Methods of treating cancer with heteroaryl-biphenylamide derivatives

Abstract

A subject in need of treatment for certain cancers is administered an effective amount of a compound of formula (I), including stereoisomers and pharmaceutically acceptable salts thereof. JPEG2023531970000019.jpg30160, wherein R 1 , R 2 , R 3 , R 4 , R a , and R b is as defined herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 042,807, filed June 23, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] STATEMENT AS TO RIGHTS TO THIS INVENTION MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT Not applicable.

[0003] Reference to a "Sequence Listing," table, or computer program listing appendix submitted on a compact disc Not applicable. [Background technology]

[0004] Programmed cell death protein 1 (PD-1) is a member of the CD28 superfamily that transmits negative signals upon interaction with its two ligands, PD-L1 and PD-L2. PD-1 and its ligands are widely expressed and exert a wide range of immunoregulatory roles in T cell activation and tolerance. PD-1 and its ligands have been implicated in attenuating infection and tumor immunity and promoting chronic infection and tumor progression. Modulation of the PD-1 pathway has therapeutic potential in various human diseases (Hyun-Tak Jin et al., Curr Top Microbiol Immunol. (2011); 350:17-37). Blockade of the PD-1 pathway has become an attractive target in cancer therapy. Therapeutic antibodies that block programmed cell death protein 1 (PD-1) prevent the downregulation of T cells and promote immune responses against cancer. Several PD-1 pathway inhibitors have shown robust activity in various phases of clinical trials (RD Harvey, Clinical Pharmacology and Therapeutics (2014); 96(2), 214-223). Agents that block the interaction of PD-L1 with either PD-1 or CD80 are desirable. Several antibodies have been developed and marketed. Several patent applications have been published disclosing non-peptide small molecules (BMS applications WO 2015 / 160641, WO 2015 / 034820, and WO 2017 / 066227 and WO 2018 / 009505, Aurigene applications WO 2015 / 033299 and WO 2015 / 033301, Incyte applications WO 2017 / 070089, U.S. Patent Application Publication No. 2017 / 0145025, WO 2017 / 106 (See, for example, U.S. Patent Application Publication No. 634, U.S. Patent Application Publication No. 2017 / 0174679, WO 2017 / 192961, WO 2017 / 222976, WO 2017 / 205464, WO 2017 / 112730, WO 2017 / 041899, and WO 2018 / 013789, Maxinovel application WO 2018 / 006795, and ChemoCentryx application WO 2018 / 005374 to the present inventor.) However, there remains a need for alternative compounds, such as small molecules as inhibitors of PD-L1, which may have advantageous characteristics in terms of oral administration, high tumor penetration, stability, bioavailability, therapeutic index, and toxicity. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Hyun-Tak Jin et al.,Curr Top Microbiol Immunol.(2011);350:17-37 [Patent Document 2] RD Harvey,Clinical Pharmacology and Therapeutics(2014);96(2),214-223 [Non-patent literature]

[0006] [Non-Patent Document 1] International Publication No. 2015 / 160641 [Non-patent document 2] International Publication No. 2015 / 034820 [Non-patent document 3] International Publication No. 2017 / 066227 [Non-patent document 4] International Publication No. 2018 / 009505 [Non-Patent Document 5] International Publication No. 2015 / 033299 [Non-patent document 6] International Publication No. 2015 / 033301 [Non-Patent Document 7] International Publication No. 2017 / 070089 [Non-patent document 8] US Patent Application Publication No. 2017 / 0145025 [Non-Patent Document 9] International Publication No. 2017 / 106634 Summary of the Invention [Means for solving the problem]

[0007] In some embodiments, a subject in need of treating cancer is administered an effective amount of a compound of formula (I) [ka] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R a , and R b is as described herein.

[0008] In some embodiments, the cancer is selected from the group consisting of colon cancer, renal cancer, colorectal cancer, gastric cancer, bladder cancer, melanoma, non-small cell lung cancer, Merkel cell carcinoma, liver cancer, breast cancer, and head and neck cancer. [Brief explanation of the drawings]

[0009] [Figure 1A] Figures 1A-1B plot the PD-1 / PD-L1 binding ELISA data (top panels) and PD-1 / PD-L1 blocking cell line assay data (bottom panels) for compound 2.001 (A) and compound 2.002 (B). [Figure 1B] Figures 1A-1B plot the PD-1 / PD-L1 binding ELISA data (top panels) and PD-1 / PD-L1 blocking cell line assay data (bottom panels) for compound 2.001 (A) and compound 2.002 (B). [Figure 2AB] Figures 2A-2C show how compound 2.001 enhances human T cell allogenic immune responses in an ex vivo mixed lymphocyte reaction (MLR) assay, showing T cell responses from three separate donors: Donor 1 (A), Donor 2 (B), and Donor 3 (C). [Figure 2C] Figures 2A-2C show how compound 2.001 enhances human T cell allogenic immune responses in an ex vivo mixed lymphocyte reaction (MLR) assay, showing T cell responses from three separate donors: Donor 1 (A), Donor 2 (B), and Donor 3 (C). [Figure 3A] Figures 3A-3C show how compound 2.002 enhances human T cell allogenic immune responses in an ex vivo mixed lymphocyte reaction (MLR) assay, showing T cell responses from three separate donors: Donor 1 (A), Donor 2 (B), and Donor 3 (C). [Figure 3BC]Figures 3A-3C show how compound 2.002 enhances human T cell allogenic immune responses in an ex vivo mixed lymphocyte reaction (MLR) assay, showing T cell responses from three separate donors: Donor 1 (A), Donor 2 (B), and Donor 3 (C). [Figure 4A] Figures 4A-4B show PBMC-mediated tumor cell killing of compound 2.002 (A, left-most column), compound 2.001 (A, middle column), and a control compound (A, right-most column). Additional control experiments used an anti-PD-L1 antibody (durvalumab) (B, left-most column), and an antibody isotype (B, right-most column). [Figure 4B] Figures 4A-4B show PBMC-mediated tumor cell killing of compound 2.002 (A, left-most column), compound 2.001 (A, middle column), and a control compound (A, right-most column). Additional control experiments used an anti-PD-L1 antibody (durvalumab) (B, left-most column), and an antibody isotype (B, right-most column). [Figure 5] Figure 5 shows that compound 2.001 and compound 2.002 induce PD-L1 dimerization, whereas the anti-PD-L1 antibodies and the controls tested do not induce PD-L1 dimerization. [Figure 6] Figure 6 shows the surface levels of PD-L1 at 4°C (lower panel) and 37°C (upper panel) under the various test conditions. This figure demonstrates that compounds 2.001 and 2.002 specifically reduce surface PD-L1 levels at 37°C, suggesting PD-L1 internalization. [Figure 7] MC38-hPD-L1 tumor model for evaluating human PD-L1 inhibitors in vivo. The engineered MC38-hPD-L1 cells are suitable for evaluating the effects of human PD-L1-specific inhibitors in vivo; hPD-L1 and mPD-L1 bind to mPD-1 with similar affinity, and the hPD-L1 inhibitors block the interaction of hPD-L1 with hPD-1 or mPD-1 with similar potency (data not shown). MC38-hPD-L1 cells induce tumor growth in mice. [Figure 8A] Figures 8A-8C show that compound 2.002 mediates tumor growth inhibition in a dose-dependent manner in the MC38-hPD-L1 tumor model. (A) Tumor volume versus days after tumor implantation is plotted. (B) Mean tumor weight after 35 days is plotted. (C) Trough plasma compound concentrations 3 days after dosing are plotted. [Figure 8B] Figures 8A-8C show that compound 2.002 mediates tumor growth inhibition in a dose-dependent manner in the MC38-hPD-L1 tumor model. (A) Tumor volume versus days after tumor implantation is plotted. (B) Mean tumor weight after 35 days is plotted. (C) Trough plasma compound concentrations 3 days after dosing are plotted. [Figure 8C] Figures 8A-8C show that compound 2.002 mediates tumor growth inhibition in a dose-dependent manner in the MC38-hPD-L1 tumor model. (A) Tumor volume versus days after tumor implantation is plotted. (B) Mean tumor weight after 35 days is plotted. (C) Trough plasma compound concentrations 3 days after dosing are plotted. [Figure 9A] Figures 9A-9C plot tumor size at the indicated days for vehicle treatment (filled circles) and API (anti-PD-L1 antibody or the indicated compound, filled squares). The APIs tested were compound 2.001 (A), compound 2.003 (B), and anti-PD-L1 antibody (C). The top panels plot the average tumor size for each treatment group, while the bottom panels plot the tumor size for each mouse in the treatment group. [Figure 9B] Figures 9A-9C plot tumor size at the indicated days for vehicle treatment (filled circles) and API (anti-PD-L1 antibody or the indicated compound, filled squares). The APIs tested were compound 2.001 (A), compound 2.003 (B), and anti-PD-L1 antibody (C). The top panels plot the average tumor size for each treatment group, while the bottom panels plot the tumor size for each mouse in the treatment group. [Figure 9C]Figures 9A-9C plot tumor size at the indicated days for vehicle treatment (filled circles) and API (anti-PD-L1 antibody or the indicated compound, filled squares). The APIs tested were compound 2.001 (A), compound 2.003 (B), and anti-PD-L1 antibody (C). The top panels plot the average tumor size for each treatment group, while the bottom panels plot the tumor size for each mouse in the treatment group. [Figure 10A] 10A-10B plot the trough plasma concentrations of compound 2.001 (A) and compound 2.003 (B) 12 hours and 6 days after dosing in the mouse model described in Biological Example 2. [Figure 10B] 10A-10B plot the trough plasma concentrations of compound 2.001 (A) and compound 2.003 (B) 12 hours and 6 days after dosing in the mouse model described in Biological Example 2. [Figure 11] Figure 11 shows human PD-L1 staining of cells treated with an anti-PD-L1 antibody (durvalumab), an isotype antibody, compound 2.001, and vehicle. The PD-L1 detection antibody used in this assay is blocked by compound 2.001 binding to PD-L1. This figure demonstrates that MC38-hPD-L1 tumors treated with compound 2.001 are almost completely occupied by compound 2.001. [Figure 12] Figure 12 shows how various treatment conditions alter the amount of tumor-infiltrating immune cells in the MC38-HPD-L1 tumor model. The bottom panel plots the amount of measured CD8+ T cells, the middle panel plots the amount of measured CD4+ T cells, and the top panel plots the amount of measured CD8+ and CD4+ T cells. DETAILED DESCRIPTION OF THE INVENTION

