Methods of treating cancer using heteroarylbiphenylamide derivatives

Abstract

The present invention provides methods for treating certain cancers, which methods comprise administering to a subject in need thereof an effective amount of a compound having formula (I), including stereoisomers and pharmaceutically acceptable salts thereof, wherein R<supgt;1< / supgt>, R<supgt;2< / supgt>, R<supgt;3< / supgt>, R<supgt;4< / supgt>, R<supgt;a< / supgt>, and R<supgt;b< / supgt> are as defined herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 63 / 042,807, filed on June 23, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Federally funded research and development

[0004] Declaration of rights to completed invention

[0005] not applicable

[0006] Reference to a "Sequence Listing", table or computer program listing annex submitted on a CD-ROM

[0007] not applicable Background Art

[0008] Programmed cell death protein-1 (PD-1) is a member of the CD28 superfamily and transmits negative signals when interacting with its two ligands (PD-L1 or PD-L2). PD-1 and its ligands are widely expressed and play a wide range of immunomodulatory roles in T cell activation and tolerance. PD-1 and its ligands are involved in attenuating infectious immunity and tumor immunity, and promoting chronic infection and tumor progression.

[0009] In a variety of human diseases, modulation of the PD-1 pathway has therapeutic potential (Hyun-Tak Jin et al., Curr Top Microbiol Immunol. [Current Topics in Microbiology and Immunology] (2011); 350: 17-37). In cancer therapy, blocking the PD-1 pathway has become an attractive target. Therapeutic antibodies that block the programmed cell death protein-1 (PD-1) immune checkpoint pathway prevent T cell downregulation and promote immune responses against cancer. In multi-phase clinical trials, several PD-1 pathway inhibitors have shown robust activity (RD Harvey, Clinical Pharmacology and Therapeutics [Clinical Pharmacology and Therapeutics] (2014); 96 (2), 214-223).

[0010] Agents that block the interaction of PD-L1 with PD-1 or CD80 are desirable. Several antibodies have been developed and commercialized. Several patent applications disclosing non-peptide small molecules have been published (WO 2015 / 160641, WO 2015 / 034820, and WO 2017 / 066227 and WO 2018 / 009505 from BMS; WO 2015 / 033299 and WO 2015 / 033301 from Aurigene; WO 2017 / 070089, US 2017 / 0145025, WO 2017 / 106634, US 2017 / 0174679, WO 2017 / 192961, WO 2017 / 222976, WO 2017 / 205464, WO 2017 / 112730, WO 2017 / 041899 and WO 2018 / 013789 from Maxinovel, WO 2018 / 006795 from Maxinovel, and WO 2018 / 005374 from ChemoCentryx, USA). However, there is still a need for alternative compounds (such as small molecules) as inhibitors of PD-L1 that may have favorable characteristics in terms of oral administration, increased tumor penetration, stability, bioavailability, therapeutic index, and toxicity. Summary of the invention

[0011] In some aspects, provided herein are methods of treating cancer, comprising administering to a subject in need thereof an effective amount of a compound having Formula (I):

[0012]

[0013] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R a , and R b As described in this article.

[0014] 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 DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A -B plots PD1 / PD-L1 binding ELISA data (upper panel) and PD-1 / PD-L1 blocking cell-based assay data (lower panel) for compounds 2.001 (A) and 2.002 (B).

[0016] Figure 2A -C shows how compound 2.001 promotes allogeneic immune responses of human T cells in an ex vivo mixed lymphocyte reaction (MLR) assay; responses of T cells from three separate donors are shown: Donor #1 (A), Donor #2 (B), and Donor #3 (C).

[0017] Figure 3A -C shows how compound 2.002 promotes allogeneic immune responses of human T cells in an ex vivo mixed lymphocyte reaction (MLR) assay; responses of T cells from three separate donors are shown: Donor #1 (A), Donor #2 (B), and Donor #3 (C).

[0018] Figure 4A -B illustrates PBMC-mediated tumor cell killing of compound 2.002 (A, leftmost column), compound 2.001 (A, middle column), and control compound (A, rightmost column). Additional control experiments used anti-PD-L1 antibody (Durvalumab) (B, leftmost column) and antibody isotypes (B, rightmost column).

[0019] Figure 5 Compounds 2.001 and 2.002 are shown to induce PD-L1 dimerization, whereas anti-PD-L1 antibodies and the controls tested do not.

[0020] Figure 6 Shown are surface levels of PD-L1 at 4°C (lower panel) and 37°C (upper panel) under multiple conditions tested. This figure demonstrates that compounds 2.001 and 2.002 reduce surface PD-L1 levels specifically at 37°C, indicating PD-L1 internalization.

[0021] Figure 7 MC38-hPD-L1 tumor model for evaluating human PD-L1 inhibitors in vivo. Engineered MC38-hPD-L1 cells are suitable for evaluating the effects of specific inhibitors of human PD-L1 in vivo: hPD-L1 and mPD-L1 bind to mPD-1 with similar affinity; current 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.

[0022] Fig. 8A -C illustrates the inhibition of tumor growth mediated by compound 2.002 in a dose-dependent manner in the MC38-hPD-L1 tumor model. (A) Tumor volume is plotted against days after tumor implantation; (B) Average tumor weight after 35 days is plotted; (C) Plasma compound trough concentration is plotted after 3 days of dosing.

[0023] Fig. 9A -C plots the tumor size of vehicle treatment (filled circles) and API (anti-PD-L1 antibody or specified compound, filled squares) on the specified days. The API tested was compound 2.001 (A), compound 2.003 (B), and anti-PD-L1 antibody (C). The upper panel plots the average tumor size of each treatment group, while the lower panel plots the tumor size of each mouse in the treatment group.

[0024] Fig. 10A -B plots the plasma trough concentrations of Compound 2.001 (A) and Compound 2.003 (B) in the mouse model described in Biological Example 2, 12 hours after dosing and 6 days after dosing.

[0025] Fig.11 Shown is human PD-L1 staining of cells when treated with anti-PD-L1 antibody (Durvalumab), isotype antibody, compound 2.001, and vehicle. The detection antibody for PD-L1 used in this analysis is blocked by compound 2.001, which binds to PD-L1. This figure demonstrates that MC38-hPD-L1 tumors treated with compound 2.001 are almost completely occupied by compound 2.001.

[0026] Fig.12 Figure 2 shows how various treatment conditions altered the amount of tumor-infiltrating immune cells in the MC38-HPD-L1 tumor model. The lower panel plots the amount of measured CD8+ T cells; the middle panel plots the amount of measured CD4 + The amount of T cells; and the upper panel plots the measured CD8 + and CD4 + The amount of T cells. DETAILED DESCRIPTION

[0027] I. General

[0028] The present disclosure provides methods of treating specific cancers using compounds of formula (I). The claimed compounds have robust anti-tumor properties and have high affinity for PD-L1. When administered, these compounds effectively disrupt PD-1 / PD-L1 signaling and, in some embodiments, induce dimerization and internalization of PD-L1 on cancer cells.

[0029] The development of PD-1 / PD-L1 small molecule modulators has been hampered by the need to balance multiple factors, including: PD-1 / PD-L1 affinity, hydrophobicity / hydrophilicity of the compound, biological clearance, and anti-target activity (e.g., CYP and hERG inhibition). In fact, to date, there are no PD-1 / PD-L1 inhibitors approved for oral administration.

