WO2023177894A1

WO2023177894A1 - Combination therapy comprising a mat2a inhibitor and an antimetabolite agent

Info

Publication number: WO2023177894A1
Application number: PCT/US2023/015548
Authority: WO
Inventors: Claire L. NEILAN; Marcus Michael FISCHER; Kimberline Yang GERRICK; Jenny Leigh LARAIO; Anthony Mazurek; Geeta Sharma
Original assignee: Ideaya Biosciences, Inc.; Glaxosmithkline Intellectual Property (No.4) Limited
Priority date: 2022-03-18
Filing date: 2023-03-17
Publication date: 2023-09-21

Abstract

Provided herein is a combination of a methionine adenosyltransferase II alpha (MAT2A) inhibitor and an antimetabolite. Also provided are methods of using such combinations to treat diseases or disorders, for example, cancer.

Description

COMBINATION THERAPY COMPRISING A MAT2A INHIBITOR AND AN ANTIMETABOLITE AGENT

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/321 ,330, filed March 18, 2022; U.S. Provisional Application No. 63/369,501 , filed July 26, 2022; and U.S. Provisional Application No. 63/373,139, filed August 22, 2022, the entire contents of which are incorporated by reference in their entirety.

BACKGROUND

Cancer is a leading cause of death throughout the world. A limitation of prevailing therapeutic approaches, e.g., chemotherapy and immunotherapy, is that their cytotoxic effects are not restricted to cancer cells and adverse side effects can occur within normal tissues.

Methionine adenosyltransferase 2A (MAT2A) is an enzyme that utilizes methionine (Met) and adenosine triphosphate (ATP) to generate s-adenosyl methionine (SAM). SAM is a primary methyl donor in cells used to methylate several substrates including DNA, RNA, and proteins. One methylase that utilizes SAM as a methyl donor is protein arginine N- methyltransferase 5 (PRMT5). While SAM is required for PRMT5 activity, PRMT5 is competitively inhibited by 5’methylthioadenosine (MTA). Since MTA is part of the methionine salvage pathway, cellular MTA levels stay low in a process initiated by methylthioadenosine phosphorylase (MTAP).

MTAP is located at a locus on chromosome 9 that is often deleted in the cells of patients with cancers from several tissues of origin including central nervous system, pancreas, esophageal, bladder and lung (cBioPortal database). Loss of MTAP results in the accumulation of MTA making MTAP-deleted cells more dependent on SAM production, and thus MAT2A activity, compared to cells that express MTAP. In an shRNA cell-line screen across approximately 400 cancer cell lines, MAT2A knockdown resulted in the loss of viability in a larger percentage of MTAP-deleted cells compare to MTAP WT cells (see McDonald et. al. 2017 Cell 170, 577-592). Furthermore, inducible knockdown of MAT2A protein decreased tumor growth in vivo (see Marjon et. al., 2016 Cell Reports 15(3), 574- 587). These results indicate that MAT2A inhibitors may provide a novel therapy for cancer patients including those with MTAP-deleted tumors.

Anti-metabolites and anti-folates are utilized for the treatment of cancer. These agents interfere with one-carbon metabolism which is involved with both the folate and methionine cycles. The by-products of the metabolism of folate and methionine are utilized for essential functions such as nucleotide biosynthesis and methylation reactions (see Newman et. Al. Br J Cancer. 2017 Jun 6; 116(12): 1499-1504).

Despite many recent advances in cancer therapies, there remains a need for more effective and/or enhanced treatment for those individuals suffering the effects of cancer.

SUMMARY

Provided herein is a combination product comprising a methionine adenosyltransferase II alpha (MAT2A) inhibitor and an antimetabolite. The combination product is useful for the treatment of a variety of cancers, including solid tumors. The combination product is also useful for the treatment of a variety of diseases or disorders treatable by inhibiting MAT2A. The combination product is also useful for treating MTAP- deficient tumors.

In an embodiment, provided herein is a combination of a MAT2A inhibitor and an antimetabolite. In an embodiment, the antimetabolite is an antifolate.

In an embodiment, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a methionine adenosyltransferase II alpha (MAT2A) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of an antimetabolite. In some embodiments, the antimetabolite is an antifolate.

In an embodiment, provided herein are methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject a combination comprising a MAT2A inhibitor and an antimetabolite, thereby treating the cancer in the subject. In an embodiment, the antimetabolite is an antifolate.

In an embodiment, provided herein are methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject a combination comprising a MAT2A inhibitor and an antimetabolite, together with at least a pharmaceutically acceptable carrier, thereby treating the cancer in the subject. In an embodiment, the antimetabolite is an antifolate.

In still another embodiment, the cancer is characterized by a reduction or absence of methylthioadenosine phosphorylase (MTAP) gene expression, absence of MTAP gene, reduced function of MTAP protein, reduced level or absence of MTAP protein, MTA accumulation, or combination thereof.

In an embodiment, provided herein are methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a MAT2A inhibitor and a therapeutically effective amount of a pharmaceutical composition comprising an antimetabolite, thereby treating the cancer in the subject. In some embodiments, the antimetabolite is an antifolate.

In an embodiment, provided herein are methods of treating a disease or disorder treatable by inhibiting MAT2A in a subject in need thereof, the methods comprising administering to the subject a combination comprising a methionine adenosyltransferase II alpha (MAT2A) inhibitor and an antimetabolite, thereby treating the disease or disorder in the subject. In an embodiment, the antimetabolite is an antifolate. In an embodiment, the disease or disorder is cancer.

In an embodiment, provided herein are methods of treating a disease or disorder treatable by inhibiting MAT2A in a subject in need thereof, the methods comprising administering to the subject a combination comprising a MAT2A inhibitor and an antimetabolite, together with at least a pharmaceutically acceptable carrier, thereby treating the disease or disorder in the subject. In an embodiment, the antimetabolite is an antifolate. In an embodiment, the disease or disorder is cancer.

In an embodiment, the MAT2A inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the variables of Formula (I) are defined below.

In another embodiment, the MAT2A inhibitor is Compound A having the following structural formula:

Compound A or a pharmaceutically acceptable salt thereof.

In another embodiment, the MAT2A inhibitor is Compound A1 having the following structural formula:

Compound A1 or a pharmaceutically acceptable salt thereof. MAT2A inhibitors for use in the combination therapy described herein are described in WO 2020/123395 (PCT/US19/65260). The generic and specific compounds described in the patent application are incorporated herein by reference and can be used to treat cancer as described herein.

In an embodiment, the antimetabolite is an antifolate.

In yet another embodiment, the antifolate is pemetrexed (Compound B) having the following structural formula:

Compound B or a pharmaceutically acceptable salt and/or hydrate thereof. The chemical name for pemetrexed is (2S)-2-({4-[2-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl]benzoyl}amino)pentanedioic acid. “Pemetrexed,” or “Compound B” also includes pharmaceutically acceptable salts and/or hydrates thereof. In an embodiment, Compound B is pemetrexed disodium. In another embodiment, Compound B is pemetrexed disodium heptahydrate (also known as Alimta®).

In still another embodiment, the antifolate is 5-fluorouracil (Compound C) having the following structural formula:

Compound C or a pharmaceutically acceptable salt thereof. The chemical name for 5-fluorouracil is 5-fluoro-1 ,3-diazinane-2, 4-dione.

In another embodiment, the antifolate is capecitabine (also known as Xeloda® and referred to herein as Compound D) having the following structural formula:

Compound D or a pharmaceutically acceptable salt thereof. The chemical name for capecitabine is pentyl [1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4- yl]carbamate.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the efficacy of Compound A and Compound B in the RT-112/84 human bladder tumor cell line.

Figure 2 shows the summary of efficacy of Compound A and Compound B in the RT112/84 human bladder tumor cell line.

Figure 3 shows the anti- tumor efficacy of Compound A and Compound C in CTG- 0707 gastric cancer patient-derived xenografts (PDX).

Figure 4 shows the efficacy of Compound A and Compound B or Compound C in the NCI-H838 human NSCLC xenograft.

Figure 5 shows plasma s-adenosyl methionine on day 5 from NCI-H838 human NSCLC xenograft.

Figure 6 shows tumor s-adenosyl methionine on day 5 from NCI-H838 human NSCLC xenograft.

Figure 7 shows Pemetrexed (Compound B) single agent growth inhibition across six MTAP-deficient cell lines.

Figure 8 shows Excess over Bliss Synergy of Compound A and Pemetrexed (Compound B) combinations across six MTAP-deficient cell lines.

Figure 9 shows the efficacy of Compound A and Compound B in the LXFA737 NSCLC patient-derived xenograft (PDX).

Figures 10A-10M show the growth inhibition of Compound A and Compound B in 13 MTAP-deficient cell lines.

Figures 11 A-11 M show the Loewe Synergy of Compound A and Compound B in 13 MTAP-deficient cell lines.

DETAILED DESCRIPTION

Provided herein is a combination therapy comprising a methionine adenosyltransferase II alpha (MAT2A) inhibitor, or a pharmaceutically acceptable salt thereof, and an antimetabolite, or a pharmaceutically acceptable salt thereof. The combination therapy is useful for the treatment of a variety of cancers. In another aspect, the combination therapy is useful for the treatment of any number of MAT2A-associated diseases. In another aspect, the combination therapy is useful for the treatment of a disease or disorder treatable by inhibiting MAT2A.

Because MTAP is an essential enzyme for the salvage pathway of adenosine synthesis, MTAP -deficient tumors are hypothesized to be sensitive to antifolate agents such as pemetrexed, due to dual blocking of the de novo and salvage pathways of adenosine synthesis. Indeed (Chen et al., Abstract 385: MTAP gene deficiency creates vulnerability to anti-folate therapy in urothelial bladder carcinoma. Cancer Res 1 July 2019; 79) (13 Supplement) Chen et al. demonstrated that both in an in vitro and in vivo settings, MTAP-deleted bladder cell lines or tumor models were more sensitive to pemetrexed than MTAP-WT models. Additionally, 100% of patients with MTAP-deficient metastatic bladder cancer responded to pemetrexed treatment, whereas only 9% of patients with MTAP-WT metastatic bladder cancer responded to this treatment. The addition of a MAT2A inhibitor to pemetrexed in the MTAP-deleted setting should further reduce DNA synthesis as a result of the reduction in SAM synthesis (a key component of the folate cycle), arising from inhibition of MAT2A.

Definitions

Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used in the specification and in the claims, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”.

The terms “combination,” “therapeutic combination,” “pharmaceutical combination,” or “combination product” as used herein refer to either a fixed combination in one dosage unit form, or non-fixed combination in separate dosage forms, or a kit of parts for the combined administration where two or more therapeutic agents may be administered independently, at the same time or separately within time intervals.

The term “combination therapy” refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single formulation having a fixed ratio of active ingredients or in separate formulations (e.g., capsules and/or intravenous formulations) for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential or separate manner, either at approximately the same time or at different times. Regardless of whether the active ingredients are administered as a single formulation or in separate formulations, the drugs are administered to the same patient as part of the same course of therapy. In any case, the treatment regimen will provide beneficial effects in treating the conditions or disorders described herein.

As used herein, the term “treating” or “treatment” refers to inhibiting a disease; for example, inhibiting a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomology of the disease, condition, or disorder (/.e., arresting further development of the pathology and/or symptomology) or ameliorating the disease; for example, ameliorating a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomology of the disease, condition, or disorder (/.e., reversing the pathology and/or symptomology) such as decreasing the severity of the disease.

As used herein, the term “prevent” or “prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of the combination therapy provided herein to prevent some or all of the symptoms associated with the disorder or disease. As used herein, the term “patient,” “individual,” or “subject” refers to a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and marine mammals. Preferably, the patient, subject, or individual is human.