[0010] I. Overview The present disclosure provides methods for treating certain cancers using compounds of formula (I). The claimed compounds have robust antitumor properties with high affinity for PD-L1. Upon administration, these compounds effectively interrupt PD-1 / PD-L1 signaling and, in some embodiments, induce PD-L1 dimerization and internalization in cancer cells.

[0011] The development of small molecule modulators of PD-1 / PD-L1 has been hampered by the need to balance various factors, including PD-1 / PD-L1 affinity, the compound's hydrophobicity / hydrophilicity, biological clearance rate, and anti-target activity (e.g., CYP and hERG inhibition). Indeed, to date, no PD-1 / PD-L1 inhibitors have been approved for oral administration.

[0012] In contrast to intravenous drug formulations, the bioavailability of orally administered compounds requires, among other things, gastric absorption and resistance to significant degradation via the portal circulation to the liver (so-called "first-pass metabolism"). In some embodiments, the methods described herein provide PD-1 / PD-L1 modulators that are unexpectedly suitable for oral administration in the treatment of certain cancers. The compounds in the described methods do not require extremely high concentrations of the compounds in the blood; instead, these compounds can elicit anti-tumor effects in the ng / mL range.

[0013] II. Abbreviations and Definitions As used herein, the terms "a," "an," or "the" include embodiments having one component as well as embodiments having more than one component. For example, the singular forms "a," "an," and "the" include plural referents unless expressly stated otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells, a reference to an "agent" includes reference to one or more agents known to those of skill in the art, and so forth.

[0014] The terms "about" and "approximately" generally refer to an acceptable degree of error for a measured quantity given the nature or accuracy of the measurement results. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" can refer to values ​​that are within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless otherwise specified, and the term "about" or "approximately" means that they can be inferred when not expressly stated.

[0015] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical having the specified number of carbon atoms (i.e., C 1~8 means 1 to 8 carbons). Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds. Examples of alkenyl groups are vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, and 3-(1,4-pentadienyl). Examples of alkynyl groups are ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "cycloalkyl" refers to a cycloalkyl group having the indicated number of ring atoms (e.g., C 3~6"Cycloalkyl" refers to a hydrocarbon ring that is fully saturated or has one or fewer double bonds between the ring vertices. "Cycloalkyl" is also meant to refer to bicyclic and polycyclic hydrocarbon rings, such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. Bicyclic or polycyclic rings can be fused, bridged, spiro, or combinations thereof. The term "heterocycloalkyl" or "heterocyclyl" refers to a cycloalkyl group containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atoms are optionally tetramerized. Heterocycloalkyl can be a monocyclic, bicyclic, or polycyclic ring system. The bicyclic or polycyclic rings can be fused, bridged, spiro, or combinations thereof. C 4~12 The recitation of heterocyclyl refers to groups having 4 to 12 ring members, where at least one of the ring members is a heteroatom. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, tetrazolone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. Heterocycloalkyl groups can be attached to the remainder of the molecule via a ring carbon or heteroatom.

[0016] The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, as embodied by -CHCHCHCH-. An alkylene group can be straight or branched. Examples of the latter are -CHC(CH)CH-, -CHC(CH)-, or -CH(CH)CHCH-. Typically, an alkyl (or alkylene) group will have from 1 to 12 carbon atoms, with those groups having 8 or fewer carbon atoms being preferred in this disclosure. Similarly, "alkenylene" and "alkynylene" refer to unsaturated forms of "alkylene" having a double or triple bond, respectively.

[0017] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively. Additionally, for dialkylamino groups, the alkyl portions can be the same or different and can also combine to form a 3- to 7-membered ring with the nitrogen atom to which each is attached. Thus, -NR a R b A group represented as: is meant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl, and the like.

[0018] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, "C 1~4 The term "haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0019] The term "hydroxyalkyl" or "alkyl-OH" refers to an alkyl group, as defined above, in which at least one (and up to three) of the hydrogen atoms have been replaced with a hydroxy group. With respect to alkyl groups, a hydroxyalkyl group is defined as any group having a C 1~6 Exemplary hydroxyalkyl groups include, but are not limited to, hydroxymethyl, hydroxyethyl (wherein the hydroxy is in the 1- or 2-position), hydroxypropyl (wherein the hydroxy is in the 1-, 2-, or 3-position), and 2,3-dihydroxypropyl.

[0020] The term "aryl," unless otherwise specified, refers to a polyunsaturated, typically aromatic, hydrocarbon group which may be a single ring or multiple rings (up to three rings) that are fused or covalently linked together. A "heteroaryl" ring refers to an aryl group (or aryl ring) containing 1 to 5 heteroatoms selected from N, O, and S, in which the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atoms are optionally tetramerized. A heteroaryl group can be attached to the remainder of the molecule via a heteroatom. C 5~10It is understood that recitation of heteroaryl refers to heteroaryl moieties having 5 to 10 ring members, where at least one of the ring members is a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolinyl, and benzoindolinyl.

[0033] Examples of aryl and heteroaryl ring systems include aryl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl, and the like. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

[0021] The terms "carbocyclic ring," "carbocycle," or "carbocyclyl" refer to a cyclic moiety having only carbon atoms as ring vertices. Carbocyclic ring moieties can be saturated or unsaturated and aromatic. Generally, carbocyclic ring moieties have 3 to 10 ring members. Carbocyclic ring moieties having multiple ring structures (e.g., bicyclic) can contain a cycloalkyl ring fused to an aromatic ring (e.g., 1,2,3,4-tetrahydronaphthalene). Thus, carbocyclic rings include cyclopentyl, cyclohexenyl, naphthyl, and 1,2,3,4-tetrahydronaphthyl. The term "heterocyclic ring" refers to both "heterocycloalkyl" and "heteroaryl" moieties. Thus, heterocyclic rings can be saturated or unsaturated and aromatic. Generally, heterocyclic rings have 4 to 10 ring members and include piperidinyl, tetrazinyl, pyrazolyl, and indolyl.

[0022] When any of the above terms (e.g., "alkyl," "aryl," and "heteroaryl") are referred to as "substituted" without further remarks regarding substituents, the substituted forms of the indicated group are as provided below.

