WO2023090850A1

WO2023090850A1 - Synergic combination of 2,3-dioxygenase inhibitor and immune checkpoint inhibitor for the treatment of cancer

Info

Publication number: WO2023090850A1
Application number: PCT/KR2022/018084
Authority: WO
Inventors: Gyu-Jin Lee; Eui-Chul LEE; Sol Park; Dong-Hoon Lee; Su-Hwa Kim; Jong-Suk Park; Ho-Woong Kang
Original assignee: Yuhan Corporation
Priority date: 2021-11-17
Filing date: 2022-11-16
Publication date: 2023-05-25
Also published as: KR20230072446A

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. And, the present invention provides a method for treating a cancer in a mammal, which comprises 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 the mammal in need thereof.

Description

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

The present invention relates to a synergic combination of an indoleamine 2,3-dioxygenase inhibitor and an immune checkpoint inhibitor useful for the treatment of cancer. More specifically, the present invention relates to a synergic combination of a selective inhibitor against indoleamine 2,3-dioxygenase 1 (IDO1) and an immune checkpoint inhibitor, particularly an inhibitor targeting PD-1/PD-L1 signaling pathway, e.g., a programmed death receptor-1 (PD-1) antagonist, 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 bring about immune escape through T cell-mediated immune tolerance or immuno-editing. Immune checkpoints are a protein which interferes with the destruction of cancer cells. Cancers activate Immune-inhibitory molecules, resulting in tumor resistance that evades 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 and suppresses T cell function to evade immunity.

Immune checkpoint inhibitors block immune evasion signals by binding directly to an immune checkpoint receptors controlling the activity of cancer-attacking T cells (e.g., CTLA-4, PD-1) or by binding to PD-L1 on the surface of cancer cells, thereby preventing the formation of immunological synapses, which leads to destroying cancer cells by the T cells which are not interfered with immune evasion. Representative immune checkpoint inhibitors include a CTLA-4 monoclonal antibody [e.g., Ipilimumab (YERVOY^TM), etc.], a PD-1 monoclonal antibody [e.g., Nivolumab (Opdivo^TM), Pembrolizumab (Keytruda^TM), etc.], a PD-L1 monoclonal antibody [e.g., Atezolizumab (TECENTRIQ^TM), Durvalumab (IMFINZI^TM), etc.]. However, it is known in the art that immune checkpoint inhibitors respond successfully to only a part of the entire cancer patients; and that it is required to overcome side effects such as autoimmune diseases.

Tryptophan is an essential amino acid for proliferation and survival of cells. Indoleamine 2,3-dioxygenase 1 (conventionally, referred to as 'IDO-1') is an intracellular heme-containing enzyme that catalyzes the first and rate-limiting step of L-tryptophan degradation to N-formyl-kynurenine. IDO-1 acts on the metabolism of L-tryptophan to degrade it into N-formyl-kynurenine, which is then 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. Therefore, IDO depletes tryptophan and produces kynurenine, thereby inhibiting 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 also 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 may lead to increased resistance in the tumor microenvironment, which results in growing cancer tissues.

It has been reported that the up-regulation of IDO-1 leads to a poor prognosis in cancer patients (Uyttenhove, C. et al. Nature Med. 9, 1269-1274 (2003)). From the test using IDO-1 gene knockout mice, it has been confirmed that IDO-1 plays a key role in 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)). Especially, it has been reported that the use of an IDO-1 inhibitor as a supplemental treating agent improves 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 strong effect of the anticancer drug imatinib (Gleevec) on solid gastrointestinal stromal tumor is derived from the inhibition of IDO-1 (Balachandran, V. P. et al. Nature Med. 17, 1094-1100 (2011)).

Therefore, IDO-1 inhibitors can effectively inhibit cancer metastasis and cancer proliferation. And, IDO-1 inhibitors can be also usefully applied 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 the pregnancy, malignant tumors, or virus-induced T cell suppression. Although the mechanism of action is not well defined, it is expected that IDO-1 inhibitors can be also applied for the treatment of patients with neuropsychiatric diseases or symptoms such as depression. The present inventors have found that a derivative having a cyclohexyl-ethylene-amino-heteroaryl moiety or pharmaceutically acceptable salt thereof not only has excellent inhibitory activity against IDO-1 but also exhibits remarkably high in vivo exposure 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 applied for preventing or treating various diseases associated with IDO-1, e.g., proliferative disorders such as cancer, viral infections and/or autoimmune diseases, etc.

The present inventors have found that administrations of a derivative having a certain cyclohexyl-ethylene-amino-heteroaryl moiety or pharmaceutically acceptable salt thereof in combination with an immune checkpoint inhibitor exhibit synergistic antitumor activities, in comparison with the administration of an immune checkpoint inhibitor alone.

Therefore, the present invention provides a pharmaceutical composition for preventing or treating a cancer, which comprises a combination of a derivative having a certain cyclohexyl-ethylene-amino-heteroaryl moiety or pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor.

In addition, the present invention provides a method for treating a cancer in a mammal, which comprises administering a derivative having a certain cyclohexyl-ethylene-amino-heteroaryl moiety or pharmaceutically acceptable salt thereof in combination with an immune checkpoint inhibitor.

According to an aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a cancer, which comprises a combination of a first compartment comprising a compound of Formula 1 or pharmaceutically acceptable salt thereof as an active ingredient and a second compartment comprising an immune checkpoint inhibitor as an active ingredient:

wherein,

R₁ is a C₁∼C₆ alkyl group,

A is a heteroaryl group selected from the group consisting of benzo[d]oxazolyl and quinazolinyl, wherein the heteroaryl group is substituted with one or two substituents selected from the group consisting of halogen and C₁∼C₆ alkyl.

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

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

It has been found by the present invention that administrations of a derivative having a certain cyclohexyl-ethylene-amino-heteroaryl moiety (i.e., the compound of Formula 1) or pharmaceutically acceptable salt thereof in combination with an immune checkpoint inhibitor exhibit synergistic antitumor activities, in comparison with the administration of an immune checkpoint inhibitor alone. Therefore, the combination of said compound of Formula 1 or pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor can be usefully applied for inhibiting cancer metastasis and cancer recurrence.

FIG. 1a shows the plasma concentration profiles obtained from the oral administrations of the compound of Example 1 and the control (BMS-986205) in rats.

FIG. 1b shows the plasma concentration profiles obtained from the oral administrations of the compounds of Examples 2 to 5 in rats, along with the virtual plasma concentration profile calculated from the oral administration of the compound of Example 1 in rats.

