Pharmacological inhibition of MGAT1, methods of treating cancer, and therapeutic compositions

Abstract

Disclosed herein are pharmaceutical agents that inhibit MGAT1 and uses in managing diseases and conditions associated with MGAT1 interactions, such as cancer. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering to a subject in need thereof an effective amount of a pharmaceutical agent that inhibits MGAT1 or suppress MGAT1 and CD73 binding interactions. In certain embodiments, the compound is N-(3-(5,6-dimethylbenzo[d]oxazol-2-yl)-4-hydroxy-5-methylphenyl)benzo[d][1,3]dioxole-5-carbox-amide (W-GTF01), derivative, prodrug, ester, or salt thereof.

Description

[0001] PHARMACOLOGICAL INHIBITION OF MGAT1, METHODS OF TREATING CANCER, AND THERAPEUTIC COMPOSITIONS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 725,474 filed November 26, 2024. The entirety of this application is hereby incorporated by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under CA258857, CA258765, and CA250110 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] BACKGROUND

[0007] Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited response to immunotherapeutic agents. PD-L1 inhibitors demonstrate promising clinical benefits in certain subsets of patients with TNBC. However, the intricacies of the tumor microenvironment, with its inherent immunosuppressive properties and varied immunogenicity profiles, limit the broad efficacy of such therapies. Thus, there is a need to identify improvements.

[0008] Alpha- 1, 3 -mannosyl-gly coprotein 2-beta-N-acetylglucosaminyltransferase (MGAT1) is an enzyme that plays a role in the process of N-linked glycosylation. MGAT1 glycosylation is implicated in a number of cancers.

[0009] Zavareh et al. report suppression of cancer progression by MGAT1 shRNA knockdown PLoS ONE, 2012, 7(9): e43721.

[0010] Li et al. report N-acetylglucosaminyltransferase I promotes glioma cell proliferation and migration through increasing the stability of the glucose transporter GLUT1. FEBS Lett, 2020, 594, 358-366.

[0011] Oliveira et al. report glycoproteome remodeling in MLL-rearranged B-cell precursor acute lymphoblastic leukemia. Theranostics, 2021, 11, 9519-9537. Liu et al. report microRNA-204-3p inhibits metastasis of pancreatic cancer via downregulating MGAT1. J Buon, 2021, 26, 2149-2156.

[0012] Yu et al. report mevalonate pathway inhibition slows breast cancer metastasis via reduced N-glycosylation abundance and branching. Cancer Res, 2021, 81, 2625-2635.

[0013] Bi et al report microRNA miR-331-3p suppresses osteosarcoma progression via the BcL 2 / Bax and Wnt / beta-Catenin signaling pathways and the epithelial-mesenchymal transition by targeting N-acetylglucosaminyltransferase I (MGAT1). Bioengineered, 2022, 13, 14159-14174.

[0014] Zhu et al. report LINC00173 promotes Wilms' tumor progression through MGAT1- mediated MUC3 A N-glycosylation. Cell Cycle, 2022, 21, 1795-1810.

[0015] Li et al. report circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression. J Exp Clin Cancer Res, 2023, 42, 313.

[0016] Yang et al. report MGAT1 / 2 and SPPL3 are potential targets for blocking cancer cell invasion. Cell Reports, 2023, 42, 112065.

[0017] Jiang et al. report a role of glycosylation-related gene MGAT1 in pancreatic ductal adenocarcinoma. Front Immunol, 2024, 15: 1438935.

[0018] Rodriguez et al. report the transcriptional landscape of glycosylation-related genes in cancer. iScience, 27, 109037, 2024.

[0019] Stevens et al. report compounds for upregulating interferon regulatory factor 3 (IRF3) activity. WO2021226129

[0020] References cited herein are not an admission of prior art.

[0021] SUMMARY

[0022] Disclosed herein are pharmaceutical agents that inhibit MGAT1 and uses in managing diseases and conditions associated with MGAT1 interactions, such as cancer. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering to a subject in need thereof an effective amount of a pharmaceutical agent that inhibits MGAT1 or suppress MGAT1 and CD73 binding interactions. In certain embodiments, the compound is N-(3- (5,6-dimethylbenzo[d]oxazol-2-yl)-4-hydroxy-5-methylphenyl)benzo[d][l,3]dioxole-5-carbox- amide (W-GTF01), derivative, prodrug, ester, or salt thereof. In certain embodiments, this disclosure relates to pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound disclosed herein that inhibits MGAT1 or MGAT1 and CD73 binding interactions, derivatives, prodrugs, esters, or salts thereof.

[0023] In certain embodiments, this disclosure relates to methods of treating or preventing diseases and conditions associated with inhibits MGAT1 inhibition or MGAT1 and CD73 binding interactions, such as cancer by administering an effective amount of a compound or pharmaceutical agent as disclosed herein to a subject in need thereof.

[0024] In certain embodiments, this disclosure relates to methods of treating or preventing any cancer, breast, lung, colon, or ovarian cancer comprising administering an effective amount of a compound or pharmaceutical agent as disclosed herein to a subject in need thereof. In certain embodiments, the subject is diagnosed with breast cancer, triple negative breast cancer, lung cancer, colon cancer, or other cancer.

[0025] In certain embodiments, this disclosure relates to the production of a medicament for use in the management of diseases and conditions associated with MGAT1 and CD73 binding interactions, such as cancer.

[0026] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0027] Figure 1A illustrates the chemical structure for the compound TW-37 N-(4-((2-(tert- butyl)phenyl)sulfonyl)phenyl)-2,3,4-trihydroxy-5-(2-isopropylbenzyl)benzamide.

[0028] Figure IB illustrates the chemical structure and name for the compound No 2. 6-(2- (anthracen-9-ylmethylene)hydrazineyl)-N2-(4-nitrophenyl)-N4-(o-tolyl)-l,3,5-triazine-2,4- diamine.

[0029] Figure 1C illustrates the chemical structures and name for the compounds No. 9, 2-amino- l-((3,4-dihydroxybenzylidene)amino)-N-(2,5-dimethylphenyl)-lH-pyrrolo[2,3-b]quinoxaline-3- carboxamide, compound No. 8, N-(3-(5,6-dimethylbenzo[d]oxazol-2-yl)-4-hydroxy-5- methylphenyl)benzo[d][l,3]dioxole-5-carboxamide, and compound No. 14, 2-(5-cyclopropyl-lH- pyrazol-3-yl)-10-methyl-ll-(l-methyl-lH-pyrazol-4-yl)-9,ll-dihydropyrazolo[4',3':5,6]pyrano [3,2-e][l,2,4]triazolo[l,5-c]pyrimidine.

[0030] Figure ID shows data indicating the inhibition of MGAT1 with compounds disclosed herein.

[0031] Figure IE shows data indicating interactions with CD73 with compounds disclosed herein. Figure 2 shows data indicating the treatment of a breast cancer tumor with compound 8 (W-GTF01) in combination with an PDL1 antibody.

[0032] DETAILED DISCUSSION

[0033] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. An “embodiment” refers to an example, and the claims are not necessarily limited to such example. The scope of the present disclosure will be limited only by the appended claims or as amended during prosecution.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0035] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0036] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0037] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0038] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. As used herein, the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In certain embodiments, about means within a standard deviation using measurements generally acceptable in the art. In certain embodiments, about means a range extending to + / - a percentage of the specified value and includes the specified value. In certain embodiments, the term “about” can include a 5 % or 10 % difference.

[0039] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0040] "Consisting essentially of' or "consists of' or the like, have the meaning ascribed to them in U.S. Patent law in that when applied to methods and compositions encompassed by the present disclosure refers to the idea of excluding certain prior art element(s) as an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.

[0041] Method of use

[0042] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a compound as disclosed herein to a subject in need thereof. In certain embodiments, the cancer is breast cancer. In certain embodiments, the subject is diagnosed with triple negative breast cancer. In certain embodiments, the compound is administered in combination with a chemotherapy agent. In certain embodiments, the chemotherapy agent is a checkpoint inhibitor.

[0043] In certain embodiments, this disclosure relates to methods of treating or preventing cancer comprising administering to a subject in need thereof an effective amount of a pharmaceutical agent that inhibits MGAT1 or suppresses MGAT1 and CD73 binding interactions. In certain embodiments, the therapeutic agent is a small molecule, antibody, or other specific binding agent.

[0044] A "subject" refers any animal, preferably a human patient, livestock, or domestic pet. The term "effective amount" refers to that amount of a compound or pharmaceutical agent described herein that is sufficient to effect the intended application including, but not limited to, disease treatment as illustrated below. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose will vary depending on, for example, the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0045] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e g. patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression.

[0046] The terms "co-administration" or "administered in combination with," and their grammatical equivalents, as used herein, encompass administration of two or more agents to a subject so that both agents and / or their metabolites are present in the subject at the same time. Coadministration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

[0047] In certain embodiments, the small molecule compound is N-(3-(5,6- dimethylbenzo[d]oxazol-2-yl)-4-hydroxy-5-methylphenyl)benzo[d][l,3]dioxole-5-carboxamide (W-GTF01), derivative, prodrug, ester, or salt thereof.

[0048] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a compound or pharmaceutical agent disclosed herein to a subject in need thereof. In certain embodiments, cancer is breast cancer. In certain embodiments, cancer is triple negative breast cancer. In certain embodiments, cancer is lung cancer. In certain embodiments, cancer is ovarian cancer or colon cancer.

[0049] "Cancer" refers any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and have many different forms in each body area. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the compound. It may also be identified by a change in relevant biomarker or gene expression profile, such as PSA for prostate cancer, HER2 for breast cancer, or others.