As used herein, the terms “effective amount,” “pharmaceutically effective amount,” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term “pharmaceutically acceptable salt” refers to derivatives of the disclosed compounds wherein a parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts described herein include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts discussed herein can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used. The phrase “pharmaceutically acceptable salt” is not limited to a mono, or 1 :1, salt. For example, “pharmaceutically acceptable salt” also includes bis-salts, such as a bis-hydrochloride salt. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the composition to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound disclosed herein, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of a compound disclosed herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. Other additional ingredients that may be included in the pharmaceutical compositions are known in the art and described, for example, in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

The term “single formulation” as used herein refers to a single carrier or vehicle formulated to deliver therapeutically effective amounts of both therapeutic agents to a patient. The single vehicle is designed to deliver a therapeutically effective amount of each of the agents, along with any pharmaceutically acceptable carriers or excipients. In some embodiments, the vehicle is a tablet, capsule, pill, or a patch. In other embodiments, the vehicle is a solution or a suspension. As used herein “methionine adenosyltransferase II alpha inhibitor” or “MAT2A inhibitor” means an agent that modulates the activity of MAT2A or inhibits the production of S-adenosylmethionine (SAM) by methionine adenosyltransferase 2A (MAT2A).

As used herein, "antimetabolite" includes, but is not limited to, 5-fluorouracil or 5-FU, capecitabine, and folic acid antagonists such as pemetrexed.

The combination of agents described herein may display a synergistic effect. The term “synergistic effect” or “synergy” as used herein, refers to action of two agents such as, for example, a MAT2A inhibitor and an antimetabolite, producing an effect, for example, slowing the symptomatic progression of cancer or symptoms thereof, which is greater than the simple addition of the effects of each drug administered by themselves. A synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981 )), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

In an embodiment, provided herein is a combination therapy comprising a therapeutically effective amount of a MAT2A inhibitor and an antimetabolite. A “therapeutically effective amount” of a combination of agents (i.e., a MAT2A inhibitor and an antimetabolite) is an amount sufficient to provide an observable improvement over the baseline clinically observable signs and symptoms of the disorders treated with the combination.

“Alkyl” means a linear saturated monovalent hydrocarbon radical of one to six carbon (i.e. C_1-6 means one to six carbons) atoms or a branched saturated monovalent hydrocarbon radical of three to six carbon atoms (i.e. C_3-6 means three to six carbons). Alkyl can include any number of carbons, such as C_1-2, C_1-3, C_1-4, C_1-5, C_1-6, C_2-3, C_2-4, C_2-5, C_2-6, C_3-4, C_3-5, C_3-6, C_4-5, C_4-6 and C_5-6. Example of alkyl groups inlcude methyl, ethyl, propyl, 2-propyl, butyl, pentyl, and the like. It will be recognized by a person skilled in the art that the term “alkyl” may include “alkylene” groups.

“Amino” means a -NH₂.

“Cycloalkyl” means a monocyclic monovalent hydrocarbon radical of three to six carbon atoms (e.g., C_3-6 cycloalkyl) which may be saturated or contains one double bond. Cycloalkyl can include any number of carbons, such as C_3-6, C_4-6, and C_5-6, Partially unsaturated cycloalkyl groups have one or more double in the ring, but cycloalkyl groups are not aromatic. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

“Halo” means fluoro, chloro, bromo, or iodo, preferably fluoro or chloro.

“Haloalkyl” means alkyl radical as defined above, which is substituted with one to five halogen atoms, such as fluorine or chlorine, including those substituted with different halogens, e.g., -CH₂CI, -CF₃, -CHF₂, -CH₂CF₃, -CF₂CF₃, -CF(CH₃)₂, and the like. When the alkyl is substituted with only fluoro, it can be referred to as fluoroalkyl. As for alkyl group, haloalkyl groups can have any suitable number of carbon atoms, such as C_1-6.

Combination Product

Provided herein is a combination product comprising a MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and an antimetabolite, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof. This combination product is also referred to herein as a combination therapy. The combination product is useful for the treatment of a variety of cancers, including solid tumors. In another aspect, the combination product is useful for the treatment of any number of MAT2A-associated diseases. In another aspect, the combination product is useful for the treatment of a disease or disorder treatable by inhibiting MAT2A. In another aspect, the combination product is useful for treating MTAP- deficient tumors.

The disclosure provides MAT2A inhibitors. In an embodiment, the MAT2A inhibitor is a compound or a pharmaceutically acceptable salt thereof of Formula (I):

wherein

X is C or N;

R³ is halo, C_1-6 haloalkyl or C_3-6 cycloalkyl;

R² is -NR⁴R⁵;

R⁴ is hydrogen or C_1-6 alkyl;

R⁵ is hydrogen, C_1-6 alkyl or C_3-6 cycloalkyl; and

R¹ is phenyl, wherein phenyl is substituted with 0-2 halo.

In an embodiment, X in Formula (I) and subembodiments thereof is C. In an embodiment, X in Formula (I) and subembodiments thereof is N. In still another embodiment, R³ in formula (I) and subembodiments thereof is halo or C_1-6haloalkyl. In an embodiment, R³ in formula (I) and subembodiments thereof is halo. In an embodiment, R³ in formula (I) and subembodiments thereof is C_1-6 haloalkyl. In an embodiment, R³ in formula (I) and subembodiments thereof is C_3-6 cycloalkyl. In an

5 embodiment, R³ in formula (I) and subembodiments thereof is chloro, fluoro, bromo, -CH₂CI, -CF₃, -CHF₂, -CH₂CF₃, -CF₂CF₃, or -CF(CH₃)₂. In an embodiment, R³ in formula (I) and subembodiments thereof is chloro or -CF₃. In an embodiment, R³ in formula (I) and subembodiments thereof is chloro. In an embodiment, R³ in formula (I) and subembodiments thereof is -CF₃.

10 In still another embodiment, R⁴ in formula (I) and subembodiments thereof is H. In an embodiment, R⁴ in formula (I) and subembodiments thereof is C_1-3alkyl. In an embodiment, R⁴ in formula (I) and subembodiments thereof is methyl, ethyl, propyl, or isopropyl.

In still another embodiment, R⁵ in formula (I) and subembodiments thereof is H. In an embodiment, R⁵ in formula (I) and subembodiments thereof is C_1-3alkyl. In an embodiment,

15 R⁵ in formula (I) and subembodiments thereof is methyl, ethyl, propyl, or isopropyl. In an embodiment, R⁵ in formula (I) and subembodiments thereof is C_3-6 cycloalkyl.

In still another embodiment, R² in formula (I) and subembodiments thereof is -NH₂, - NHC_1-3alkyl, or -N(Ci-3alkyl)2. In an embodiment, R² in formula (I) and subembodiments thereof is NH₂, -NHMe, or -N(Me)2. In an embodiment, R² in formula (I) and subembodiments

20 thereof is NH₂. In an embodiment, R² in formula (I) and subembodiments thereof is -NHMe.

In still another embodiment, R¹ in formula (I) and subembodiments thereof is unsubstituted phenyl. In an embodiment, R¹ in formula (I) and subembodiments thereof is phenyl substituted with 1 halo. In an embodiment, R¹ in formula (I) and subembodiments thereof is phenyl substituted 1 halo selected fluoro and chloro. In an embodiment, R¹ in

25 formula (I) and subembodiments thereof is phenyl substituted chloro. In an embodiment, R 1 in formula (I) and subembodiments thereof is phenyl substituted 2 halo.

In yet another embodiment, the MAT2A inhibitor is selected from the group consisting of a compound from Table 1 , or a pharmaceutically acceptable salt thereof.

Table 1.

12

In another embodiment, the MAT2A inhibitor is Compound A:

Compound A or a pharmaceutically acceptable salt thereof.

Compound A1 or a pharmaceutically acceptable salt thereof.

The preparation and activity of the MAT2A inhibitors provided herein are disclosed in PCT/US2019/065260 (WO 2020/123395), the entire contents of which are hereby incorporated by reference in their entirety.

In an embodiment, the antimetabolite is an antifolate. In yet another embodiment, the antifolate is selected from the group consisting of a compound in Table 2. Table 2

or a pharmaceutically acceptable salt and/or a hydrate thereof.

The preparation and activity of the antifolates provided herein are described in US 3,682,917 (Compound C); US 5,472,949 (Compound D); US 7,053,065 (Compound B); and US 7,772,209 (Compound B), the entire contents of which are hereby incorporated by reference in their entirety.

In another aspect, provided herein is a combination product comprising Compound A or a pharmaceutically acceptable salt thereof, and Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof. In an embodiment, Compound B is pemetrexed disodium. In another embodiment, Compound B is pemetrexed disodium heptahydrate (also known as Alimta®).

In another aspect, provided herein is a combination product comprising Compound A, or a pharmaceutically acceptable salt thereof, and Compound C, or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a combination product comprising Compound A, or a pharmaceutically acceptable salt thereof, and Compound D, or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a combination product comprising Compound A1 , or a pharmaceutically acceptable salt thereof, and Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof. In an embodiment, Compound B is pemetrexed disodium. In another embodiment, Compound B is pemetrexed disodium heptahydrate. In another aspect, provided herein is a combination product comprising Compound A1 , or a pharmaceutically acceptable salt thereof, and Compound C, or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a combination product comprising Compound A1 , or a pharmaceutically acceptable salt thereof, and Compound D, or a pharmaceutically acceptable salt thereof.

Methods of Treatment

In an embodiment, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a MAT2A inhibitor and administering to the subject a therapeutically effective amount of an antimetabolite, thereby treating the cancer in the subject. In an embodiment, the antimetabolite is an antifolate.

In an embodiment, provided herein are methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject a combination comprising a MAT2A inhibitor and an antimetabolite, together with at least a pharmaceutically acceptable carrier, thereby treating the cancer in the subject. In some embodiments, the antimetabolite is an antifolate.

In an embodiment, provided herein are methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a MAT2A inhibitor and a therapeutically effective amount of a pharmaceutical composition comprising an antimetabolite, thereby treating the cancer in the subject. In an embodiment, the antimetabolite is an antifolate.

In an embodiment, use of a combination of a MAT2A inhibitor and an antimetabolite for the manufacture of a medicament is provided. In some embodiments, the antimetabolite is an antifolate. In one embodiment, the MAT2A inhibitor is Compound A. In one embodiment, the MAT2A inhibitor is Compound A1. In an embodiment, the antifolate is Compound B. In another embodiment, the antifolate is Compound C. In yet another embodiment, the antifolate is Compound D. In one embodiment, provided is a combination of Compound A and Compound B for the manufacture of a medicament. In one embodiment, provided is a combination of Compound A and Compound C for the manufacture of a medicament. In one embodiment, provided is a combination of Compound A and Compound D for manufacture of a medicament. In one embodiment, provided is a combination of Compound A1 and Compound B for the manufacture of a medicament. In one embodiment, provided is a combination of Compound A1 and Compound C for the manufacture of a medicament. In one embodiment, provided is a combination of Compound A1 and Compound D for manufacture of a medicament.