FIG. 2 shows the results obtained by evaluating the inhibitory activities against tumor growth according to the administrations of the compound of Example 1 in combination with the anti-PD-1 antibody.

FIG. 3 shows a Waterfall plot showing tumor growth inhibitions on Day 11.

FIG. 4 shows survival curves for each group of C57BL/6 mice bearing MC38-implanted tumors.

FIGs. 5a to 5c show the tumor volume-tracking curves for the surviving rats exhibiting complete cure.

The present invention provides a pharmaceutical composition for preventing or treating a cancer, which comprises a combination of a first compartment comprising a compound of Formula 1 or pharmaceutically acceptable salt thereof as an active ingredient and a second compartment comprising an immune checkpoint inhibitor as an active ingredient:

wherein,

R₁ is a C₁∼C₆ alkyl group,

Preferably, the compound of Formula 1 in the first compartment may be selected from the group consisting of:

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]oxazol-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 in the first compartment may be 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 of the present invention may be in a pharmaceutically acceptable salt form. The salt may be a conventional acid addition salt form, which includes e.g., salts derived from an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid or nitric acid; and salts derived from an organic acid such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, citric acid, maleic acid, malonic 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 conventional metal salt forms, e.g., salts derived from metals such as lithium, sodium, potassium, magnesium, or calcium. The acid addition salt or metal salt may be prepared according to conventional methods.

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

X-A

wherein, R₁ and A are the same as defined in the above; and X is halogen.

The compound of Formula 3 is commercially available. The reaction between the compound of Formula 2 or salt thereof (e.g., 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, and the like, and the solvent may be an organic solvent such as N,N-dimethylformamide, 1,4-dioxane, tetrahydrofuran, ethanol, or isopropyl alcohol. In addition, the reaction may be carried out at room temperature to 100℃.

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

In the Reaction Scheme 1, R₁ is the same as defined in the above.

The compound of Formula 5 may be prepared through the Suzuki reaction between the compound of Formula 4 (which is commercially available) and 4-chloro-6-fluoroquinoline. The reaction may be carried out using a palladium catalyst, such as palladium(II) acetate (Pd(OAc)₂), tris(dibenzylideneacetone)dipalladium (Pd₂(dba)₃), tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl₂), etc. In addition, said reaction may be carried out in the presence of a ligand and a base, in addition to the palladium catalyst. The ligand includes (S)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP), 1,1'-bis(diphenylphosphino)ferrocene (dppf), tri(o-tolyl)phosphine (P(o-Tol)₃), etc. The base includes an inorganic base such as cesium carbonate (Cs₂CO₃), sodium carbonate (Na₂CO₃), potassium carbonate (K₂CO₃), potassium fluoride (KF), cesium fluoride (CsF), sodium hydroxide (NaOH), potassium phosphate (K₃PO₄), sodium tert-butoxide (tert-BuONa), potassium tert-butoxide (tert-BuOK) etc. Said reaction may be carried out in a non-polar organic solvent such as benzene or toluene or in a polar organic solvent such as 1,4-dioxane, tetrahydrofuran, acetonitrile, 1,2-dimethoxyethane, or N,N-dimethylformamide, at 50℃ to 150℃, preferably 80℃ to 110℃. Other reaction conditions including a reaction time may be determined according to known methods for the Suzuki reaction (Barbara Czako and Laszlo Kurti, STRATEGIC APPLICATIONS of NAMED REACTIONS in ORGANIC SYNTHESIS, 2005).

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

The reduction of the compound of Formula 6 may be carried out using lithium aluminium hydride in an organic solvent such as tetrahydrofuran or dichloromethane. The reduction may be carried out typically at -78℃ to room temperature.

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

The compound of Formula 9 may be prepared by condensing the compound of Formula 8 with (S)-(-)-2-methyl-2-propanesulfinamide. The condensation may be carried out 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℃ 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, deprotection of the compound of Formula 10 may give the compound of Formula 2 or salt thereof (eg, hydrochloride). The deprotection may be carried out 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 may be carried out using a trifluoroacetic acid or hydrochloric acid solution, in an organic solvent such as dichloromethane, 1,4-dioxane, or ethyl acetate, at room temperature.

In the pharmaceutical composition of the present invention, the immune checkpoint inhibitor may be an inhibitor targeting programmed death receptor-1 (PD-1) / programmed death ligand-1 (PD-L1) signaling pathway. The immune checkpoint inhibitor may be preferably an anti-CTLA-4 antibody (e.g., Ipilimumab, etc.), an anti-PD-1 antibody (e.g., Nivolumab, Pembrolizumab, etc.), or an anti-PD-L1 antibody (Atezolizumab, Durvalumab, etc.), more preferably an anti-PD-1 antibody (e.g., Nivolumab, Pembrolizumab, etc.), but not limited thereto. The antibody may be a monoclonal antibody or an antigen-binding fragment thereof. The antigen-binding fragment refers to a fragment having the function capable of binding to an antigen of a monoclonal antibody, such as Fab, Fab', F(ab')2, scFv (scFv)2, scFv-Fc, and Fv, 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 myeloid leukemia, multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer, hepatic 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 not limited thereto.

In the pharmaceutical composition of the present invention, the first compartment may be a compartment for oral administration; and the second compartment may be a compartment for injection. Accordingly, the first compartment may comprise a pharmaceutically acceptable carrier, such as diluents (e.g., lactose, corn starch, etc.), disintegrants, lubricants (e.g., magnesium stearate, etc.), which is conventionally used in the art. The first compartment may be formulated to an oral dosage form such as tablets, capsules, powders, granules, suspensions, emulsions, or syrups, according to conventional methods. The first compartment in an oral dosage form may be dosage forms for single administration or for multiple administrations. The second compartment may be in a formulation suitable for parenteral administration such as an injection, for example in a form suitable for intramuscular, intraperitoneal, subcutaneous or intravenous administration. The second compartment may be dosage forms for single administration or for multiple administrations. For said injection, sterile solutions of the immune checkpoint inhibitor are usually prepared, and the pH of the solutions should be suitably adjusted and buffered. For intravenous administration, the total concentration of solutes should be controlled in order to render the preparation isotonic. The second compartment may be in the form of an aqueous solution containing pharmaceutically acceptable carriers, e.g., saline having a pH level of 7.4.