[0050] The cancer to be treated in the context of the present disclosure may be any type of cancer or tumor. These tumors or cancer include, and are not limited to, tumors of the hematopoietic and lymphoid tissues or hematopoietic and lymphoid malignancies, tumors that affect the blood, bone marrow, lymph, and lymphatic system. Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells; the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas, lymphocytic leukemias, and myeloma are from the lymphoid line, while acute and chronic myelogenous leukemia, myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

[0051] Also contemplated are malignancies located in the colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, hypophysis, testicles, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax and genito-urinary apparatus and, more particularly, childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocellular cancer, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant tumors, anal cancer, astrocytoma, cancer of the biliary tract, cancer of the bladder, bone cancer, brain stem glioma, brain tumors, breast cancer, cancer of the renal pelvis and ureter, primary central nervous system lymphoma, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma, cancer of the cervix, childhood (primary) hepatocellular cancer, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial germ cell tumors, childhood Hodgkin's disease, childhood Hodgkin's lymphoma, childhood visual pathway and hypothalamic glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin's lymphoma, childhood supratentorial primitive neuroectodermal and pineal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia, cancer of the colon, cutaneous T-cell lymphoma, endocrine pancreatic islet cells carcinoma, endometrial cancer, ependymoma, epithelial cancer, cancer of the esophagus, Ewing's sarcoma and related tumors, cancer of the exocrine pancreas, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic biliary tract cancer, cancer of the eye, breast cancer in women, Gaucher's disease, cancer of the gallbladder, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumor, head and neck cancer, hepatocellular cancer, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, Kaposi's sarcoma, cancer of kidney, cancer of the larynx, cancer of the lip and mouth, cancer of the liver, cancer of the lung, lymphoproliferative disorders, macroglobulinemia, breast cancer in men, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, occult primary metastatic squamous neck cancer, primary metastatic squamous neck cancer, metastatic squamous neck cancer, multiple myeloma, multiple myeloma / plasmatic cell neoplasia, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, paranasal sinus and nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma during pregnancy, nonmelanoma skin cancer, non-small cell lung cancer, metastatic squamous neck cancer with occult primary, buccopharyngeal cancer, malignant fibrous histiocytoma, malignant fibrous osteosarcoma / histiocytoma of the bone, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, cancer of the penis, phaeochromocytoma, hypophysis tumor, neoplasia of plasmatic cells / multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell cancer, cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, cancer of the salivary glands, sarcoidosis, sarcomas, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous neck cancer, stomach cancer, pineal and supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional renal pelvis and ureter cancer, trophoblastic tumors, cell cancer of the renal pelvis and ureter, cancer of the urethra, cancer of the uterus, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, cancer of the vulva, Waldenstrom's macroglobulinemia, Wilms' tumor and any other hyperproliferative disease, as well as neoplasia, located in the system of a previously mentioned organ.

[0052] In certain embodiments, methods of treatment include administering a pharmaceutical agent that inhibits MGAT1 or suppresses MGAT1 and CD73 interactions that is a small molecule compound, peptide, or antibody that specifically binds MGAT1. In certain embodiments, the MGAT1 inhibitor is administered in combination with another anticancer agent. In certain embodiments, the anticancer agent is a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is anti-PD-1, anti-PD-Ll, or anti-CTLA4 antibody or combinations thereof. In certain embodiments, the antibody is durvalumab, atezolizumab, avelumab, ipilimumab, tremelimumab, nivolumab, pembrolizumab, cemiplimab, or combinations thereof. In certain embodiments, anticancer agent is an anti-CD73 antibody. In certain embodiments, the anticancer agent anticancer agent atezolizumab or avelumab.

[0053] In certain embodiments, agents disclosed herein may be administered in combination with another pharmaceutical agent or anticancer agent. A “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent,” or the like, refer to molecules that are recognized to aid in the treatment of a cancer. Contemplated examples include the following molecules or derivatives such as abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfdzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa- 2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5 -fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5 -fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MVAC).

[0054] In certain embodiments, the chemotherapy agent is an anti-PD-1, anti-PD-Ll anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti-PDl (e.g., nivolumab, pembrolizumab, cemiplimab) and anti-PD-Ll (e.g., atezolizumab, avelumab, durvalumab).

[0055] In certain embodiments, the method of administration is in a subject with a lymphodepleted environment due to prior or concurrent administration of lymphodepl eting agents. In certain embodiments, lymphodepleting agents (e.g., cyclophosphamide and fludarabine).

[0056] In certain embodiments, the method of administration is in combination with a therapeutic antibody such as, antibodies selected from the group consisting of abagovomab, abciximab, abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, afutuzumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, andecaliximab, anetumab, anifrolumab, anrukinzumab, apolizumab, aprutumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atlizumab, atorolimumab, avelumab, azintuxizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, belantamab, belimumab, bemarituzumab, belimumab, bemarituzumab, benralizumab, berlimatoxumab, bersanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, birtamimab, bivatuzumab, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab, briakinumab, brodalumab, brolucizumab, brontictuzumab, burosumab, cabiralizumab, camidanlumab, camrelizumab, canakinumab, cantuzumab, caplacizumab, capromab, carlumab, carotuximab, catumaxomab, cedelizumab, cemiplimab, cergutuzumab, certolizumab, cetrelimab, cetuximab, cibisatamab, citatuzumab, cixutumumab, clazakizumab, clenoliximab, clivatuzumab, codrituzumab, cofetuzumab, coltuximab, conatumumab, concizumab, cosfroviximab, crenezumab, crizanlizumab, crotedumab, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab, daratumumab, dectrekumab, demcizumab, denintuzumab, denosumab, depatuxizumab, derlotuximab, detumomab, dezamizumab, dinutuximab, diridavumab, domagrozumab, dorlimomab, drozitumab, duligotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab, enavatuzumab, enfortumab, enlimomab, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etigilimab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faricimab, farletuzumab, fasinumab, felvizumab fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frunevetmab, fulranumab, futuximab, galcanezumab, galiximab, gancotamab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gemtuzumab, gevokizumab, gilvetmab, gimsilumab, girentuximab, glembatumumab, golimumab, gomiliximab, gosuranemab, guselkumab, ianalumab, ibalizumab, ibritumomab, icrucumab, idarucizumab, ifabotuzumab, igovomab, iladatuzumab, imalumab, imaprelimab, imciromab, imgatuzumab, inclacumab, indatuximab, indusatumab, inebilizumab, inflectra, infliximab, intetumumab, inolimomab, inotuzumab, ipilimumab, iratumumab, isatuximab, iscalimab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, lacnotuzumab, ladiratuzumab, lampalizumab, lanadelumab, landogrozumab, laprituximab, larcaviximab, lebrikizumab, lemalesomab, lendalizumab, lenvervimab, lenzilumab, lerdelimumab, leronlimab, lesofavumab, letolizumab, lexatumumab, libivirumab, lifastuzumab, ligelizumab, loncastuximab, losatuxizumab, lilotomab, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab, lucatumumab, lulizumab, lumiliximab, lumretuzumab, lupartumab, lutikizumab, mapatumumab, margetuximab, marstacimab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mirikizumab, mirvetuximab, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, mosunetuzumab, motavizumab, moxetumomab, nacolomab, namilumab, naptumomab, naratuximab, narnatumab, natalizumab, navicixizumab, navivumab, naxitamab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomab, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, oleclumab, olendalizumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab, oregovomab, orticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pankomab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab, pintumomab, placulumab, plozalizumab, pogalizumab, polatuzumab, ponezumab, porgaviximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab, raxibacumab, ravagalimab, ravulizumab, refanezumab, regavirumab, remtolumab, reslizumab, rilotumumab, rinucumab, risankizumab, rituximab, rivabazumab, robatumumab, roledumab, romilkimab, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab, rovelizumab, rozanolixizumab, ruplizumab, sacituzumab, samalizumab, samrotamab, sapelizumab, sarilumab, satralizumab, satumomab, secukinumab, selicrelumab, seribantumab, setoxaximab, setrusumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirtratumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tabalumab, tacatuzumab, tadocizumab, talacotuzumab, talizumab, tamtuvetmab, tanezumab, taplitumomab, tarextumab, tavolimab, tefibazumab, telimomab, telisotuzumab, tenatumomab, teneliximab, teplizumab, tepoditamab, teprotumumab, tesidolumab, tetulomab, tezepelumab, tibulizumab, tildrakizumab, tigatuzumab, timigutuzumab, timolumab, tiragotumab, tislelizumab, tisotumab, tocilizumab, tomuzotuximab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, tregalizumab, tremelimumab, trevogrumab, tucotuzumab, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vanalimab, vandortuzumab, vantictumab, vanucizumab, vapaliximab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, vobarilizumab, volociximab, vonlerolizumab, vopratelimab, vorsetuzumab, votumumab, vunakizumab, xentuzumab, zalutumumab, zanolimumab, zenocutuzumab, ziralimumab, zolbetuximab, and zolimomab.

[0057] In certain embodiments, this disclosure relates to methods of screening a test compound for the ability to inhibit MGAT1 or suppress MGAT1 and CD73 interactions binding interactions comprising contacting a compound such as N-(3-(5,6-dimethylbenzo[d]oxazol-2-yl)-4-hydroxy- 5-methylphenyl)benzo[d][l,3]dioxole-5-carboxamide (W-GTF01), or derivative as disclosed herein optionally conjugated to a label and a test compound and detecting whether the test compound suppresses MGAT1 and CD73 binding interactions. Detecting whether the test compound suppresses binding interactions can be accomplished in multiple ways known in art.

[0058] In certain embodiments, whether the test compound suppresses MGAT1 and CD73 binding or non-binding interactions is recorded on a non-transitory computer readable medium, e g. in the form of a detection value or non-detection value, measured or quantitative value.

[0059] Binding assays typically include quantitative / qualitative detection of binding partners as well as the resulting binding complexes. For this purpose, either the target biomolecule, ligands or both may be labelled. Numerous labelling / detection techniques may be used in binding assays including radioligand assays, affinity chromatography, surface plasmon resonance, isothermal titration calorimetry and light-based techniques using absorbance, fluorescence, or luminescence readouts. Commercially available kits utilize typically utilize light detection modes such as absorbance, fluorescence, luminescence, FRET (Fluorescence Resonance Energy Transfer), Fluorescence polarization (FP), TRF (time-resolved fluorescence), TR-FRET (time-resolved fluorescence energy transfer). Absorbance ELISA binding assays typically utilize antibody pairs specific for the binding partners, e.g., MGAT1 and CD73. While one antibody is used to bind an antigen, e.g., MGAT1 or CD73 to a surface, e.g., microtiter plate surface, the other antibody binds the other binding partner. The binding complex is typically detected by a colorimetric enzymatic reaction coupled to the second antibody, while any unbound binding partners in solution are optionally removed by washing step(s). In certain embodiments, identifying the suppression of MG ATI and CD73 binding interactions can be accomplished using in vitro immunofluorescence staining and proximity ligation assays.