In another embodiment, use of a combination of a MAT2A inhibitor and an antimetabolite for the treatment of cancer is provided. In an embodiment, the antimetabolite is an antifolate. In one embodiment, the MAT2A inhibitor is a compound of Formula (I). In one embodiment, the MAT2A inhibitor is Compound A. In one embodiment, the MAT2A inhibitor is Compound A1. In an embodiment, the antifolate is Compound B. In another embodiment, the antifolate is Compound C. In yet another embodiment, the antifolate is Compound D. In one embodiment, provided is a combination of Compound A and Compound B for the treatment of cancer. In one embodiment, provided is a combination of Compound A and Compound C for the treatment of cancer. In one embodiment, provided is a combination of Compound A and Compound D for the treatment of cancer. In one embodiment, provided is a combination of Compound A1 and Compound B for the treatment of cancer. In one embodiment, provided is a combination of Compound A1 and Compound C for the treatment of cancer. In one embodiment, provided is a combination of Compound A1 and Compound D for the treatment of cancer.

In an embodiment of the methods, the MAT2A inhibitor is a compound or a pharmaceutically acceptable salt thereof of Formula (I):

wherein the variables are defined supra.

In another embodiment of the methods, the MAT2A inhibitor is selected from the group consisting of a compound from Table 1 , or a pharmaceutically acceptable salt thereof.

In yet another embodiment of the methods, the MAT2A inhibitor is Compound A or a pharmaceutically acceptable salt thereof.

In yet another embodiment of the methods, the MAT2A inhibitor is Compound A1 or a pharmaceutically acceptable salt thereof.

In an embodiment of the methods, the antimetabolite is an antifolate.

In an embodiment of the methods, the antifolate is selected from the group consisting of a compound in Table 2, or a pharmaceutically acceptable salt and/or a hydrate thereof. In another embodiment of the methods, the antifolate is Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof. In an embodiment, Compound B is pemetrexed disodium. In another embodiment, Compound B is pemetrexed disodium heptahydrate.

In yet another embodiment of the methods, the antifolate is Compound C, or a pharmaceutically acceptable salt thereof.

In still another embodiment of the methods, the antifolate is Compound D, or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and administering to the subject a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof. In an embodiment, Compound B is pemetrexed disodium. In another embodiment, Compound B is pemetrexed disodium heptahydrate.

In yet another aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and administering to the subject a therapeutically effective amount of Compound C, or a pharmaceutically acceptable salt thereof.

In still another aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and administering to the subject a therapeutically effective amount of Compound D, or a pharmaceutically acceptable salt thereof.

In an aspect, provided herein is a method of treating bladder cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and administering to the subject a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt and/or hydrate thereof.

In another aspect, provided herein is a method of treating gastric cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and administering to the subject a therapeutically effective amount of Compound C, or a pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a method of treating non-small cell lung cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and administering to the subject a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt and/or hydrate thereof.

In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and administering to the subject a therapeutically effective amount of Compound C, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided is a product containing a MAT2A inhibitor and an antimetabolite as a combined preparation for simultaneous, separate, or sequential use in medicine. In one embodiment, the MAT2A inhibitor is a compound of Formula (I). In one embodiment, the MAT2A inhibitor is a compound in Table 1. In one embodiment, the MAT2A inhibitor is Compound A. In one embodiment, the MAT2A inhibitor is Compound A1. In an embodiment, the antimetabolite is an antifolate. In one embodiment, the antifolate is Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof. In an embodiment, Compound B is pemetrexed disodium. In another embodiment, Compound B is pemetrexed disodium heptahydrate. In one embodiment, the antifolate is Compound C, or a pharmaceutically acceptable salt thereof. In one embodiment, the antifolate is Compound D, or a pharmaceutically acceptable salt thereof.

In one embodiment, provided is a product containing Compound A and Compound B as a combined preparation for simultaneous, separate, or sequential use in medicine. In one embodiment, provided is a product containing Compound A and Compound C as a combined preparation for simultaneous, separate, or sequential use in medicine. In one embodiment, provided is a product containing Compound A and Compound D as a combined preparation for simultaneous, separate, or sequential use in medicine.

In another embodiment, provided is a product containing a MAT2A inhibitor and an antimetabolite as a combined preparation for simultaneous, separate, or sequential use in treating cancer in a subject. In one embodiment, the MAT2A inhibitor is a compound of Formula (I). In one embodiment, the MAT2A inhibitor is a compound in Table 1. In one embodiment, the MAT2A inhibitor is Compound A. In one embodiment, the MAT2A inhibitor is Compound A1. In an embodiment, the antimetabolite is an antifolate. In one embodiment, the antifolate is Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof. In an embodiment, Compound B is pemetrexed disodium. In another embodiment, Compound B is pemetrexed disodium heptahydrate. In one embodiment, the antifolate is Compound C, or a pharmaceutically acceptable salt thereof. In one embodiment, the antifolate is Compound D, or a pharmaceutically acceptable salt thereof.

In one embodiment, provided is a product containing Compound A and Compound B as a combined preparation for simultaneous, separate, or sequential use in treating cancer in a subject. In one embodiment, provided is a product containing Compound A and Compound C as a combined preparation for simultaneous, separate, or sequential use in treating cancer in a subject. In one embodiment, provided is a product containing Compound A and Compound D as a combined preparation for simultaneous, separate, or sequential use in treating cancer in a subject.

In yet another embodiment, the cancer is selected from the group consisting of leukemia, glioma, melanoma, pancreatic, non-small cell lung cancer, bladder cancer, astrocytoma, osteosarcoma, head and neck cancer, myxoid chondrosarcoma, ovarian cancer, endometrial cancer, breast cancer, anal cancer, stomach cancer, colon cancer, colorectal cancer, soft tissue sarcoma, non-Hodgkin lymphoma, gastric cancer, esophagogastric cancer, esophageal cancer, malignant peripheral nerve sheath tumor, and mesothelioma.

In an embodiment, the cancer is mesothelioma. In an embodiment, the cancer is non-small cell lung cancer. In another embodiment, the cancer is nonsquamous non-small cell lung cancer. In one embodiment, the cancer is cancer of the colon or rectum. In an embodiment, the cancer is adenocarcinoma of the colon or rectum. In an embodiment, the cancer is breast cancer. In an embodiment, the cancer is adenocarcinoma of the breast. In an embodiment, the cancer is gastric cancer. In an embodiment, the cancer is gastric adenocarcinoma. In an embodiment, the cancer is pancreatic cancer. In an embodiment, the cancer is pancreatic adenocarcinoma. In an embodiment, the cancer is bladder cancer.

In an embodiment, the cancer is characterized as being MTAP-null.

In an embodiment, the cancer is characterized as being MTAP-deficient.

In still another embodiment, the cancer is a solid tumor. In still another embodiment, the cancer is a MTAP-deleted solid tumor. In still another embodiment, the cancer is a metastatic MTAP-deleted solid tumor.

In still another embodiment, the cancer is metastatic.

In still another embodiment, the cancer is a solid malignant tumor.

In still another embodiment, the cancer is MTAP-deficient lung or MTAP- deficient pancreatic cancer, including MTAP-deficient NSCLC or MTAP-deficient pancreatic ductal adenocarcinoma (PDAC) or MTAP-deficient esophageal cancer.

In another embodiment, the cancer is a tumor having an MTAP gene deletion.

In any one of the embodiments herein, the cancer is a solid tumor or a haematological cancer. In one embodiment, the tumor is deficient in MTAP. In another embodiment, the tumor is normal in its expression of MTAP.

In still another embodiment, the cancer is NSCLC, mesothelioma, squamous carcinoma of the head and neck, salivary gland tumors, urothelial cancers, sarcomas, or ovarian cancer. In still another embodiment, the cancer is NSCLC, esophagogastric and pancreatic cancers. In still another embodiment, the cancer is bladder cancer or gastrointestinal cancer.

In still another embodiment, the cancer is characterized by a reduction or absence of MTAP gene expression, absence of MTAP gene, reduced function of MTAP protein, reduced level or absence of MTAP protein, MTA accumulation, or combination thereof.

In still another embodiment, the cancer is characterized by a reduction or absence of MTAP gene expression.

In still another embodiment, the cancer is characterized by reduced function of MTAP protein.

In still another embodiment, the cancer is characterized reduced level or absence of MTAP protein.

In still another embodiment, the cancer is characterized by MTA accumulation.

In still another embodiment, the cancer is a tumor having a GNAQ gene mutation.

In still another embodiment, the cancer is a tumor having a GNA11 gene mutation.

In still another embodiment, the cancer is a tumor having PRKC fusions.

In an embodiment, the MAT2A inhibitor and the antimetabolite are in separate dosage forms.

In another embodiment, the MAT2A inhibitor and the antimetabolite are in the same dosage form.

In another embodiment, the treatment comprises administering the MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and the antimetabolite, or a pharmaceutically acceptable salt thereof, at substantially the same time. In yet another embodiment, the treatment comprises administering the MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and the antimetabolite, or a pharmaceutically acceptable salt thereof, at different times.

In still another embodiment, the MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, is administered to the subject, followed by administration of the antimetabolite, or a pharmaceutically acceptable salt thereof. In an embodiment, the antimetabolite, or a pharmaceutically acceptable salt thereof, is administered to the subject, followed by administration of MAT2A inhibitor, or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the method comprises administering to the subject in need thereof a MAT2A inhibitor.

In still another embodiment, the method comprises administering to the subject in need thereof an antimetabolite. In some embodiments, the antimetabolite is an antifolate.

In an embodiment, the MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and the antimetabolite, or a pharmaceutically acceptable salt thereof, are administered orally. In another embodiment, the cancer to be treated is selected from the group consisting of leukemia, glioma, melanoma, pancreatic, non-small cell lung cancer, bladder cancer, astrocytoma, osteosarcoma, head and neck cancer, myxoid chondrosarcoma, ovarian cancer, endometrial cancer, breast cancer, anal cancer, stomach cancer, colon cancer, colorectal cancer, soft tissue sarcoma, non-Hodgkin lymphoma, gastric cancer, esophagogastric cancer, esophageal cancer, malignant peripheral nerve sheath tumor, and mesothelioma.

In an aspect, provided herein is a MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and an antimetabolite, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof, for use in therapy.

In an embodiment, the MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and the antimetabolite, or a pharmaceutically acceptable salt thereof, are for use in the treatment of cancer in a subject in need thereof.

Exemplary lengths of time associated with the course of the treatment methods disclosed herein include: about one week; about two weeks; about three weeks; about four weeks; about five weeks; about six weeks; about seven weeks; about eight weeks; about nine weeks; about ten weeks; about eleven weeks; about twelve weeks; about thirteen weeks; about fourteen weeks; about fifteen weeks; about sixteen weeks; about seventeen weeks; about eighteen weeks; about nineteen weeks; about twenty weeks; about twenty-one weeks; about twenty-two weeks; about twenty-three weeks; about twenty four weeks; about seven months; about eight months; about nine months; about ten months; about eleven months; about twelve months; about thirteen months; about fourteen months; about fifteen months; about sixteen months; about seventeen months; about eighteen months; about nineteen months; about twenty months; about twenty one months; about twenty-two months; about twenty-three months; about twenty-four months; about thirty months; about three years; about four years; and about five years.

In an embodiment of the methods, the method involves the administration of a therapeutically effective amount of a combination or composition comprising compounds provided herein, or pharmaceutically acceptable salts thereof, to a subject (including, but not limited to a human or animal) in need of treatment (including a subject identified as in need).

In another embodiment of the methods, the treatment includes co-administering the amount of the MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and the amount of the antimetabolite, or a pharmaceutically acceptable salt thereof. In an embodiment, the amount of the MAT2A inhibitor or a pharmaceutically acceptable salt thereof and the amount of antimetabolite or a pharmaceutically acceptable salt thereof are in a single formulation or unit dosage form. In still other embodiments, the amount of MAT2A inhibitor or a pharmaceutically acceptable salt thereof and the amount of antimetabolite or a pharmaceutically acceptable salt thereof are in a separate formulations or unit dosage forms.