[0023] Substituents for alkyl groups (including those groups often referred to as alkylene, alkenyl, alkynyl, and cycloalkyl) can be a variety of groups selected from -halogen, -OR", -NR'R", -SR', -SiR'R"R'", -OC(O)R', -C(O)R', -COR', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R'", -NR"C(O)R', -NH-C(NH)=NH, -NR'C(NH)=NH, -NH-C(NH)=NR', -S(O)R', -S(O)R', -S(O)NR'R", NR'S(O)R", -CN, and -NO in number ranging from 0 to (2m'+1), where m' is the total number of carbon atoms in such group. R', R'', and R'" are each independently hydrogen, unsubstituted C 1~8 Alkyl, unsubstituted heteroalkyl, unsubstituted aryl, 1-3 halogens, unsubstituted C 1~8 Alkyl groups, unsubstituted C 1~8 Alkoxy group or unsubstituted C 1~8 aryl substituted with a thioalkoxy group or unsubstituted aryl-C 1~4 refers to an alkyl group. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl. The term "acyl" used by itself or as part of another group refers to an alkyl group in which two substituents on the carbon nearest to the point of attachment to the group have been replaced with the substituent =0 (e.g., -C(O)CH3, -C(O)CH2CH"OR', and the like).

[0024] Similarly, substituents for the aryl and heteroaryl groups vary and generally include -halogen, -OR', -OC(O)R', -NR'R", -SR', -R', -CN, -NO2, -C02R', CONR'R", C(O)R', -OC(O)NR'R", -NR"C(O)R', -NR"C(O)2R', -NR'-C(O)NR"R"', NH-C(NH2)=NH, -NR' selected from C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R", -NR'S(O)2R", -N3, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, in a number ranging from 0 to the total number of open valences in the aromatic ring system, and R', R", and R"' are independently selected from hydrogen, C 1~8 Alkyl, C 3~6 Cycloalkyl, C 2~8 Alkenyl, C 2~8 Alkynyl, unsubstituted aryl and unsubstituted heteroaryl, (unsubstituted aryl)-C 1~4 Alkyl and unsubstituted aryloxy-C 1~4 Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene chain of 1 to 4 carbon atoms.

[0025] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring optionally have the formula -TC(O)-(CH) q -U-, where T and U are independently -NH-, -O-, -CH2-, or a single bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by a substituent of the formula A-(CH2) rA and B may be replaced with a substituent of the formula -B-, where A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 3. One of the single bonds in the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH2) s -X-(CH2) t wherein s and t are independently an integer of 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituent R' in -NR'- and -S(O)2NR'- is hydrogen or an unsubstituted C 1~6 alkyl.

[0026] As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

[0027] The present disclosure further relates to prodrugs and bioisosteres thereof. Suitable bioisosteres include, for example, carboxylate substitutes (phosphate, phosphinic acid, sulfonic acid, sulfinic acid, and acidic heterocyclic groups such as tetrazole). Suitable prodrugs include conventional groups known to hydrolyze and / or oxidize under physiological conditions to provide compounds of Formula I.

[0028] The terms "patient" and "subject" include primates (especially humans), domestic companion animals (such as dogs, cats, horses, and the like), and livestock (such as cows, pigs, sheep, and the like).

[0029] As used herein, the term "treating" or "treatment" encompasses both disease-modifying and symptomatic treatments, either of which may be prophylactic (i.e., prior to onset of symptoms to prevent, delay, or reduce the severity of symptoms) or therapeutic (after onset of symptoms to reduce the severity and / or duration of symptoms).

[0030] The term "pharmaceutically acceptable salts" is intended to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When a compound of the present disclosure contains a relatively acidic functionality, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable bases include salts of primary, secondary, and tertiary amines, including substituted, cyclic, and naturally occurring amines, and the like, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When compounds of the present disclosure contain a relatively basic functionality, acid addition salts can be obtained by contacting the neutral form of such compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, monohydrogen sulfate, hydroiodic acid, or phosphorous acid, and the like, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as alginate and the like, and salts of organic acids such as glucuronic acid or galactunoric acid and the like (see, e.g., Berge, S.M., et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0031] The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of this disclosure.

[0032] Certain compounds of the present disclosure can exist in unsolvated forms and solvated forms, including hydrated forms. Generally, solvated forms are equivalent to unsolvated forms and are intended to be included within the scope of the present disclosure. Certain compounds of the present disclosure can exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0033] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, positional isomers, and individual isomers (e.g., individual enantiomers) are all intended to be encompassed within the scope of the present invention. When a stereochemical diagram is shown, it is meant to refer to a compound in which one of the isomers is present and substantially free of the other isomer. "Substantially free of" another isomer indicates at least an 80 / 20 ratio of the two isomers, more preferably 90 / 10, or 95 / 5 or more. In some embodiments, one of the isomers is present in an amount of at least 99%.

[0034] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may contain, for example, tritium ( 3 H), iodine-125( 125 I), or carbon-14 ( 14 C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are intended to be encompassed within the scope of the present disclosure. For example, a compound may be radiolabeled with a radioactive isotope, such as deuterium ( 2 The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. Unnatural proportions of isotopes may be defined as a range from the amount found in nature of the atom in question to an amount consisting of 100%. For example, compounds may contain, for example, tritium ( 3 H), iodine-125( 125 I) or carbon-14 ( 14 Radioactive isotopes such as C, or deuterium ( 2 H) or carbon-13 ( 13Non-radioactive isotopes, such as CI, ...

[0035] III. EMBODIMENTS OF THE DISCLOSURE Treatment method In some embodiments, a subject in need of treating cancer is administered an effective amount of a compound of formula (I) [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 2 are each independently selected from the group consisting of F, Cl, CH3, and CF3; R 3 is selected from the group consisting of F, Cl, CH3, CF3, -O-CH3, and -O-CF3; R 4 But -Y and -X 1 -Y, wherein each X 1 C 1~4 alkylene, and Y is C 3~6 C having 1 to 3 heteroatom ring vertices independently selected from the group consisting of cycloalkyl, N, O, and S 4~6 heterocycloalkyl, and 5-6 membered heteroaryl having 1-3 heteroatom ring vertices independently selected from the group consisting of N, O, and S, each of which is unsubstituted or selected from oxo, OH, C 1~4 Alkyl, C1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, and C 1~4 hydroxyalkoxy; and R a and R b are independent, H, C 1~3 Alkyl, and C 1~4 Provided herein are methods wherein the alkyl group is selected from the group consisting of haloalkyl.

[0036] In some embodiments, R 1 is selected from the group consisting of Cl and CH. In some embodiments, R 1 is Cl. In some embodiments, R 1 is CH3.

[0037] In some embodiments, R 2 is selected from the group consisting of Cl and CH. In some embodiments, R 2 is Cl. In some embodiments, R 2 is CH3.

[0038] In some embodiments, R 3 is selected from the group consisting of —O—CH and —O—CF. In some embodiments, R 3 is —O—CH. In some embodiments, R 3 is -O-CF3.

[0039] In some embodiments, R a is selected from the group consisting of H, CH, and CF. In some embodiments, R a is CH3.

[0040] In some embodiments, R b is selected from the group consisting of H, CH, and CF. In some embodiments, Rb is CH3.

[0041] In some embodiments, the compound of Formula I has the formula (Ia): [ka] or a pharmaceutically acceptable salt thereof.

[0042] In some embodiments, —NH(R 4 )teeth, [ka] is selected from the group consisting of:

[0043] In some embodiments, —NH(R 4 )teeth, [ka] is selected from the group consisting of:

[0044] In some embodiments, -NHR 4 teeth, [ka] is selected from the group consisting of:

[0045] In some embodiments, -NHR 4 teeth, [ka] is.

[0046] In some embodiments, the compounds of formula (I) are optically pure or enriched isomers.

[0047] In some embodiments, the compound of formula (I) is selected from the compounds in Table 1.

[0048] As described herein, the disclosed methods for treating certain cancers do not require extremely high concentrations of the compound of Formula (I) in the blood. In fact, these compounds are sufficiently potent to provide therapeutic benefit at lower plasma concentrations. Thus, in some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of 1,000 ng / mL or less. In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of 750 ng / mL or less. In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of 500 ng / mL or less. In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of 400 ng / mL or less. In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of 300 ng / mL or less. In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of 200 ng / mL or less. In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of 100 ng / mL or less.

[0049] In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of about 2 ng / mL to 1,000 ng / mL. In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of about 5 ng / mL to 500 ng / mL. In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of about 10 ng / mL to 400 ng / mL. In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of about 20 ng / mL to 300 ng / mL. In some embodiments, an effective amount of a compound of Formula (I) maintains a trough plasma concentration of about 40 ng / mL to 200 ng / mL.