[0030] Compared to IV drug formulations, the bioavailability of orally administered compounds requires, among other things, gastric absorption and resistance to significant degradation via portal circulation to the liver (known as "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 methods do not require extremely high concentrations of the compounds in the blood; rather, these compounds can elicit their anti-tumor effects in the ng / mL range.

[0031] II. Abbreviations and definitions

[0032] As used herein, the terms "a," "an," or "the" include not only aspects of one member, but also aspects of more than one member. For example, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the agent" includes reference to one or more agents known to those skilled in the art, etc.

[0033] The terms "about" and "approximately" generally mean an acceptable degree of error for a quantity measured given the nature or precision of the measurement. A typical, exemplary degree of error is within 20% (%) of a given value or range of values, preferably within 10%, and more preferably within 5%. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean a value within an order of magnitude of a given value, preferably within 5 times, and more preferably within 2 times. Unless otherwise stated, numerical quantities given herein are approximate, meaning that the term "about" or "approximately" can be inferred when not explicitly stated.

[0034] Unless otherwise stated, the term "alkyl" by itself or as part of another substituent means a radical having the specified number of carbon atoms (i.e., C 1-8 The term "alkynyl" 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 include vinyl, 2-propenyl, butenyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl and 3-(1,4-pentadienyl). Examples of alkynyl groups include ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologues and isomers. The term "cycloalkyl" refers to an unsaturated alkyl group having a specified number of ring atoms (e.g., C 3-6"Cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings, such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. The bicyclic or polycyclic rings may be fused, bridged, spirocyclic, or a combination thereof. The term "heterocycloalkyl" or "heterocyclyl" refers to a cycloalkyl group containing from one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and one or more nitrogen atoms are optionally quaternized. Heterocycloalkyl may be a monocyclic, bicyclic, or polycyclic ring system. The bicyclic or polycyclic rings may be fused, bridged, spirocyclic, or a combination thereof. It is understood that C 4-12 The description of heterocyclyl refers to a group having from 4 to 12 ring members, at least one of which is a heteroatom. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolinone, tetrazolylone, 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, etc. The heterocycloalkyl group can be attached to the rest of the molecule through a ring carbon or a heteroatom.

[0035] The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane (as exemplified by -CH2CH2CH2CH2-). Alkylene groups can be linear or branched. Examples of the latter are -CH2C(CH3)2CH2-, -CH2C(CH3)2-, or -CH(CH3)CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 12 carbon atoms, with those having 8 or fewer carbon atoms being preferred in the present disclosure. Similarly, "alkenylene" and "alkynylene" refer to unsaturated forms of "alkylene" having double or triple bonds, respectively.

[0036] The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to those 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 moieties may be the same or different and may also be combined with the nitrogen atom to which they are attached to form a 3-7 membered ring. Thus, the alkyl group represented by -NR a R b The group is meant to include piperidinyl, pyrrolidinyl, morpholinyl, azepanyl and the like.

[0037] Unless otherwise stated, the term "halo" or "halogen" alone or as part of another substituent means a fluorine, chlorine, bromine, or iodine atom. In addition, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C 1-4 "Haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0038] The term "hydroxyalkyl" or "alkyl-OH" refers to an alkyl group as defined above in which at least one (and up to three) hydrogen atoms are replaced by a hydroxyl group. As with alkyl groups, the hydroxyalkyl group may have any suitable number of carbon atoms, such as C 1-6 Exemplary hydroxyalkyl groups include, but are not limited to, hydroxymethyl, hydroxyethyl (wherein the hydroxyl group is at the 1- or 2-position), hydroxypropyl (wherein the hydroxyl group is at the 1-, 2-, or 3-position), and 2,3-dihydroxypropyl.

[0039] Unless otherwise indicated, the term "aryl" means a polyunsaturated, typically aromatic hydrocarbon group which may be a single ring or multiple rings (up to three rings) fused together or covalently linked. The term "heteroaryl" refers to an aryl group (or ring) containing from one to five heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized and one or more nitrogen atoms are optionally quaternized. The heteroaryl group may be attached to the rest of the molecule via a heteroatom. It should be understood that C 5-10 The description of heteroaryl refers to heteroaryl moieties having from 5 to 10 ring members, at least one of which 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, isobenzofuranyl, isoindolyl, and the like. Substituents for each of the above mentioned aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

[0040] The term "carbocyclic ring (carbocyclic ring or carbocyclic)" or "carbocyclyl" refers to a ring portion having only carbon atoms as ring vertices. The carbocyclic portion is saturated or unsaturated and can be aromatic. Typically, the carbocyclic portion has from 3 to 10 ring members. The carbocyclic portion (e.g., bicyclic) with multiple ring structures can include a cycloalkyl ring (e.g., 1,2,3,4-tetrahydronaphthalene) fused to an aromatic ring. Therefore, carbocyclic rings include cyclopentyl, cyclohexenyl, naphthyl, and 1,2,3,4-tetrahydronaphthyl. The term "heterocycle" refers to both "heterocycloalkyl" and "heteroaryl" portions. Therefore, heterocycles are saturated or unsaturated and can be aromatic. Typically, heterocycles have 4 to 10 ring members and include piperidinyl, tetrazinyl, pyrazolyl and indolyl.

[0041] When any of the above terms (eg, "alkyl," "aryl," and "heteroaryl") are referred to as 'substituted' without further annotation of the substituent, the substituted form of the designated group will be provided as follows.

[0042] Substituents for alkyl groups (including those groups commonly referred to as alkylene, alkenyl, alkynyl, and cycloalkyl) may be various groups selected from the group consisting of -halogen, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O) )2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R", -NR'S(O)2R", -CN and -NO2, ranging from zero to (2m'+1), where m' is the total number of carbon atoms in such a group. R', R" and R'" each independently refer to hydrogen, unsubstituted C 1-8 alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl substituted with 1 to 3 halogens, unsubstituted C 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 A thioalkoxy group, or an unsubstituted aryl-C 1-4Alkyl groups. 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 alone or as part of another group refers to an alkyl group in which both substituents on the carbon closest to the point of attachment of the group are replaced by the substituent =0 (e.g., -C(O)CH3, -C(O)CH2CH2OR', etc.).

[0043] Similarly, substituents for aryl and heteroaryl groups are varied and are generally selected from: -halogen, -OR', -OC(O)R', -NR'R", -SR', -R', -CN, -NO2, -CO2R', -CONR'R", -C(O)R', -OC(O)NR'R", -NR"C(O)R', -NR"C(O)2R', -NR'-C(O)NR"R"', -NH-C( wherein 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 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 via an alkylene chain of 1-4 carbon atoms.

[0044] Two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by substituents having the formula -TC(O)-(CH2) q -U-, wherein T and U are independently -NH-, -O-, -CH2- or a single bond, and q is an integer from 0 to 2. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced by a substituent having the formula -A-(CH2) r-B-, wherein 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 of the new ring so formed may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by a double bond having the formula -(CH2) s -X-(CH2) t -, wherein s and t are independently integers from 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 selected from hydrogen or unsubstituted C 1-6 alkyl.

[0045] As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

[0046] The disclosure herein further relates to prodrugs and bioisosteres thereof. Suitable bioisosteres, for example, will include carboxylate substitutes (phosphonic acid, hypophosphorous acid, sulfonic acid, sulfinic acid, and acidic heterocyclic groups such as tetrazole). Suitable prodrugs will include those conventional groups known to hydrolyze and / or oxidize under physiological conditions to provide compounds of Formula I.