In the foregoing methods, the treatment can include administering the amount of MAT2A inhibitor or a pharmaceutically acceptable salt thereof and the amount of antimetabolite or a pharmaceutically acceptable salt thereof at substantially the same time or administering the amount of MAT2A inhibitor or a pharmaceutically acceptable salt thereof and the amount of antimetabolite or a pharmaceutically acceptable salt thereof at different times. In some embodiments of the foregoing methods, the amount of MAT2A inhibitor or a pharmaceutically acceptable salt thereof and/or the amount of antimetabolite or a pharmaceutically acceptable salt thereof is administered at dosages that would not be effective when one or both of MAT2A inhibitor or a pharmaceutically acceptable salt thereof and antimetabolite or a pharmaceutically acceptable salt thereof is administered alone, but which amounts are effective in combination.

In another embodiment of the methods, the treatment includes co-administering the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof. In an embodiment, the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof, are in a single formulation or unit dosage form. In still other embodiments, the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof, are in a separate formulations or unit dosage forms.

In another embodiment of the methods, the treatment includes co-administering the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound C, or a pharmaceutically acceptable salt thereof. In an embodiment, the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound C, or a pharmaceutically acceptable salt thereof, are in a single formulation or unit dosage form. In still other embodiments, the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound C, or a pharmaceutically acceptable salt thereof, are in a separate formulations or unit dosage forms.

In another embodiment of the methods, the treatment includes co-administering the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound D, or a pharmaceutically acceptable salt thereof. In an embodiment, the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound D, or a pharmaceutically acceptable salt thereof, are in a single formulation or unit dosage form. In still other embodiments, the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound D, or a pharmaceutically acceptable salt thereof, are in a separate formulations or unit dosage forms.

In the foregoing methods, the treatment can include administering the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound

B, or a pharmaceutically acceptable salt and/or a hydrate thereof, at substantially the same time or administering the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof, at different times. In some embodiments of the foregoing methods, the amount of Compound A, or a pharmaceutically acceptable salt thereof, and/or the amount of Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof, is administered at dosages that would not be effective when one or both of Compound A, or a pharmaceutically acceptable salt thereof, and Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof, is administered alone, but which amounts are effective in combination.

C, or a pharmaceutically acceptable salt thereof, at substantially the same time or administering the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound C, or a pharmaceutically acceptable salt thereof, at different times. In some embodiments of the foregoing methods, the amount of Compound A, or a pharmaceutically acceptable salt thereof, and/or the amount of Compound C, or a pharmaceutically acceptable salt thereof, is administered at dosages that would not be effective when one or both of Compound A, or a pharmaceutically acceptable salt thereof, and Compound C, or a pharmaceutically acceptable salt thereof, is administered alone, but which amounts are effective in combination.

D, or a pharmaceutically acceptable salt thereof, at substantially the same time or administering the amount of Compound A, or a pharmaceutically acceptable salt thereof, and the amount of Compound D, or a pharmaceutically acceptable salt thereof, at different times. In some embodiments of the foregoing methods, the amount of Compound A, or a pharmaceutically acceptable salt thereof, and/or the amount of Compound D, or a pharmaceutically acceptable salt thereof, is administered at dosages that would not be effective when one or both of Compound A, or a pharmaceutically acceptable salt thereof, and Compound D, or a pharmaceutically acceptable salt thereof, is administered alone, but which amounts are effective in combination. Pharmaceutical Compositions

In an aspect, provided herein is a pharmaceutical composition comprising a MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, an antimetabolite, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof, and at least one pharmaceutically acceptable carrier.

In an embodiment, a pharmaceutical composition comprising a therapeutically effective amount of a MAT2A inhibitor or a pharmaceutically acceptable salt thereof, and a second pharmaceutical composition comprising a therapeutically effective amount of antimetabolite or a pharmaceutically acceptable salt thereof and/or a hydrate thereof is provided.

In another aspect, provided herein is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of a MAT2A inhibitor or a pharmaceutically acceptable salt thereof, and a second pharmaceutical composition comprising a therapeutically effective amount of an antimetabolite or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

In an embodiment, the MAT2A inhibitor is a compound or a pharmaceutically acceptable salt thereof of Formula (I):

wherein the variables are defined supra.

In another embodiment, the MAT2A inhibitor is selected from the group consisting of a compound from Table 1 , or a pharmaceutically acceptable salt thereof.

In an embodiment, the antimetabolite is an antifolate. In another embodiment, the antifolate is selected from the group consisting of a compound in Table 2, or a pharmaceutically acceptable salt and/or a hydrate thereof.

In another embodiment, the MAT2A inhibitor is Compound A:

Compound A or a pharmaceutically acceptable salt thereof. In another embodiment, the MAT2A inhibitor is Compound A1 :

Compound A1 or a pharmaceutically acceptable salt thereof. In another embodiment, the antifolate is Compound B:

or a pharmaceutically acceptable salt and/or a hydrate thereof. In an embodiment, Compound B is pemetrexed disodium. In another embodiment, Compound B is pemetrexed disodium heptahydrate.

In another embodiment, the antifolate is Compound C:

Compound C or a pharmaceutically acceptable salt thereof.

In another embodiment, the antifolate is Compound D, or a pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and a second pharmaceutical composition comprising a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof.

In still another aspect, provided herein is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and a second pharmaceutical composition comprising a therapeutically effective amount of Compound C, or a pharmaceutically acceptable salt thereof. In an aspect, provided herein is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and a second pharmaceutical composition comprising a therapeutically effective amount of Compound D, or a pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of Compound A1 , or a pharmaceutically acceptable salt thereof; and a second pharmaceutical composition comprising a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof.

In still another aspect, provided herein is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of Compound A1 , or a pharmaceutically acceptable salt thereof; and a second pharmaceutical composition comprising a therapeutically effective amount of Compound C, or a pharmaceutically acceptable salt thereof.

In an aspect, provided herein is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of Compound A1 , or a pharmaceutically acceptable salt thereof; and a second pharmaceutical composition comprising a therapeutically effective amount of Compound D, or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof; and a pharmaceutically acceptable carrier.

In yet another aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; Compound C, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

In still another aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; Compound D, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of Compound A1, or a pharmaceutically acceptable salt thereof; Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof; and a pharmaceutically acceptable carrier. In yet another aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of Compound A1, or a pharmaceutically acceptable salt thereof; Compound C, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

In still another aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of Compound A1, or a pharmaceutically acceptable salt thereof; Compound D, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

In an embodiment, the pharmaceutical composition is for use in the treatment of cancer in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of a solid tumor in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of a solid malignant tumor in a patient. In still another embodiment, the cancer is MTAP-deficient lung or MTAP- deficient pancreatic cancer, including MTAP- deficient NSCLC or MTAP-deficient PDAC or MTAP-deficient esophageal cancer.

In any one of the embodiments herein, the cancer is a solid tumor or a haematological cancer. In still another embodiment, the cancer is NSCLC, mesothelioma, squamous carcinoma of the head and neck, salivary gland tumors, urothelial cancers, sarcomas, or ovarian cancer. In still another embodiment, the cancer is NSCLC, esophagogastric and pancreatic cancers. In still another embodiment, the cancer is bladder cancer or gastrointestinal cancer. In still another embodiment, the cancer is bladder (urothelial) cancer or gastrointestinal cancer. In an embodiment, the cancer is bladder cancer. In another embodiment, the cancer is urothelial cancer. In yet another embodiment, the cancer is gastrointestinal cancer.

In an embodiment, the pharmaceutical composition is for use in the treatment of mesothelioma in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of non-small cell lung cancer in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of nonsquamous non-small cell lung cancer in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of colon cancer in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of rectal cancer in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of colon or rectal adenocarcinoma of the colon or rectum in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of breast cancer in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of breast adenocarcinoma in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of gastric cancer in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of gastric adenocarcinoma in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of pancreatic cancer in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of pancreatic adenocarcinoma in a patient. In an embodiment, the pharmaceutical composition is for use in the treatment of bladder cancer in a patient.

Administration / Dosage / Formulations

In another aspect, provided herein is a pharmaceutical composition or pharmaceutical combination comprising the compounds disclosed herein, together with a pharmaceutically acceptable carrier.

In an embodiment of the combination product, MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and antimetabolite, or a pharmaceutically acceptable salt thereof, are in the same formulation. In another embodiment of the combination product, MAT2A inhibitor and antimetabolite, are in separate formulations. In a further embodiment of this embodiment, the formulations are for simultaneous or sequential administration.

Administration of the combination includes administration of the combination in a single formulation or unit dosage form, administration of the individual agents of the combination concurrently but separately, or administration of the individual agents of the combination sequentially by any suitable route. The dosage of the individual agents of the combination may require more frequent administration of one of the agent(s) as compared to the other agent(s) in the combination. Therefore, to permit appropriate dosing, packaged pharmaceutical products may contain one or more dosage forms that contain the combination of agents, and one or more dosage forms that contain one of the combination of agents, but not the other agent(s) of the combination.

Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

In particular, the selected dosage level will depend upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could begin administration of the pharmaceutical composition to dose the disclosed compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of the disclosed compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the disclosed compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a disclosed compound for the treatment of pain, a depressive disorder, or drug addiction in a patient.

In one embodiment, the compounds provided herein are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a disclosed compound and a pharmaceutically acceptable carrier.

The optimum ratios, individual and combined dosages, and concentrations of the drug compounds that yield efficacy without toxicity are based on the kinetics of the active ingredients’ availability to target sites, and are determined using methods known to those of skill in the art.

Routes of administration of any of the compositions discussed herein include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. In one embodiment, the preferred route of administration is oral.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions are not limited to the particular formulations and compositions that are described herein. For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gel caps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

For parenteral administration, the disclosed compounds may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing or dispersing agents may be used.

Kits

In an aspect, the present disclosure provides a kit for treating cancer comprising a MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and an antimetabolite, or a pharmaceutically acceptable salt thereof.

In certain embodiments, the kit comprises a pharmaceutical product comprising a pharmaceutical composition comprising a MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent; and a pharmaceutical composition comprising an antimetabolite, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

In some embodiments, the kit comprises a pharmaceutical composition comprising a MAT2A inhibitor, or a pharmaceutically acceptable salt thereof; an antimetabolite, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier or diluent.

In additional embodiments, pharmaceutical kits are provided. The kit includes a sealed container approved for the storage of pharmaceutical compositions, the container containing one of the above-described pharmaceutical compositions. In some embodiments, the sealed container minimizes the contact of air with the ingredients, e.g. an airless bottle. In other embodiments, the sealed container is a sealed tube. An instruction for the use of the composition and the information about the composition are to be included in the kit. In a particular embodiment, the compounds of the combination can be dosed on the same schedule, whether by administering a single formulation or unit dosage form containing all of the compounds of the combination, or by administering separate formulations or unit dosage forms of the compounds of the combination. However, some of the compounds used in the combination may be administered more frequently than once per day, or with different frequencies that other compounds in the combination. Therefore, in one embodiment, the kit contains a formulation or unit dosage form containing all of the compounds in the combination of compounds, and an additional formulation or unit dosage form that includes one of the compounds in the combination of agents, with no additional active compound, in a container, with instructions for administering the dosage forms on a fixed schedule.

The kits provided herein include prescribing information, for example, to a patient or health care provider, or as a label in a packaged pharmaceutical formulation. Prescribing information may include for example efficacy, dosage and administration, contraindication and adverse reaction information pertaining to the pharmaceutical formulation.

In all of the foregoing the combination of compounds of the invention can be administered alone, as mixtures, or with additional active agents.