[0050] Many cancers can be treated using the methods described herein. In some embodiments, the cancer is melanoma, glioblastoma, esophageal tumor, nasopharyngeal tumor, uveal melanoma, lymphoma, lymphocytic lymphoma, primary central nervous system lymphoma, T-cell lymphoma, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, prostate cancer, castration-resistant prostate cancer, chronic myeloid leukemia, Kaposi's fibrosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, angiosarcoma, lymphangiosarcoma, synovium, meningioma, leiomyosarcoma, rhabdomyosarcoma, soft tissue sarcoma, sarcoma, sepsis, biliary tract tumor, basal cell carcinoma, thymus tumor, thyroid cancer. , parathyroid cancer, uterine cancer, adrenal cancer, liver infection, Merkel cell carcinoma, nerve tumors, follicular center lymphoma, colon cancer, Hodgkin's disease, non-Hodgkin's lymphoma, leukemia, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, ovarian tumors, myelodysplastic syndrome, cutaneous or intraocular malignant melanoma, renal cell carcinoma, small cell lung cancer, lung cancer, mesothelioma, liver cancer, breast cancer, squamous non-small cell lung cancer (SCLC), non-squamous NSCLC, colorectal cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic cancer carcinoma), pancreatic cancer cancer), pancreatic ductal cancer, head and neck squamous cell carcinoma, head and neck cancer, gastrointestinal tract, gastric cancer, HIV, hepatitis A, hepatitis B, hepatitis C, hepatitis D, herpesvirus, papillomavirus, influenza, bone cancer, skin cancer, rectal cancer, anal area. cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, tongue cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, urethral cancer, penile cancer, bladder cancer, renal cancer, ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, tumor angiogenesis, chordoma (spinal) axis tumor, brain stem glioma, pituitary adenoma, epidermoid carcinoma, asbestosis, carcinoma, adenocarcinoma, papillary carcinoma, cystadenocarcinoma, bronchogenic carcinoma, renal cell carcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, pleomorphic adenoma, hepatocellular papilloma, renal tubular adenoma, cystadenoma, papilloma, adenoma, leiomyoma, rhabdomyoma, hemangioma, lymphangioma, osteoma, chondroma, lipoma, and fibroma. In some embodiments, each of the listed cancers is a PD-L1 positive cancer.

[0051] In some embodiments, the cancer is colon cancer, renal cancer, colorectal cancer, gastric cancer, bladder cancer, melanoma, non-small cell lung cancer, Merkel cell carcinoma, liver cancer, breast cancer, and head and neck cancer. In some embodiments, each of the listed cancers is a PD-L1 positive cancer.

[0052] In some embodiments, the disease or disorder is colon cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is breast cancer. In some embodiments, each of the listed cancers is a PD-L1 positive cancer.

[0053] In some embodiments, an effective amount of one or more additional therapeutic agents is further administered to the subject. In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of cytotoxic agents, gene expression modulating agents, chemotherapeutic agents, anti-cancer agents, anti-angiogenic agents, immunotherapeutic agents, anti-hormonal agents, radiation therapy, radiotherapeutic agents, anti-tumor agents, and anti-proliferative agents. In some embodiments, the one or more additional therapeutic agents are antagonists of chemokine and / or chemoattractant receptors, including, but not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CCR12, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, C3aR, and / or C5aR. Antagonists of chemokine and / or chemoattractant receptors are known in the art and are described, for example, in WO 2007 / 002667, WO 2007 / 002293, WO 2003 / 105853, WO 2007 / 022257, WO 2007 / 059108, WO 2007 / 044804, WO 2007 / 115232, WO 2007 / 115231, WO 2008 / 147815, WO 2010 / 030815, WO 2010 / 075257, WO 2011 / 163 640, International Publication No. 2010 / 054006, International Publication No. 2010 / 051561, International Publication No. 2011 / 035332, International Publication No. 2013 / 082490, International Publication No. 2013 / 082429, International Publication No. 2014 / 085490, International Publication No. 2014 / 100735, International Publication No. 2014 / 089495, International Publication No. 2015 / 084842, International Publication No. 2016 / 187393, International Publication No. 2017 / 127409, International Publication No. 2017 / 087607, International Publication No. 2017 / 087610,International Publication No. 2017 / 176620, International Publication No. 2018 / 222598, International Publication No. 2018 / 222601, International Publication No. 2013 / 130811, International Publication No. 2006 / 076644, International Publication No. 2008 / 008431, International Publication No. 2009 / 038847, International Publication No. 2008 / 008375, International Publication No. These are described in WO 2008 / 008374, WO 2008 / 010934, WO 2009 / 009740, WO 2005 / 112925, WO 2005 / 112916, WO 2005 / 113513, WO 2004 / 085384 and WO 2004 / 046092. Chemokine and / or chemoattractant receptor antagonists also include CCX354, CCX9588, CCX140, CCX872, CCX598, CCX6239, CCX9664, CCX2553, CCX3587, CCX3624, CCX2991, CCX282, CCX025, CCX507, CCX430, CCX765, CCX224, CCX662, CCX650, CCX832, CCX168, CCX168-M1, CCX3022, and / or CCX3384.

[0054] The treatment methods provided herein generally involve administering to a patient an effective amount of one or more compounds provided herein. Suitable patients include those suffering from or susceptible to the disorders or diseases identified herein (i.e., prophylactic treatment). Typical patients for treatment as described herein include mammals, particularly primates, and especially humans. Other suitable patients include domestic companion animals such as dogs, cats, horses, and the like, or livestock such as cows, pigs, sheep, and the like.

[0055] Route of administration and dosage Routes of administration contemplated in the present disclosure include those known in the art for delivering active agents for the treatment of cancer, including, but not limited to, oral administration, intratumoral injection, intravenous administration, and subcutaneous injection. In some embodiments, an effective amount of a compound of Formula (I) is administered orally. In some embodiments, an effective amount of a compound of Formula (I) is administered via intratumoral injection. In some embodiments, an effective amount of a compound of Formula (I) is administered intravenously. In some embodiments, an effective amount of a compound of Formula (I) is administered via subcutaneous injection.

[0056] Generally, the treatment methods provided herein involve administering to a patient an effective amount of a compound of Formula (I) or one or more compounds provided herein. An effective amount can be an amount sufficient to modulate PD-1 / PD-L1 interaction, slow tumor growth, inhibit tumor growth, and / or reduce tumor size in a subject. Preferably, the amount administered is sufficient to produce a plasma concentration of the compound (or, if the compound is a prodrug, its active metabolite) high enough to sufficiently modulate PD-1 / PD-L1 interaction. Treatment dosing regimens can vary depending on the compound used and the particular condition being treated, with a dosing frequency of four times daily or less being preferred for the treatment of most disorders. Generally, a twice-daily dosage regimen is more preferred, with once-daily dosing being particularly preferred. It will be understood, however, that the specific dose level and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the patient's age, body weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combinations (i.e., other drugs being administered to the patient), and the severity of the particular disease being treated, as well as the judgment of the prescribing medical professional. Generally, use of the lowest dose sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using medical or veterinary criteria appropriate to the condition being treated or prevented.

[0057] Dosage levels on the order of about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in treating or preventing conditions involving PD-1 / PD-L1 interaction (about 0.5 mg to about 7 g per human patient per day). The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Unit dosage forms generally contain from about 1 mg to about 500 mg of active ingredient. For compounds administered orally, transdermally, intravenously, or subcutaneously, it is preferred that the amount of compound administered be sufficient to achieve a plasma concentration of 5 ng (nanogram) / mL to 1 μg (microgram) / mL plasma, more preferably, sufficient compound should be administered to achieve a plasma concentration of 20 ng to 0.5 μg / mL plasma, and most preferably, sufficient compound should be administered to achieve a plasma concentration of 30 ng / mL to 200 ng / mL plasma.

[0058] The frequency of administration may also vary depending on the compound used, the route of administration, and the specific disease being treated.However, for the treatment of most disorders, a dosage regimen of four times a day, three times a day, or less is preferred, with a dosage regimen of once a day or twice a day being particularly preferred.However, it will be understood that the specific dose level for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet (food), time of administration, route of administration, and excretion rate, drug combinations (i.e., other drugs administered to the patient), the severity of the specific disease being treated, and other factors, including the judgment of the prescribing medical professional.

[0059] Pharmaceutical Composition When administered to a subject, Formula (I) is typically in a pharmaceutical composition. As used herein, the term "composition" is intended to encompass any product containing the specified ingredients in the specified amounts, and any product that results directly or indirectly from the combination of the specified ingredients in the specified amounts. By "pharmaceutically acceptable" is meant that the carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient.

[0060] Pharmaceutical compositions for administering the compounds of the present disclosure can be conveniently presented in unit dosage form for oral administration and can be prepared by any of the methods well known in the art of pharmacy and drug delivery.All methods include the step of bringing the active ingredient together with a carrier, which constitutes one or more accessory ingredients.In general, pharmaceutical compositions can be prepared by uniformly and comminutedly dissolving the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.In this pharmaceutical composition, the active object compound is included in an amount sufficient to produce the desired effect in response to the disease process or condition.

