Inhibiting exosome biogenesis for the treatment of cancer

Abstract

Disclosed are compounds, pharmaceutical compositions, and methods for inhibiting exosome biogenesis in the treatment of cancer. Exosome biogenesis inhibitors include compounds of Formula (A) and Formula (B), pharmaceutically acceptable salts thereof, and prodrugs thereof, in particular, hypoxia‑activated prodrugs (HAPs): (A) (B).

Description

Attorney Docket No. 134675-875134TITLE INHIBITING EXOSOME BIOGENESIS FOR THE TREATMENT OF CANCER CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 748,639, filed January 23, 2025, the entire contents of which are incorporated herein by reference.FIELD OF THE DISCLOSURE

[0002] The present disclosure generally relates to compounds, compositions, and methods for treating cancers by inhibiting exosome biogenesis.BACKGROUND

[0003] Cancer remains a leading cause of morbidity and mortality worldwide. Over the past decade, immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxicT-lymphocyte-associated protein 4 (CTLA-4), and other pathways have transformed the treatment landscape for several malignancies, including melanoma, lung cancer, and certain other solid tumors. These agents can unleash anti-tumor T-cell responses and have produced durable clinical benefit in subsets of patients. However, only a fraction of patients are eligible for checkpoint blockade based on target expression, and even among eligible patients, objective response rates and long-term survival remain limited. Many patients either do not respond initially or develop acquired resistance, and systemic immune activation can lead to immune-related adverse events that restrict broader use. Accordingly, there is a continuing need to identify additional therapeutic strategies that address mechanisms of immune evasion beyond classical checkpoint pathways.

[0004] In addition to direct cell-cell interactions, tumors communicate with their microenvironment and distant tissues through extracellular vesicles, including exosomes. Exosomes are nanoscale membrane vesicles that originate from the endosomal system and are released when multivesicular bodies fuse with the plasma membrane. They can carry a diverse cargo of proteins, lipids, and nucleic acids, and have been implicated in modulating immune responses, remodeling the tumor microenvironment, promoting angiogenesis, and facilitating metastatic spread.1108531467.1Attorney Docket No. 134675-875134Tumor-derived exosomes (TDEs) can contribute to immune suppression by, for example, carrying inhibitory ligands, enzymes, or microRNAs that dampen effector cell function or promote regulatory cell populations. Elevated levels of circulating exosomes and specific exosomal cargo have been associated with poor prognosis, treatment resistance, and increased metastatic potential in multiple cancer types.

[0005] Exosome biogenesis is a multistep process involving endosomal sorting and membrane remodeling. Early endosomes mature into multivesicular bodies (MVBs), within which the limiting membrane invaginates to form intraluminal vesicles that later become exosomes upon secretion. Both ESCRT-dependent and ESCRT-independent pathways contribute to this process. Lipid metabolism, and in particular the generation of ceramide from sphingomyelin, has been identified as an important driver of inward budding and vesicle formation. Sphingomyelinases, including neutral and acid sphingomyelinase, catalyze the hydrolysis of sphingomyelin to ceramide and thus serve as key regulators of membrane curvature and exosome formation.

[0006] Solid tumors frequently exhibit regions of hypoxia due to abnormal vasculature and rapid proliferation outpacing oxygen supply. Tumor hypoxia is associated with aggressive behavior, resistance to radiotherapy and chemotherapy, and poor clinical outcome. Hypoxia-activated prodrugs (HAPs) have been developed to exploit this feature by remaining relatively inert under normoxic conditions and becoming activated in low-oxygen environments through reductive metabolism. Several HAPs, including nitroaromatic and related scaffolds, have advanced into clinical trials; however, challenges in achieving sufficient tumor selectivity, optimizing protective groups, and appropriately selecting patients based on hypoxia biomarkers have limited their success to date.SUMMARY OF THE DISCLOSURE

[0007] The present disclosure provides methods, compounds, and compositions for treating cancer by directly inhibiting exosome biogenesis, including with small -molecule inhibitors and hypoxia-activated prodrugs (HAPs) (collectively referred to as exosome biogenesis (EB) inhibitors) that target acid key enzymes involved in exosome formation and function, such as sphingomyelinase (aSMase). By suppressing the production and release of tumor-derived exosomes, these agents2108531467.1Attorney Docket No. 134675-875134disrupt exosome-mediated immune suppression and tumor protection, thereby enhancing anti-tumor immune responses and reducing metastatic spread.