[0060] In certain embodiments, it is contemplated that MGAT1 or CD73 or fragments are conjugated to a label e.g., a fluorophore and the other contains an alternative fluorophore or quencher. The fluorophores and / or quencher are attached within binding sequences so that so that they are within close proximity when bound together and fluorescence intensity is quenched or is heightened. In certain embodiments, when the test compound interruptus binding MGAT1 and CD73 to each other, the fluorophore physically separates from the quencher, allowing a fluorescence or other label signal to be emitted, e g., upon excitation, the signal intensity changes e.g., is heightened or diminished. Thus, detection of a change in the fluorescent signal provides an indication that the test compound suppresses or interrupts or weakens the ability MGAT1 and CD73 to bind together, and when the signal is generated.

[0061] A "label" refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a "label receptor" refers to incorporation of a heterologous polypeptide in the receptor. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moi eties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as18F,35S or131I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0062] Pharmaceutical agents for MGAT1 inhibition

[0063] In certain embodiments, this disclosure relates to compounds and other specific binding agent that inhibit MGAT1 or suppress MG ATI and CD73 binding. In certain embodiments, the compound is N-(3-(5,6-dimethylbenzo[d]oxazol-2-yl)-4-hydroxy-5-methylphenyl)benzo [d][l,3]dioxole-5-carboxamide (W-GTF01, compound 8), derivative, prodrug, ester, or salt thereof.

[0064] In certain embodiments, the compound has the following formula or salt thereof wherein n is 1 or 2;

[0065] X is NH, O, S, or CH2;

[0066] Y is CH2or CH2CH2;

[0067] Z is O, S, NH, or NR12,

[0068] R1, R2, R3, R4, R3, R6, R7, R8, R9, R10, R11, or R12are each, the same or different, hydrogen, alkyl, halogenated alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, glycol, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, or R12are optionally substituted with one or more, the same or different, R13; R13is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R13is optionally substituted with one or more, the same or different, R14; and

[0069] R14is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N- ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethyl sulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

[0070] In certain embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, or R12are each, the same or different, hydrogen, halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N- methyl-N-ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methyl sulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxy carbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

[0071] In certain embodiments, X is NH.

[0072] In certain embodiments, Y is CH2.

[0073] In certain embodiments, Z is O.

[0074] In certain embodiments, R1is H.

[0075] In certain embodiments, R2is H or alkyl.

[0076] In certain embodiments, R3is H or alkyl.

[0077] In certain embodiments, R4is H.

[0078] In certain embodiments, R5is H or hydroxy. In certain embodiments, R6is H or alkyl.

[0079] In certain embodiments, R7is H.

[0080] In certain embodiments, R8is H.

[0081] In certain embodiments, R9is H. In certain embodiments, R10is H.

[0082] In certain embodiments, R11is H.

[0083] In certain embodiments, R12is H or alkyl.

[0084] In certain embodiments, X is NH, Y is CH2 and Z is O.

[0085] In certain embodiments, Z is O, R1is H, R2is alkyl R3is alkyl, and R4is H. In certain embodiments, X is NH, R5is hydroxy, R6is alkyl, R7is H, and R11is H.

[0086] In certain embodiments, Y is CH2, n is 1, R8is H, R9is H, and R10is H.

[0087] In certain embodiments, the compound is 2-amino-l-((3,4-dihydroxybenzylidene)amino)- N-(2,5-dimethylphenyl)-lH-pyrrolo[2,3-b]quinoxaline-3-carboxamide (Compound 9), derivative, prodrug, ester, or salt thereof.

[0088] In certain embodiments, the compound has the following formula or salt thereof wherein

[0089] X is O, S, NH, or NR18, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, or R18, or are each, the same or different, hydrogen, alkyl, halogenated alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, glycol, alkylthio, alkylamino, (alkyl)2amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R1, R2, R3, R4, R\ R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, or R18are optionally substituted with one or more, the same or different, R19;

[0090] R19is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R19is optionally substituted with one or more, the same or different, R20; and R20is halogen, nitro, cyano, hydroxy, trifluoromethoxy, tri fluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N- ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

[0091] In certain embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, or R18are each, the same or different, hydrogen, halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, acetyloxy, N- methylcarbamoyl, acetylamino, acetyloxy, N-ethylcarbamoyl, acetylamino, acetyloxy, N,N- dimethylcarbamoyl, acetylamino, acetyloxy, N,N-diethylcarbamoyl, acetylamino, acetyloxy, N- methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N- ethyl sulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

[0092] In certain embodiments, R1is H.

[0093] In certain embodiments, R2is H.

[0094] In certain embodiments, R3is H or hydroxy.

[0095] In certain embodiments, R4is H or hydroxy.

[0096] In certain embodiments, R5is H.

[0097] In certain embodiments, R6is H.

[0098] In certain embodiments, R7is H.

[0099] In certain embodiments, R8is H.

[0100] In certain embodiments, R9is H.

[0101] In certain embodiments, R10is H.

[0102] In certain embodiments, R11is H. In certain embodiments, R12is H.

[0103] In certain embodiments, R13is H or alkyl.

[0104] In certain embodiments, R14is H.

[0105] In certain embodiments, R15is H.

[0106] In certain embodiments, R16is H or alkyl.

[0107] In certain embodiments, R17is H.

[0108] In certain embodiments, R18is H.

[0109] In certain embodiments, the compound is 2-(5-cyclopropyl-lH-pyrazol-3-yl)-10-methyl- ll-(l-methyl-lH-pyrazol-4-yl)-9,l l-dihydropyrazolo[4',3,:5,6]pyrano[3,2-e][l,2,4]triazolo[l,5- c]pyrimidine (compound 14), derivative, prodrug, ester, or salt thereof.

[0110] In certain embodiments, the compound has the following formula or salt thereof wherein

[0111] X is O, S, NH, or NR11;

[0112] R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, or R11are each, the same or different, hydrogen, alkyl, halogenated alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, glycol, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, or R11are optionally substituted with one or more, the same or different, R12; R12is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R12is optionally substituted with one or more, the same or different, R13; and

[0113] R13is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N- ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethyl sulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

[0114] In certain embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, or R11are each, the same or different, hydrogen, halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N- ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methyl sulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxy carbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

[0115] In certain embodiments, R1is H.

[0116] In certain embodiments, R2is H or carbocyclyl.

[0117] In certain embodiments, R3is H.

[0118] In certain embodiments, R4is H.

[0119] In certain embodiments, R5is H.

[0120] In certain embodiments, R6is H or alkyl.

[0121] In certain embodiments, R7is H.

[0122] In certain embodiments, R8is H or alkyl. In certain embodiments, R9is H.

[0123] In certain embodiments, R10is H.

[0124] In certain embodiments, R11is H.

[0125] In certain embodiments, the compound is 6-(2-(anthracen-9-ylmethylene)hydrazineyl)- N2-(4-nitrophenyl)-N4-(o-tolyl)-l,3,5-triazine-2,4-diamine (Compound 2) derivative, prodrug, ester, or salt thereof.

[0126] X is O, S, or NH;

[0127] Y is O, S, or NH; Z is O, S, or NH;

[0128] R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, or R18, or are each, the same or different, hydrogen, alkyl, halogenated alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, glycol, alkylthio, alkylamino, (alkyl)2amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, or R18are optionally substituted with one or more, the same or different, R19; R19is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R19is optionally substituted with one or more, the same or different, R20; and

[0129] R20is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N- ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethyl sulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

[0130] In certain embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, or R18are each, the same or different, hydrogen, halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, acetyloxy, N- methylcarbamoyl, acetylamino, acetyloxy, N-ethylcarbamoyl, acetylamino, acetyloxy, N,N- dimethylcarbamoyl, acetylamino, acetyloxy, N,N-diethylcarbamoyl, acetylamino, acetyloxy, N- methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methylsulfmyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N- ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

[0131] In certain embodiments, R1is H.

[0132] In certain embodiments, R2is H.

[0133] In certain embodiments, R3is H.

[0134] In certain embodiments, R4is H.

[0135] In certain embodiments, R5is H.

[0136] In certain embodiments, R6is H.

[0137] In certain embodiments, R7is H. In certain embodiments, R8is H.

[0138] In certain embodiments, R9is H.

[0139] In certain embodiments, R10is H.

[0140] In certain embodiments, R11is H.

[0141] In certain embodiments, R12is H.

[0142] In certain embodiments, R13is H or alkyl.

[0143] In certain embodiments, R14is H.

[0144] In certain embodiments, R15is H.

[0145] In certain embodiments, R16is H or nitro.

[0146] In certain embodiments, R17is H.

[0147] In certain embodiments, R18is H.

[0148] In certain embodiments, the compound is N-(4-((2-(tert-butyl)phenyl)sulfonyl)phenyl)- 2,3,4-trihydroxy-5-(2-isopropylbenzyl)benzamide (TW-37) derivative, prodrug, ester, or salt thereof.

[0149] As used herein, “alkyl” means a noncyclic straight chain or branched, unsaturated or saturated hydrocarbon such as those containing from 1 to 10 carbon atoms, typically 1 to 6 carbon atoms. Within any embodiments, herein alkyl may refer to an alkyl with 1 to 6 carbons (Ci-ealkyl). Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n- hexyl, n-septyl, n-octyl, n-nonyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an “alkenyl” or “alkynyl,” respectively). Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1 -pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2- butenyl, 2,3- dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3- methyl-1- butynyl, and the like.