[0061] Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions and self-emulsifying agents as described in U.S. Patent Application Publication No. 2002 / 0012680, hard or soft capsules, syrups, elixirs, liquids, buccal patches, oral gels, chewing gums, chewable tablets, effervescent powders, and effervescent tablets. Compositions intended for oral use may be prepared by any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents, antioxidants, and preservatives to provide a medicament with a clean and pleasant taste. Tablets contain the active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients can be, for example, inert diluents such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as PVP, cellulose, PEG, starch, gelatin, or acacia; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets can be uncoated or enteric- or otherwise coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate can be employed. Tablets can also be coated by the techniques described in U.S. Pat. Nos. 4,256,108, 4,166,452, and 4,265,874 to form osmotic therapeutic tablets for controlled release.

[0062] Formulations for oral use may also be presented as hard gelatin capsules, in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, polyethylene glycols (PEG) of various average diameters (e.g., PEG400, PG4000), and certain surfactants, such as Cremophor or Solutol, or soft gelatin capsules, in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil. Additionally, emulsions may be prepared with water-immiscible ingredients, such as oils, and stabilized with surfactants, such as monoglycerides or diglycerides, PEG esters, and the like.

[0063] Aqueous suspensions contain the active ingredient in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, and the dispersing or wetting agent may be a natural phosphatide such as lecithin, or a condensation product of an alkylene oxide with a fatty acid such as polyoxyethylene stearate, or a condensation product of ethylene oxide with a long-chain fatty alcohol such as heptadecaethyleneoxycetanol, or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride such as polyoxyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweeteners such as sucrose or saccharin.

[0064] Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil, such as peanut oil, olive oil, sesame oil, or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions can contain thickening agents such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners and flavoring agents, such as those described above, can be added to provide a chewable oral preparation. These compositions can be preserved by the addition of an antioxidant, such as ascorbic acid.

[0065] Dispersible powders or granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are embodied by those already mentioned above. Additional excipients, for example, sweeteners, flavoring agents, and coloring agents, may also be present.

[0066] The pharmaceutical composition of the present disclosure can also be in the form of an oil-in-water emulsion.The oil phase can be vegetable oil such as olive oil or peanut oil, or mineral oil such as liquid paraffin, or a mixture thereof.Suitable emulsifiers can be natural gums such as gum acacia or gum tragacanth, natural phosphatides such as soybean, lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of the partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.Emulsifying agents can also contain sweeteners and flavoring agents.

[0067] Syrups and elixirs can be formulated with sweeteners such as glycerol, propylene glycol, sorbitol, or sucrose. Such formulations can also contain demulcents, preservatives, and flavoring and coloring agents. Oral solutions can be prepared with, for example, cyclodextrins, PEGs, and surfactants.

[0068] The compounds of the present disclosure may also be coupled to carriers that are polymers suitable for targetable drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds of the present disclosure may be coupled to biodegradable polymers useful for achieving controlled drug release, including carriers that are a class of crosslinked or amphiphilic block copolymers of polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and hydrogels. Polymers and semipermeable polymer matrices may be formed into shaped articles, such as valves, stents, tubing, prostheses, and the like. In one embodiment of the present disclosure, the compounds of the present disclosure are coupled to a polymer or semipermeable polymer matrix formed as a stent or stent-graft device.

[0069] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of antibacterial agents, antiviral agents, cytotoxic agents, gene expression modulating agents, chemotherapeutic agents, anticancer agents, antiangiogenic agents, immunotherapeutic agents, antihormonal agents, antifibrotic agents, radiation therapy, radiotherapeutic agents, antitumor agents, and antiproliferative agents. In some embodiments, the one or more additional therapeutic agents are antagonists of chemokine and / or chemoattractant receptors, including, but not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CCR12, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, C3aR, and / or C5aR. Antagonists of chemokine and / or chemoattractant receptors are known in the art and are described, for example, in WO 2007 / 002667, WO 2007 / 002293, WO 2003 / 105853, WO 2007 / 022257, WO 2007 / 059108, WO 2007 / 044804, WO 2007 / 115232, WO 2007 / 115231, WO 2008 / 147815, WO 2010 / 030815, WO 2010 / 075257 ... International Publication No. 2011 / 163640, International Publication No. 2010 / 054006, International Publication No. 2010 / 051561, International Publication No. 2011 / 035332, International Publication No. 2013 / 082490, International Publication No. 2013 / 082429, International Publication No. 2014 / 085490, International Publication No. 2014 / 100735, International Publication No. 2014 / 089495, International Publication No. 2015 / 084842, International Publication No. 2016 / 187393, International Publication No. 2017 / 127409, International Publication No. 2017 / 087607,International Publication No. 2017 / 087610, International Publication No. 2017 / 176620, International Publication No. 2018 / 222598, International Publication No. 2018 / 222601, International Publication No. 2013 / 130811, International Publication No. 2006 / 076644, International Publication No. 2008 / 008431, International Publication No. 2009 / 038847, International Publication No. 2008 / 008375 These are described in WO 2008 / 008374, WO 2008 / 010934, WO 2009 / 009740, WO 2005 / 112925, WO 2005 / 112916, WO 2005 / 113513, WO 2004 / 085384 and WO 2004 / 046092. Chemokine and / or chemoattractant receptor antagonists also include CCX354, CCX9588, CCX140, CCX872, CCX598, CCX6239, CCX9664, CCX2553, CCX3587, CCX3624, CCX2991, CCX282, CCX025, CCX507, CCX430, CCX765, CCX224, CCX662, CCX650, CCX832, CCX168, CCX168-M1, CCX3022, and / or CCX3384. [Example]

[0070] The following examples illustrate various methods of making compounds of the present disclosure, including compounds of Formula (I) or Formula (Ia). The following examples are offered to illustrate, but not to limit, the claimed disclosure.

[0071] Reagents and solvents used below can be obtained from commercial sources such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA). 1H-NMR spectra were recorded on a Varian Mercury 400 MHz NMR spectrometer. Significant peaks were assigned relative to TMS and tabulated in order of multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet) and proton number. Mass spectrometry results are reported as mass-to-charge ratios. In this example, a single m / z value is reported for the M+H (or, where noted, MH) ion, which contains the most common atomic isotope. The isotope pattern corresponds to the expected formula in all cases. Electrospray ionization (ESI) mass spectrometry was performed on a Hewlett-Packard MSD electrospray mass spectrometer using an HP1100 HPLC for sample delivery. Typically, the analyte was dissolved at 0.1 mg / mL in methanol or CH3CN, and 1 microliter was injected into the mass spectrometer with the delivery solvent, scanning from 100 to 1000 daltons. All compounds could be analyzed in either positive or negative ESI mode using acetonitrile / water with 1% formic acid as the delivery solvent.

[0072] The following abbreviations are used in the examples and throughout the specification of this disclosure: TLC means thin layer chromatography.

[0073] Compounds within the scope of the present disclosure can be synthesized using a variety of reactions known to those of skill in the art, as described below. Those skilled in the art will also recognize that alternative methods may be employed to synthesize target compounds of the present disclosure, and that the approaches described within the body of this document are not exhaustive, but provide broadly applicable and practical routes to compounds of interest.

[0074] Certain molecules claimed in this patent can exist in different enantiomeric and diastereomeric forms, and all such variants of these compounds are claimed unless a specific enantiomer is specified.

[0075] Detailed descriptions of the experimental procedures used to synthesize key compounds in this document are linked to molecules described by physical data that identify the compounds and by structural diagrams associated with the compounds.

[0076] Those skilled in the art will also recognize that acids and bases are frequently used during standard workup procedures in organic chemistry. Salts of parent compounds are sometimes prepared during the experimental procedures described within this patent, if they have the necessary inherent acidity or basicity.

[0077] Example 1: N-(2'-chloro-3'-(5-((((3R,4R)-3-hydroxytetrahydro-2H-pyran-4-yl)amino)methyl)-6-methoxypyridin-2-yl)-2-methyl-[1,1'-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide [ka] Step a: To a mixture of 1,3-dimethyl-N-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (3.6 g, 9.0 mmol), 1,3-dibromo-2-chlorobenzene (6.9 g, 25.5 mmol), and KCO (3.8 g, 27.5 mmol) in p-dioxane (40 mL) and DI H2O (6 mL) was added Pd(dppf)Cl2 complex with dichloromethane (912 mg, 1.12 mmol). The reaction mixture was degassed (N2) for 2 min and stirred at 90 °C under N2 for 2 h. The reaction mixture was diluted with EtOAc, filtered through Celite, washed with brine, and dried over MgSO4. The solvent was removed under reduced pressure and the residue was purified by silica gel flash chromatography (5 to 100% EtOAc in hexanes followed by 0 to 5% MeOH in EtOAc) to give N-(3'-bromo-2'-chloro-2-methyl-[1,1'-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide. MS: (ES) m / z C 20 H 18 BrClN3O3[M+H] + The calculated value is 462.0, and the measured value is 462.0.