[0008] In certain embodiments, the exosome biogenesis inhibitor is built into a HAP scaffold that remains inert under normal oxygen conditions and is selectively activated in the hypoxic tumor microenvironment via reductases that are upregulated in specific cancers, such as malignant pleural mesothelioma, providing improved tumor selectivity and reduced systemic toxicity.

[0009] The disclosed EB inhibitors (e.g., compounds and prodrugs), and compositions can be administered alone or in combination with existing modalities, including immunotherapy, chemotherapy, radiation therapy, and surgery, to improve response rates and overcome treatment resistance across a broad range of solid and hematologic malignancies.

[0010] The present disclosure is drawn to a method for inhibiting, ameliorating, or treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an exosome biogenesis (EB) inhibitor. The EB inhibitors are preferably selected from small molecule compounds and prodrugs thereof. The EB inhibitors are useful for inhibiting tumor growth and / or reducing tumor volume.

[0011] Nonlimiting examples of cancers include malignant pleural mesothelioma (MPM); lung cancer, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC); head and neck cancer, including squamous cell carcinoma; gastrointestinal cancer, including esophageal, gastric, colorectal, pancreatic, hepatobiliary, hepatocellular carcinoma (HCC), and cholangiocarcinoma; genitourinary cancer, including renal cell carcinoma (RCC), bladder cancer, and prostate cancer; gynecologic cancer, including ovarian, endometrial, and cervical cancer; breast cancer, including triple-negative breast cancer (TNBC); skin cancer, including melanoma, basal cell carcinoma, and squamous cell carcinoma; sarcoma, including osteosarcoma and soft tissue sarcoma; central nervous system cancer, including glioblastoma; hematologic malignancy, including leukemia, lymphoma (Hodgkin and non-Hodgkin), and myeloma; thyroid carcinoma; and rare cancers.

[0012] In one aspect, the present disclosure relates compounds of Formula (A) and Formula (B), and pharmaceutically acceptable salts thereof; and to methods for3108531467.1Attorney Docket No. 134675-875134inhibiting, ameliorating, or treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds of Formula (A) and / or Formula (B), or pharmaceutically acceptable salts thereof:Re is an optionally substituted thiadiazolyl.

[0013] In a preferred embodiment Re is selected from:4108531467.1Attorney Docket No. 134675-875134

[0014] The optionally substituted C4-C5 heterocyclyl is an optionally substituted oxazolidin-2-one or an optionally substituted oxetane. Nonlimiting examples of C4-C5heterocyclyls include the following, which may be optionally substituted:

[0015] Nonlimiting examples of compounds of Formula (A) or Formula (B) include compounds 1-50, or pharmaceutically acceptable salts thereof.5108531467.1Attorney Docket No. 134675-8751346108531467.1Attorney Docket No. 134675-8751347108531467.1Attorney Docket No. 134675-8751348108531467.1Attorney Docket No. 134675-8751349108531467.1Attorney Docket No. 134675-87513410108531467.1Attorney Docket No. 134675-875134

[0016] Preferred compounds of Formula (A) and Formula (B) include compounds 41 and 42, and pharmaceutically acceptable salts thereof.

[0017] Further preferred compounds of Formula (A) and Formula (B) include compounds 43-50, and pharmaceutically acceptable salts thereof.