[0150] Non-aromatic mono or polycyclic alkyls are referred to herein as "carbocycles" or "carbocyclyl" groups. Representative saturated carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated carbocycles include cyclopentenyl and cyclohexenyl, and the like. "Heterocarbocycles" or heterocarbocyclyl" groups are carbocycles which contain from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur which may be saturated or unsaturated (but not aromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Heterocarbocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0151] "Aryl" means an aromatic carbocyclic monocyclic or polycyclic ring such as phenyl or naphthyl. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic.

[0152] As used herein, "heteroaryl" refers an aromatic heterocarbocycle having 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, and containing at least 1 carbon atom, including both mono- and polycyclic ring systems. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic. Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that the use of the term "heteroaryl" includes N-alkylated derivatives such as a 1-methylimidazol- 5-yl substituent.

[0153] As used herein, "heterocycle" or "heterocyclyl" refers to mono- and polycyclic ring systems having 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, and containing at least 1 carbon atom. The mono- and polycyclic ring systems may be aromatic, non-aromatic or mixtures of aromatic and non-aromatic rings. Heterocycle includes heterocarbocycles, heteroaryls, and the like.

[0154] "Alkylthio" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through a sulfur bridge. An example of an alkylthio is methylthio, (i.e., -S-CH3).

[0155] "Alkoxy" refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n- pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, s-butoxy, t-butoxy. " Alkylamino" refers an alkyl group as defined above attached through an amino bridge. An example of an alkylamino is methylamino, (i.e., -NH-CH3).

[0156] "Alkanoyl" refers to an alkyl as defined above attached through a carbonyl bride (i.e., -(C=O)alkyl).

[0157] “Alkoxycarbonyl” refers to an alkyl as defined above attached through a carboxy bridge (i.e., -(C=O)Oalkyl.

[0158] “Alkylcarbamoyl” refers to an alkyl as defined above attached through a carbonyl bridge (i.e., -(C=O)NHalkyl).

[0159] “Alkanoyl” refers to an alkyl as defined above attached through a carbonyl bridge (i.e., -(C=O)alkyl).

[0160] "Alkylsulfonyl" refers to an alkyl as defined above attached through a sulfonyl bridge (i.e., -S(=O)2alkyl) such as mesyl and the like, and "Aryl sulfonyl" refers to an aryl attached through a sulfonyl bridge (i.e., - S(=O)2aryl).

[0161] "Alkylsulfonamide" refers to an alkyl as defined above attached through a sulfamoyl bridge (i.e., -S(=O)2NHalkyl), and an "Aryl sulfonamide" refers to an alkyl attached through a sulfamoyl bridge (i.e., (i.e., - S(=O)2NHaryl).

[0162] "Alkylsulfinyl" refers to an alkyl as defined attached through a sulfinyl bridge (i.e. -S(=O)alkyl).

[0163] “Glycol” refers to an alkyl group substitute with terminal oxygen atoms, and the term is intended to include repeating glycol units, e.g., polyethylene glycol -O(CH2CH2O)nX, wherein X is typically an H, alkyl, alkoxy, halogenated alkoxy, or alkanoyl, and n is typically 1-50, 1-100, or 1-1,000.

[0164] The terms “halogen” and “halo” refer to fluorine, chlorine, bromine, and iodine.

[0165] The term “halogenated alkyl” refers to an alkyl partially or entirely substituted with halogens, e.g., CF3.

[0166] The term "substituted" refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are "substituents." The molecule may be multiply substituted. In the case of an oxo substituent ("=O"), two hydrogen atoms are replaced. Example substituents within this context may include halogen, hydroxy, alkyl, alkoxy, halogenated alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarb ocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaNRb, -NRaC(=O)ORb, - NRaSChRb, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRb, -OC(=O)NRaRb, -ORa, -SRa, -SORa, - S(=O)2R , -OS(=O)2Ra and -S(=O)2ORa. Ra and Rb in this context may be the same or different and independently hydrogen, halogen hydroxyl, alkyl, alkoxy, halogenated alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl.

[0167] The term "optionally substituted," as used herein, means that substitution is optional and therefore it is possible for the designated atom to be unsubstituted.

[0168] As used herein, "salts" refer to derivatives of the disclosed compounds where the parent compound is modified making acid or base salts thereof. Examples of salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkylamines, or dialkylamines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. In certain embodiment the salts are conventional nontoxic pharmaceutically acceptable salts including the quaternary ammonium salts of the parent compound formed, and non-toxic inorganic or organic acids. Contemplated salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

[0169] The term “prodrug” refers to an agent that is converted into a biologically active form in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of an alcohol or acetamide, formamide and benzamide derivatives of an amine functional group in the active compound and the like.

[0170] As used herein, "pharmaceutically acceptable esters" include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, arylalkyl, and cycloalkyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids, and boronic acids.

[0171] As used herein, the term “small molecule” refers to any variety of covalently bound molecules with a molecular weight of less than 1000 or 1500. Typically, the majority of atoms include carbon, hydrogen, oxygen, nitrogen, and to a lesser extent sulfur and / or a halogen. Examples include steroids, short peptides, mono or polycyclic aromatic or non-aromatic, heterocyclic compounds.

[0172] As used herein, the term “derivative” refers to a structurally similar compound that retains sufficient functional attributes of the identified analogue. The derivative may be structurally similar because it is lacking one or more atoms, substituted, a salt, in different hydration / oxidation states, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing a hydrogen with a deuterium, oxygen atom with a sulfur atom or replacing an amino group with a hydroxyl group. The derivative may be a prodrug. Derivatives may be prepared by any variety of synthetic methods or appropriate adaptations presented in synthetic or organic chemistry textbooks, such as those provide in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) Michael B. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze hereby incorporated by reference.

[0173] Pharmaceutical compositions

[0174] In certain embodiments, this disclosure relates to pharmaceutical agents that inhibit MGAT1 or suppress MGAT1 and CD73 binding interactions; thus, useful in the context of managing diseases and conditions associated with these binding interactions, such as cancer.

[0175] In certain embodiments, pharmaceutical compositions comprise a compound as reported herein and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition is in the form of a pill, tablet, capsule, gel, gel capsule, granule, powder, lotion, or cream. In certain embodiments, the pharmaceutical composition is in the form of a liquid optionally isotonic comprising pH buffering agents and salts, and / or saccharide or polysaccharide.

[0176] In certain embodiments, the the pharmaceutically acceptable excipient is selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N-pyrrolidone, crospovidone, ethyl cellulose, povidone, methyl and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, corn starch, carnauba wax, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, iron oxide, triacetin, acacia gum, esters, or salts thereof.

[0177] Pharmaceutical compositions typically comprise an effective amount of compounds and a suitable pharmaceutical acceptable excipient or carrier. The preparations can be prepared in a manner known per se, which usually involves mixing the compounds according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences. Pharmaceutically acceptable salts, solvates, and hydrates of the compounds listed are also useful in the method of the disclosure and in pharmaceutical compositions of the disclosure.

[0178] In certain embodiments, a composition comprising a pharmaceutical agent disclosed herein in enantiomeric excess, diastereomeric excess, or racemic mixture or salt thereof as reported herein of the present disclosure can be administered to a subject either alone or as a part of a pharmaceutical composition.

[0179] In certain embodiments, the isomer may be present in a composition with enantiomeric excess or diastereomeric excess of greater than 60%. In certain embodiments, the R isomer may be present in enantiomeric excess or diastereomeric excess of greater than 70%. In certain embodiments, the isomer may be present in enantiomeric excess or diastereomeric excess greater than 80%. In certain embodiments, the isomer may be present in enantiomeric excess, diastereomeric excess, of greater than 90%. In certain embodiments, the isomer may be present in enantiomeric excess diastereomeric excess of greater than 95%.

[0180] In certain embodiments, the pharmaceutical composition is in the form of a tablet, pill, capsule, powders, granules, gel, gel capsule, granule, or cream. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or: (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, as for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia gum, (c) humectants, as for example, glycerol (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.

[0181] Solid dosage forms can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. In certain embodiments, pharmaceutical composition is in solid form surrounded by an enteric coating. In certain embodiments, the enteric coating comprises methyl acrylate-methacrylic acid copolymers, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, or combinations thereof.

[0182] In certain embodiments, this disclosure contemplates an intravenous formulation with pH buffering agents and tonicity in a range representing physiological values (pH 7 to 8) or for bolus administration, e.g., containing normal saline or dextrose optionally containing pH buffering agents. In certain embodiments, the pharmaceutical composition is in the form of a sterilized pH buffered aqueous salt solution or a saline phosphate buffer between a pH of 6 to 8, optionally comprising a saccharide or polysaccharide.

[0183] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable (such as olive oil, sesame oil) and injectable organic esters such as ethyl oleate.

[0184] These compositions may also contain preserving, emulsifying, and dispensing agents. Prevention of the action of microorganisms may be controlled by addition of any of various antibacterial and antifungal agents, example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0185] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, com germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.

[0186] The pharmaceutical compositions of the present disclosure can be administered to subjects either topically to the skin, orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), intraci stemally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments, or drops), or as a buccal or nasal spray.

[0187] In certain embodiments, the pharmaceutical compositions are in a form for inhalation. In certain embodiments, the pharmaceutical composition comprises a compound disclosed herein and a propellant. In certain embodiments, an aerosolizing propellant is compressed air, ethanol, nitrogen, carbon dioxide, nitrous oxide, hydrofluoroalkanes (HFAs), or combinations thereof.

[0188] In certain embodiments, the disclosure contemplates a pressurized or unpressurized container comprising a compound herein. In certain embodiments, the container is a manual pump spray, inhaler, meter-dosed inhaler, dry powder inhaler, nebulizer, vibrating mesh nebulizer, jet nebulizer, or ultrasonic wave nebulizer.

[0189] Pharmaceutical compositions typically comprise an effective amount of a composition or preparation that can be prepared in a manner known per se, which usually involves mixing the at least one compound according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences. It is well known that ester prodrugs are readily degraded in the body to release the corresponding alcohol. See e.g., Imai, Drug Metab Pharmacokinet, 2006, 21 (3): 173- 85, entitled “Human carboxylesterase isozymes: catalytic properties and rational drug design.