[0047] The terms "patient" and "subject" include primates (especially humans), domestic companion animals (eg, dogs, cats, horses, etc.), and livestock (eg, cattle, pigs, sheep, etc.).

[0048] As used herein, the terms "treating" or "treatment" encompass both disease-modifying and symptomatic treatments, either of which can be prophylactic (i.e., prior to the onset of symptoms, to prevent, delay or reduce the severity of symptoms) or therapeutic (i.e., after the onset of symptoms, to reduce the severity and / or duration of symptoms).

[0049] The term "pharmaceutically acceptable salt" is meant to include salts of the active compounds, which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When the compounds of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base (either pure or in a suitable inert solvent). Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganese, manganese divalent, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, including substituted amines, cyclic amines, 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, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and the like, and salts derived from relatively nontoxic 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-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids (e.g., arginine salts, and the like) and salts of organic acids such as glucuronic acid or galactunoric acid, and the like (see, e.g., Berge, SM et al., "Pharmaceutical Salts" Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present disclosure contain both basic and acidic functionalities, which allow the compounds to be converted into either base or acid addition salts.

[0050] The neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties (e.g., solubility in polar solvents), but in other respects, these salts are equivalent to the parent form of the compound for the purposes of this disclosure.

[0051] Some compounds of the present disclosure can exist in non-solvated form and solvated form (including hydrated form). Generally speaking, the solvated form is equivalent to the non-solvated form and is intended to be included in the scope of the present disclosure. Some compounds of the present disclosure can exist in multiple crystals or amorphous forms. Generally speaking, all physical forms are equivalent for the purposes contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0052] Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, positional isomers, and individual isomers (e.g., isolated enantiomers) are all intended to be encompassed within the scope of the present invention. When stereochemical depictions are shown, it refers to a compound in which one isomer is present and substantially free of another isomer. "Substantially free" of another isomer indicates at least an 80 / 20 ratio of two isomers, more preferably 90 / 10, or 95 / 5 or greater. In some embodiments, one isomer will be present in an amount of at least 99%.

[0053] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more atoms that constitute such compounds. For example, the compounds may be treated with radioactive isotopes such as, for example, tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C) radiolabeled. All radioactive or non-radioactive isotopic variants of the compounds disclosed herein are intended to be included within the scope of the disclosure. For example, compounds can be prepared such that any number of hydrogen atoms are replaced by deuterium ( 2 H) isotope substitution. The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes on one or more atoms that constitute such compounds. Unnatural proportions of isotopes can be defined as the amount of atoms considered from the amount found in nature to the amount of the constituent element being 100%. For example, the compounds may incorporate radioactive isotopes, such as, for example, tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C), or non-radioactive isotopes such as deuterium ( 2 H) or carbon-13 ( 13 C). Such isotopic variants may provide additional utility to those described elsewhere in this application. For example, isotopic variants of the compounds of the present disclosure may find additional utility, including but not limited to as diagnostic and / or imaging agents, or as therapeutic agents for cytotoxicity / radiotoxicity. In addition, isotopic variants of the compounds of the present disclosure may have altered pharmacokinetic and pharmacodynamic characteristics that may contribute to enhanced safety, tolerability or efficacy during treatment. All radioactive or non-radioactive isotopic variants of the compounds of the present disclosure are intended to be encompassed within the scope of the present disclosure.

[0054] III. Embodiments of the Disclosure

[0055] Treatment

[0056] In some aspects, provided herein are methods of treating cancer, comprising administering to a subject in need thereof an effective amount of a compound having Formula (I):

[0057]

[0058] or a pharmaceutically acceptable salt thereof, wherein:

[0059] R 1 and R 2 Each independently selected from the group consisting of: F, Cl, CH3, and CF3;

[0060] R 3 selected from the group consisting of: F, Cl, CH3, CF3, –O–CH3, and –O–CF3;

[0061] R 4 Choose between –Y and –X 1 –Y groups, where each X 1 It is C 1-4 alkylene, and Y is selected from the group consisting of: C 3-6 Cycloalkyl, having 1 to 3 heteroatoms independently selected from the group consisting of N, O, and S at the vertices of the C 4-6 heterocycloalkyl, and 5-6 membered heteroaryl having 1 to 3 heteroatom ring vertices independently selected from the group consisting of N, O, and S, each of which is unsubstituted or substituted with one to two substituents independently selected from the group consisting of 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

[0062] R a and R b Independently selected from the group consisting of: H, C 1-3 Alkyl, and C 1-4 Halogenated alkyl.

[0063] In some embodiments, R 1 Selected from the group consisting of Cl and CH3. In some embodiments, R 1 is Cl. In some embodiments, R 1 It is CH3.

[0064] In some embodiments, R 2 Selected from the group consisting of Cl and CH3. In some embodiments, R 2 is Cl. In some embodiments, R 2 It is CH3.

[0065] In some embodiments, R 3 is selected from the group consisting of -O-CH3 and -O-CF3. 3 is -O-CH3. In some embodiments, R 3 It is –O–CF3.

[0066] In some embodiments, R a is selected from the group consisting of: H, CH3, and CF3. In some embodiments, R a It is CH3.

[0067] In some embodiments, R b is selected from the group consisting of: H, CH3, and CF3. In some embodiments, R b It is CH3.

[0068] In some embodiments, the compound having Formula I has Formula (Ia):

[0069]

[0070] or a pharmaceutically acceptable salt thereof.

[0071] In some embodiments, -NH(R 4 ) is selected from the group consisting of:

[0072]

[0073] In some embodiments, -NH(R 4 ) Choose Free

[0074] Composed of groups.

[0075] In some embodiments, –NHR 4 Selected from the group consisting of:

[0076]

[0077] In some embodiments, –NHR 4 yes

[0078]

[0079] In some embodiments, the compound of formula (I) is an optically pure or isomerically enriched.

[0080] In some embodiments, the compound having formula (I) is selected from the compounds in Table 1.

[0081] As described herein, the disclosed methods for treating certain cancers do not require extremely high concentrations of compounds of formula (I) in the blood. Instead, these compounds are sufficient to effectively provide therapeutic benefits at lower plasma concentrations. Therefore, in some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough concentration of no more than 1,000 ng / mL. In some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough concentration of no more than 750 ng / mL. In some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough concentration of no more than 500 ng / mL. In some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough concentration of no more than 400 ng / mL. In some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough concentration of no more than 300 ng / mL. In some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough concentration of no more than 200 ng / mL. In some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough concentration of no more than 100 ng / mL.

[0082] In some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough 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 plasma trough concentration of about 5 ng / mL to 500 ng / mL. In some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough concentration of about 10 ng / mL to 400 ng / mL. In some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough concentration of about 20 ng / mL to 300 ng / mL. In some embodiments, an effective amount of a compound of formula (I) maintains a plasma trough concentration of about 40 ng / mL to 200 ng / mL.