A kit provided herein can be designed for conditions necessary to properly maintain the components housed therein (e.g., refrigeration or freezing). A kit can contain a label or packaging insert including identifying information for the components therein and instructions for their use (e.g., dosing parameters, clinical pharmacology of the active ingredient(s), including mechanism(s) of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.).

Each component of the kit can be enclosed within an individual container, and all of the various containers can be within a single package. Labels or inserts can include manufacturer information such as lot numbers and expiration dates. The label or packaging insert can be, e.g., integrated into the physical structure housing the components, contained separately within the physical structure, or affixed to a component of the kit (e.g., an ampule, syringe or vial).

Non-Limiting Exemplary Embodiments:

In further embodiments 1 to 34 below, the present disclosure includes:

1 . In an embodiment, provided is a combination product comprising a MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and an antimetabolite, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof. 2. In embodiment 2, the MAT2A inhibitor of embodiment 1 is a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

3. In embodiment 3, the MAT2A inhibitor of embodiment 1 or 2 is selected from the group consisting of a compound in Table 1 , or a pharmaceutically acceptable salt thereof.

4. In embodiment 4, the MAT2A inhibitor of embodiment 3 is Compound A, or a pharmaceutically acceptable salt thereof.

5. In embodiment 5, the MAT2A inhibitor of any one of the embodiments 1 to 3 is Compound A1 , or a pharmaceutically acceptable salt thereof.

6. In embodiment 6, the antimetabolite of any one of embodiments 1 to 5 is an antifolate or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

7. In embodiment 7, the antifolate of embodiment 6 is selected from the group consisting of a compound in Table 2, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

8. In embodiment 8, the antifolate of embodiment 6 or 7 is Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof.

9. In embodiment 9, the antifolate of embodiment 6 or 7 is Compound C, or a pharmaceutically acceptable salt thereof.

10. In embodiment 10, the antifolate of embodiment 6 or 7 is Compound D, or a pharmaceutically acceptable salt thereof.

11. In embodiment 11 , provided is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of a MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and a second pharmaceutical composition comprising a therapeutically effective amount of an antimetabolite, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

12. In embodiment 12, the MAT2A inhibitor of embodiment 11 is a compound of Formula (I), or a pharmaceutically acceptable salt thereof. 13. In embodiment 13, the MAT2A inhibitor of embodiment 11 or 12 is selected from the group consisting of a compound in Table 1 , or a pharmaceutically acceptable salt thereof.

14. In embodiment 14, the antimetabolite of any one of embodiment 11 to 13 is an antifolate, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

15. In embodiment 15, the antifolate of embodiment 14 is selected from the group consisting of a compound in Table 2, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

16. In embodiment 16, provided is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and a second pharmaceutical composition comprising a therapeutically effective amount of Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof.

17. In embodiment 17, provided is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and a second pharmaceutical composition comprising a therapeutically effective amount of Compound C, or a pharmaceutically acceptable salt thereof.

18. In embodiment 18, provided is a combination product comprising a first pharmaceutical composition comprising a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof; and a second pharmaceutical composition comprising a therapeutically effective amount of Compound D, or a pharmaceutically acceptable salt thereof.

19. In embodiment 19, provided is a MAT2A inhibitor for use in treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with an antimetabolite.

20. In embodiment 20, the MAT2A inhibitor of embodiment 19 is a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

21. In embodiment 21 , the MAT2A inhibitor of embodiment 19 or 20 is selected from the group consisting of a compound in Table 1 , or a pharmaceutically acceptable salt thereof. 22. In embodiment 22, the antimetabolite of any one of embodiments 19 to 21 is an antifolate.

23. In embodiment 23, the antifolate of embodiment 22 is selected from the group consisting of a compound in Table 2, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

24. In embodiment 24, provided is a MAT2A inhibitor for use in treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with an antifolate; wherein the MAT2A inhibitor is Compound A, or a pharmaceutically acceptable salt thereof; and the antifolate is Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof.

25. In embodiment 25, provided is a MAT2A inhibitor for use in treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with an antifolate; wherein the MAT2A inhibitor is Compound A, or a pharmaceutically acceptable salt thereof; and the antifolate is Compound C, or a pharmaceutically acceptable salt thereof.

26. In embodiment 26, provided is a MAT2A inhibitor for use in treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with an antifolate; wherein the MAT2A inhibitor is Compound A, or a pharmaceutically acceptable salt thereof; and the antifolate is Compound D, or a pharmaceutically acceptable salt thereof.

27. In embodiment 27, provided is a use of a MAT2A inhibitor in the manufacture of a medicament for treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with an antimetabolite.

28. In embodiment 28, the MAT2A inhibitor of embodiment 27 is a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

29. In embodiment 29, the MAT2A inhibitor of embodiment 27 or 28 is selected from the group consisting of a compound in Table 1 , or a pharmaceutically acceptable salt thereof.

30. In embodiment 30, the antimetabolite of any one of the embodiments 27 to 29 is an antifolate. 31 . In embodiment 31 , the antifolate of embodiment 30 is selected from the group consisting of a compound in Table 2, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

32. In embodiment 32, provided is a use of a MAT2A inhibitor in the manufacture of a medicament for treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with an antifolate; wherein the MAT2A inhibitor is Compound A, or a pharmaceutically acceptable salt thereof; and the antifolate is Compound B, or a pharmaceutically acceptable salt and/or hydrate thereof.

33. In embodiment 33, provided is a use of a MAT2A inhibitor in the manufacture of a medicament for treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with an antifolate; wherein the MAT2A inhibitor is Compound A, or a pharmaceutically acceptable salt thereof; and the antifolate is Compound C.

34. In embodiment 34, provided is a use of a MAT2A inhibitor in the manufacture of a medicament for treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with an antifolate; wherein the MAT2A inhibitor is Compound A, or a pharmaceutically acceptable salt thereof; and the antifolate is Compound D, or a pharmaceutically acceptable salt thereof.

Additional non-limiting embodiments include:

1 . A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a MAT2A inhibitor and administering to the subject a therapeutically effective amount of an antimetabolite, wherein the MAT2A inhibitor is a compound of Formula (I) or a pharmaceutically acceptable salt thereof:

wherein

X is N or C;

R³ is C_1-6 haloalkyl, halo, or C_3-6 cycloalkyl;

R² is -NR⁴R⁵;

R⁴ is hydrogen or C_1-6 alkyl; R⁵ is hydrogen, C_1-6 alkyl or C_3-6 cycloalkyl; and

R¹ is phenyl, wherein phenyl is substituted with 0-2 halo.

2. The method of embodiment 1 , wherein X is N.

3. The method of embodiment 1 , wherein X is C.

4. The method of any one of embodiments 1 to 3, wherein R⁴ is hydrogen and R⁵ is hydrogen or C_1-3 alkyl.

5. The method of any one of embodiments 1 to 4, wherein R⁵ is hydrogen or methyl.

6. The method of any one of embodiments 1 to 5, wherein R¹ is phenyl substituted with chloro.

7. The method of any one of embodiments 1 to 6, wherein R³ is C_1-3haloalkyl or halo.

8. The method of any one of embodiments 1 to 7, wherein R³ is trifluromethyl or chloro.

9. The method of any one of embodiments 1 to 6, wherein R³ is cyclopropyl.

10. The method of any one of embodiments 1 to 9, wherein the MAT2A inhibitor is selected from the group consisting of a compound from Table 1 , or a pharmaceutically acceptable salt thereof.

11. The method of any one of embodiments 1 to 10, wherein the MAT2A inhibitor is Compound A:

Compound A or a pharmaceutically acceptable salt thereof.

12. The method of any one of embodiments 1 to 10, wherein the MAT2A inhibitor is Compound A1 :

Compound A1 or a pharmaceutically acceptable salt thereof. 13. The method of any one of embodiments 1 to 12, wherein the antimetabolite is an antifolate.

14. The method of embodiment 13, wherein the antifolate is selected from the group consisting of

or a pharmaceutically acceptable salt and/or a hydrate thereof.

15. The method of embodiment 14, wherein the antifolate is Compound B:

Compound B or a pharmaceutically acceptable salt and/or a hydrate thereof.

16. The method of embodiment 14, wherein the antifolate is Compound C

Compound C or a pharmaceutically acceptable salt thereof.

17. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of an antimetabolite, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

18. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound A1 , or a pharmaceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of an antimetabolite, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

19. The method of embodiment 18 or 19, wherein the antimetabolite is an antifolate.

20. The method of embodiment 19, wherein the antifolate is Compound B, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

21. The method of embodiment 19, wherein the antifolate is Compound C, or a pharmaceutically acceptable salt thereof.

22. The method of any one of embodiment 1 to 21 , wherein the cancer is selected from the group consisting of leukemia, glioma, lung cancer, esophageal cancer, MTAP-deficient pancreatic ductal adenocarcinoma (PDAC), melanoma, pancreatic, non-small cell lung cancer, bladder cancer, astrocytoma, osteosarcoma, head and neck cancer, myxoid chondrosarcoma, ovarian cancer, endometrial cancer, breast cancer, anal cancer, stomach cancer, colon cancer, colorectal cancer, soft tissue sarcoma, non-Hodgkin lymphoma, gastric cancer esophagogastric cancer, oesophageal cancer, malignant peripheral nerve sheath tumor, mesothelioma, salivary gland tumors, urothelial cancers, gastrointestinal cancer, and sarcomas.

23. The method of any one of embodiments 1 to 22, wherein the cancer is a solid tumor or a haematological cancer.

24. The method of any one of embodiments 1 to 23, wherein the cancer is a solid malignant tumor.

25. The method of claim any one of embodiments 1 to 24, wherein the cancer is characterized by a reduction or absence of MTAP gene expression, absence of MTAP gene, reduced function of MTAP protein, reduced level or absence of MTAP protein, MTA accumulation, or combination thereof.

26. The method of any one of embodiments 1 to 25, wherein the MAT2A inhibitor and the antimetabolite are in separate dosage forms.

27. The method of any one of embodiments 1 to 25, wherein the MAT2A inhibitor and the antimetabolite are in the same dosage form.

28. A method of inhibiting tumor growth or slowing the rate of tumor growth in a subject with an MTAP deleted cancer, the method comprising: a) sequential administration of a MAT2A inhibitor and an antimetabolite, wherein i) the MAT2A inhibitor is administered prior to the antimetabolite, or ii) the antimetabolite is administered prior to the MAT2A inhibitor; or b) simultaneous administration of a MAT2A inhibitor and an antimetabolite.

29. The method of embodiment 28, wherein tumor growth is measured by change in tumor volume from a first time point to a second time point.

30. The method of embodiment 29, wherein the tumor volume at the second time point shows no increase when compared to the first time point.

31. The method of embodiment 30, wherein tumor volume decreases between the first time point and the second time point. 32. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a MAT2A inhibitor and an antimetabolite, wherein the subject has undergone a previous cancer treatment regimen without a MAT2A inhibitor.

33. The method of claim 32, wherein the MAT2A inhibitor and antimetabolite are either sequentially or simultaneously administered.

34. The method of claim 32 or 33, wherein the previous cancer treatment regimen comprised antimetabolite treatment without a MAT2A inhibitor.

35. The method of claim 34, where treatment with the MAT2A inhibitor and antimetabolite decreases the rate of tumor growth when compared to the treatment with the antimetabolite only for a similar time period.

36. The method of any one of claims 32 to 35, wherein the previous cancer treatment regimen comprised a surgical intervention.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings of the present disclosure as set forth. EXAMPLES

The compounds and methods disclosed herein are further illustrated by the following examples, which should not be construed as further limiting. The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of organic synthesis, cell biology, cell culture, and molecular biology, which are within the skill of the art.