In an embodiment of the pharmaceutical composition of the present invention, the first compartment may be orally administered at a dosage of 5 to 200 mg/kg of the compound of Formula 1 or pharmaceutically acceptable salt thereof, 1 to 4 times a day. In a preferable embodiment, the first compartment may be orally administered at a dosage of 10 to 20 mg/kg of the compound of Formula 1 or pharmaceutically acceptable salt thereof, 1 to 2 times a day. Of course, the dosage may be changed 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 dosage of 0.005 to 10 mg/kg of the immune checkpoint inhibitor. In a preferable embodiment, the second compartment may be injected 1 to 5 times a week at a dosage of 1 to 10 mg/kg of an anti-PD-1 antibody. Of course, the dosage may be changed according to the patient's condition, age, severity of cancer, and the like.

The present invention also provides a method for treating a cancer in a mammal, which comprises administering a therapeutically effective amount of a compound of Formula 1 or pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of an immune checkpoint inhibitor to the mammal in need thereof:

wherein,

R₁ is a C₁∼C₆ alkyl group,

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

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

In the method of the present invention, the compound of Formula 1 or pharmaceutically acceptable salt thereof, the immune checkpoint inhibitor, and the types of cancer are the same as those described with respect to the pharmaceutical composition of the present invention.

In an embodiment of the method of the present invention, the compound of Formula 1 or pharmaceutically acceptable salt thereof may be orally administered and the immune checkpoint inhibitor may be administered by injection. Accordingly, the compound of Formula 1 or pharmaceutically acceptable salt thereof may be administered in a form for oral administration, which is the same as described with respect to the first compartment of the pharmaceutical composition of the present invention. In addition, the immune checkpoint inhibitor may be administered in the form of an injection. Parenteral dosage forms, such as an injection, are the same as described with respect to the second compartment of the pharmaceutical composition of the present invention.

In an embodiment of the method of the present invention, the compound of Formula 1 or pharmaceutically acceptable salt thereof may be orally administered at a dosage of 5 to 200 mg/kg, 1 to 4 times a day. In a preferable embodiment, the compound of Formula 1 or 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 may be changed according to the patient's condition, age, severity of cancer, and the like.

In another embodiment of the method of the present invention, the immune checkpoint inhibitor may be injected at a dosage of 0.005 to 10 mg/kg, 1 to 5 times a week. In a preferable embodiment, an anti-PD-1 antibody may be injected at a dosage of 1 to 10 mg/kg, 1 to 5 times a week. Of course, the dosage may be changed according to the patient's condition, age, severity of cancer, and the like.

The present invention also provides a use of a combination of a compound of Formula 1 or pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor for the manufacture of a medicament for treating a cancer in a mammal. In the use of the present invention, the compound of Formula 1 or pharmaceutically acceptable salt thereof, the immune checkpoint inhibitor, and the types of cancer are the same as those described with respect to the pharmaceutical composition of the present invention. In addition, the forms comprising each of the compound of Formula 1 or pharmaceutically acceptable salt thereof and the immune checkpoint inhibitor; dosages; administration methods, etc. are the same as those described in relation to the method of the present invention. For example, the compound of Formula 1 or pharmaceutically acceptable salt thereof may be in the form of a pharmaceutical formulation for oral administration and the immune checkpoint inhibitor may be in the form of a pharmaceutical formulation for injection.

The following examples and experimental examples are provided for illustration purposes only, and are not intended to limit the scope of the invention.

The analyses of the compounds prepared in the following Examples were carried out as follows: Nuclear magnetic resonance (NMR) spectrum analysis was carried out using Bruker 400 MHz spectrometer and chemical shifts thereof were analyzed in ppm. Column chromatography was carried out on silica gel (Merck, 70-230 mesh) (W.C. Still, J. Org. Chem., 43, 2923, 1978). Each starting material is a known compound which was synthesized according to literatures or purchased commercially, e.g., from Sigma-Aldrich.

Example 1: 6-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)benzo[d]oxazol-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)Cl₂) (675 mg) was added to the solution, which was then stirred at 95℃ overnight. The reaction mixture was concentrated and then ethyl acetate was added thereto. The mixture was washed with distilled water, dried over anhydrous magnesium sulfate and then concentrated under reduced pressure to obtain a yellow oil residue. The residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate = 1/1, v/v) to give 4.40 g of the titled compound as a white solid. (Yield: 82.4 %)

¹H-NMR (CDCl₃) 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

A solution of ethyl 2-(4-(6-fluoroquinolin-4-yl) cyclohex-3-en-1-yl)acetate (4.40 g) prepared in

Step

1 and 10% Pd/C (440 mg), and acetic acid (0.16 ml) in methanol (30 ml) was stirred for 12 hours under a hydrogen atmosphere. The reaction mixture was dried and then filtered. The resulting residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate = 1/1, v/v) to give 4.17 g of the titled compound as a white solid. (Yield: 94.2 %)

¹H-NMR (CDCl₃) 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)ethan-1-ol

A solution of ethyl 2-(4-(6-fluoroquinolin-4-yl)cyclohexyl)acetate (2.27 g) prepared in Step 2 in tetrahydrofuran (24 ml) was stirred at 0℃ for 10 minutes and then lithium aluminium hydride (355 mg) was slowly added thereto. The reaction mixture was stirred at room temperature for 6 hours. The reaction was quenched by adding water to the reaction mixture, which was then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting filtrate was concentrated under reduced pressure. Two stereoisomers confirmed by TLC were collected and then purified by silica gel column chromatography (n-hexane/ethyl acetate/=1/2, v/v) to give 1.53 g of the titled compound as a white solid. (Yield: 73.2%)

¹H-NMR (DMSO-d₆) 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

A solution of 2-(4-(6-fluoroquinolin-4-yl)cyclohexyl)ethan-1-ol (15.14 g) prepared in Step 3 in dichloromethane (185 ml) was stirred at 0℃ for 30 minutes and then Dess-Martin oxidizing agent (35.2 g) was slowly added thereto. The reaction mixture was stirred at room temperature for 5 hours. The reaction was quenched by adding water to the reaction mixture, which was then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting filtrate was concentrated under reduced pressure. The lower material of the two stereoisomers confirmed by TLC in the resulting residue was purified by silica gel column chromatography (n-hexane/ethyl acetate/=3/1, v/v) to prepare 8.72 g of the titled compound as a white solid. (Yield: 57.9%)

¹H-NMR (CDCl₃) 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-methylpropan-2-sulfinamide