[0190] The pharmaceutical preparations of the disclosure are preferably in a unit dosage form, and can be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which can be properly labeled); optionally with one or more leaflets containing product information and / or instructions for use. Generally, such unit dosages will contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the disclosure e g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

[0191] In certain embodiments, production processes are contemplated which two components, compounds disclosed herein and a pharmaceutical carrier, are provided already in a combined dry form ready to be reconstituted together. In other embodiments, it is contemplated that compounds disclosed herein and a pharmaceutical carrier are admixed to provide a pharmaceutical composition.

[0192] Providing a pharmaceutic composition is possible in a one-step process, simply by adding a suitable pharmaceutically acceptable diluent to the composition in a container. In certain embodiments, the container is preferably a syringe for administering the reconstituted pharmaceutical composition after contact with the diluent. In certain embodiments, the coated compounds can be filled into a syringe, and the syringe can then be closed with the stopper. A diluent is used in an amount to achieve the desired end-concentration. The pharmaceutical composition may contain other useful component, such as ions, buffers, excipients, stabilizers, etc.

[0193] A "dry" pharmaceutical composition typically has only a residual content of moisture, which may approximately correspond to the moisture content of comparable commercial products, for example, has about 12% moisture as a dry product. Usually, the dry pharmaceutical composition according to the present invention has a residual moisture content preferably below 10% moisture, more preferred below 5% moisture, especially below 1% moisture. The pharmaceutical composition can also have lower moisture content, e.g. 0.1% or even below. In certain embodiments, the pharmaceutical composition is provided in dry in order to prevent degradation and enable storage stability.

[0194] A container can be any container suitable for housing (and storing) pharmaceutically compositions such as syringes, vials, tubes, etc. The pharmaceutical composition may then preferably be applied via specific needles of the syringe or via suitable catheters. A typical diluent comprises water for injection, and NaCl (preferably 50 to 150 mM, especially 110 mM), CaC12 (preferably 10 to 80 mM, especially 40 mM), sodium acetate (preferably 0 to 50 mM, especially 20 mM) and mannitol (preferably up to 10% w / w, especially 2% w / w). Preferably, the diluent can also include a buffer or buffer system so as to buffer the pH of the reconstituted dry composition, preferably at a pH of 6.2 to 7.9, especially at pH of 6.9 to 7.1.

[0195] In certain embodiments, the diluent is provided in a separate container. This can preferably be a syringe. The diluent in the syringe can then easily be applied to the container for reconstitution of the dry compositions. If the container is also a syringe, both syringes can be finished together in a pack. It is therefore preferred to provide the dry compositions in a syringe, which is finished with a diluent syringe with a pharmaceutically acceptable diluent for reconstituting, said dry and stable composition.

[0196] In certain embodiments, this disclosure contemplates a kit comprising a pharmaceutical composition disclosed herein and a container with a suitable diluent. Further components of the kit may be instructions for use, administration means, such as syringes, catheters, brushes, etc. (if the compositions are not already provided in the administration means) or other components necessary for use in medical (surgical) practice, such as substitute needles or catheters, extra vials or further wound cover means. In certain embodiments, the kit comprises a syringe housing the dry and stable hemostatic composition and a syringe containing the diluent (or provided to take up the diluent from another diluent container).

[0197] Development of MGAT1 inhibitor to block CD73 glycosylation in cancer therapy

[0198] MGAT1 was identified as a prevalent regulator among immune-cold triple negative breast cancers (TNBCs), where its expression correlates with tumor immune evasion. CD73 was identified as a substrate of MGAT1 on Golgi bodies, and that glycosylation of CD73 in the cytosol alters its membrane abundance by regulating CD73 translocation. Although it is not intended that embodiments, of this disclosure be limited by any particular mechanism, it is contemplated that glycosylation of CD73 by MGAT1 triggers the CD73 dimerization that, in turn, facilitates its loading onto VAMP3 for membrane fusion and translocation wherein THBS1 is an upstream etiological factor orchestrating the MGAT1-CD73-VAMP3 -adenosine axis in suppressing CD8+ T cell antitumor activity. W-GTF01 was identified as having immunomodulatory activity due to its ability to inhibit CD73 glycosylation and dimerization, which prevents CD73 membrane translocation. In preclinical models of TNBC, a combination of W-GTF01 and PD-L1 blockade was particularly efficient in suppressing immune-cold TNBC tumors. The inhibition of MG ATI -mediated CD73 glycosylation can be favorably combined with PD-L1 blockade to achieve superior immuno- infiltration of cold tumors, as well as improved tumor growth suppression.

[0199] “MGAT1” or “Mannosyl(alpha-l,3-)-glycoprotein beta-1, 2-N-acetylglucosaminyl transferase” and the like refer to an enzyme that processes N-linked glycosylation, a common post- translational modification of proteins. MGAT1 can promote cancer through its role in N-linked glycosylation, resulting altered cell signaling, immune evasion, enhanced metastatic potential, and therapeutic resistance. The ability of MGAT1 to promote cancer through these mechanisms allows for evaluation of targeted cancer therapies. It is contemplated that inhibiting MGAT1 or modulating its activity disrupt glycosylation patterns that cancer cells rely on for proliferation, immune evasion, and metastasis. MGAT1 specific inhibitors can be used to enhance the effectiveness of existing cancer therapies and to counteract therapeutic resistance.

[0200] In silico virtual screening was used to identify compounds that were tested in vitro HTS assay wherein the enzymatic activity of MGAT1 translates into a measurable light signal. Thus, the assay can be used to identify therapeutic potential of various glycosyltransferases and glycosidases implicated in cancer.

[0201] Experiments indicate that overexpression of MGAT1 and CD73 are correlated with poor prognosis of several types of cancers, including triple-negative breast. MGAT1 catalyzes CD73 glycosylation and strengthens its dimerization and translocation to plasma membrane. MGAT1- mediated CD73 spatiotemporal distribution and dynamics at the plasma membrane which leads to an immunosuppressive environment and decreased tumor immunogenicity. MGATlhigh / CD73high is an “immune-cold” signature that dampens the efficacy of immunotherapy.

[0202] W-GTF01 is small molecule identified to inhibit MGAT1 catalytic function with high specificity and low toxicity. Although it is not intended that embodiments of this disclosure be limited by any particular mechanism, it is contemplated that W-GTF01 occupies the sugar donor binding domain, significantly inhibits CD73 glycosylation and reduces CD73 dimerization and membrane-bound CD73 abundance. In in vitro and in vivo models indicate that W-GTF01 enhances the efficacy of inflammatory breast cancer (IBC) TBC therapy in immune-cold triplenegative inflammatory breast cancer (TNBC) cells by restoring the capacity of CD8+ INF-y- producing T cells to elicit a response against the tumor cells.

[0203] It is contemplated that W-GTF01 suppresses MGAT1 -mediated CD73 glycosylation. This specific targeting can potentially reduce issues related to drug specificity and resistance. It is contemplated that W-GTF01 can restored an immune response, e.g., W-GTF01 not only inhibits CD73 dimerization and membrane translocation but also rejuvenates the suppressed cytotoxic CD8+ T cell function, thereby tilting the balance from an immunosuppressive to a more immunoreactive tumor environment.

[0204] It is contemplated that W-GTF01 can be used in conjunction with PD-L1 blockade, e.g., as a potent combination therapy for TNBC, especially in cases with high MGAT1 and CD73 expression. It is contemplated that W-GTF01 targets glycosyltransferase MGAT1 and represents a therapeutic strategy for treating triple negative breast cancer (TNBC) patients, i.e., cancer cells from a patient that fail to express, or express at abnormally low concentrations, estrogen receptors, progesterone receptors and HERZ. In certain embodiments, the subject is diagnosed "negative" on all 3 tests using test kits or methods approved by a drug regulatory agency. It is contemplated that by interrupting immunosuppressive mechanism, W-GTF01 has the potential to restore immune response and enhance the effectiveness of existing immunotherapies, opening a new avenue for TNBC and other types of cancer treatment, especially TNBC patients who do not currently respond to standard treatments.

[0205] MGATl-Mediated Glycosylation Orchestrates Immune Checkpoints and Antitumor Immunity

[0206] Using multiomic spatial analyses and experimental validation MGAT1, a glycosyltransferase, was identified as a factor governing tumor immune responses. Experiments indicate that overexpression of MGAT1 leads to immune evasion due to aberrant elevation of CD73 membrane translocation, which suppresses CD8+ T cell function, especially in immune- cold triple-negative breast cancer (TNBC). The addition of N-acetylglucosamine to CD73 by MGAT1 enables the CD73 dimerization necessary for CD73 loading onto VAMP3, ensuring membrane fusion. THBS1 was identified as an upstream etiological factor orchestrating the MGAT1-CD73-VAMP3 -adenosine axis in suppressing CD8+ T cell antitumor activity. Spatial transcriptomic profiling reveals spatially resolved features of interacting malignant and immune cells pertaining to expression levels of MGAT1 and CD73. In preclinical models of TNBC, W- GTF01 specifically blocked the MGAT1 -catalyzed CD73 glycosylation, sensitizing refractory tumors to anti-PD-Ll therapy via restoring capacity to elicit a CD8+ IFNy-producing T cell response. These results indicate a strategy for targeting the immunosuppressive molecule CD73 by inhibiting MGAT1.

[0207] MGAT1 is a glycosyltransferase involved in the synthesis and maturation of complex N- linked glycan structures. The addition of N-acetylglucosamine (GlcNAc) to growing glycan chains catalyzed by MGAT1 ensures the completion of glycan chain assembly on target proteins, resulting in the orchestration of various cellular processes such as protein folding, protein translocation, and cell-cell communication. Elevated MGAT1 expression in cancer cells leads to increased branching of N-linked glycan structures on cell surface glycoproteins, facilitating a number of tumorigenic processes, including enhanced cell adhesion, migration, and invasion. Silencing MGAT1 can impede cancer cell progression and metastasis.