Example 1. Efficacy of Compound A in Combination with Compound B in RT-112/84 Xenografts

The effect of Compound A and Compound B (Alimta®) as single-agent anti-tumor agents and in combination was assessed in the RT112/84 human bladder tumor cell line. Cells were expanded in RPMI-1640 (Gibco, Catalog Number 1 1875093) with 10% fetal bovine serum. These cells were free of mycoplasma and authenticated by STR profiling. One to Three million cells in log growth phase were resuspended in Hanks Balanced Salt Solution containing 50% Matrigel and implanted subcutaneously into the flank of each recipient female Crl:NU-Foxn1 nu mouse. Mice were housed in microisolator cages with corn cob bedding with additional enrichment consisting of sterile nesting material (Innovive) and Bio-huts (Bio-Serv). Water (Innovive) and diet (Teklad Global 19% Protein Extruded Diet 2919, Irradiated) were provided ad libitum. The environment was maintained on a 12-hour light cycle at approximately 68-72 °F and 40-60% relative humidity.

Tumor Volume (TV) was calculated using the following formula: TV (mm³) = (width x width x length)/2. No dose holidays were provided for during the study and all mice were euthanized following the final dose on Day fourteen. Tumor growth inhibition (TGI) was calculated by [(TV controlfinal - TV treatedfi_nai)/(TV controlfinal - TV contro nitiai) x 100]. TV was analyzed for statistical significance utilizing GraphPad Prism version 9.1.0. Repeated Measures 2-Way ANOVA with Tukey’s Multiple Comparisons was utilized, and P-values were presented from the final tumor measurement and were considered statistically significant if less than 0.05.

Compound A and Compound B were found to result in no single agent activity in RT112/84. When Compound A was combined with Compound B, the combination provided significant anti-tumor activity.

Study 1 :

Mean tumor volume at dosing start was approximately 156 mm³, with ten mice randomized to each treatment group. Mice were dosed orally, once per day, with Vehicle, Compound A at 10 mg/kg, or dosed for 5 consecutive days followed by two days holiday by intraperitoneal (IP) injection of Compound B at 75 mg/kg, or Compound A at 10 mg/kg and Compound B at 75 mg/kg. The vehicle group was a combination of Vehicle A (for Compound A, 0.5% 400 cps methylcellulose with 0.5% Tween-80 in sterile water) and Vehicle B (for Compound B, Saline).

Treatment with Compound A resulted in -4% TGI, while Compound B alone resulted in 14% TGI. The combination of Compound A and Compound B at resulted in 71% TGI, see Error! Reference source not found, and Error! Reference source not found.. The combination of Compound A and Compound B significantly inhibited tumor growth.

Table 3 Summary of Efficacy of Compound A and Compound B in RT112/84

Study 2:

Mean tumor volume at dosing start was approximately 250 mm³, with eight mice randomized to each treatment group. Mice were dosed orally, once per day, with Vehicle, Compound A at 10 mg/kg, or dosed for 5 consecutive days followed by two days holiday by intraperitoneal (IP) injection of Compound B at 75 mg/kg, or Compound A at 10 mg/kg and Compound B at 75 mg/kg. The vehicle group was a combination of Vehicle A (for Compound A, 0.5% 400 cps methylcellulose with 0.5% Tween-80 in sterile water) and Vehicle B (for Compound B, Saline).

Treatment with Compound A resulted in 26% TGI, while Compound B alone resulted in 21% TGI. The combination of Compound A and Compound B at resulted in 79% TGI, see Table 4 and Error! Reference source not found.. The combination of Compound A and Compound B significantly inhibited tumor growth.

Table 4 Summary of Efficacy of Compound A and Compound B in RT112/84

The MTAP-deleted bladder CDX model RT-112/84 was found to be resistant to either Compound A or Compound B. Compound A when combined with Compound B provides significant anti-tumor activity.

Example 2. Efficacy of MAT2A inhibitor Compound A in combination with anti- metabolite Compound C in CTG-0707 Gastric cancer PDX

The anti-tumor activity of Compound A and Compound C as single-agents and in combination was assessed in the CTG-0707 Gastric cancer patient-derived xenograft model (PDX).

Tumor Volume (TV) was calculated using the following formula: TV (mm3) = (width x width x length)/2. Tumor growth inhibition (TGI) was calculated by [(TV controlfinal - TV treatedfinal)/(TV controlfinal - TV controlinitial) x 100]. TV was analyzed for statistical significance utilizing GraphPad Prism version 9.1.0. Repeated Measures 2-Way ANOVA with Tukey’s Multiple Comparisons was utilized, and P-values were presented from the final tumor measurement and were considered statistically significant if less than 0.05.

Mean tumor volume at dosing start was approximately 213 mm3, with nine mice randomized to each treatment group. Mice were dosed orally, once per day, with Vehicle, Compound A at 3 mg/kg or 10 mg/kg, or once weekly with 50 mg/kg IP Compound C, or Compound A at 3 mg/kg or 10 mg/kg and Compound C at 50 mg/kg IP. The vehicle group was a combination of Vehicle A (for Compound A, 0.5% 400 cps methylcellulose with 0.5% Tween-80 in sterile water) and Vehicle B (for Compound C, Saline).

Treatments as monotherapies resulted in 9% TGI for 3 mg/kg QD Compound A, 81% TGI for 3 mg/kg QD Compound A, and 11% TGI for Compound C. The combination of Compound A at 3 mg/kg QD and Compound C produced 66% TGI. The combination of Compound A at 10 mg/kg QD and Compound C produced 68% TGI, See Table 5 and Figure 3. The combination of Compound A and Compound C produced significant tumor growth inhibition when Compound A was administered at the sub-maximally efficacious dose of 3 mg/kg QD.

Table 5 Summary of Efficacy of Compound A and Compound C in CTG-0707

The MTAP-deleted Gastric PDX CTG-0707 was found to be sensitive to Compound A, but not Compound C. When Compound A was administered at a sub-maximally efficacious dose of 3 mg/kg QD and combined with Compound C, a significant anti-tumor growth response occurred. The response observed with Compound A at 3 mg/kg QD when combined with Compound C was similar to that observed for Compound A at 10 mg/kg QD as a monotherapy.

Compound C was not found to result in single agent activity. Compound A resulted in significant efficacy at 10 mg/kg. When Compound A was combined with Compound C, the combination provided significant tumor growth inhibition compared to when 3 mg/kg Compound A was combined with Compound C. Compound A at 10 mg/kg QD produced the maximal tumor growth inhibition and no further combination benefit was determined. The combination of Compound A and Compound C produced significant tumor growth inhibition when Compound A was administered at the sub-maximally efficacious dose of 3 mg/kg QD.

Example 3. Efficacy of MAT2A inhibitor Compound A in combination with anti- metabolite Compound B or Compound C in NCI-H838 NSCLC xenografts Study 1 :

The anti-tumor effect of Compound A, Compound B (Alimta®), and Compound C as single agents and in combination was assessed in the NCI-H838 human NSCLC xenograft. Cells were expanded in RPMI-1640 with 10% fetal bovine serum. These cells were free of mycoplasma and authenticated by STR profiling. Ten million cells in log growth phase were resuspended in RPMI containing 50% Matrigel and implanted subcutaneously into the flank of each recipient female BALB/c nude mouse.

Tumor Volume (TV) was calculated using the following formula: TV (mm³) = (width x width x length)/2. Tumor growth inhibition (TGI) was calculated by [(TV controlfinal - TV treatedfinai)/(TV controlfinal - TV controlinitial) x 100]. TV was analyzed for statistical significance utilizing GraphPad Prism version 9.1.0. Repeated Measures 2-Way ANOVA with Tukey’s Multiple Comparisons was utilized, and P-values were presented from the final tumor measurement and were considered statistically significant if less than 0.05.

Mean tumor volume at dosing start was approximately 145 mm³, with ten mice randomized to each treatment group. Mice were dosed orally, once per day, with Vehicle, Compound A at 10 mg/kg, or dosed for 5 consecutive days followed by two days holiday by intraperitoneal (IP) injection of Compound B at 75 mg/kg, or once weekly with 50 mg/kg IP Compound C, or Compound A at 10 mg/kg and Compound B at 75 mg/kg, or Compound A at 10 mg/kg and Compound C at 50 mg/kg. The vehicle group was a combination of Vehicle A (for Compound A, 0.5% 400 cps methylcellulose with 0.5% Tween-80 in sterile water) and Vehicle B (for Compound B and C, Saline).

Treatments as monotherapies resulted in 94% TGI for Compound A, 56% TGI for Compound B and 74% TGI for Compound C. The combination of Compound A and B produced a mean TGI of 87%. In addition, the combination of Compound A and Compound B increased the rate of mice with tumor regressions (a reduction in tumor volume beyond that of the tumor volume at the initiation of treatment; e.g. >100% TGI) from 20% to 30%. The combination of Compound A and Compound C resulted in 98% TGI and increased the tumor regressions to 40%, See Table 6 and Error! Reference source not found.. The combination of Compound A and Compound B or Compound C enriched for mice with regressions and resulted in significant tumor inhibition.

Table 6 Summary of Efficacy of Compound A and Compound B or Compound C in H838

Study 2:

The pharmacodynamic modulation of s-adenosyl methionine (SAM) by administration of Compound A, Compound B, or Compound C as single-agent anti-tumor agents and in combination was assessed in the NCI-H838 human NSCLC tumor xenograft. Cells were expanded in RPMI-1640 with 10% fetal bovine serum. These cells were free of mycoplasma and authenticated by STR profiling. Ten million cells in log growth phase were resuspended in RPMI containing 50% Matrigel and implanted subcutaneously into the flank of each recipient female BALB/c nude mouse.

Mean tumor volume at dosing start was approximately 477 mm³, with 9 mice randomized to each treatment group. Mice were dosed orally, once per day, with Vehicle, Compound A at 10 mg/kg, or dosed for 5 consecutive days by intraperitoneal (IP) injection of Compound B at 75 mg/kg, or twice with 50 mg/kg IP Compound C (day 1 and day 5), or Compound A at 10 mg/kg and Compound B at 75 mg/kg, or Compound A at 10 mg/kg and Compound C at 50 mg/kg. The vehicle group was a combination of Vehicle A (for Compound A, 0.5% 400 cps methylcellulose with 0.5% Tween-80 in sterile water) and Vehicle B (for Compound B and C, Saline). On Day 5, plasma and tumor were collected following 2-hours, 8-hours, or 24-hours post the last dose on day 5. Repeated Measures 2-Way ANOVA with Tukey’s Multiple Comparisons was utilized, and P-values were presented from the absolute individual SAM measurements at indicated time-points and were considered statistically significant if less than 0.05.

Treatment with Compound B or Compound C significantly increased SAM levels in plasma and tumor as compared to vehicle alone. Treatment with the combination of Compound A and Compound B or Compound C resulted in decreased SAM in both plasma (Table 7) and tumor homogenates (Table 8). The combination of Compound A with Compounds B or C maintained SAM reduction for 24-hours post the final dose on Day 5 in both plasma (Figure 5) and tumor (Figure 6).

Table 7. Plasma s-adenosyl methionine modulation in NCI-H838 xenografts

Table 8. Tumor s-adenosyl methionine modulation in NCI-H838 xenografts

The MTAP-deleted NSCLC xenograft model NCI-H838 was found to be sensitive to Compound A, Compound B, and Compound C. Compound A when combined with Compound B or Compound C provided an improvement for the individual response rate and additionally benefitted the response by providing early disease stabilization. Administration of Compounds B and C alone caused an increase in SAM in both tumor and plasma, while a sustained decrease in plasma and tumor SAM was observed upon administration of Compound A either as a single agent or in combination with Compound B or C.