A solution of 2-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)acetaldehyde (4.50 g) prepared in Step 4 and (S)-(-)-2-methyl-2-propanesulfinamide (4.02 g) in dichloromethane (55 ml) was stirred at 0℃ for 10 minutes and then titanium(IV) isopropoxide (9.82 ml) was slowly added thereto. The reaction mixture was stirred at room temperature for 8 hours. The reaction was quenched by adding water to the reaction mixture, which was then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting 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 give 5.22 g of the titled compound as a white solid. (Yield: 84.0%)

¹H-NMR (CDCl₃) 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

A solution of (S)-N-(2-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethylidene)-2-methylpropan-2-sulfinamide (2.54 g) prepared in Step 5 in dichloromethane (23 ml) was stirred at 0℃ for 10 minutes and then a solution of methylmagnesium bromide in diethyl ether (3.0 M, 4.6 ml) was slowly added thereto. The reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched by adding a saturated ammonium chloride solution to the reaction mixture, which was then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting filtrate was concentrated under reduced pressure. The resulting material was dissolved in ethyl acetate (25 ml) and then a solution of hydrochloric acid in 1,4-dioxane (4N, 2 ml) was slowly added thereto. The reaction mixture was stirred at room temperature for 8 hours. The resulting solid was filtered under reduced pressure and then washed with ethyl acetate to give 1.67 g of the titled compound as a white solid. (Yield: 86.0%)

¹H-NMR (DMSO-d₆) 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 (22 mg) prepared in

Step

6 and 2,6-dichlorobenzoxazole (15 mg) were dissolved in 1,4-dioxane (1.0 ml) and then N,N-diisopropylethylamine (40 μL) was slowly added thereto. The reaction mixture was stirred at 80℃ for 12 hours and then cooled to room temperature. The reaction was quenched by adding water to the reaction mixture, which was then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting 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 give 14.7 mg of the titled compound. (Yield: 43.7 %)

¹H-NMR (CDCl₃) 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]oxazol-2-amine

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

A solution of (S)-N-(2-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethylidene)-2-methylpropan-2-sulfinamide (3.0 g) prepared in Step 5 of Example 1 in dichloromethane (30 ml) was stirred at -20℃ for 10 minutes and then a solution of ethylmagnesium bromide in tetrahydrofuran (1.0 M, 5.34 ml) was slowly added thereto. The reaction mixture was stirred at -20℃ for 6 hours and then raised to room temperature. The reaction was quenched by adding a saturated ammonium chloride solution to the reaction mixture, which was then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting filtrate was concentrated under reduced pressure. The resulting material was dissolved in ethyl acetate (30 ml) and then a solution of hydrochloric acid in 1,4-dioxane (4N, 8 ml) was slowly added thereto. The reaction mixture was stirred at room temperature for 8 hours. The resulting solid was filtered under reduced pressure and then washed with ethyl acetate to give 2.33 g of the titled compound as a white solid. (Yield: 86.0%)

¹H-NMR (DMSO-d₆) 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 (70 mg) prepared in

Step

1, 2,6-dichlorobenzoxazole (32 mg), and potassium carbonate (57 mg) were dissolved in N,N-dimethylformamide (1.0 ml) and then triethylamine (30 μL) was slowly added thereto. The reaction mixture was stirred at 80℃ for 12 hours and then cooled to room temperature. The reaction was quenched by adding water to the reaction mixture, which was then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting 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 give 42 mg of the titled compound. (Yield: 44.7 %)

¹H-NMR (CDCl₃) 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]oxazol-2-amine

(R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-amine hydrochloride (30 mg) prepared in Step 6 of Example 1 and 2-chloro-6-fluoro-1,3-benzoxazole (16 mg) were dissolved in 1,4-dioxane (1.0 ml) and then N,N-diisopropylethylamine (50 μL) was slowly added thereto. The reaction mixture was stirred at 100℃ for 12 hours and then cooled to room temperature. The reaction was quenched by adding water to the reaction mixture, which was then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting 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 give 3.9 mg of the titled compound. (Yield: 9.5 %)

¹H-NMR (CDCl₃) 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 (50 mg) prepared in Step 6 of Example 1 and 2-chloro-6,7-difluoro-1,3-benzoxazole (33 mg) were dissolved in tetrahydrofuran (1.0 ml). The resulting solution was placed in a sealed tube and then triethylamine (110 μL) was slowly added thereto. After sealing the sealed tube, the reaction mixture was stirred at 120℃ for 12 hours and then cooled to room temperature. The reaction was quenched by adding water to the reaction mixture, which was then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting 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 give 51.5 mg of the titled compound. (Yield: 75.6 %)

¹H-NMR (CDCl₃) 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-methylquinazolin-4-amine

(R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-amine hydrochloride (50 mg) prepared in Step 6 of Example 1 and 2,4-dichloro-7-methylquinazoline (37 mg) were dissolved in tetrahydrofuran (1.0 ml). The resulting solution was placed in a sealed tube and then triethylamine (110 μL) was added thereto. After sealing the sealed tube, the reaction mixture was stirred at 80℃ for 12 hours and then cooled to room temperature. The reaction was quenched by adding water to the reaction mixture, which was then extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting 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 give 49.5 mg of the titled compound as a pale yellow liquid. (Yield: 69.3 %)

¹H-NMR (CDCl₃) 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)

Experimental Example 1: IDO1 enzyme assay

IDO enzyme analyses of the compounds prepared in Examples were carried out with a IDO1 fluorogenic inhibitor screening assay kit (catalog #72037 BPS Bioscience). 5 μl of each test solution (prepared by dissolving the compounds of Examples in 10 μl of DMSO, respectively; and then mixing with 90 μl of phosphate-buffered saline (PBS)) was put in a black 384 well plate and then 5 μl of the IDO1 His-tag enzyme (40 ng/μl in IDO1 assay buffer) was added thereto. The mixture was pre-incubated at 37℃ for 2 hours. The IDO1 fluorogenic reaction solution (90 μl per well) was added to the mixture, which was then reacted at room temperature for 1 hour. The fluorescence solution (20 μl per well) was added to the mixture, which was reacted at 37℃ for 4 hours and then cooled at room temperature for 10 minutes. Fluorescence was measured at 400 nm of excitation and 510 nm of emission with a fluorometer. For analysis of the results, the fluorescence (Ft) of the well containing the IDO protein without treatment of the test compound was set to 100%; and the fluorescence (Fb) of the well containing no IDO protein without treatment of the test material was set to 0%. The % fluorescence was calculated according to the following equation:

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

(F = fluorescence of the well treated with test material)

The results obtained by calculating the 50% inhibitory concentration (IC₅₀) of each test material, from the fluorescence values obtained as described above, are shown in Table 1 below.