[0083] A variety of cancers can be treated using the methods described herein. In some embodiments, the cancer is selected from the group consisting of melanoma, glioblastoma, esophageal tumor, nasopharyngeal carcinoma, uveal melanoma, lymphoma, lymphocytic lymphoma, primary CNS 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 sarcoma, fibrosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, angiosarcoma, lymphangiosarcoma, synovioma, meningioma, leiomyosarcoma, rhabdomyosarcoma, soft tissue sarcoma, sarcoma, sepsis, bile duct tumor, basal cell carcinoma, thymic neoplasm, thyroid cancer, parathyroid cancer, uterine cancer, adrenal cancer, Liver infection, Merkel cell carcinoma, neural tumors, follicle center lymphoma, colon cancer, Hodgkin's disease, non-Hodgkin's lymphoma, leukemia, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), multiple myeloma, ovarian tumors, myelodysplastic syndrome, malignant melanoma of the skin or eye, 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, stomach cancer, hepatocellular carcinoma, pancreatic cancer (pancreatic carcinoma and pancreatic cancer), pancreatic ductal adenocarcinoma, head and neck squamous cell carcinoma, head and neck cancer, gastrointestinal tract gastric cancer, HIV, hepatitis A, hepatitis B, hepatitis C, hepatitis D, herpes virus, papillomavirus, influenza, bone cancer, skin cancer, rectal cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, urethra cancer, penis cancer, bladder cancer, kidney cancer, ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, tumor angiogenesis, spinal cord axis tumors, brain stem gliomas, pituitary adenomas, epidermoid carcinomas, asbestosis, carcinomas, adenocarcinomas, papillary carcinomas, cystadenocarcinomas, bronchogenic carcinomas, renal cell carcinomas, transitional cell carcinomas, choriocarcinomas, seminoma, embryonal carcinomas, Wilms' tumors, pleomorphic adenomas, hepatocellular papilloma, renal tubular adenomas, cystadenomas, papilloma, adenoma, leiomyoma, rhabdomyomas, hemangiomas, lymphangiomas, osteomas, chondromas, lipomas, and fibromas. In some embodiments, each of the listed cancers is a PD-L1 positive cancer.

[0084] In some embodiments, the cancer is colon cancer, kidney 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.

[0085] In some embodiments, the disease or disorder is colon cancer. In some embodiments, the cancer is kidney 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.

[0086] In certain embodiments, one or more other therapeutic agents of effective amount are further applied to the subject. In certain embodiments, one or more other therapeutic agents are selected from the group consisting of: cytotoxic agent, gene expression regulator, chemotherapeutic agent, anticancer agent, antiangiogenic agent, immunotherapeutic agent, antihormonal agent, radiotherapy, radiotherapeutic agent, antitumor agent and antiproliferative agent. In certain embodiments, one or more other therapeutic agents are antagonists of chemokine and / or chemotactic receptor, 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. Chemokine and / or chemokine receptor antagonists 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 / 163640, WO 2010 / 054006, WO 2010 / 051561, WO 2011 / 035332, WO 2013 / 082490、WO 2013 / 082429、WO 2014 / 085490、WO 2014 / 100735、WO 2014 / 089495、WO 2015 / 084842、WO 2016 / 187393、WO 2017 / 127409、WO 2017 / 087607、WO 2017 / 087610、WO 2017 / 176620、WO 2018 / 222598、WO 2018 / 222601、WO 2013 / 130811、WO 2006 / 076644、WO 2008 / 008431、WO 2009 / 038847、WO 2008 / 008375、WO 2008 / 008374、WO 2008 / 010934、WO 2009 / 009740、WO 2005 / 112925、WO 2005 / 112916、WO 2005 / 113513、WO 2004 / 085384、WO 2004 / 046092.Chemokine and / or chemokine receptor antagonists also include CCX354, CCX9588, CCX140, CCX872, CCX598, CCX6239, CCX9664, CCX2553, CCX3587, CCX3624, CCX 2991, CCX282, CCX025, CCX507, CCX430, CCX765, CCX224, CCX662, CCX650, CCX832, CCX168, CCX168-M1, CCX3022 and / or CCX3384.

[0087] Typically, the therapeutic methods provided herein include administering to a patient an effective amount of one or more compounds provided herein. Suitable patients include those suffering from or susceptible to (i.e., preventive treatment) the disorder or disease identified herein. 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, etc.) or livestock animals (e.g., cattle, pigs, sheep, etc.).

[0088] Route of administration and dosage

[0089] The administration routes contemplated by the present disclosure include those known in the art for delivering active agents for treating cancer. This includes, but is 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.

[0090] Generally, the treatment methods provided herein include administering to the patient an effective amount of a compound having formula (I) or one or more compounds provided herein. An effective amount may be an amount sufficient to regulate PD-1 / PD-L1 interactions, slow tumor growth, inhibit tumor growth, and / or reduce tumor size in a subject. Preferably, the amount administered is sufficient to produce a sufficiently high plasma concentration of the compound (or its active metabolite, if the compound is a prodrug) to fully regulate the PD-1 / PD-L1 interaction. The treatment regimen may vary depending on the compound used and the specific condition to be treated; for the treatment of most disorders, a frequency of administration of 4 times a day or less is preferred. Generally, a dosage regimen of 2 times a day is more preferred, and once-a-day administration is particularly preferred. However, it should be understood that the specific dosage level and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination (ie, other drugs administered to the patient) and the severity of the specific disease undergoing therapy, and the judgment of the practitioner who prescribes the prescription. Generally, it is preferred to use the minimum dose sufficient to provide effective therapy. Patients can generally be monitored for effectiveness of treatment using medical or veterinary standards applicable to the condition to be treated or prevented.

[0091] Dosage levels of about 0.1 mg to about 140 mg / day / kg body weight are useful in treating or preventing disorders involving PD-1 / PD-L1 interactions (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 on the host being treated and the specific mode of administration. The unit dosage form will generally contain an active ingredient between about 1 mg and about 500 mg. For compounds administered orally, transdermally, intravenously, or subcutaneously, it is preferred that a sufficient amount of the compound be administered to achieve a plasma concentration of 5 ng (nanogram) / mL-1 μg (microgram) / mL plasma, more preferably, a sufficient amount of the compound should be administered to achieve a plasma concentration of 20 ng-0.5 μg / ml plasma, and most preferably, a sufficient amount of the compound should be administered to achieve a plasma concentration of 30 ng / ml-200 ng / ml plasma.

[0092] The frequency of dosage can also vary according to the compound used, the route of administration, and the specific disease being treated. However, for the treatment of most disorders, 4 times a day, 3 times a day or less treatment regimens are preferred, with once a day or 2 times a day treatment regimens being particularly preferred. However, it should be understood that the specific dosage level for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, and excretion rate, drug combination (i.e., other drugs administered to the patient), the severity of the specific disease undergoing therapy, and other factors, including the judgment of the practitioner who prescribes the prescription.

[0093] Pharmaceutical composition

[0094] When administered to a subject, Formula (I) is typically in a pharmaceutical composition. As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product obtained directly or indirectly from a combination of the specified ingredients in the specified amounts. "Pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with the other components of the formulation and not harmful to the recipient thereof.

[0095] The pharmaceutical composition for administering the compound of the present disclosure can be routinely present in a unit dosage form for oral administration, and can be prepared by any method known in the field of pharmacy and drug delivery. All methods include the step of associating the active ingredient with a carrier constituting one or more auxiliary components. Generally, the pharmaceutical composition is prepared by the following steps: the active ingredient is uniformly and closely associated with a liquid carrier or a finely dispersed solid carrier or both, and then, if necessary, the product is formed into a desired formulation. The active target compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect on the process or condition of the disease.