Example 4: In Vitro Proliferation Screen Identifies Combination Benefit with MAT2A and Pemetrexed Inhibition

Materials and Methods

A 10-day proliferation screen was performed in a panel of 10 MTAP-deficient pancreatic, non-small cell lung, and bladder cancer cell lines. Optimal cell seeding for all cell lines was determined by assessing the growth over a range of seeding densities in a 384- well format to identify conditions that permitted proliferation for 10 days. Cells were then plated at the optimal seeding density in the presence of 20-150 nM of Compound A or DMSO vehicle control. Cells were incubated at 37°C with 5% CO2 for 4 days to allow for target engagement of the pre-treatment compound. Maintaining the pre-treatment condition, cells were also treated with an 11 -point, three-fold titration series of compounds from a chemically diverse library of 424 compounds. The combination compound concentrations ranged from 0.2 nM to 14,679 nM. A plate of cells was harvested at the time of combination compound addition to quantify the number of cells at the start of the combination (To). For cell quantification, the harvested cells were lysed with Promega CellTiter-Glo (CTG) reagent according to the manufacturer’s protocol and the chemiluminescent signal was detected on a Synergy Neo plate reader. CTG estimates cell number through detection of cellular ATP levels. Cells were incubated with the drug combinations at 37°C with 5% CO2 for an additional 6 days for a total of a 10-day assay including the pre-treatment. Cells were then lysed with CTG and the chemiluminescent signal was measured.

Analysis

CTG values obtained after the 10-day treatment were background subtracted, expressed as a percent of the To value, and plotted against compound concentration. Data were fit with a four-parameter equation to generate concentration response curves. Growth IC50 values and maximal growth inhibition were compared between the DMSO and the Compound A pre-treated cells for each combination titration. Combination hit calling was based on the observation of >2-fold shift in growth IC50 and/or greater than 20% decrease in maximal growth inhibition.

Results

Pemetrexed (Compound B) was tested in combination with Compound A in 10 MTAP-deficient cancer cell lines. In four of these cell lines, i.e., UMUC5, NCI-H2228, SW900 and NCI-H2170 cell lines, the combination showed 3.3-, 2.3-, 2.2- and 5.2-fold shifts in growth IC₅₀, respectively (Table 9). Pemetrexed (Compound B) in combination with Compound A was also examined in the NCI-H838 cell line where two different pre-treatment doses of Compound A were tested in separate experiments. In the first experiment, a 9.2- fold shift in the Pemetrexed growth IC₅₀ was observed when cells were pre-treated with 20nM Compound A. In the second experiment, a 4.7-fold shift in the Pemetrexed growth IC₅₀ was observed when cells were pre-treated with 150nM Compound A (Table 9).

Table 9: Growth IC50 values with and without Compound A pre-treatment in 10 cancer cell lines

Example 5: In Vitro Synergistic Growth Inhibition with MAT2A Inhibitor and Pemetrexed Combination

Materials and Methods

A 10-day proliferation assay was performed in a panel of MTAP-deficient pancreatic, non-small cell lung, and bladder cancer cell lines. Optimal cell seeding for all cell lines was determined by assessing the growth over a range of seeding densities in a 384-well format to identify conditions that permitted proliferation for 10 days. Cells were then plated at the optimal seeding density and treated with a double titration of a 16-point, two-fold dilution series of Compound A and a 16-point, two-fold dilution series of Pemetrexed (Compound B). The double titrations were compared to 16-point, two-fold dilution series of each single agent. Concentrations tested for Compound A and Pemetrexed alone or in combination ranged from 0.6 nM to 19,250 nM. An untreated plate of cells was harvested at the time of dosing to quantify the number of cells at the beginning of the combination (To) using CTG as described in example 1. Combination plates were incubated at 37 °C in 5% CO2for a total of 10 days prior to cell lysis and CTG measurement.

Analysis

CTG values obtained after the 10-day treatment were background subtracted and expressed as a percent of the To value. Data were plotted against compound concentration and fit with a four-parameter equation to generate single agent concentration response curves. Percent inhibition was determined for each combination and single agent concentration. Synergistic growth inhibition was assessed using the excess over Bliss model for the combinations of compound doses across the full dose range tested. The observed growth inhibition was compared to the predicted inhibition based on the additive activity of each single agent. Predicted inhibition was calculated using (Ea+Eb) - (Ea*Eb) where E equals the effect (inhibition) of each single agent (a and b). A difference between the observed inhibition percentage and the predicted inhibition percentage greater than 10% was considered synergistic. An excess over Bliss score was only calculated for combinations eliciting >20% growth inhibition. Since excess over Bliss is calculated based on growth inhibition, scores within cytotoxic dose ranges are not relevant.

Results

Synergistic growth inhibition was observed with Compound A in combination with Pemetrexed across numerous combination concentrations as assessed by excess over Bliss in 6/6 MTAP-deficient cell lines (Figure 8 and Tables 10A-10F). Due to the sharp dose response curves observed with single agent Pemetrexed, the window available for observation of combination synergy within this assay design was narrow for some cell lines. For example, 38nM single agent Pemetrexed is sufficient to maximally inhibit cell growth in the SW900 cell line (Figure 7). Therefore, the dose range in which combination synergy can be observed in SW900 is below the 38nM Pemetrexed dose. Despite this limited window, combination synergy was observed at doses below that needed for maximal single agent inhibition in all six MTAP-deficient cell lines. Beyond the combination doses where synergy was observed, additive growth inhibition was observed at additional concentrations in all /WTAP-deficient cell lines tested.

Synergy is measured at all tested concentrations of Compound A across all concentrations of Pemetrexed using excess over Bliss analyses for the NCI-H838 (A), SW900 (B), UMUC5 (C), UMUC11 (D), HuP-T4 (E) and Pane 03.27 MTAP CRISPR Knock- out Clone 31 (F) cell lines. Scores between 1 and 10 were considered additive, scores >10 were considered synergistic. Excess over Bliss scores were only calculated for combinations exhibiting greater than 20% growth inhibition. All reported values are an average of two biological replicates.

Table 10A: Excess over Bliss Synergy of Compound A and Pemetrexed Co-treatment Combinations in NCI-H838 cell line.

Table 10A (cont’d)

Table 10B: Excess over Bliss Synergy of Compound A and Pemetrexed Co-treatment Combinations in SW900 cell line

Table 10B (cont’d)

Table 10C: Excess over Bliss Synergy of Compound A and Pemetrexed Co-treatment Combinations in UMUC5 cell line

Table 10C (cont’d)

Table 10D: Excess over Bliss Synergy of Compound A and Pemetrexed Co-treatment Combinations in UMUC11 cell line

Table 10D (cont’d)

Table 10E: Excess over Bliss Synergy of Compound A and Pemetrexed Co-treatment Combinations in HuP-T4 cell line

Table 10E (cont’d)

Table 10F: Excess over Bliss Synergy of Compound A and Pemetrexed Co-treatment Combinations in Pane 03.27 MTAP CRISPR Knock-Out Clone 31 cell line

Table 10F (cont’d)

Example 6: Efficacy of Compound A in Combination with Compound B in MTAP deleted Lung Tumor PDX model LXFA737

The anti-tumor efficacy of Compound A and Compound B as single agent and in combination was assessed in MTAP deleted NSCLC PDX mouse model - LXFA737. Tumors were implanted subcutaneously (s.c.) in one flank of NMRI nu/nu mice. Animals were housed in individually ventilated cages (TECNIPLAST Sealsafe-IVC-System, TECNIPLAST, Hohenpeissenberg, Germany). They were kept under a 14L:10D artificial light cycle. The temperature inside the cages was maintained at 22 -26 °C with a relative humidity of 45-65% and 60-65 air changes/hour in the cage.

The animals were fed autoclaved Teklad Global Extruded 19% Protein Rodent Diet from Envigo RMS SARL and had access to sterile filtered and acidified (pH 2.5) tap water that was changed twice weekly. Feed and water were provided ad libitum. All materials were autoclaved prior to use

Mice were housed in microisolator cages with corn cob bedding with additional enrichment consisting of sterile nesting material (Innovive) and Bio-huts (Bio-Serv). Water (Innovive) and diet (Teklad Global 19% Protein Extruded Diet 2919, Irradiated) were provided ad libitum

Tumor Volume (TV) was calculated using the following formula: TV (mm³) = (width x width x length)/2. No dose holidays were provided for during the study and all mice were euthanized following the final dose on day fourteen. Tumor growth inhibition (TGI) was calculated by [(TV control_final - TV treated_final)/(TV control_final - TV control_initial) x 100]. TV was analyzed for statistical significance utilizing GraphPad Prism version 9.1.0. Repeated Measures 2-Way ANOVA with Tukey’s Multiple Comparisons was utilized, and P-values were presented from the final tumor measurement and were considered statistically significant if less than 0.05.

Study:

The antitumor efficacy of Compound A and B was assessed in monotherapy and in combination in the lung tumor model LXFA737 implanted s.c. in NMRI nu/nu mice. Compound A monotherapy slowed LXFA737 tumor growth almost to stasis with a TGI value of 91.4% (Figure 9, Table 11). The reduction in tumor volume compared to the control group was statistically significant (Kruskal-Wallis test/Dunn’s post test). Compound B monotherapy slowed LXFA737 tumor growth with a TGI value of 64.8% without a statistically significant reduction in tumor volume compared to the control group (Figure 9, Table 11). Compounds A and B in combination inhibited LXFA737 tumor growth with a TGI value of 105.4% and a tumor regression of 66.6% (Figure 9, Table 11). The reduction in tumor volume compared to the control group and pemetrexed monotherapy was statistically significant (Kruskal-Wallis test/Dunn’s post test).

Table 11: Summary of Efficacy of Compound A and Compound B in LXFA737

Example 7: MAT2A and Pemetrexed Demonstrate Synergistic Growth Inhibition in In Vitro MTAP-Deficient Models

Materials and Methods

A panel of 13 MTAP-deficient non-small cell lung cancer (NSCLC), bladder, pancreatic, gastric, esophageal, and head and neck squamous cell carcinoma (HNSCC) cancer cell lines was used to assess the combinatory effect of a MAT2A inhibitor (Compound A) and pemetrexed (Compound B) using Horizon Discovery’s High Throughput Screening platform. To determine the optimal concentration range of each compound in each cell line for the combination screen, cells were seeded at 150 cells/well for Compound A or 500 cells/well for Compound B in a 384-well plate, and 24 hrs later, treated with a 9- point titration of each compound as a single agent for 6 days. For the combination screen, cells were seeded at 150 cells/well in a 384-well plate, and 24 hrs later, co-treated with a 5- point titration of each compound in an optimized 6x6 dose matrix for 6 days. Cells were subsequently lysed with Promega CellTiter-Glo (CTG) 2.0 reagent according to the manufacturer's protocol and the chemiluminescent signal was measured using a PerkinElmer EnVision plate reader for cell number quantification. A plate of untreated cells was harvested at the time of compound addition (To or time zero) for cell number quantification using CTG. Each data point was run in technical triplicate.

The percent of growth inhibition is calculated as: lf T<V₀ : 100*(1-(T-V₀)/V₀) lf T> V₀ : 100*(1-(T-V_o)/(V-V₀)) where T is the signal measure for a test article, V is the vehicle-treated control measure, and V₀ is the vehicle control measure at To). This formula is derived from the growth inhibition calculation used in the National Cancer Institute’s NCI-60 high throughput screen. The percent growth inhibition is used to generate dose-response curves and Gl₅₀ calculations for single drug activity and drug combination synergy in Horizon’s Chalice Analyzer software. 100% and > 100% growth inhibition represented cytostasis and cytotoxicity, respectively.