Experimental Example 2: IDO1 HeLa cell assay

Hela cells were seeded at　a density of 20000 cells/well in 100 μl of a culture medium (EMEM supplemented with 10% FBS, penicillin 100 U/ml, and streptomycin 100 μg/ml) in a tissue culture-treated 96 well plate. The cells were cultured in a 5% carbon dioxide incubator for 24 hours, treated with recombinant human interferon gamma at the concentration of 50 ng/ml, and then cultured for 48 hours in a 5% carbon dioxide incubator to induce IDO expression.

The activities were measured using IDO1 cellular activity-measuring substrate (IDO1 cellular activity quickDetect supplements catalog: #62000-2, BPS Bioscience). Specifically, an assay medium was prepared by diluting IDO1 assay medium supplement 1 and IDO1 assay medium supplement 2 in a culture medium at a ratio of 1:100, respectively. The test compounds (the compounds prepared in Examples) were diluted by 1/3 from 1 μM in the fresh assay medium so as to prepare 10 concentrations thereof. After removing the culture medium using a multi-pipet, 200 μl of each assay medium containing the test material was added into the respective well, followed by incubating in a 5% carbon dioxide incubator for 24 hours. The next day, 140 μl of culture medium from each well was transferred to a new 96-well plate. 10 μl of 6.1N trichloroacetic acid was added into each well, which was then cultured in a 50℃ incubator for 30 minutes. The plate was centrifuged at 2500 rpm for 10 minutes to settle any sediment. A detection reagent solution was prepared by diluting the detection reagent (component D kit, catalog: #62000-2, BPS Bioscience) 50-fold in acetic acid. 100 μl of the supernatant taken from the centrifuged plate was transferred to a new transparent 96-well plate and then 100 μl of the detection reagent solution was added thereto. After reacting at room temperature for 10 minutes, absorbance was measured at 480 nm wavelength. For analysis of the results, the absorbance (At) of the well containing Hela cells in which IDO protein expression was induced without treatment of an inhibitor was set to 100%; and the absorbance (Ab) of the well containing Hela cells in which IDO protein expression was not induced was set to 0%. The % absorbance was calculated according to the following equation: % Absorbance = (A-Ab)/(At-Ab), A = absorbance of the well treated with the test material.

The results obtained by calculating the 50% inhibitory concentration (IC₅₀) of each test material, from the absorbance values obtained as described above, are shown in Table 1 below.

Experimental Example 3: IDO1 HEK293 cell assay

HEK293 cells were seeded at　a density of 30000 cells/well in 100 μl of a culture medium (DMEM supplemented with 10% FBS, penicillin 100 U/ml, and streptomycin 100 μg/ml) in a tissue culture-treated 96 well plate. The cells were cultured in a 5% carbon dioxide incubator for 24 hours, transfected with the IDO1 expression vector (component A in IDO1 cell-based assay kit, catalog #72031, BPS Bioscience) using Lipofectamine 2000 (Life Technologies, #11668027), and then cultured for 24 hours in a 5% carbon dioxide incubator to express the IDO1 protein.

The activities were measured using a IDO1 cell-based assay kit (catalog #72031, BPS Bioscience). Specifically, an assay medium was prepared by diluting IDO1 assay medium supplement 1 and IDO1 assay medium supplement 2 in a culture medium at a ratio of 1:100, respectively. The test compounds (the compounds prepared in Examples) were diluted by 1/3 from 1 μM in the fresh assay medium so as to prepare 10 concentrations thereof. After removing the culture medium using a multi-pipet, 200 μl of each assay medium containing the test material was added into respective well, followed by incubating in a 5% carbon dioxide incubator for 24 hours. The next day, 140 μl of culture medium from each well was transferred to a new 96-well plate. 10 μl of 6.1N trichloroacetic acid was added into each well, which was then cultured in a 50℃ incubator for 30 minutes. The plate was centrifuged at 2500 rpm for 10 minutes to settle any sediment. A detection reagent solution was prepared by diluting the detection reagent (component D in IDO1 cell-based assay kit, catalog #72031 BPS Bioscience) 50-fold in acetic acid. 100 μl of the supernatant taken from the centrifuged plate was transferred to a new transparent 96-well plate and then 100 μl of the detection reagent solution was added thereto. After reacting at room temperature for 10 minutes, absorbance was measured at 480 nm wavelength. For analysis of the results, the absorbance (At) of the well containing HEK293 cells in which IDO protein was expressed without treatment of an inhibitor was set to 100%; and the absorbance (Ab) of the well containing HEK293 cells in which IDO protein was not expressed was set to 0%. The % absorbance was calculated according to the following equation: % Absorbance = (A-Ab)/(At-Ab), A = absorbance of the well treated with the test material.

Inhibitory Activity (IC₅₀, nM)

	IDO1 enzyme assay (Experimental Example 1)	IDO1 HeLa cell assay (Experimental Example 2)	IDO1 HEK293 cell assay (Experimental 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.

Experimental Example 4-1: Comparative study of pharmacokinetics through oral administration in normal rats

The pharmacokinetics of the compound of Example 1 and BMS-986205 (control) were measured in rats, respectively. The compound of Example 1 and BMS-986205 (control) were each suspended in 0.5% methyl cellulose containing 0.2% Tween 80 and then orally administered to rats at a dose of 10 mg/kg/5 mL, respectively. Plasma samples were collected from the rats in predetermined times. The concentrations of each compound in the samples were analyzed to obtain the plasma concentration profiles (FIG. 1a). The pharmacokinetic parameters obtained therefrom are shown in Table 2 below.

	Maximum plasma concentration (ng/mL)	Time to maximum plasma concentration (hr)	Area under the curve (ng·hr/mL)	Bioavailability (%)
BMS-986205	656.8	1	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 exhibits 3.3 times higher Cmax; and 4.0 times higher AUC, compared to the control (BMS-986205), in normal rats. These results show that the compound of Example 1 exhibits remarkably high in vivo exposure. Accordingly, the compound of Example 1 is expected to show excellent drug efficacy by exhibiting significantly higher in vivo exposure than BMS-986205 at the same dose. In addition, the compound of Example 1 is expected to show excellent safety since they can obtain similar in vivo exposure to BMS-986205 even when administered at a low dose.

