active ingredient or a pharmaceutical composition for treatment.
<Formula 1>

during the ceremony,
R ₁ is a C ₁ -C ₆ alkyl group;
A is a heteroaryl group selected from the group consisting of benzo[d]oxazolyl and quinazolinyl, and the heteroaryl group is substituted with one or two substituents selected from the group consisting of halogen and C ₁ -C ₆ alkyl. The method of claim 1, wherein the compound of formula 1
6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazol-2-amine ;
6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)butan-2-yl)benzo[d]oxazol-2-amine ;
6-fluoro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazol-2- amine;
6,7-difluoro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazole -2-amine; and
2-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)-7-methylquinazolin-4-amine
A pharmaceutical composition, characterized in that selected from the group consisting of. The method of claim 1, wherein the compound of Formula 1 is 6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propane-2- 1) A pharmaceutical composition, characterized in that benzo [d] oxazol-2-amine. The pharmaceutical composition according to claim 1, wherein the immune checkpoint inhibitor is a PD-1/PD-L1 signaling inhibitor. The pharmaceutical composition according to claim 1, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody. The pharmaceutical composition according to claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. The method of claim 1, wherein the cancer is squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myelogenous leukemia, multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer Cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, bladder cancer, breast cancer, colon cancer A pharmaceutical composition, characterized in that at least one selected from the group consisting of species, urothelial cancer and head and neck cancer. The pharmaceutical composition according to any one of claims 1 to 7, wherein the first compartment is an oral administration compartment and the second compartment is an injection compartment. A method for treating cancer comprising administering to a mammal a therapeutically effective amount of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of an immune checkpoint inhibitor.
<Formula 1>

during the ceremony,
R ₁ is a C ₁ -C ₆ alkyl group;
A is a heteroaryl group selected from the group consisting of benzo[d]oxazolyl and quinazolinyl, and the heteroaryl group is substituted with one or two substituents selected from the group consisting of halogen and C ₁ -C ₆ alkyl. 10. The method of claim 9, wherein the compound of Formula 1 is
6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazol-2-amine ;
6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)butan-2-yl)benzo[d]oxazol-2-amine ;
6-fluoro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazol-2- amine;
6,7-difluoro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazole -2-amine; and
2-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)-7-methylquinazolin-4-amine
A treatment method, characterized in that selected from the group consisting of. The method of claim 9, wherein the compound of Formula 1 is 6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propane-2- 1) A treatment method characterized in that benzo [d] oxazol-2-amine. 10. The method of claim 9, wherein the immune checkpoint inhibitor is a PD-1/PD-L1 signaling inhibitor. 10. The method of claim 9, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody. 10. The method of claim 9, wherein said immune checkpoint inhibitor is an anti-PD-1 antibody. 10. The method of claim 9, wherein the cancer is squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myelogenous leukemia, multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian Cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, bladder cancer, breast cancer, colon cancer A method of treatment, characterized in that at least one species is selected from the group consisting of urothelial cancer and head and neck cancer. 16. The method according to any one of claims 9 to 15, wherein the compound of Formula 1 or a pharmaceutically acceptable salt thereof is administered orally and the immune checkpoint inhibitor is injected.

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