Synergistic growth inhibition was assessed using the Loewe additivity model. The observed growth inhibition at each dose was compared to the predicted inhibition based on the additive activity of either drug combined with itself. The calculation for Loewe additivity is: I_{L oewe} that satisfies (X/X_I) + (Y/Y_I) = 1 , where X_I and Y_I are the single agent effective concentrations for the observed combination effect I. A difference between the observed and predicted growth inhibition > 20% was considered synergistic.

The strength of synergy was quantified using Horizon’s Synergy Score, which is calculated as:

Synergy Score = log f_x log f_Y Z max(0,l_data)(l_data - I _Loewe)

The fractional inhibition for each component agent and combination point in the matrix is calculated relative to the median of all untreated/vehicle-treated control wells. The Synergy Score equation integrates the observed growth inhibition each at point in the dose matrix in excess over predicted growth inhibition and dilution factors of individual compounds. The inclusion of positive inhibition gating or an l_data multiplier removes noise near the zero-effect level, and biases results for synergistic interactions at that occur at high activity levels. Combinations with higher maximum growth inhibition effects or those which are synergistic at low concentrations will have higher Synergy Scores. A Synergy Score of > 3.0 was considered synergistic, given that it corresponded with high growth inhibition and > 20% excess over Loewe additivity model at multiple dose points. Results

The combination of Compound A and B was synergistic (Synergy Score > 3) in 5/13 MTAP-deficient cell lines, NCI-H838 (NSCLC), RT112 (bladder), RT4 (bladder), KP4 (pancreatic), and MIAPACA-2 (pancreatic) (Table 12). This is supported by the enhanced growth inhibition observed at multiple evaluated doses compared to the effect of either agent alone, which is reflected in the Loewe additivity model of synergy (Figures 10A-10G and 11A-11G). Additionally, while this combination was synergistic across models of multiple tumor types, it was synergistic in all evaluated models of bladder and pancreatic cancer, suggesting that patients having these tumor types may be sensitive to this combination therapy.

For Figures 10A-10G, the growth inhibition is presented as a percent of TO for each cell line in a 6x6 dose matrix. 50-100% growth inhibition is highlighted in light gray, with 100% growth inhibition representing cytostasis. Greater than 100% growth inhibition is highlighted in dark gray and represents cytotoxicity.

For Figures 11 A-11G, synergy is measured at all tested concentrations of Compound A and Compound B using the Loewe additivity model for each cell line. Scores >20 are highlighted in dark gray and are considered synergistic.

Table 12: Synergy scores for the combination of Compound A and either Compound B or D in 13 MTAP-deficient cell lines.

Scores > 3.0 are considered synergistic.

Particular embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Upon reading the foregoing, description, variations of the disclosed embodiments may become apparent to individuals working in the art, and it is expected that those skilled artisans may employ such variations as appropriate. Accordingly, it is intended that the invention be practiced otherwise than as specifically described herein, and that the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto 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 otherwise clearly contradicted by context.

All patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls. Other embodiments are within the following claims.

Claims

1 . A combination therapy comprising a MAT2A inhibitor and an antimetabolite for use in the treatment of cancer in a subject in need thereof, wherein the MAT2A inhibitor is a compound of Formula (I) or a pharmaceutically acceptable salt thereof:

wherein

X is N or C;

R³ is C_1-6 haloalkyl, halo, or C_3-6 cycloalkyl;

R² is -NR⁴R⁵;

R⁴ is hydrogen or C_1-6 alkyl;

R⁵ is hydrogen, C_1-6 alkyl, or C_3-6 cycloalkyl; and

R¹ is phenyl, wherein phenyl is substituted with 0-2 halo.

2. The combination therapy for use according to claim 1 , wherein X is N.

3. The combination therapy for use according to claim 1 , wherein X is C.

4. The combination therapy for use according to any one of claims 1 to 3, wherein R⁴ is hydrogen and R⁵ is hydrogen or C_1-3alkyl .

5. The combination therapy for use according to any one of claims 1 to 4, wherein R⁵ is hydrogen or methyl.

6. The combination therapy for use according to any one of claims 1 to 5, wherein R¹ is phenyl substituted with chloro.

7. The combination therapy for use according to any one of claims 1 to 6, wherein R³ is C1-3 haloalkyl or halo.

8. The combination therapy for use according to any one of claims 1 to 7, wherein R³ is trifluromethyl or chloro.

9. The combination therapy for use according to any one of claims 1 to 6, wherein R³ is cyclopropyl.

10. The combination therapy for use according to any one of claims 1 to 9, wherein the

5 MAT2A inhibitor is selected from the group consisting of a compound from Table 1 , or a pharmaceutically acceptable salt thereof.

11. The combination therapy for use according to any one of claims 1 to 10, wherein the MAT2A inhibitor is Compound A:

10

Compound A or a pharmaceutically acceptable salt thereof.

12. The combination therapy for use according to any one of claims 1 to 10, wherein the

15 MAT2A inhibitor is Compound A1 :

Compound A1 or a pharmaceutically acceptable salt thereof.

20 13. The combination therapy for use according to any one of claims 1 to 12, wherein the antimetabolite is an antifolate.

14. The combination therapy for use according to claim 13, wherein the antifolate is selected from the group consisting of

62

or a pharmaceutically acceptable salt and/or a hydrate thereof.

15. The combination therapy for use according to claim 14, wherein the antifolate is Compound B:

or a pharmaceutically acceptable salt and/or a hydrate thereof.

16. The combination therapy for use according to claim 14, wherein the antifolate is Compound C

or a pharmaceutically acceptable salt thereof.

17. A combination therapy comprising Compound A, or a pharmaceutically acceptable salt thereof, and an antimetabolite, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof, for use in the treatment of cancer.

18. A combination therapy comprising Compound A1 , or a pharmaceutically acceptable salt thereof, and an antimetabolite, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof, for use in the treatment of cancer.

19. The combination therapy for use according to claim 18 or 19, wherein the antimetabolite is an antifolate.

20. The combination therapy for use according to claim 19, wherein the antifolate is Compound B, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

21. The combination therapy for use according to claim 19, wherein the antifolate is Compound C, or a pharmaceutically acceptable salt thereof.

22. The combination therapy for use according to any one of claims 1 to 21 , wherein the cancer is selected from the group consisting of leukemia, glioma, lung cancer, esophageal cancer, MTAP-deficient pancreatic ductal adenocarcinoma (PDAC), melanoma, pancreatic, non-small cell lung cancer, bladder cancer, astrocytoma, osteosarcoma, head and neck cancer, myxoid chondrosarcoma, ovarian cancer, endometrial cancer, breast cancer, anal cancer, stomach cancer, colon cancer, colorectal cancer, soft tissue sarcoma, non-Hodgkin lymphoma, gastric cancer, esophagogastric cancer, oesophageal cancer, malignant peripheral nerve sheath tumor, mesothelioma, salivary gland tumors, urothelial cancers, gastrointestinal cancer, and sarcomas.

23. The combination therapy for use according to any one of claims 1 to 22, wherein the cancer is a solid tumor or a haematological cancer.

24. The combination therapy for use according to any one of claims 1 to 23, wherein the cancer is a solid malignant tumor.

25. The combination therapy for use according to claim any one of claims 1 to 24, wherein the cancer is characterized by a reduction or absence of MTAP gene expression, absence of MTAP gene, reduced function of MTAP protein, reduced level or absence of MTAP protein, MTA accumulation, or combination thereof.

26. The combination therapy for use according to any one of claims 1 to 25, wherein the MAT2A inhibitor and the antimetabolite are in separate dosage forms.

27. The combination therapy for use according to any one of claims 1 to 25, wherein the MAT2A inhibitor and the antimetabolite are in the same dosage form.

28. A combination product comprising a MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, and an antimetabolite, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

29. The combination product of claim 28, wherein the antimetabolite is an antifolate.

30. The combination product of claim 28 or 29, wherein the MAT2A inhibitor is a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and the antifolate is selected from the group consisting of Compound B, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof, and Compound C, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

31. The combination product of claim 30, wherein the MAT2A inhibitor is selected from a group consisting of Compound A, or a pharmaceutically acceptable salt thereof, and Compound A1 , or a pharmaceutically acceptable salt thereof, and the antifolate is selected from the group consisting of Compound B, or a pharmaceutically acceptable salt and/or a hydrate thereof, and Compound C, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof.

32. A MAT2A inhibitor for use in treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with an antimetabolite.

33. The MAT2A inhibitor for use of claim 32, wherein the antimetabolite is an antifolate.

34. The MAT2A inhibitor for use of claim 32 or 33, wherein the MAT2A inhibitor is a compound of Formula (I), or a pharmaceutically acceptable salt thereof; and the antifolate is selected from the group consisting of Compound B, or a pharmaceutically acceptable salt and/or hydrate thereof, and Compound C, or a pharmaceutically acceptable salt thereof and/or hydrate thereof.

35. The MAT2A inhibitor for use of claim 34, wherein the MAT2A inhibitor is Compound A, or a pharmaceutically acceptable salt thereof, or Compound A1, or a pharmaceutically acceptable salt thereof.

36. Use of a MAT2A inhibitor in the manufacture of a medicament for treating cancer, wherein the MAT2A inhibitor is to be administered simultaneously or sequentially with an antimetabolite.

37. The use of claim 36, wherein the antimetabolite is an antifolate.

38. The use of claim 36 or 37, wherein the MAT2A inhibitor is a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and the antifolate is selected from the group consisting of Compound B, or a pharmaceutically acceptable salt thereof and/or a hydrate thereof, and Compound C, or a pharmaceutically acceptable salt thereof and/or hydrate thereof.

39. The use of claim 38, wherein the MAT2A inhibitor is Compound A or a pharmaceutically acceptable salt thereof, or Compound A1 , or a pharmaceutically acceptable salt thereof.

40. A combination therapy for use in inhibiting tumor growth or slowing the rate of tumor growth in a subject with an MTAP deleted cancer, the therapy comprising: a) sequential administration of a MAT2A inhibitor and an antimetabolite, wherein i) the MAT2A inhibitor is administered prior to the antimetabolite, or ii) the antimetabolite is administered prior to the MAT2A inhibitor; or b) simultaneous administration of a MAT2A inhibitor and an antimetabolite.

41. The combination therapy for use according to claim 40, wherein tumor growth is measured by change in tumor volume from a first time point to a second time point.

42. The combination therapy for use according to claim 41 , wherein the tumor volume at the second time point shows no increase when compared to the first time point.

43. The combination therapy for use according to claim 42, wherein tumor volume decreases between the first time point and the second time point.

44. A combination therapy for use in treating cancer in a subject in need thereof, the therapy comprising administering to the subject a MAT2A inhibitor and an antimetabolite, wherein the subject has undergone a previous cancer treatment regimen without a MAT2A inhibitor.

45. The combination therapy for use according to claim 44, wherein the MAT2A inhibitor and antimetabolite are either sequentially or simultaneously administered.

46. The combination therapy for use according to claim 44 or 45, wherein the previous cancer treatment regimen comprised antimetabolite treatment without a MAT2A inhibitor.

47. The combination therapy for use according to claim 46, where treatment with the MAT2A inhibitor and antimetabolite decreases the rate of tumor growth when compared to the treatment with the antimetabolite only for a similar time period.

48. The combination therapy for use according to any one of claims 44 to 47, wherein the previous cancer treatment regimen comprised a surgical intervention.