Experimental Example 4-2: Comparative study 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 each suspended in 0.5% methyl cellulose containing 0.2% Tween 80 and then orally administered to rats at a dose of 3 mg/kg/5 mL, respectively. Plasma samples were collected from the rats in predetermined times. The concentration of the compound in each sample was analyzed to obtain the plasma concentration profiles (FIG. 1b). The pharmacokinetic parameters obtained therefrom are shown in Table 3 below.

	Maximum plasma concentration (ng/mL)	Time to maximum plasma concentration (hr)	Area under the curve (ng·hr/mL)	Bioavailability (%)
BMS-986205 (equivalent to 3mg/kg of dose)	197.1	1	1193	39.5
Example 1 (equivalent to 3mg/kg of 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	1	2835	46.8

As can be seen from the results of Table 3, the compounds of Examples 2 to 5 exhibit 1.8∼2.8 times higher AUC, compared to the control (BMS-986205), in normal rats. These results show that the compounds of Examples 2 to 5 exhibit high in vivo exposure. Accordingly, the compounds of Examples 2 to 5 are expected to show excellent drug efficacy by exhibiting higher in vivo exposure than BMS-986205 at the same dose. In addition, the compounds of Examples 2 to 5 are expected to show excellent safety since they can obtain similar in vivo exposure to BMS-986205 even when administered at a low dose.

Experimental Example 5-1: Studies on biomarker changes and pharmacokinetics according to the administration of the compound of Example 1 in a mouse subcutaneous MC38 tumor model

The studies on biomarker changes and pharmacokinetic according to the administration of the compound of Example 1 were carried out in the C57BL/6 mice model subcutaneously implanted with MC38 colorectal tumor cells. BMS-986205 was also evaluated as a control.

(1) Cell culture

MC38 tumor cells were cultured as monolayer culture in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 1% antibiotics-antimycotics, under the conditions of 37℃, 5% CO₂. The tumor cells were subcultured twice weekly by trypsin-EDTA treatment. Subcultures were carried out while maintaining about 70-80% confluency. After counting the cell number using Adam, an automatic cell counting equipment, 1 x 10⁶ cells were transferred to a T-75 flask and then maintained and incubated. Finally, the growing cells in the exponential growth phase were harvested and then counted for tumor implantation.

(2) Tumor implantation and Animal grouping

Each mouse was subcutaneously implanted in the right upper flank with MC38 cells (5 x 10⁶ cells in 0.1 mL of PBS) for tumor development. When the mean tumor volume reached about 114 mm³ at about 2 weeks after the tumor implantation, group separation was carried out so that the mean tumor volume for each group was similar. The test materials were repeated administered for 1 day and 3 days according to the experimental design of Table 4 below. Plasma samples were collected at 0, 2, 12, 14, and 24 hours. Tumor tissue samples were collected at 24 hours. The concentrations of the drug, tryptophan, and kynurenine were measured.

Group	Treatment	Dose	Administration	Administration route	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 day	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 the repeated administrations for 1 day and 3 days as described above, the results obtained by analyzing the concentrations of tryptophan and kynurenine in the tumor tissues are shown in Table 5 below, and the pharmacokinetic parameters of the compound of Example 1 obtained from the plasma concentration profiles, the dose proportionalities and the accumulations in the body calculated therefrom are shown in Table 6.

Treatment	Tryptophan in tumor (㎍/g)		Kynurenine in tumor (㎍/g)		K/T ratio		% Inhibition of kynurenine
Treatment	Mean	SD	Mean	SD	Mean	SD	% Inhibition of kynurenine
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)	Day 1			Day 3
Dose (mg/kg)	50 (QD)	100 (QD)	100 (BID)	50 (QD)	100 (QD)	100 (BID)
C_max (㎍/ml)	9.5	12.9	12.7	8.3	12.4	19.1
C_trough (㎍/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)
Day 3/ Day 1 (C_max)	-	-	-	0.9	1.0	1.5
Day 3/ Day 1 (AUC_last)	-	-	-	1.1	1.1	1.7
Dose proportionality (Fold increase)
Dose	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 administrations of the compound of Example 1 for 3 days showed significantly reduced levels of kynurenine irrespective of the administered concentrations, in comparison with the administrations thereof for 1 day. And, the repeated administrations of the compound of Example 1 for 3 days also showed significantly reduced kynurenine concentrations in the tumor tissues. Especially, when the compound of Example 1 was repeatedly administered at 100 mg/kg BID for 3 days, the kynurenine concentration in the tumor tissue was almost completely reduced. In addition, the pharmacokinetic parameters obtained from the plasma concentration profile according to the administration of the compound of Example 1 are shown in Table 6, which shows low systemic exposure compared to the dose proportionality. The accumulation according to repeated administrations of 50 mg/kg QD and 100 mg/kg QD was hardly observed, although the accumulation (1.7-fold) according to repeated administrations of 100 mg/kg BID was observed (Table 6).

Experimental Example 5-2: Studies on biomarker changes and pharmacokinetics according to the administration of the compounds of Examples 1 to 5 in a mouse subcutaneous MC38 tumor model

The studies on biomarker changes and pharmacokinetic according to the administration of the compounds of Examples 1 to 5 were carried out in the C57BL/6 mice model subcutaneously implanted with MC38 colorectal tumor cells.

(1) Cell culture

(2) Tumor implantation and Animal grouping

Each mouse was subcutaneously implanted in the right upper flank with MC38 cells (5 x 10⁶ cells in 0.1 mL of PBS) for tumor development. When the mean tumor volume reached about 171 mm³ at about 2 weeks after the tumor implantation, group separation was carried out so that the mean tumor volume for each group was similar. The test materials were repeated administered for 3 days according to the experimental design of Table 7 below. Plasma samples were collected at 0, 2, 4, 8, 14, and 24 hours. Tumor tissue samples were collected at 24 hours. The concentrations of the drug, tryptophan, and kynurenine were measured.

Group	Treatment	Dose	Administration	Administration route	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 the repeated administrations for 3 days as described above, the results obtained by analyzing the concentrations of tryptophan and kynurenine in the plasma and tumor tissues are shown in Tables 8 and 9 below, respectively, and the pharmacokinetic parameters of the compounds of Examples 1 to 5 obtained from the plasma concentration profiles are shown in Table 10.