[0096] The pharmaceutical composition containing the active ingredient can be in a form suitable for oral use, for example, as tablets, sugars, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions and self-emulsifications (as described in U.S. Patent Application 2002-0012680), hard or soft capsules, syrups, elixirs, solutions, oral patches, oral gels, chewing gums, chewable tablets, effervescent powders and effervescent tablets. Compositions intended for oral use can be prepared according to any method known in the art for making pharmaceutical compositions, and in order to provide pharmaceutically refined and palatable preparations, such compositions can contain one or more medicaments selected from the group consisting of sweeteners, flavoring agents, coloring agents, antioxidants and preservatives. Tablets contain the active ingredient, which is mixed with non-toxic pharmaceutically acceptable excipients suitable for producing 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 agents and disintegrants such as corn starch or alginic acid; binders such as PVP, cellulose, PEG, starch, gelatin or gum arabic 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 and thereby provide a lasting effect over a longer period of time. For example, time-delay materials such as monostearate or distearate can be used. They 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.

[0097] Formulations for oral use may also be presented in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, or kaolin), polyethylene glycols (PEG) of various average sizes (e.g., PEG400, PEG4000), and certain surfactants (e.g., Cremophor or solutol), or in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin, or olive oil). In addition, emulsions may be prepared with water-immiscible ingredients (e.g., oils) and stabilized with surfactants (e.g., mono- or di-glycerides, PEG esters, etc.).

[0098] Aqueous suspensions contain the active substance in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth and gum arabic; dispersants or wetting agents may be naturally occurring phosphatides (e.g., lecithin), or condensation products of olefin oxides with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). The aqueous suspension 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 sweetening agents, such as sucrose or saccharin.

[0099] Oily suspensions can be prepared 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 thickeners, such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners such as those described above and flavoring agents can be added to provide a palatable oral formulation. These compositions can be preserved by adding an antioxidant such as ascorbic acid.

[0100] Dispersible powders and granules suitable for preparing aqueous suspensions provide the active ingredient mixed with a dispersant or wetting agent, a suspending agent and one or more preservatives by adding water. Suitable dispersing or wetting agents and suspending agents are such as those already mentioned above. Additional excipients such as sweeteners, flavorings and coloring agents may also be present.

[0101] The pharmaceutical composition of the present disclosure can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil (e.g., olive oil or peanut oil), or a mineral oil (e.g., liquid paraffin), or a mixture of these substances. Suitable emulsifiers can be naturally occurring gums, such as gum arabic or tragacanth, naturally occurring phospholipids, such as soybeans, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as monooleic acid sorbitan, and the condensation product of the partial ester and ethylene oxide, such as polyoxyethylene monooleic acid sorbitan. Emulsions can also contain sweeteners and flavorings.

[0102] Syrups and elixirs can be formulated with sweeteners such as glycerol, propylene glycol, sorbitol or sucrose. Such preparations may also contain demulcents, preservatives and flavoring and coloring agents. Oral solutions can be prepared in combination with, for example, cyclodextrins, PEG and surfactants.

[0103] The compound of the present disclosure can also be coupled with the carrier of a suitable polymer as a targeted drug carrier.Such polymers can include polyvinyl pyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxyethyl-aspartamide (aspartamide)-phenol or polyethylene oxide-polylysine substituted with palmitoyl residues.In addition, the compound of the present disclosure can be coupled to a carrier, which is a class of biodegradable polymers for realizing the controlled release of drugs, such as polylactic acid, polyglycolic acid, polylactic acid and polyglycolic acid copolymers, poly-ε caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and hydrogel crosslinking or amphiphilic block copolymers.Polymer and semipermeable polymer matrix can form shaped articles, such as valves, stents, pipelines, prostheses, etc.In one embodiment of the present disclosure, the compound of the present disclosure is coupled with a polymer and a semipermeable polymer matrix forming a stent or stent graft device.

[0104] In certain embodiments, the pharmaceutical composition further comprises one or more other therapeutic agents. In certain embodiments, one or more other therapeutic agents are selected from the group consisting of: antimicrobial agents, antiviral agents, cytotoxic agents, gene expression regulators, chemotherapeutic agents, anticancer agents, antiangiogenic agents, immunotherapeutic agents, antihormonal agents, antifibrotic agents, radiotherapy, radiotherapeutic agents, antitumor agents and antiproliferative agents. In certain embodiments, one or more other therapeutic agents are antagonists of chemokines and / or chemotactic 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. Chemokine and / or chemokine receptor antagonists 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 / 163640, WO 2010 / 054006, WO 2010 / 051561, WO 2011 / 035332, WO 2013 / 082490, WO 2013 / 082429, WO 2014 / 085490, WO 2014 / 100735, WO 2014 / 089495, WO 2015 / 084842, WO 2016 / 187393, WO 2017 / 127409, WO 2017 / 087607, WO 2017 / 087610, WO 2017 / 176620, WO 2018 / 222598, WO 2018 / 222601, WO 2013 / 130811, WO 2006 / 076644, WO 2008 / 008431, WO 2009 / 038847, WO 2008 / 008375, WO 2008 / 008374, WO 2008 / 010934, WO2009 / 009740, WO 2005 / 112925, WO 2005 / 112916, WO 2005 / 113513, WO 2004 / 085384, WO2004 / 046092.Chemokine and / or chemokine receptor antagonists also include CCX354, CCX9588, CCX140, CCX872, CCX598, CCX6239, CCX9664, CCX2553, CCX3587, CCX3624, CCX 2991, CCX282, CCX025, CCX507, CCX430, CCX765, CCX224, CCX662, CCX650, CCX832, CCX168, CCX168-M1, CCX3022 and / or CCX3384.

[0105] Examples

[0106] The following examples illustrate various methods of preparing compounds of the present disclosure, including compounds having formula (I) or (Ia).The following examples are provided to illustrate, but not to limit, the claimed disclosure.

[0107] The reagents and solvents used below can be obtained from commercial sources, such as Aldrich Chemical Co (Milwaukee, WI, USA). Spectra were recorded on a Varian Mercury 400 MHz NMR spectrometer. 1 H-NMR spectrum. Significant peaks relative to TMS are provided and tabulated in the following order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet) and number of protons. Mass spectrometry results are reported as mass-to-charge ratios. In the examples, single m / z values ​​of M+H (or, as described, MH) ions containing the most common atomic isotopes are reported. In all cases, the isotope pattern corresponds to the expected formula. Electrospray ionization (ESI) mass spectrometry analysis was performed on a Hewlett-Packard MSD electrospray mass spectrometer using an HP1100 HPLC (for sample delivery). Typically, the analyte was dissolved in methanol or CH3CN at 0.1 mg / mL, and 1 microliter of the mixture was infused into the mass spectrometer with the delivery solution, which scanned from 100 to 1000 Daltons. All compounds can be analyzed in positive or negative ESI mode using acetonitrile / water with 1% formic acid as the delivery solution.

[0108] The following abbreviations are used in the examples and throughout this disclosure: TLC means thin layer chromatography.

[0109] Compounds within the scope of this disclosure can be synthesized using a variety of reactions known to those skilled in the art as described below. Those skilled in the art will also recognize that alternative methods can be employed to synthesize the target compounds of this disclosure, and that the methods described in the body of this document are not exhaustive, but do provide broadly applicable and practical pathways to the target compounds.

[0110] Certain molecules claimed in this patent may exist in different enantiomeric and diastereomeric forms and all such variations of these compounds are claimed unless a specific enantiomer is specified.

[0111] Detailed descriptions of the experimental procedures used to synthesize key compounds herein result in descriptions of the molecules through identification of their physical data and structural depictions associated with the molecules.