[0078] Step b: To a mixture of N-(3'-bromo-2'-chloro-2-methyl-[1,1'-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (1.4 g, 3.03 mmol), pinacoldiborane (1.0 g, 3.94 mmol), and KOAc (1.2 g, 10.2 mmol) in p-dioxane (18 mL) was added Pd(dppf)Cl2 complex with dichloromethane (350 mg, 0.43 mmol). The reaction mixture was degassed (N2) for 2 minutes and stirred at 90 °C under N2 for 3 hours. The reaction mixture was diluted with EtOAc, filtered through Celite, washed with brine, and dried over MgSO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel flash chromatography (10→60% EtOAc in hexanes) to give N-(2'-chloro-2-methyl-3'-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-[1,1'-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide. MS: (ES) m / z C 26 H 30 BClN3O5[M+H] + The calculated value is 510.2, and the measured value is 510.1.

[0079] Step c: To a mixture of N-(2'-chloro-2-methyl-3'-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-[1,1'-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (400 mg, 0.78 mmol), 6-chloro-2-methoxynicotinaldehyde (200 mg, 1.17 mmol), and KCO (350 mg, 2.53 mmol) in p-dioxane (10 mL) and DI H2O (2 mL) was added Pd(dppf)Cl2 complex with dichloromethane (70 mg, 0.086 mmol). The reaction mixture was degassed (N2) for 2 min and stirred at 95 °C under N2 for 2 h. The reaction mixture was diluted with EtOAc, filtered through Celite, washed with brine, and dried over MgSO. The solvent was removed under reduced pressure, and the residue was purified by silica gel flash chromatography (10->65% EtOAc in hexanes) to give N-(2'-chloro-3'-(5-formyl-6-methoxypyridin-2-yl)-2-methyl-[1,1'-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide. MS: (ES) m / z C 27 H 24 ClN4O5[M+H] + Calculated value: 519.1, measured value: 519.1.

[0080] Step d: To a stirred solution of N-(2'-chloro-3'-(5-formyl-6-methoxypyridin-2-yl)-2-methyl-[1,1'-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (40 mg, 0.077 mmol) and (3R,4R)-4-aminotetrahydro-2H-pyran-3-ol hydrochloride (24 mg, 0.154 mmol) in dichloroethane (2 mL) and ethanol (1 mL), triethylamine (2 drops) was added, followed by acetic acid (2 drops). The reaction mixture was stirred at 70°C for 1 hour. The mixture was then cooled to 0°C, and NaCNBH (10 mg, 0.154 mmol) was slowly added. The mixture was stirred at 0°C for 10 minutes. The mixture was passed through a syringe filter and then purified by preparative HPLC (0→40%→100% acetonitrile / HO) to give N-(2′-chloro-3′-(5-((((3R,4R)-3-hydroxytetrahydro-2H-pyran-4-yl)amino)methyl)-6-methoxypyridin-2-yl)-2-methyl-[1,1′-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide. 1 H NMR(400MHz,CD3OD)δ 11.18(s,1H),8.63(s,1H),8.13-8.06(m,1H),7.88(d,J=7.6Hz,1H),7.61 (dd,J=7.6,1.7Hz,1H),7.49(t,J=7.6Hz,1H),7.39-7.25(m,3H),7.00(d,J =7.7Hz,1H),4.35(d,J=13.3Hz,1H),4.24(d,J=13.2Hz,1H),4.11-3.93(m ,6H),3.61-3.36(m,10H),2.13(s,4H),1.87(d,J=12.4Hz,1H).MS:(ES)m / z C 32 H 34 ClNO[M+H] + Calculated value: 620.2, measured value: 620.2.

[0081] Example 2: (S)—N-(2′-chloro-3′-(6-methoxy-5-((((5-oxopyrrolidin-2-yl)methyl)amino)methyl)pyridin-2-yl)-2-methyl-[1,1′-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide [ka] Using the same procedure as in Example 1, the title compound was prepared from N-(2'-chloro-3'-(5-formyl-6-methoxypyridin-2-yl)-2-methyl-[1,1'-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide and (S)-5-aminomethylpyrrolidin-2-one hydrochloride. The crude product was purified by reverse-phase HPLC (C18 column, acetonitrile / HO with 0.1% TFA as eluent) to give the desired product (S)—N-(2′-chloro-3′-(6-methoxy-5-((((5-oxopyrrolidin-2-yl)methyl)amino)methyl)pyridin-2-yl)-2-methyl-[1,1′-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide. 1 H NMR(400MHz,CD3OD)δ 8.63(d,J=1.1Hz,1H),8.12(dd,J=7.9,1.3Hz,1H),7.88(d,J=7.6Hz,1H),7.61(d,J =7.7Hz,1H),7.50(t,J=7.6Hz,1H),7.41-7.25(m,3H),7.00(d,J=7.5Hz,1H),4.34( d,J=2.0Hz,2H),4.13-4.00(m,4H),3.55(d,J=1.0Hz,3H),3.39(d,J=1.0Hz,3H),3. 34-3.22(m,2H),2.49-2.32(m,3H),2.13(s,3H),1.92(q,J=7.5Hz,1H).MS:(ES)m / z C 32 H 33 ClN6O5[M +H] + Calculated value: 617.2, measured value: 617.2.

[0082] Example 3: (S)—N-(2,2′-dichloro-3′-(6-methoxy-5-((((5-oxopyrrolidin-2-yl)methyl)amino)methyl)pyridin-2-yl)-[1,1′-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide [ka] Using a procedure similar to that in Example 1, the title compound was prepared from N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide and (S)-5-(aminomethyl)pyrrolidin-2-one hydrochloride. The crude product was purified by preparative HPLC (C18 column, MeCN / HO with 0.1% TFA as eluent) to give (S)—N-(2,2′-dichloro-3′-(6-methoxy-5-((((5-oxopyrrolidin-2-yl)methyl)amino)methyl)pyridin-2-yl)-[1,1′-biphenyl]-3-yl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide. 1 H NMR(400MHz,CD3OD)δ 11.69(s,1H),8.66(s,1H),8.54(d,J=8.3Hz,1H),7.93-7.85(m,1H),7.65(dd,J=7.8,1. 8Hz,1H),7.51(dd,J=7.7,7.7Hz,1H),7.45-7.35(m,3H),7.10(d,J=7.6Hz,1H),4.34(s, 2H),4.14-4.01(m,4H),3.56(d,J=1.6Hz,3H),3.39(d,J=1.8Hz,3H),3.30-3.20(m,3H), 2.40(dd,J=11.8,11.1Hz,2H),2.03(d,J=1.7Hz,1H),1.92(d,J=6.9Hz,1H).MS:(ES)m / z C 31 H 31 Cl2N6O5[M+H] +Calculated value: 637.2, measured value: 637.2.

[0083] Biological Example 1: Enzyme-Linked Immunosorbent Assay - ELISA A 96-well plate was coated with 1 μg / mL human PD-L1 (obtained from R&D) in PBS overnight at 4°C. The wells were then blocked with 2% (w / v) BSA in PBS containing 0.05% Tween 20 at 37°C for 1 hour. The plate was washed three times with PBS / 0.05% Tween 20, and the compounds were serially diluted (1:5) in dilution medium and added to the ELISA plate. Human PD-1 and biotin at 0.3 μg / mL (ACRO Biosystems) were added and incubated at 37°C for 1 hour, followed by washing three times with PBS / 0.05% Tween 20. A second block was performed with 2% BSA (w / v) / 0.05% Tween 20 in PBS at 37°C for 10 minutes, and the plate was washed three times with PBS / 0.05% Tween 20. Streptavidin-HRP was added for 1 hour at 37°C, and then the plate was washed three times with PBS / 0.05% Tween 20. TMB substrate was added and reacted for 20 minutes at 37°C. Stop solution (2N H2SO4 aqueous solution) was added. Absorbance was read at 450 nm using a microplate spectrophotometer. The results are shown in Table 1. IC 50 Values ​​are provided as follows: 1000-10,000 nM (+), 10-1000 nM (++), less than 10 nM (+++).

[0084] [Table 1]

[0085] Biological Example 2: Antitumor activity of Compound 2.001, Compound 2.002, and Compound 2.003 This example demonstrates the biological anti-tumor activity of compounds 2.001, 2.002, and 2.003 disclosed herein.