Treatment	Tryptophan in plasma (μg·hr/mL)		Kynureninein plasma (μg·hr/mL)		K/T ratio		% Inhibition of kynurenine
Treatment	Mean	SD	Mean	SD	Mean	SD	% Inhibition of kynurenine
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

Treatment	Tryptophan in tumor (㎍/g)		Kynurenine in tumor (㎍/g)		K/T ratio		% Inhibition of kynurenine
Treatment	Mean	SD	Mean	SD	Mean	SD	% Inhibition of kynurenine
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
Dose (mg/kg)	100 (QD)	100 (QD)	100 (QD)	100 (QD)	100 (QD)
C_max (㎍/ml)	17.10	10.96	6.17	5.05	16.48
^*T_max (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
Concentration in plasma (㎍/ml, at 24 hr)	8.87	3.10	0.58	1.10	9.01
Concentration in tumor (㎍/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

* Mean

As can be seen from the results in Tables 8 and 9, the administrations of the compounds of Examples 1 to 5 for 3 days showed reduced similar levels of kynurenine in the plasma and tumor tissues; and excellent T/P ratios (Table 10).

Experimental Example 6: In vivo evaluation of anti-tumor efficacy according to combination administration in a mouse subcutaneous MC38 tumor model

In vivo evaluation of anti-tumor efficacy according to combination administrations of the compound of Example 1 and anti-PD-1 antibody (clone RMP1-14 (BE0146), BioXcell) was carried out in a C57BL/6 mouse subcutaneous MC38 colorectal tumor model. BMS-986205 was also evaluated as a control. The MC38 tumor cells were cultured in the same method as in Experimental Example 5 (1).

(1) Tumor implantation and Animal grouping

Each mouse was subcutaneously implanted in the right upper flank with MC38 cells (1 x 10⁶ cells in 0.1 mL of PBS) for tumor development. When the mean tumor volume reached about 50 mm³, the administrations of test materials were initiated. Since there is a difference in the rate of tumor development in the mice, tumor volume in each mouse was measured daily. And each test substance was administered individually when it reached 50 mm³. Each mouse had a different time of drug administration; and thus the first administration date was set to 'Day 1' (the first day). The number of mice in the experimental design was 20 in each group. However, internal sacrifice for ex vivo immune activation tests was carried out with 5 mice in each group; and thus the remaining 15 mice were subject to the evaluations on tumor growth inhibition and survival rate. When the tumor did not grow or the mouse died due to cannibalism, the evaluations were carried out except those mice. The experimental design for the in vivo efficacy evaluation according to the combination administration of the test materials is shown in Table 11 below.

Experimental design for combination administration

Group	Number of mice	Treatment	Dose	Administration route	Administration
1	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
4	13	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
6	14	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
8	15	Anti-PD-1 antibody	10 mg/kg	i.p.	BIW (1,4,8,11)

(2) Tumor measurements and endpoints

The tumor volume was measured daily in two-dimension using vernier calipers and the volume thereof was calculated in mm³ using the following formula:

V (volume) = 0.52a x b²

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

The tumor volume for each mouse according to the predetermined schedule was input into Prism and the average value was calculated therefrom. On Day 11 (when the evaluation on tumor growth inhibition was finished), 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 mean tumor volume of the control group mice (vehicle-administered group) on Day 11, and Tn is the mean tumor volume of each treatment group mice on Day 11. Animals were sacrificed when tumor volume reached about 2,500 mm³ and the time to reach this endpoint was used for Kaplan-Meier survival analysis.

(3) Statistical analysis

Statistical analysis of differences in tumor volume among the groups was performed on data obtained at Day 11 after the initiation of administration. When significant F-statistics (ratio of treatment variance to error variance) were obtained according to performing Two-way ANOVA, comparisons between the groups were carried out using Tukey's multiple comparisons test. All data were analyzed using Prism 9.0. It was considered statistically significant from p<0.05: *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001.

Statistical analysis of differences in survival rates among the groups was performed on survival data up to Day 50 after the initiation of administration. Kaplan-Meier test was carried out and all data were analyzed using GraphPad Prism 9.0. Comparisons among the groups were carried out with Log-rank test. It was considered statistically significant from p<0.05: *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001.

(4) Results

Tumor growth curves in each treatment group of C57BL/6 mice implanted with MC38 tumor cells are shown in FIG. 2. FIG. 3 is a Waterfall plot showing tumor growth inhibition on Day 11. TGI (Tumor Growth Inhibition) is a value calculated as a percentage of the difference in mean tumor volume of each treatment group when the mean tumor volume of the control group (vehicle-administered group) mice is set to 100%. And, the tumor volume and the statistical analysis results thereof on Day 11 are shown in Table 12 below.

Group of Treatment	Tumor volume on Day 11 ± SEM (mm³)	T/C (%)	TGI (%)	*p value
Control group (vehicle administered 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, tumor growth was significantly inhibited in all administration groups (although the TGIs thereof were different), compared to the control group (vehicle administration group). The combination administration groups of the IDO inhibitor and the anti-PD-1 antibody showed statistically significant inhibitions against tumor growth (for each group, p<0.0001). Especially, combination administration groups of the compound of Example 1 (100 mpk, BID) and the anti-PD-1 antibody showed remarkably higher (i.e., synergistic) inhibitory activity (86.7%) against tumor growth, showing markedly reduced tumor volume compared to the other groups (**** P < 0.0001).

FIG. 4 shows survival curves for each treatment group of MC38 tumor bearing C57BL/6 mice. The animals were sacrificed when tumor volume reached more than 2,500 mm³. Based on the results of FIG. 4, the statistical analysis results of the survival curves of each treatment group with respect to the control group (vehicle-administered group) are shown in Table 13 below. Using a log-rank test, comparisons of each treatment group were carried out with respect to the control group (vehicle-administered group).

Group of Treatment	Median survival (day)	P-value for log-rank test	Tumor-free mice on Day 50
Control group (vehicle administered 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 reached	<0.0001	5/15

As can be seen from the results in Table 13, compared to the control group (vehicle-administered group), the survival rates of the test animals were significantly extended in all groups [i.e., the group administered with the anti-PD-1 antibody alone; the group administered with BMS-986205 (125 mpk, QD) alone; the group administered with the combination of BMS-986205 (125 mpk, QD) and the anti-PD-1 antibody; the groups administered with the compound of Example 1 (50 mpk, BID / 100 mpk, BID) alone; and the groups administered with the combination of the compound of Example 1 (50 mpk, BID / 100 mpk, BID) and the anti-PD-1 antibody] (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 a log-rank test are shown in Table 14 below.