[0208] CD73, also called ecto-5 '-nucleotidase (NT5E), a membrane-bound enzyme, acts in association with CD39 in breaking down extracellular ATP to immunosuppressive adenosine. Adenosinergic signaling regulates tumor immunity, suppressing cytotoxic T cells and creating an immunosuppressive tumor microenvironment. The oncogenic role of CD73 in advancing tumor progression involves interacting with cancer-associated fibroblasts through adenosine receptors (AIR, A2AR, A2BR, and A3R) on various types of immune cells such as regulatory (Foxp3+) T cells (Tregs), effector T cells, natural killer (NK) cells, myeloid-derived suppressor cells (MDSCs), B cells, and macrophages. HIF-la, estrogen receptor, and certain inflammation factors have been linked to the regulation of CD73 in a transcriptional manner. CD73 is a fast-turnover protein whose abundance is governed by the interplay between ubiquitin E3 ligase TRIM21 and deubiquitinase OTETD4. CD73 is an N-glycosylated protein. The upstream glycosyltransferase(s) governing CD73 glycosylation and the biochemical or cell biological consequences of CD73 glycosylation in relation to its immunosuppressive function remain largely unknown.

[0209] It has been discovered that MGAT1 has a role of in regulating tumor immune response and has clinical relevance in breast tumor immune evasion. Overexpression of MGAT1 leads to immune evasion through uncontrolled membrane trafficking and translocation of CD73, resulting in elevation of adenosine production. The addition of GlcNAc to CD73 at the Golgi apparatus by MGAT1 enables CD73 dimerization and ensures its membrane translocation. The molecular MGATl-CD73-adenosine axis is regulated in response to THBS1. Clinically, the MGATllow / CD731ow signature in a subset of human breast malignancies was associated with a favorable immune profile. W-GTF01 was identified as a pharmacological inhibitor of MGAT1 which balances CD73 dimerization and restores CD8+ IFNy-producing T cell responses providing a strategy for targeting immunosuppressive CD73 in treating immune-cold breast cancers.

[0210] Accumulation of MGAT1, a glycosyltransferase, is associated with an unfavorable tumor immune response and prognosis in immune-cold breast cancers

[0211] To search for potential therapeutic targets for immune-cold breast cancer, a bioinformatic analysis of a proteomic data set was performed on approximately 112 immune-relevant proteins from 935 breast cancer patients in The Cancer Genome Atlas (TCGA). Elevated levels of immunerelevant proteins were identified, including VTCN1, CD274, and CD73, specifically within the basal subtype of breast cancer (TNBC), compared to other subtypes. In addition, hierarchical clustering analysis suggested two distinct subgroups within TNBC patients, each characterized by a unique immune-related protein expression profile. Further investigation via differential enriched pathway analysis, employing single-sample gene set enrichment analysis (ssGSEA), indicated that one subgroup of TNBC patients exhibited an increased negative regulation of tumor immunity pathways. This regulation coincided with a distinct molecular signature of enhanced N-glycan processing and the prevalence of N-glycosylated proteins. Pearson correlation analysis was performed to identify if specific N-glycan processing-related genes are implicated in this particular TNBC subgroup. Multiple enzymes involved in N-glycosylation correlated positively with genes driving immune suppression. MGAT1 emerged as the most significantly associated enzyme, topping the list of genes linked to this effect within the TNBC subgroup.

[0212] To further confirm the association between MGAT1 up-regulation and TNBC-related immune evasion, Spearman correlation analysis of CPTAC proteomic data was performed, which indicated a strong positive correlation between MGAT1 and several immunosuppressive proteins, including CD39 / CD73, B7H3, and CXCL12. To validate the findings immunohistochemical analysis was conducted on a tissue microarray (TMA) containing 110 unique breast tissue samples. There was a significant elevation of MGAT1 protein levels in the majority of TNBC specimens. Further systematic protein expression analysis across an extensive array of breast cancer cell lines confirmed that MGAT1 protein was prevalent in TNBC cell lines, reinforcing the potential link to immune escape mechanisms in TNBC. An unbiased approach was used to evaluate MGAT1 protein expression within tumor cells and its correlation with CD8+ T cell presence by employing multiplex immunohistochemistry. Spatial analysis unveiled heightened interactions between CD8+ T cells and MGATllo tumor cells, as well as between CD8+Ki67+ T cells and MGATllo tumor cells, compared to interactions with MGATlhi tumor cells. These experiments indicated that lower levels of MGAT1 in tumors are linked to enhanced infiltration of CD8+ T cells.

[0213] A Gene Ontology Biological Process analysis was conducted using the TCGAbreast cancer database. The evaluation indicated a strong correlation between MGAT1 expression and aspects of the adaptive immune response, including T cell activation and their antitumor functions. An increase in MG ATI expression was correlated with the down-regulation of immune response, T cell activation, and proliferation. Furthermore, the results from immunohistochemical analysis with the TMA demonstrated a significant negative correlation of MGAT1 protein levels with the CD8+ T cell infiltration in TNBC patient specimens. Additionally, data derived from TIDE, a computational model designed to assess gene correlations with T cell dysfunction, identified MGAT1 as a top-ranking N-glycan biosynthesis gene associated with a T cell dysfunction score.

[0214] To further determine the prognostic value of MGAT1 expression in TNBC patients, a cohort of 156 TNBC samples were analyzed from the TCGA database. A strong correlation between elevated MGAT1 expression and negative overall survival outcomes was observed. Moreover, when evaluating cumulative survival with respect to both MGAT1 expression levels and the CD8+ T cell population, it was evident that patients with a high CD8+ T cell presence coupled with reduced MGAT1 expression exhibited a significant survival advantage, and patients with low CD8+ T cell and high MGAT1 expression were associated with worse prognosis. This data links increased MGAT1 expression to a worse prognosis in the context of immunosuppressive TNBC.

[0215] MGAT1 regulates CD8+ T cell function in 2D and 3D tumor / immune cell coculture systems

[0216] To determine the impact of MGAT1 in regulating tumor immune response, 2D coculture analyses with TNBC breast cancer cells and pre-activated peripheral blood mononuclear cells (PBMCs), was performed followed by 3D coculture analysis using TNBC tumor spheroids and PBMCs. To this end, Flag-tagged MGAT1 overexpression (OE) and MGAT1 stable knockdown (KD) in MDA-MB-468 cells was done based on a lentivirus and CRISPR / Cas9 system. In the 2D coculture, the impact of MGAT1 in regulating CD8+ T cell function that, in turn, affects the cancer cell survival was measured by flow cytometry. MGAT1 OE in tumor cells conferred resistance to immune cell-mediated cell death, whereas tumor cells with KD of MGAT1 were more vulnerable to immune cell-mediated killing. Simultaneously, the flow cytometric evaluation of immune cells indicated that the up-regulation of MGAT1 in cancer cells dramatically inhibited the expression of TNFa, IFNy, and Ki-67 in CD8+ T cells, while depletion of MGAT1 dramatically enriched the expression of TNFa, IFNy, Granzyme B, and Ki-67, indicating a potent role for MGAT1 in modulating T cell antitumor function and proliferation. Because of the advantage of a 3D coculture system in mimicking the tumor microenvironment that represents a more physiological scenario, tumor spheroids were established based on TNBC cells. They were cocultured with stained PBMCs. The infiltration of stained immune cells in the 3D cancer cell spheroids was visualized with Z-stack imaging followed by Z-projection. Intriguingly, depletion of MGAT1 significantly boosted immune cell infiltration into MGAT1-KD TNBC spheroids, whereas elevated expression of MGAT1 in tumor cells drastically suppressed the infiltration of immune cells in the MGAT1- OE TNBC spheroids. Furthermore, flow cytometry analysis further revealed similar immune suppression of T cells cocultured with MGAT1-OE tumor cells and hyperactivation of T cells with MGAT1-KD tumor cells. Consistent with the PBMC coculture system, the coculture of MGAT1 stable cell lines with purified CD8+ T cells leads to a similar consequence on CD8+ T cell inactivation. Collectively, these results indicate that the glycosyltransferase MGAT1 acts as a crucial negative regulator of CD8+ T cells in immune cold TNBC, whose accumulation could contribute to tumor immune evasion through suppressing cytotoxic IFNy-secreting CD8+ T cell function.

[0217] Identification of CD73, an immune checkpoint protein, as a putative substrate of MGAT1 that mediates MG ATI-initiated immune suppression

[0218] To identify the downstream substrates of MGAT1 that facilitate its immune suppression, tandem affinity purification was conducted coupled with mass spectrometry to isolate the MGAT1 interactome. Stable Flag-HA-tagged hMGATl protein was expressed in MDA-MB468 cells, and the MGAT1 protein complex was then purified by affinity capture in parallel with control cells, followed by mass spectrometry analysis. This led to the identification of CD73 as a biochemical binding partner and putative downstream substrate of MGAT1 in TNBC cells. The interactome of MGAT1 resolved by the mass spectrometry analysis led to several tumor immune-responsive proteins, including antigen-presenting proteins and CD73. The results of the initial validation using co-immunoprecipitation drew attention to CD73. To further decipher the physiological relevance of the interaction between MGAT1 and CD73, a series of immunoprecipitation analyses and immunostaining for colocalization were performed. Endogenous CD73 co-immunoprecipitated with MGAT1 was observed, and MGAT1 coimmunoprecipitated with CD73. To examine the cellular compartmentalization of the interaction between MGAT1 and CD73, immunofluorescence staining to co-stain MGAT1 and CD73 was conducted followed by imaging with confocal microscopy and stimulated emission depletion microscopy. Surprisingly, MGAT1 colocalized with CD73 in the Golgi body verified by GM- 130, a Golgi body indicator. The CD73 fluorescent dots were colored based on their distance to MGAT1 in the 3D reconstructed Z-stack imaging. A proximity ligation assay was conducted to corroborate the colocalization of MGAT1 and CD73 in the Golgi body. To ascertain the physiological relevance of the CD73 and MGAT1 colocalization, adenosine production was measured with adenosine assay kits in both human and mouse TNBC breast cancer cells (MDA-MB231 and MDA-MB468) in response to altered MGAT1 expression. Overexpression (OE) of MGAT1 in breast cancer cells significantly increased adenosine production, whereas MGAT1 KD resulted in a reduction of adenosine production. Consistent with the above results, Spearman's rank correlation analysis confirmed the correlation between MGAT1 protein levels with several immune regulators, especially CD73 (NT5E). These experiments indicate a strong positive correlation between CD73 protein levels and several N-glycan biosynthesis genes, including MGAT1. In addition, in a TNBC, elevated MGAT1 protein levels were positively correlated with overexpression of CD73. Taken together, these results indicate that CD73 is a substrate of MGAT1 in mediating MGAT1 -initiated immune suppression.