[0112] Those skilled in the art will also recognize that acids and bases are commonly used during standard work-up procedures in organic chemistry. During the experimental procedures described in this patent, salts of parent compounds were sometimes produced if the parent compounds possessed the necessary inherent acidity or basicity.

[0113] 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

[0114]

[0115] 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 H0 (6 mL) was added Pd(dppf)Cl complex (912 mg, 1.12 mmol) with dichloromethane. The reaction mixture was degassed (N) for 2 min and stirred at 90 °C under N 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 flash silica gel 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. 20 H 18 MS: (ES) m / z calculated for BrClN3O3: [M+H] + 462.0, Found value: 462.0.

[0116] 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), pinacol diborane (1.0 g, 3.94 mmol), and KOAc (1.2 g, 10.2 mmol) in p-dioxane (18 mL) was added Pd(dppf)Cl complex (350 mg, 0.43 mmol) with dichloromethane. The reaction mixture was degassed (N2) for 2 min and stirred at 90°C under N2 for 3 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 flash silica gel chromatography (10% to 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. 26 H 30 MS of BClN3O5: (ES) m / z calculated value: [M+H] + 510.2, found value: 510.1.

[0117] 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 K2CO3 (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 MgSO4. The solvent was removed under reduced pressure and the residue was purified by flash silica gel chromatography (10% to 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. 27 H 24MS of ClN4O5: (ES) m / z calculated value: [M+H] + 519.1, Found value: 519.1.

[0118] 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) was added triethylamine (2 drops) 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 NaCNBH3 (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% to 40% to 100% acetonitrile / H2O) 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)δ11.18(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). C 32 H 34 MS: (ES) m / z calculated for ClN5O6: [M+H] + 620.2, Found value: 620.2.

[0119] 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

[0120]

[0121] The title compound was prepared using the same procedure as in Example 1 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 / H2O 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) δ8.63 (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). C 32 H 33 MS of ClN6O5: (ES) m / z calculated value: [M+H] + 617.2, Found value: 617.2.

[0122] 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

[0123]

[0124] The title compound was prepared using a procedure similar to Example 1 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 / H2O 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) δ11.69(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=7.6 Hz,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). C 31 H 31 MS: (ES) m / z calculated for Cl2N6O5: [M+H] + 637.2, Found value: 637.2.

[0125] Biological Example 1: Enzyme-Linked Immunosorbent Assay – ELISA

[0126] At 4°C, 96-well plates were coated with 1 μg / mL of human PD-L1 (obtained from R&D) in PBS overnight. The wells were then blocked with 2% BSA (W / V) in PBS with 0.05% Tween-20 for 1 hour at 37°C. The plates were washed 3 times with PBS / 0.05% Tween-20 and the compounds were serially diluted (1:5) in dilution medium and added to the ELISA plates. Human PD-1 and biotin 0.3 μg / mL (ACRO Biosystems) were added and incubated at 37°C for 1 hour, followed by 3 washes with PBS / 0.05% Tween-20. A second block was performed at 37°C with 2% BSA (W / V) / 0.05% Tween-20 in PBS for 10 min, and the plates were washed 3 times with PBS / 0.05% Tween-20. Streptavidin-HRP was added for 1 hour at 37°C, and the plate was then washed 3 times with PBS / 0.05% Tween-20. TMB substrate was added and reacted for 20 min at 37°C. Stop solution (2N aqueous H2SO4) was added. The absorbance was read at 450 nm using a microplate spectrophotometer. The results are shown in Table 1: IC 50 Values ​​are provided as follows: from 1000 to 10,000 nM (+); from 10 to 1000 nM (++); less than 10 nM (+++).

[0127] Table 1

[0128]

[0129] Biological Example 2: Antitumor Effects of Compounds 2.001, 2.002, and 2.003

[0130] This example demonstrates the biological antitumor effects of compounds 2.001, 2.002, and 2.003 disclosed herein.

[0131] ELISA: This assay was performed essentially as described in Biological Example 1.

[0132] Cell lines and cell culture: CHO cells constitutively expressing TCR agonists and PD-L1 were grown with Ham's solution supplemented with 10% FBS and used for cell-based assays. A luciferase reporter T lymphoid cell line (Jurkat) (effector cells, EC) (Jurkat PD-1) modified to constitutively express PD-1 and carry a TCR-inducible NFAT response element driven was grown in RPMI supplemented with 10% FBS and 1X penicillin-streptomycin and used for cell-based assays. Human melanoma cell line A375 and 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 internally and grown in RPMI supplemented with 10% FBS and 1X penicillin-streptomycin.

[0133] PD-1 / PD-L1 blocking cell-based assay: 6x10 4 Cho PD-L1 cells were seeded in 96-well plates overnight. After washing the cells with PBS 1X, 40 μl of compound diluted in 1% FBS RPMI (starting concentration 5 μM, followed by 1:5 dilution) and 40 μl of T Jurkat PD-1 (1x10 6 Cells / ml) were 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, Wisconsin) was added to the culture medium and relative light units (RLU) were measured on a FlexStation 3 plate reader at a speed of 500 ms / well. When the two cells were co-cultured together, the PD-1 / PD-L1 interaction inhibited TCR signaling and NFAT-RE-mediated luminescence. Anti-PD-1 or anti-PD-L1 antibodies / compounds that block PD-1 / PD-L1 interactions release inhibitory signals and lead to TCR activation and NFAT-RE-mediated luminescence.

[0134] PBMC isolation: StemCell SepMate with Ficoll-Paque Plus (Sigma Aldrich Inc., St. Louis, MO) was used. TM Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors by density gradient centrifugation in LRS chambers (leukoreduction system) in ELISA-50 tubes (STEMCELL Technologies, Vancouver, Canada).

[0135] Generation of monocyte-derived dendritic cells: by using human CD14 + Microbeads (MACS Miltenyi Biotech, Bergisch Gladbach, Germany) and Pro Isolator Isolation of CD14 from PBMCs by Magnetic Separation + Monocytes. The isolated monocytes were cultured at 1x10 6 Cells / ml were plated and differentiated into dendritic cells by adding GM-CSF (100 ng / ml) and IL-4 (50 ng / ml) for 6 days. Fresh medium with cytokine supplements was added on days 0 and 2. Mature dendritic cells were induced on day 6 by adding IL-6 (2000 IU / ml), IL-1B (400 IU / ml) (Peprotech, Inc., Rocky Hill, NJ), TNFα (2000 IU / ml) and PGE2 (2 ug / ml) (Sigma Aldrich) and cultured for 24 hours.

[0136] Preparation of human effector cells: using human CD4 + Microbeads (MACS, Miltenyi Biotec) and Pro Separator Isolation of CD4 from PBMCs by Magnetic Separation + T cells.

[0137] Mixed lymphocyte reaction (MLR): DCs and CD4 + T cells were cultured together at a ratio of 1:10 for 5 days on 96-well flat-bottom plates (Thermo Scientific). Test compounds were added at a starting concentration of 1 μM with a 1:4 dilution with DMSO as indicated. PD-L1 antibody (AZ Medi4736 analog) and isotype control (human IgG1, κ isotype control) (Crown Bio, Beijing) were used as positive and negative controls, respectively. Supernatants were harvested after 5 days of incubation and human IFNg was detected by ELISA using human IFN-γDuoSet ELISA (R&D Systems, Minneapolis) according to the manufacturer's instructions.