Group of Treatment	Median survival (day)	P-value for log-rank test	Tumor-free mice on 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 survival rate of the test animals was not extended in the group administered with the combination of BMS-986205 (125 mpk, QD) and the anti-PD-1 antibody (p=0.2813). In contrast, the survival rates of the test animals were significantly extended in the groups administered with the combination of the compound of Example 1 (50 mpk, BID / 100 mpk, BID) and the anti-PD-1 antibody (p = 0.0008 and p < 0.0001, respectively). In addition, in regard to tumor-free mice on the 50th day of administration of the test materials, 2 mice in 14 mice showed Complete Response (CR) in the group administered with the combination of the compound of Example 1 (50 mpk, BID) and the anti-PD-1 antibody; and 5 mice in 15 mice showed Complete Response (CR) in the group administered with the combination of the compound of Example 1 (100 mpk, BID) and the anti-PD-1 antibody.

In addition, Table 15 below shows the statistical analysis results of the survival curve for the group administered with the combination of the compound of Example 1 (100 mpk, BID) and the anti-PD-1 antibody with respect to the group administered with the compound of Example 1 (100 mpk, BID) alone using a log-rank test.

Group of Treatment	Median survival (day)	P-value for log-rank test	Tumor-free mice on 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 reached	<0.0001	5/15

As can be seen from the results in Table 15, the group administered with the combination of the compound of Example 1 (100 mpk, BID) and the anti-PD-1 antibody showed significantly higher anti-tumor effects and significantly extended survival rates of the test animals (p<0.0001), in comparison with the group administered with the compound of Example 1 (100 mpk, BID) alone. In regard to tumor-free mice on the 50th day of administration of the test materials, 5 mice in 15 mice showed Complete Response (CR) in the group administered with the combination of the compound of Example 1 (100 mpk, BID) and the anti-PD-1 antibody, while 1 mouse in 13 mice showed Complete Response (CR) in the group administered with the compound of Example 1 (100 mpk, BID) alone.

FIGs. 5a to 5c show the tumor volume tracking curves for the mice which showed Complete Response in the surviving mice. As can be seen from the results of FIGs. 5a to 5c, no tumor recurrence occurred even after the drug administration was stopped.

From the above results, it can be confirmed that the combination administration of the compound of Example 1 and the anti-PD-1 antibody showed distinct synergistic anti-tumor activities in the MC38 colorectal tumor animal model and significantly extended survival rates thereof.

Claims

A pharmaceutical composition for preventing or treating a cancer, which comprises a combination of a first compartment comprising a compound of Formula 1 or pharmaceutically acceptable salt thereof as an active ingredient and a second compartment comprising an immune checkpoint inhibitor as an active ingredient:

<Formula 1>

wherein,

R₁ is a C₁∼C₆ alkyl group,

A is a heteroaryl group selected from the group consisting of benzo[d]oxazolyl and quinazolinyl, wherein the heteroaryl group is substituted with one or two substituents selected from the group consisting of halogen and C₁∼C₆ alkyl.
The pharmaceutical composition as claimed in claim 1, wherein the compound of Formula 1 is selected from the group consisting of:

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]oxazol-2-amine; and

2-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)-7-methylquinazolin-4-amine.
The pharmaceutical composition as claimed in claim 1, 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.
The pharmaceutical composition as claimed in claim 1, wherein the immune checkpoint inhibitor is an inhibitor targeting programmed death receptor-1 (PD-1) / programmed death ligand-1 (PD-L1) signaling pathway.
The pharmaceutical composition as claimed in 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 as claimed in claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
The pharmaceutical composition as claimed in claim 1, wherein the cancer is selected from the group consisting of squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer, hepatic 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, and head and neck cancer.
The pharmaceutical composition as claimed in any one of claims 1 to 7, wherein the first compartment is a compartment for oral administration; and the second compartment is a compartment for injection.
A method for treating a cancer in a mammal, which comprises administering a therapeutically effective amount of a compound of Formula 1 or pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of an immune checkpoint inhibitor to the mammal in need thereof:

<Formula 1>

wherein,

R₁ is a C₁∼C₆ alkyl group,

A is a heteroaryl group selected from the group consisting of benzo[d]oxazolyl and quinazolinyl, wherein the heteroaryl group is substituted with one or two substituents selected from the group consisting of halogen and C₁∼C₆ alkyl.
The method as claimed in claim 9, wherein the compound of Formula 1 is selected from the group consisting of:

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]oxazol-2-amine; and

2-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)-7-methylquinazolin-4-amine.
The method as claimed in 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.
The method as claimed in claim 9, wherein the immune checkpoint inhibitor is an inhibitor targeting programmed death receptor-1 (PD-1) / programmed death ligand-1 (PD-L1) signaling pathway.
The method as claimed in claim 9, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.
The method as claimed in claim 9, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
The method as claimed in claim 9, wherein the cancer is selected from the group consisting of squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer, hepatic 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, and head and neck cancer.
The method as claimed in any one of claims 9 to 15, wherein the compound of Formula 1 or pharmaceutically acceptable salt thereof is orally administered and the immune checkpoint inhibitor is administered by injection.
Use of a combination of a compound of Formula 1 or pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor for the manufacture of a medicament for treating a cancer in a mammal:

<Formula 1>

wherein,

R₁ is a C₁∼C₆ alkyl group,

A is a heteroaryl group selected from the group consisting of benzo[d]oxazolyl and quinazolinyl, wherein the heteroaryl group is substituted with one or two substituents selected from the group consisting of halogen and C₁∼C₆ alkyl.
The use as claimed in claim 17, wherein the compound of Formula 1 is selected from the group consisting of:

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]oxazol-2-amine; and

2-chloro-N-((R)-1-((cis)-4-(6-fluoroquinolin-4-yl)cyclohexyl)propan-2-yl)-7-methylquinazolin-4-amine.
The use as claimed in claim 17, 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.
The use as claimed in claim 17, wherein the immune checkpoint inhibitor is an inhibitor targeting programmed death receptor-1 (PD-1) / programmed death ligand-1 (PD-L1) signaling pathway.
The use as claimed in claim 17, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.
The use as claimed in claim 17, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
The use as claimed in claim 17, wherein the cancer is selected from the group consisting of squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer, hepatic 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, and head and neck cancer.
The use as claimed in any one of claims 17 to 23, wherein the compound of Formula 1 or pharmaceutically acceptable salt thereof is in the form of a pharmaceutical formulation for oral administration and the immune checkpoint inhibitor is in the form of a pharmaceutical formulation for injection.