[0219] MGAT1 catalyzes CD73 glycosylation that, in turn, triggers CD73 dimerization, ensuring its translocation from the cytosol to the cell membrane

[0220] To further dissect the biochemical consequence of CD73 glycosylation by MGAT1, CD73 protein turnover, cellular localization, and interplay were systematically analyzed with other biochemical modifications such as ubiquitination. MGAT1 OE and KD were engineered in MDA- MB468 and MDA-MB231 cells. While a minor change in CD73 abundance in response to MGAT1 OE was observed, reduced glycosylation of CD73 (running smaller molecular mass about 60 kDa) was clearly detected when MGAT1 was knocked down, confirming the aforementioned CD73 glycosylation catalyzed by MGAT1. The effect of endoglycosidase H (specifically cleaving high mannose and hybrid N-glycans) and PNGase F (an N-glycosidase removing all N-linked glycans) were also tested. N-glycan is the major type of asparagine-linked carbohydrate attached to CD73, but without sufficient MGAT1, a fraction of less glycosylated CD73 is covered with high mannose and hybrid N-glycan. Also observed was a significant increase in the membrane fraction of CD73 in MGAT1 elevated expression but a decrease in MGAT1 depletion. Similar results were observed by alternative flow cytometry measurements.

[0221] In response to MGAT1 -mediated CD73 modification, a dramatic change in CD73 dimerization was observed. The dimerization of CD73 was confirmed when the CD73 complex was stabilized in the presence of a crosslinking agent, and dimerization was further confirmed by running native gels. While CD73 dimer was robustly measured for wild-type CD73 in native gels, replacement of Asn 53, 311, 333, and 403 by Gin (CD73-4NQ mutants) led to a significant decrease in CD73 dimerization on native PAGE. The CD73 dimerization interface was further identified based on molecular docking of CD73 monomers in both open and closed conformations and thereafter engineered a dimerization-deficient mutant of CD73. Deletion of amino acids 480- 537 on CD73 diminished CD73 dimerization as measured by native PAGE. Similarly, MGAT1 KD dramatically decreased CD73 dimerization and increased the fraction of the CD73 monomer, confirming the biochemical role of MGAT1 -mediated glycosylation in regulating CD73 dimerization.

[0222] To visualize the CD73 dimer in cells, split-GFP assays were conducted to validate the influence of glycosylation on CD73 dimerization. Plasmids of CD73 with GFP 1-10 or GFPllx7 extensions were engineered and co-transfected into HEK293T cells, and the green fluorescence was visualized with a confocal microscope. The strongest green fluorescence for WT CD73 were captured, while the signal from CD73-4NQ and dimer-deficient mutant CD73 was largely diminished. This observation was further confirmed by stably expressing CD73 with GFP 1-10 or GFP 11x7 in MDA-MB231 and MD-MB468 cells. To evaluate how MGAT1 expression affects the CD73 dimerization, the WT CD73 plasmids with GFP 1-10 or GFP 11x7 extensions were transformed into the MGAT1-WT / OE / KD MDA-MB468 stable lines, and the intensity of green fluorescence was measured by flow cytometry. MGAT1 OE significantly enhanced the dimerization of CD73, whereas MGAT1 KD decreased the CD73 dimerization. In addition, the impact of MGAT1 on the total cellular glycan distribution was measured by flow cytometry with fluorophore-conjugated lectins. MGAT1 OE dramatically increased the membrane complex type glycan recognized by PLA-H, while MGAT1 KD decreased the membrane distribution of complex glycan and sialic acid.

[0223] To elucidate the mechanism by which MGAT1 -mediated CD73 glycosylation and dimerization orchestrates CD73 membrane translocation from the trans-Golgi network to the plasma membrane, mass spectrometry was conducted to identify CD73 binding partners in TNBC cells. In theory, the spatial distribution of cellular organelles within exocytic pathways is distinctive, and they communicate via a complex vesicle tubular transport system. This process is orchestrated by RAB and v-SNARE proteins, which manage consecutive transport stages including vesicle formation, movement, and docking at target sites. Indeed, several vesicle- associated proteins were detected including RAB 8 A, RAB 13, and VAMP3, tightly interacting with CD73, with validation by coimmunoprecipitation and proximity ligation assays. The molecular detail on the functional interaction between CD73 and VAMP3 (a member of the vesicle-associated membrane protein family that facilitates vesicle exocytosis, docking, and fusion) were deciphered. Binding of CD73 to VAMP3 enables CD73 membrane fusion and translocation, whereas deficiency of glycosylation-mediated CD73 dimerization resulted in failure to translocate to the membrane. These experiments indicate that MGAT1 catalyzes CD73 glycosylation that, in turn, triggers CD73 dimerization, ensuring its translocation from the cytosol to the membrane via the VAMP3-RAB cascade.

[0224] To investigate the upstream signaling that potentially modulates MGAT1 in the context of tumor invasion, Spearman's rank correlation analysis was conducted and a tight correlation was found between elevated expression of MGAT1 and up-regulation of THBS1 and THBS2 signaling pathways. To confirm this observation, the effect of THBSland THBS2 on the MGAT-CD73- adenosine cascade was validated. It was observed that stimulation of TNBC cells with THBS1 leads to increased MGAT1 abundance and up-regulated membrane-bound CD73 levels as well as enhanced adenosine production, suggesting the overactivation of MGAT1 in tumor immune invasion could be due to abnormal THBS1 signaling. Development of a pharmacological inhibitor of MGAT1 that restores tumor immune responses in immune-cold breast cancer cells

[0225] To determine whether blocking the aberrant accumulation of CD73 on the tumor cell surface could revive tumor immune response in immune-cold breast cancers, an in vitro high- throughput screening assay was developed to monitor the effect on MGAT1 enzymatic activity from a library of compounds to identify inhibitors that could block the assembling glycan chain. The inhibitor TW-37 was initially the most potent candidate for inhibiting MGAT1 activity (See Figures 1A-1D).

[0226] A computational model was used to search for more potent and specific MGAT1 inhibitors based on the structure of TW-37. Collectively, virtual screening yielded a selection of compounds for experimental validation. The 14 lead compounds from the virtual screening were initially validated in an MGAT1 enzymatic activity assay. Several compounds, including No. 2, No. 8, No. 9, and No.14, showed potent inhibition of MGAT1 catalytic function. The effect of these candidate compounds on CD73 membrane translocation in cancer cells were evaluated. The candidate compounds were added to the growth medium of MDA-MB231 and MDA-MB468 cells, and the membrane-bound CD73 abundance was measured with flow cytometry and immunostaining. The adenosine production response to identified compounds was measured as well. The results demonstrated No. 2, No. 8, and No. 9, are candidates based on the dual criteria of MGAT1 activity inhibition and suppression of membrane translocation of CD73. They all showed dose-dependent inhibition of MGAT1 catalytic function.

[0227] These candidates were further evaluated using cancer and immune cell coculture to assess the effect on the antitumor immune response. Cancer cell survival was examined with image-based methods for determining viability, and CD8+ T cell function was detected using flow cytometry. Based on evaluation standards from multiple layers, W-GTF01 (No. 8 compound) was confirmed as a potent inhibitor of MGAT1 activity and stimulator of CD8+ IFNy-producing T-cell response. To investigate the mechanism by which W-GTF01 leads to superior efficacy in inhibiting MGAT1 , W-GTF01 was docked onto human MGAT. The computed binding affinity for W-GTF01 was determined to be -10.4 kcal / mol. Modulation of MGATl-mediated CD73 membrane translocation affects tumor growth and capacity to elicit antitumor CD8+ T cell responses

[0228] To evaluate the role of MGAT1 in (in vivo) tumor growth, murine cell lines were created with modified MGAT1 expression. MGAT1-OE and MGAT1-KD 4T1 and EO771 cells were developed, with an empty vector as a control. The impact on tumor growth was then assessed using a syngeneic mouse model. Engineered 4T1 control and 4T1 MGAT1 OE breast cancer cells were subcutaneously injected into the mammary fat pad of female BALB / C mice. The volume of mammary tumors was measured with calipers and calculated using the formula: V = (W2 x L) / 2, where V is the tumor volume, W is the tumor width, and L is the tumor length. A significant increase in both the tumor size and tumor weight was observed 25 days post-injection of the MGAT1-OE tumor cells. Furthermore, consistent with the aforementioned in vitro results, MGAT1 OE resulted in an increase in membrane-bound CD73 on tumor cells from tumor-bearing mice. Similar results were observed in E0771 and E0771 MGAT1 OE models. These data support a tumor-promoting role of the MGAT1-CD73 axis in vivo.

[0229] To analyze the impact of MG ATI in modulating tumor immunity, the tumor immune infiltrates were examined using a high-dimensional spectral flow cytometry panel that incorporated hallmark markers for all major immune populations. The dimensionality reduction tool was employed to compare 4T1 control and 4T1 MGAT1 OE tumors. Live intact single cells gated from CD45+ tumor infiltrates could be clearly grouped into distinct subsets, including CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), proliferating CD8+ T cells (CD3+CD8+Ki- 67+), CD8+ tissue resident memory T cells (CD69+CD103+, TRM), exhausted CD8+ T cells (PD- 1+TIM3+, TEX), stem-like progenitors of exhausted CD8+ T cells (CD69+LylO8+, TPEX), Treg (CD3+CD4+ Foxp3+CD25+), non-Treg CD4+ T cells (CD3+CD4+ Foxp3-), B cells (CD19+CD3-), dendritic cells (MHC-II+CDllc+CDllb+, DCs), tumor-associated macrophages (Grl- F4 / 80+CDllb+, TAMs), polymorphonuclear myeloid-derived suppressor cells (Ly6G+ Ly6CloCDl lb+, PMN-MDSC), monocytic MDSCs (Ly6Glo Ly6C+CDl lb+, M-MDSCs), NK (CD3- NKp46+), and NKT (CD3+NKp46+) cells. Frequencies of exhausted CD8+ T cells, Tregs, monocytic MDSCs, and TAMs among CD45+ tumor infiltrates in 4T1 MGAT1 OE tumor-bearing mice were significantly increased compared to control mice. Furthermore, terminal effector-like TEX cells were more abundant in MGAT1 OE tumors as indicated by increased expression levels of CX3CR1, CD101, and TOX. MGAT1 OE in tumor cells enhanced a tumor-promoting M2-like phenotype in TAMs, usually defined by the expression of CD 163. Furthermore, there was a significant reduction in IFNy and TNFa secretion by infiltrating CD8+ T cells in MGAT1 OE tumors.