[0086] ELISA: This assay was performed essentially as described in Biological Example 1.

[0087] Cell lines and cell culture: CHO cells constitutively expressing TCR agonists and PD-L1 were grown in Ham's solution supplemented with 10% FBS and used for cell-based assays. A T lymphoid cell line (Jurkat) (effector cells, ECs) constitutively expressing PD-1 and engineered to carry a luciferase reporter gene driven by a TCR-inducible NFAT response element (Jurkat PD-1) was grown in RPMI supplemented with 10% FBS and 1x penicillin-streptomycin and used for cell-based assays. The human melanoma cell line A375 and the human breast cancer cell line MDA-MB-231 were obtained from ATTC and grown in DMEM supplemented with 10% FBS and 1x penicillin-streptomycin. Human PBMCs were isolated in-house and grown in RPMI supplemented with 10% FBS and 1× penicillin-streptomycin.

[0088] PD-1 / PD-L1 Blockade Cell Line Assay: 6×10 4 Cho PD-L1 cells were seeded in a 96-well plate overnight at 37°C. The cells were washed with 1x PBS and then incubated with 40μl of TJurkat PD-1 (1x10 6 40 μl of compound diluted in 1% FBS RPMI containing 1% PD-L1 (a starting concentration of 5 μM followed by a 1:5 dilution) was added to each well and incubated at 37°C for 6 hours. After cooling the cells to room temperature, 80 μl of Bio-Glo Reagent (Promega, Madison, WI) was added to the medium, and relative light units (RLU) were measured at a rate of 500 ms / well using a FlexStation 3 plate reader. When the two cells are co-cultured together, PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-RE-mediated luminescence. Upon addition, anti-PD-1 or anti-PD-L1 antibodies / compounds that block PD-1 / PD-L1 interaction release an inhibitory signal, resulting in TCR activation and NFAT-RE-mediated luminescence.

[0089] PBMC isolation: Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from LRS chambers (leukocyte reduction systems) from healthy donors by density gradient centrifugation using StemCell SepMate™-50 tubes (STEMCELL Technologies, Vancouver, CA) containing Ficoll-Paque Plus (Sigma Aldrich Inc., St. Louis, MO).

[0090] Generation of monocyte-derived dendritic cells: CD14 + Monocytes were isolated from PBMCs and human CD14 + Monocytes were isolated by magnetic separation using MicroBeads (MACS Miltenyi Biotech, Bergisch Gladbach, Germany) and an autoMACS® Pro Separator. Isolated monocytes were collected at 1 × 10 6 The cells were seeded at a concentration of 100 cells / ml and differentiated into dendritic cells by adding GM-CSF (100 ng / ml) and IL-4 (50 ng / ml) for 6 days. Fresh medium containing cytokine supplements was added on days 0 and 2. Mature dendritic cells were induced after 6 days by adding IL-6 (2000 IU / ml), IL-1B (400 IU / ml) (Peprotech, Inc., Rocky Hill, NJ), TNF-alpha (2000 IU / ml), and PGE2 (2 μg / ml) (Sigma-Aldrich, Inc.) and cultured for 24 hours.

[0091] Preparation of human effector cells: CD4 + T cells were isolated from PBMCs by magnetic separation using human CD4+ MicroBeads (MACS Miltenyi Biotech) and an autoMACS® Pro Separator.

[0092] Mixed lymphocyte reaction (MLR): DC cells and CD4 from mismatched donors +T cells were cultured together at a 1:10 ratio in 96-well flat-bottom plates (Thermo Scientific) for 5 days. Test compounds were added at a starting concentration of 1 μM, diluted 1:4 in DMSO, as indicated. PD-L1 antibody (AZ Medi4736 analog) and isotype control (human IgG1, kappa isotype control) (CrownBio, Beijing) were used as positive and negative controls, respectively. Supernatants were harvested 5 days after inoculation, and human IFNg was detected by ELISA using the Human IFN-gamma DuoSet ELISA (R&D System, Minneapolis) according to the manufacturer's instructions.

[0093] In vitro immunotherapy efficacy assay: A375-eGFP-Puro cells (ATCC) were grown in complete medium (DMEM + 10% FBS + P / S 1x) containing 1 μg / ml puromycin. Human peripheral blood mononuclear cells (hPBMCs) were isolated from buffy coats from healthy donors using an LRS chamber (leukocyte reduction system) by density gradient centrifugation using StemCell SepMate™-50 tubes (STEMCELL Technologies, Vancouver, CA) containing Ficoll-Paque Plus (Sigma Aldrich Inc., St. Louis, MO). Freshly isolated hPBMCs were stimulated with 100 ng / ml Staphylococcal Enterotoxin B (SEB) (EMD Millipore, Cat. No. 324798) for 3 days. Cells were washed twice and resuspended in regular growth medium. 3x10 4A375-eGFP-Puro cells were seeded in a final volume of 100 μl in a 96-well, clear-bottom, black, TC-treated plate (Corning). Test compounds or anti-human PD-L1 antibodies (AZ Medi4736 analog, CrownBio, Beijing) were added to the wells at different concentrations. SEB-stimulated hPBMCs were added to the wells at a 2:1 effector:target ratio (E:T). The mixed cells were incubated for 96–120 hours at 37°C in 5% CO2. The medium was carefully aspirated, and 100 μl of PBS 1x was added to each well. Fluorescence from the A375-eGFP cells was detected using a FlexStation3 plate reader.

[0094] Dimerization assay PD-L1 protein dimerization was assessed in vitro by chemiluminescence detection using the PathHunter® Dimerization Assay (DiscoverX, Fremont, CA). The assay was performed according to the manufacturer's protocol. 2 × 10 4 U2OS cells were seeded in a final volume of 100 μl into a 96-well white-bottom TC-treated plate (Costar, San Jose, CA). ChemoCentryx compounds or anti-human PD-L1 antibodies (AZ Medi4736 analog, CrownBio, Beijing) were added to the experimental cells at different concentrations and incubated for 16 hours at 37°C and 5% CO2. 110 μl of PathHunter Flash detection reagent (DiscoverX) was added to each well and incubated for 1 hour in the dark at room temperature. Chemiluminescent signals were measured using a FlexStation3 plate reader (Molecular Devices, San Jose, CA) at a speed of 100 ms / well.

[0095] Internalization assay: MC38-hPD-L1 cells (GenOway SA, France) and RKO cells (ATCC) grown at 37°C and 5% CO were detached and resuspended in cold FACS buffer (PBS 1x containing 10% FBS and 0.1% azide) and plated in a 96-well assay plate (V-bottom) (Axygen, Union City, CA) at 10 × 10 4 Cells were added at a concentration of 10 ...

[0096] Generation and culture of MC38-hPD-L1 cells: The compound is only known to cross-react with human PD-L1; therefore, a syngeneic tumor model with mouse MC-38 colon tumor cells expressing human PD-L1 (MC38-hPD-L1 tumor model) was used. MC38-hPD-L1 cells were generated by GenOway. Endogenous mouse PDL1 was first knocked out using CRISPR technology, and then human PDL1 was stably transfected into these mouse PD-L1 knockout MC38 cells. MC38-hPD-L1 cells were cultured under standard conditions for MC38 cells (DMEM containing 10% fetal bovine serum and penicillin / streptomycin) with G418 to maintain transgene expression. Two days before inoculation into mice, the cells were trypsinized and plated without antibiotics.

[0097] In vivo testing: 5 × 10 5MC38-hPD-L1 cells were injected subcutaneously in the right flank. Nine days after tumor inoculation, mice were randomly assigned to treatment groups based on tumor size. Only mice that developed measurable tumors were enrolled in the study. Anti-PD-L1 (durvalumab) or isotype control was administered intraperitoneally at 100 μg per dose per mouse, twice weekly for 2 weeks. Compound 2.001 and compound 2.002, suspended in 1% HPMC, were administered orally daily at the indicated doses in a volume of 100 μl per mouse. Control animals were administered the vehicle, 1% HPMC, at the same volume and frequency.

[0098] Tumor volumes were measured three times a week using a digital caliper (width 2 × length / 2). 3 When the stool was reached, the mice were sacrificed.

[0099] The width (W) and length (L) of the tumor were measured three times a week with a calisper, and the tumor volume was calculated using the formula V = (W(2) × L) / 2. 3 When the tumor size reached 100 μg / kg, the mice were sacrificed and the tumors were excised for further analysis.

[0100] Cellular phenotyping of tumor infiltrates: The excised tumor was finely pulverized with a blade and sieved through a 200 μm sieve. The cells were then sieved through a 70 μM sieve. The cells were washed and resuspended in FACS buffer (PBS 1× containing 10% FBS and 0.1% azide).