[0138] In vitro immunotherapy efficacy assay: A375-eGFP-Puro cells (ATCC) were grown in complete medium (DMEM + 10% FBS + P / S 1X) containing 1 ug / ml puromycin. StemCell SepMate with Ficoll-Paque Plus (Sigma-Aldrich, St. Louis, MO) was used.TM Human peripheral blood mononuclear cells (hPBMCs) were isolated from healthy donors from LRS chambers (leukocyte removal system) by density gradient centrifugation in 50-50 tubes (Stem Cell Technologies, Vancouver, Canada). Freshly isolated hPBMCs were stimulated with 100 ng / ml of Staphylococcus enterotoxin B (SEB) (EMD Millipore, Cat. No. 324798) for three days. The cells were washed twice and resuspended in regular growth medium. 3x10 4 A375-eGFP-Puro cells were seeded in 96-well clear bottom black TC treated plates (final volume of 100ul) (Corning). Test compounds or anti-human PD-L1 antibodies (AZ Medi4736 analogs, Crown Bio, Beijing) were added to the wells at different concentrations. SEB stimulated hPBMCs were added to the wells at an E:T (effector cell: target cell) ratio of 2:1. The mixed cells were incubated for 96 to 120 hours at 37°C in 5% CO2. The culture medium was carefully aspirated and 100μl of PBS 1X was added to each well. Fluorescence from A375-eGFP cells was detected using a FlexStation 3 plate reader.

[0139] Dimerization assay using Dimerization assay (DiscoverX, Fremont, CA) PD-L1 protein dimerization was assessed in vitro by chemiluminescent detection. The assay was performed according to the supplier's protocol. 2x10 50 μL of ... 4 U2OS cells. ChemoCentryx compounds or anti-human PD-L1 antibodies (AZ Medi4736 analogs, Crown Bio, Beijing) were added to the experimental cells at different concentrations and incubated at 37°C in 5% CO2 for 16 hours. 110ul of PathHunter rapid detection reagent (Discavorex) was added to each well and incubated in the dark at room temperature for 1 hour. The chemiluminescent signal was measured on a FlexStation 3 plate reader (Molecular Devices, San Jose, CA) at a speed of 100ms / well.

[0140] Internalization assay: MC38-hPD-L1 cells (GenOway, Aubagne, France) and RKO cells (ATCC) grown at 37°C in 5% CO2 were detached, resuspended in cold FACS buffer (PBS 1X with 10% FBS and 0.1% azide) and incubated at 10x104 Cells were added to a 96-well assay plate (V bottom) (Axygen, Union City, CA) at a concentration of 10 cells / well. ChemoCentryx compounds or anti-human PD-L1 antibodies (AZMedi4736 analogs) were added to the wells at different concentrations and incubated at 37°C or at 4°C for 2 hours. The cells were washed twice with ice-cold FACS buffer and stained with recombinant rabbit monoclonal anti-human PD-L1 antibody ([28-8](PE)(ab209962), Abcam) or recombinant rabbit monoclonal IgG isotype control ([EPR25A](PE)(ab209478), Abcam) on ice for 30 minutes. Cells were washed twice with FACS buffer before FACS analysis. Data were analyzed using FlowJo software.

[0141] Generation and culture of MC38-hPD-L1 cells: These compounds are only known to cross-react with human PD-L1, so a syngeneic tumor model with murine MC-38 clonal tumor cells expressing human PD-L1 (MC38-hPD-L1 tumor model) was used. MC38-hPD-L1 cells were generated by GenOway. Endogenous mouse PD-L1 was first knocked out in MC38 cells using CRISPR technology, and then human PDL1 was stably transfected in these mouse PD-L1 knockout MC38 cells. MC38-hPD-L1 cells were cultured as MC38 cells with G418 (DMEM with 10% fetal bovine serum and penicillin / streptomycin) under standard conditions to maintain transgene expression. Two days before these cells were inoculated into mice, the cells were trypsinized and inoculated without antibiotics.

[0142] In vivo studies: 8-week-old female C57BL / 6 mice were injected subcutaneously on the right flank with 5 x 10 5 MC38-hPD-L1 cells. Nine days after tumor inoculation, mice were randomly assigned to treatment groups based on tumor size. Only mice with measurable tumors were included in the study. Anti-PD-L1 (Durvalumab) or isotype control was administered intraperitoneally twice a week at a dose of 100ug / mouse / a for 2 weeks. Compound 2.001 and Compound 2.002 suspended in 1% HPMC were orally administered daily at the specified dose, with a volume of 100μl per mouse. The vehicle (1% HPMC) was administered to the control animals at the same volume and frequency.

[0143] Tumor volume was measured three times weekly using a digital caliper and calculated as (width 2 *length / 2). According to IACUC guidelines, when the tumor volume reaches 2,000 mm 3 The mice were killed.

[0144] Tumor width (W) and length (L) were measured three times a week using calipers, and tumor volume was calculated using the formula V = (W (2) x L) / 2. 3 Mice were sacrificed and tumors were excised for further analysis.

[0145] Cellular phenotype of tumor infiltrates: Excised tumors were minced with a razor blade and passed through a 200 um mesh. The cells were then filtered through a 70 μM mesh. The cells were washed and resuspended in FACS buffer (PBS 1X with 10% FBS and 0.1% azide).

[0146] Antibodies used for flow cytometry were obtained from BioLegend (San Diego, CA). The flow cytometric panel included CD45 in FITC, PD-L1 in PE, CD8 in APC, CD4 in APC-Cy7. Flow cytometric data were obtained using a FACSCanto II (BD Biosciences, San Jose, CA) cytometer and analyzed using FlowJO v10.2 (FlowJo, Ashland, OR).

[0147] result:

[0148] In an enzyme-linked immunosorbent assay (ELISA), both compounds 2.001 and 2.002 effectively inhibited the direct interaction of PD-L1 with PD-1. The average IC values ​​of 2.001 and 2.002 from multiple assays were 50 The average EC values ​​of compounds 2.001 and 2.002 were 0.3 nM and 0.4 nM, respectively (Figure 1). In a cell-based assay assessing PD-1-mediated downstream signaling, these compounds enhanced luciferase expression driven by the NFAT promoter, which is inhibited by the PD-L1 / PD-1 interaction. In this assay, the average EC values ​​of compounds 2.001 and 2.002 were 50 52nM and 46nM respectively.

[0149] In the mixed lymphocyte reaction (MLR) assay (Figures 2 and 3), compound 2.001 and compound 2.002 dose-dependently increased the release of INFγ from human T cells. T cells from different donors responded differently, but both compounds showed an EC of less than 100 nM for different T cells. 50 .

[0150] In the presence of pre-stimulated primary human PBMCs, compounds 2.001 and 2.002 promoted the killing of GFP-labeled human cancer cell line A375 ( Figure 4AIn this study, durvalumab (FDA-approved anti-PD-L1 antibody) was used as a positive control and comparator ( Figure 4B ).

[0151] In the pathhunter assay (dimerization assay), dimerization of two PD-L1 molecules brings the two enzyme subunits together and forms a functional enzyme that produces a bioluminescent signal. Both compounds 2.001 and 2.002 strongly induced dimerization signals, while control compounds and anti-PD-L1 antibodies did not induce such signals ( Figure 5 ).

[0152] Surface PD-L1 was measured on tumor cell lines by flow cytometry. Binding of the test antibodies to PD-L1 was not affected by small molecule inhibitors, as shown by minimal changes in stained PD-L1 with 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 the surface level of PD-L1. These facts suggest that compounds 2.001 and 2.002 promote PD-L1 internalization.