[0230] Conversely, MGAT1 KD in either 4T1 or EO771 murine breast cancer cell lines hindered tumor development compared to the control cell line (with empty vectors) as determined by both tumor size and tumor weight measurements. Additionally, there was decreased fraction of Tim3+ / PD1+ and increased fraction of CD8+ T cells expressing a higher level of Ki67 in MGAT1 knockdown tumor-bearing mice compared to that in the control mice. To evaluate the importance of the physical interaction between MG ATI and CD73 for tumor growth, both MGAT1 and CD73 WT (MGAT1 OE + CD73WT) or MGAT1 and CD73-4NQ (MGAT1 OE + CD734NQ) in 4T1 and EO771 murine TNBC cell lines were overexpressed. EO771, EO771-MGAT1-CD73WT, and EO771-MGAT1-CD734NQ cells were subcutaneously injected into the mammary fat pad of female C57BL / 6 mice. While there was no significant difference in tumor growth between mice with EO771-MGAT1-CD73 4NQ and EO771 vehicle control, tumor growth was accelerated in EO771-MGAT1-CD73WT tumor-bearing mice. MGAT1-CD73 co-overexpression resulted in a significant increase in membrane-bound CD73 on tumor cells followed by immune-suppression from tumor-bearing mice, but it is not observed in E0771-MGAT1- CD734NQ. Similar results were observed in 4T1 models. These results underscore the importance of MGAT1 -mediated glycosylation of CD73 for tumor growth.

[0231] Pharmacological blockade of the MGAT1-CD73 axis promotes tumor immunogenicity and inhibits tumor progression in immune-cold breast cancers

[0232] To determine the therapeutic relevance of our newly developed inhibitor W-GTF01, the antitumor effect of W-GTF01 by single treatment or in combination with the anti-hPD-Ll drug durvalumab was examined using a TNBC 4T1 model where the endogenous mouse PD-L1 was knocked out and replaced with the human counterpart. W-GTF01 was injected twice per week at the dose of 5 mg / kg and PD-L1 antibody durvalumab was injected three times at the dose of 5 mg / kg. PBS and IgG were used in control groups. Combining W-GTF01 with durvalumab led to enhanced suppression in tumor growth and prolonged survival without evident toxic effects and consistent mouse body weights. To assess the spatial interaction of MGAT1 and CD73 in tumor cells with immune signaling programs within the tumor microenvironment, spatially indexed transcript profiling (GeoMx™ digital spatial profiling) of tumor tissue cores (3 per patient) from 50 TNBC patients was conducted. Regions of interest (ROIs) per tissue section were profiled. Each region further segmented into epithelial versus non-epithelial areas based on staining. Co-staining of MGAT1 and CD73 was used subsequently for spatial segmentation analysis. These regions were scanned to construct digital maps of cellular content and ROIs containing heterocellular populations representative of the whole section or regions exclusively containing immune cells. Individual ROIs were subject to independent transcriptional analysis by next-generation sequencing. The major cell types were classified using the CIBERSORT™ digital cytometry platform. ROIs contained a diverse, spatially variable population of constituent cells. There were relatively higher average fractions of immune system cell types in the non-epithelial stromal areas as compared to PanCK+ tumor areas. Although the non-tumor regions displayed similar proportions of endothelial cells, fibroblasts, and individual immune subsets between MGATllo and MGATlhi areas or between MGATlloCD731o (DL) and MGATlhiCD73hi areas (DH), CD8+ memory T cells, memory B cells, and plasma cells were enriched at higher levels in MGATllo areas relative to MGATlhi areas or DL relative to DH areas within the non-epithelial stromal compartment.

[0233] A positive correlation between MGAT1 and membrane CD73 expression was validated in malignant epithelial areas across all regions per tumor using multiplexed immunohistochemistry (mIHC). Notably, the subsequent spatial analysis revealed a greater abundance of CD8+ T cells touching DL tumor cells compared with that touching DH tumor cells. Using gene pathway analysis (KEGG / Reactome / Gene ontology) based on spatial transcriptomes, the glycoprotein pathways pertaining to CD73 glycosylation modulation were found to be particularly enriched in DH compared to DL tumor areas, further supporting a specific role of MGAT1 in regulating CD73 glycosylation and protein expression in tumor cells.

[0234] Innate immune response, B cell receptor signaling and B cell differentiation, T cell activation, and TCR signaling pathways were found to be related closely to the tumor immune response and significantly enriched in non-tumor compartments from DL areas compared to that from DH areas. In addition, Gene Set Enrichment Analysis (GSEA) revealed the significantly enriched pathways of TGF-P signaling and IL-10 signaling in non-tumor compartments from DH areas compared to that from DL areas. Similar results are also obtained from comparison between MGATllo and MGATlhi areas in non-tumor compartments across all ROIs. These findings indicate that distinct immune features of the DH areas are implicated in the suppression of antitumor responses and the promotion of tumor growth, further supporting the importance of MGAT1 for CD73 glycosylation in tumor immune evasion.

Claims

CLAIMSWhat is claimed is:1 . A compound that binds MGAT1 inhibiting bind to CD73.

2. The compound of claim 1 which is N-(3-(5,6-dimethylbenzo[d]oxazol-2-yl)-4-hydroxy-5- methylphenyl)benzo[d][l,3]dioxole-5-carboxamide (W-GTFOl / Compound No. 8) or salt thereof.

3. The compound of claim 1 have the following formula.or salt thereof wherein n is 1 or 2;X is NH, O, S, or CH2;Y is CH2or CH2CH2;Z is O, S, NH, or NR12,R1, R2, R3, R4, R3, R6, R7, R8, R9, R10, R11, or R12are each, the same or different, hydrogen, alkyl, halogenated alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, glycol, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkyl sulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, or R12are optionally substituted with one or more, the same or different, R13;R13is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, alkoxy, halogenated alkoxy, alkylthio, alkylamino, (alkyl)2amino,alkyl sulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R13is optionally substituted with one or more, the same or different, R14; andR14is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N- ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methyl sulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.In certain embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, or R12are each, the same or different, hydrogen, halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, acetylamino, acetyloxy, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N- methyl-N-ethylamino, acetylamino, acetyloxy, N-methylcarbamoyl, acetylamino, acetyloxy, N- ethylcarbamoyl, acetylamino, acetyloxy, N,N-dimethylcarbamoyl, acetylamino, acetyloxy, N,N- diethylcarbamoyl, acetylamino, acetyloxy, N-methyl-N-ethylcarbamoyl, acetylamino, acetyloxy, methylthio, ethylthio, methyl sulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethyl sulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

4. The compound of claim 3 wherein X is NH, Y is CH2, and Z is O.

5. The compound of claim 3 wherein Z is O, R1is H, R2is alkyl R3is alkyl, and R4is H.

6. The compound of claim 3 wherein X is NH, R5is hydroxy, R6is alkyl, R7is H, and R11isH.

7. The compound of claim 3 wherein Y is CH2, n is 1, R8is H, R9is H, and R10is H.

8. The compound of claim 1 which is 2-amino-l -((3,4-dihydroxybenzylidene)amino)-N-(2,5- dimethylphenyl)-lH-pyrrolo[2,3-b]quinoxaline-3-carboxamide (compound 9) or salt thereof.

9. The compound of claim 1 which is 2-(5-cyclopropyl-lH-pyrazol-3-yl)-10-methyl-ll-(l- methyl-lH-pyrazol-4-yl)-9,ll-dihydropyrazolo[4',3':5,6]pyrano[3,2-e][l,2,4]triazolo[l,5- c]pyrimidine (compound 14) or salt thereof.

10. The compound of claim 1 which is 6-(2-(anthracen-9-ylmethylene)hydrazineyl)-N2-(4- nitrophenyl)-N4-(o-tolyl)-l,3,5-triazine-2,4-diamine (Compound 2) or salt thereof.

11. The compound of claim 1 which is N-(4-((2-(tert-butyl)phenyl)sulfonyl)phenyl)-2,3,4- trihydroxy-5-(2-isopropylbenzyl)benzamide (TW-37) or salt thereof.

12. A pharmaceutical composition comprising a compound as in any of claims 1-11 and a pharmaceutically acceptable excipient.

13. The pharmaceutical composition of claim 12 in the form of a pill, tablet, capsule, gel, gel capsule, powder, lotion, or cream.

14. The pharmaceutical composition of claim 12, in the form of a liquid optionally isotonic comprising pH buffering agents and salts, and / or saccharide or polysaccharide.

15. The pharmaceutical composition of claim 12, wherein the pharmaceutically acceptable excipient is selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N-pyrrolidone, crospovidone, ethyl cellulose, povidone, methyl and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, corn starch, carnauba wax, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, iron oxide, triacetin, acacia gum, esters, or salts thereof.

16. A method of treating cancer comprising administering an effective amount of a compound as in any of claims 1-11 to a subject in need thereof.

17. The method of claim 16, wherein the cancer is breast cancer.

18. The method of claim 17, wherein the subject is diagnosed with triple negative breast cancer.

19. The method of claim 16, wherein the compound is administered in combination with a chemotherapy agent.

20. The method of claim 19, wherein the chemotherapy agent is a checkpoint inhibitor.

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