[0101] Antibodies for flow cytometry were obtained from BioLegend (San Diego, CA). The flow cytometry panel included CD45 in FITC, PD-L1 in PE, CD8 in APC, and CD4 in APC-Cy7. Flow cytometry data were acquired on a FACSCanto II (BD Biosciences, San Jose, CA) cytometer and analyzed using FlowJO version 10.2 (FlowJo, Ashland, OR).

[0102] result: In enzyme-linked immunosorbent assays (ELISAs), both compounds 2.001 and 2.002 potently inhibited the direct interaction of PD-L1 with PD-1. The mean IC values ​​for 2.001 and 2.002 from multiple assays were 50 are 0.3 nM and 0.4 nM, respectively (Figure 1). In a cell-based assay assessing PD-1-mediated downstream signaling, these compounds enhance NFAT promoter-driven luciferase expression, which is suppressed by PD-L1 / PD-1 interaction. The mean EC 50 are 52 nM and 46 nM, respectively.

[0103] In mixed lymphocyte reaction (MLR) assays (Figures 2 and 3), compounds 2.001 and 2.002 dose-dependently increased the release of INF-gamma from human T cells. Although the responsiveness of T cells from different donors varied, both compounds had EC values ​​of less than 100 nM in different T cells. 50 It presents.

[0104] In the presence of pre-stimulated primary human PBMCs, compounds 2.001 and 2.002 promoted killing of the GFP-labeled human cancer cell line A375 (Figure 4A). The FDA-approved anti-PD-L1 antibody durvalumab was used as a positive control and comparator in this study (Figure 4B).

[0105] In the pathway hunter assay (dimerization assay), dimerization of two PD-L1 molecules brings the two enzyme subunits together to form a functional enzyme, which generates a bioluminescent signal. Compounds 2.001 and 2.002 both strongly induced a dimerization signal, whereas the control compound and anti-PD-L1 antibody did not induce such a signal (Figure 5).

[0106] Surface PD-L1 on tumor cell lines was measured by flow cytometry. Binding of the detection antibody to PD-L1 was unaffected by small molecule inhibitors, as indicated by minimal changes in PD-L1 staining upon compound treatment at 4°C. At 37°C, a temperature that allows receptor internalization, compounds 2.001 and 2.002 significantly reduced surface PD-L1 levels on the cell surface (Figure 6). Anti-PD-L1 antibodies had no effect on PD-L1 surface levels. These findings suggest that compounds 2.001 and 2.002 promote PD-L1 internalization.

[0107] We used the murine tumor cell line MC38, in which murine PD-L1 was replaced with a human PD-L1 transgene, to induce tumor growth in mice (Figure 7). We confirmed that human and murine PD-L1 bind to murine PD-1 with similar affinity, and our PD-L1 inhibitor blocks the interaction of human PD-L1 with murine PD-1 with similar efficacy (data not shown).

[0108] In this model, orally administered compound 2.002 inhibited tumor growth in a dose-dependent manner (Figures 8A-8C). Eight of 10 mice treated with 30 mg / kg compound 2.002 (twice daily) achieved complete tumor eradication (Figure 8A). Final tumor weights were consistent with tumor size measurements, and eradicated tumors were not included in the tumor weight graph (Figure 8B). Plasma compound concentrations also demonstrated dose-dependence (Figure 8C).

[0109] Compound 2.001 and compound 2.003, each administered orally at 30 mg / kg (bid), also led to tumor inhibition similar to that of the anti-PD-L1 antibody, durvalumab (compare Figures 9A, 9B, and 9C). Figure 9A plots tumor growth when mice are administered compound 2.001, Figure 9B plots tumor growth when mice are administered compound 2.003, and Figure 9C plots tumor growth when mice are administered the anti-PD-L1 antibody, durvalumab. The top panel in each figure is the average tumor size from 10 mice in each group, and the bottom graph is the tumor progression of individual animals. Six animals in the anti-PD-L1 treatment group, four in the compound 2.001-treated group, and four in the compound 2.001-treated group achieved complete regression.

[0110] In the above referenced model, plasma concentrations for Compound 2.001 and Compound 2.003 (each dosed twice daily at 30 mg / kg) were measured in each mouse 6 days after dosing, and trough plasma concentrations are plotted in Figure 10.

[0111] To examine the extent to which compound 2.001 occupies PD-L1 on tumor cells, we utilized another PD-L1 detection antibody to stain cells isolated from these tumors. This detection antibody does not bind to PD-L1 once bound by compound 2.001 or treated anti-PD-L1 (durvalumab). Cells from compound 2.001-treated tumors completely lacked PD-L1 staining by this detection antibody, demonstrating near-complete PD-L1 occupancy by compound 2.001 (Figure 11).

[0112] Each treatment condition in the mouse model referenced above was analyzed for tumor-infiltrating immune cells. + T cells and CD4 + T cells are increased by compound 2.001 treatment in both anti-PD-L1 treated tumors as well (Figure 12).

[0113] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the disclosed embodiments may be apparent to those skilled in the art upon reading the foregoing description, and it is expected that such variations will be adaptable by those skilled in the art. Accordingly, it is intended that the invention be practiced as otherwise specifically described herein, including all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

[0114] All publications, patent applications, accession numbers, and other references cited herein are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The present invention provides, for example, the following items. (Item 1) 1. A method for treating cancer selected from the group consisting of colon cancer, renal cancer, colorectal cancer, gastric cancer, bladder cancer, melanoma, non-small cell lung cancer, Merkel cell carcinoma, liver cancer, breast cancer, and head and neck cancer, comprising administering to a subject in need thereof an effective amount of a compound of formula (I) [ka] or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 are each independently F, Cl, or CH 3 , and CF 3 is selected from the group consisting of R 3 However, F, Cl, CH 3 , C.F. 3 , -O-CH 3 , and -O-CF 3 is selected from the group consisting of R 4 But -Y and -X 1 -Y, wherein each X 1 C 1~4 alkylene, and Y is C 3~6 C having 1 to 3 heteroatom ring vertices independently selected from the group consisting of cycloalkyl, N, O, and S 4~6 heterocycloalkyl, and 5-6 membered heteroaryl having 1-3 heteroatom ring vertices independently selected from the group consisting of N, O, and S, each of which is unsubstituted or selected from oxo, OH, C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, and C 1~4 hydroxyalkoxy; and R a and R b are independent, H, C 1~3 Alkyl, and C 1~4 haloalkyl. (Item 2) Item 10. The method of item 1, wherein the effective amount of the compound of formula (I) is administered orally. (Item 3) R 1 But Cl and CH 3 The method according to item 1 or 2, selected from the group consisting of: (Item 4) R 1 The method according to item 1 or 2, wherein is Cl. (Item 5) R 1 is CH 3 The method according to item 1 or 2, wherein (Item 6) R 2 Cl and CH 3 6. The method according to any one of items 1 to 5, selected from the group consisting of: (Item 7) R 2 The method according to any one of items 1 to 5, wherein is Cl. (Item 8) R 2 is CH 3 6. The method according to any one of items 1 to 5, wherein (Item 9) R 3 But -O-CH 3 and -O-CF 3 The method according to any one of items 1 to 8, selected from the group consisting of: (Item 10) R 3 -O-CH 3 The method according to any one of items 1 to 8, wherein (Item 11) R 3 Ga-O-CF 3 The method according to any one of items 1 to 8, wherein (Item 12) R a But, H, CH 3 , and CF 3 12. The method according to any one of items 1 to 11, selected from the group consisting of: (Item 13) R a is CH 3 12. The method according to any one of items 1 to 11, wherein (Item 14) R b But, H, CH 3 , and CF 3 14. The method according to any one of items 1 to 13, selected from the group consisting of: (Item 15) R b is CH 3 14. The method according to any one of items 1 to 13, wherein (Item 16) The compound of formula I is of formula (Ia)

change

change

change

change

change

Claims

1. 1. A composition for treating colon cancer in a subject by slowing tumor growth, inhibiting tumor growth, and / or reducing tumor size, the composition comprising an effective amount of 【Chemistry 1】 A composition comprising a compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

2. The composition of claim 1 , wherein the composition is administered orally.

3. 3. The composition of claim 1 or 2, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the subject in combination with an effective amount of one or more additional therapeutic agents.

4. 4. The composition of claim 3, wherein the one or more additional therapeutic agents are selected from the group consisting of cytotoxic agents, gene expression modulating agents, chemotherapeutic agents, anti-cancer agents, anti-angiogenic agents, immunotherapeutic agents, anti-hormonal agents, radiation therapy, radiotherapeutic agents, anti-tumor agents, and anti-proliferative agents.

Images