[0153] A mouse tumor cell line (MC38) in which mouse PD-L1 was replaced by a human PD-L1 transgene was used to induce tumor growth in mice ( Figure 7 We confirmed that human and mouse PD-L1 bind to mouse PD-1 with similar affinity and that our PD-L1 inhibitor blocks the interaction of human PD-L1 with mouse PD-1 with similar potency (data not shown).

[0154] In this model, oral administration of compound 2.002 inhibited tumor growth in a dose-dependent manner ( Fig. 8A -C). Treatment of eight of ten mice with 30 mg / kg (twice daily) of compound 2.002 achieved complete eradication of the tumor ( Fig. 8A Final tumor weights were consistent with tumor size measurements, and eradicated tumors were not included in the tumor weight graphs ( Figure 8B Plasma compound concentrations also demonstrated dose dependence ( Figure 8C ).

[0155] Compound 2.001 and Compound 2.003, each administered orally at 30 mg / kg twice daily, also resulted in similar tumor inhibition as the PD-L1 antibody (Durvalumab) (compare, Fig. 9A , Fig. 9B and Fig. 9C ). Fig. 9A The tumor growth when compound 2.001 was administered to mice was plotted. Fig. 9B Plotted is the tumor growth when compound 2.003 was administered to mice, Fig. 9C Tumor growth when anti-PD-L1 antibody (Durvalumab) was administered to mice is plotted. The upper panel in each figure is the average tumor size of 10 mice in each group, and the lower panel is the tumor progression of individual animals. Six animals in the anti-PD-L1 treatment group, four animals in the compound 2.001 treatment group, and four animals in the compound 2.001 treatment group achieved complete regression.

[0156] In the above model, the plasma concentrations of compound 2.001 and compound 2.003 (each orally administered at 30 mg / kg, twice daily) were measured in each mouse 6 days after administration. The plasma trough concentrations are plotted in FIG10 .

[0157] To examine the extent to which compound 2.001 occupies PD-L1 on tumor cells, we stained cells isolated from these tumors with an additional PD-L1 detection antibody. Once compound 2.001 or therapeutic anti-PD-L1 (Durvalumab) binds to PD-L1, this detection antibody does not bind to it. Cells from tumors treated with compound 2.001 completely lack PD-L1 stained by this detection antibody, demonstrating that compound 2.001 almost completely occupies PD-L1 ( Fig.11 ).

[0158] Tumor-infiltrating immune cells were analyzed for each treatment condition in the above mouse models. Compound 2.001 treatment increased CD8 + and CD4 + T cells, which is similar to tumors treated with anti-PD-L1 ( Fig.12 ).

[0159] Specific embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. After reading the foregoing description, variations of the disclosed embodiments will become apparent to those skilled in the art, and it is expected that such variations may be appropriately adopted by those skilled in the art. Therefore, it is intended to practice the present invention in a manner different from that specifically described herein, and the present invention includes all modifications and equivalent forms of the subject matter recorded in the appended claims as permitted by applicable law. In addition, unless otherwise indicated herein or clearly contradictory to the context, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof.

[0160] All publications, patent applications, accession numbers, and other references cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An effective amount of a compound of formula (I): or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating cancer by slowing tumor growth, inhibiting tumor growth and / or reducing tumor size, the cancer being selected from the group consisting of 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, wherein: R 1 and R 2 Each independently selected from the group consisting of: F, Cl, CH3, and CF3; R 3 selected from the group consisting of: –O–CH3, and –O–CF3; –NH(R 4 ) is selected from the group consisting of: and R a and R b Independently selected from the group consisting of: C 1-3 Alkyl and C 1-4 Halogenated alkyl.

2. The use according to claim 1, wherein the effective amount is administered orally.

3. The use as claimed in claim 1, wherein R 1 Selected from the group consisting of Cl and CH3.

4. The use as claimed in claim 1, wherein R 1 It is Cl.

5. The use as claimed in claim 1, wherein R 1 It is CH3.

6. The use as claimed in claim 1, wherein R 2 Selected from the group consisting of Cl and CH3.

7. The use as claimed in claim 1, wherein R 2 It is Cl.

8. The use as claimed in claim 1, wherein R 2 It is CH3.

9. The use as claimed in claim 1, wherein R 3 is –O–CH3.

10. The use according to claim 1, wherein R 3 It is –O–CF3.

11. The use as claimed in claim 1, wherein R a Selected from the group consisting of: CH3, and CF3.

12. The use as claimed in claim 1, wherein R a It is CH3.

13. The use as claimed in claim 1, wherein R b Selected from the group consisting of: CH3, and CF3.

14. The use as claimed in claim 1, wherein R b It is CH3.

15. The use according to claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is a compound of formula (Ia): or a pharmaceutically acceptable salt thereof.

16. The use according to claim 1, wherein -NH(R 4 ) Choose Free The group composed of.

17. The use according to claim 1, wherein -NHR 4 Selected from the group consisting of:

18. The use according to claim 1, wherein -NHR 4 yes 19. The use as claimed in claim 1, wherein the compound of formula (I) is an optically pure or enriched isomer.

20. The use of claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is or a pharmaceutically acceptable salt thereof.

21. The use of claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is or a pharmaceutically acceptable salt thereof.

22. The use of claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is or a pharmaceutically acceptable salt thereof.

23. The use of any one of claims 1 to 22, wherein the effective amount maintains a plasma trough concentration of about 2 ng / mL to about 1,000 ng / mL.

24. The use of any one of claims 1 to 22, wherein the effective amount maintains a plasma trough concentration of about 5 ng / mL to about 500 ng / mL.

25. The use of any one of claims 1 to 22, wherein the effective amount maintains a plasma trough concentration of about 20 ng / mL to about 300 ng / mL.

26. The use of any one of claims 1 to 22, wherein the effective amount maintains a plasma trough concentration of about 30 ng / mL to about 200 ng / mL.

27. The use of claim 1, wherein the cancer is colon cancer.

28. The use of claim 1, wherein the cancer is colorectal cancer.

29. The use of claim 1, wherein the cancer is breast cancer.

30. The use of claim 1, wherein the cancer is liver cancer.

31. The use of claim 1, wherein the cancer is melanoma.

32. The use of claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered together with an effective amount of one or more additional therapeutic agents.

33. The use of claim 32, wherein the one or more additional therapeutic agents is a cytotoxic agent.

34. The use of claim 32, wherein the one or more additional therapeutic agents are gene expression regulators.

35. The use of claim 32, wherein the one or more additional therapeutic agents is a chemotherapeutic agent.

36. The use of claim 32, wherein the one or more additional therapeutic agents are anti-cancer agents.

37. The use of claim 32, wherein the one or more additional therapeutic agents is an anti-angiogenic agent.

38. The use of claim 32, wherein the one or more additional therapeutic agents are immunotherapeutic agents.

39. The use of claim 32, wherein the one or more additional therapeutic agents is an anti-hormonal agent.

40. The use of claim 32, wherein the one or more additional therapeutic agents is a radiotherapeutic agent.

41. The use of claim 32, wherein the one or more additional therapeutic agents are anti-tumor agents.

42. The use of claim 32, wherein the one or more additional therapeutic agents are anti-proliferative agents.

43. The use of claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered together with an effective amount of radiation therapy.

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