[0018] In another aspect, the present disclosure relates compounds of Formula (I) and Formula (II), and pharmaceutically acceptable salt thereof; and to methods for inhibiting, ameliorating, or treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds of Formula (I) and Formula (II), or pharmaceutically acceptable salts thereof:11108531467.1Attorney Docket No. 134675-875134wherein:Ri and R2are interconnected to form an optionally substituted C4-C5heterocyclyl;R3is selected from: -C(O)-NH-OH, -C(O)-NH-S(O)2-R4, -(CH2)nC(0)-NH- OH, -(CH2)nC(0)-NHS(0)2-R4, and -C(O)-R6;R4 is hydrogen or an optionally substituted Ci-Ce alkyl;n is o, 1, or 2; andRe is an optionally substituted heterocycle.

[0019] In certain embodiments, in Formula (I), when R3is -C(O)-NH-OH, Ri and R2are not :

[0020] In further embodiments, the compounds of Formula (I) are preferably selected from compounds of Formula (I-A) and Formula (I-B), or pharmaceutically acceptable salts thereof:12108531467.1Attorney Docket No. 134675-875134(I-A) (I-B) wherein:R3is -C(O)-NH-OH, -C(O)-NH-S(O)2-R4, or -C(O)-R6; andR4is hydrogen or an optionally substituted C1-C6 alkyl.

[0021] R3is preferably -C(O)-NH-OH.

[0022] In a further embodiment, the compounds of Formula (I) are selected from compounds of Formula (I-C):(I-C)wherein R3is -C(O)-NH-OH, -C(O)-NH-S(O)2R4, or -C(O)-R6, where R6is as defined above as an optionally substituted heterocycle, and wherein R3is preferably -C(O)-NH-OH.13108531467.1Attorney Docket No. 134675-875134

[0023] In another embodiment, the compounds of Formula (II) are preferably selected from compounds of Formula (II-A) and pharmaceutically acceptable salts thereof:(II-A)wherein R3is -C(O)-NH-OH, -C(0)-NHS(0)2R4, or -C(O)-R6, where Re is as defined above as an optionally substituted heterocycle, and wherein R3is preferably -C(O)-NH-OH.

[0024] In certain embodiments, Re is an optionally substituted thiadiazolyl. Nonlimiting examples include:

[0025] The EB inhibitors are preferably administered to subject, preferably a human subject, in a pharmaceutical composition containing the one or more EB inhibitors and one or more a pharmaceutically acceptable carriers. Moreover, the EB inhibitors may be administered to the subject in conjunction with one or more additional anti-cancer therapies, for example selected from immunotherapy, chemotherapy, radiation therapy, and surgery.14108531467.1Attorney Docket No. 134675-875134BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Implementation of the present technology is described, by way of example only, with reference to the attached figure, wherein:FIG. 1 illustrates inhibition of acid sphingomyelinase (aSMase) activity by selected exosome biogenesis (EB) inhibitors in a cell-free assay, including comparison to a reference aSMase inhibitor.FIG. 2 illustrates immunoblot analysis of the exosome marker CD63 in conditioned media from tumor cells treated with an EB inhibitor (a) and immunoblot analysis of the exosome marker TSG101 in conditioned media from tumor cells treated with an EB inhibitor (b).FIG. 3 illustrates flow cytometric analysis of exosome release from tumor cells following treatment with an EB inhibitor.FIG. 4 illustrates nanoparticle tracking analysis (NTA)quantification of exosome release from HCC1806 tumor cells following treatment with an EB inhibitor (a) and nanoparticle tracking analysis (NTA) quantification of exosome release from MDA-MB-231 tumor cells following treatment with an EB inhibitor (b).FIG. 5 illustrates nanoparticle tracking analysis (NTA) of exosome concentration in conditioned media from HCC1806 tumor cells treated with an EB inhibitor (a) and nanoparticle tracking analysis (NTA) of exosome concentration in conditioned media from MDA-MB-231 tumor cells treated with an EB inhibitor (b).FIG. 6 illustrates tumor growth inhibition in an in vivo tumor model following administration of an EB inhibitor.FIG. 7 illustrates body weight measurements over time in subjects treated with an EB inhibitor, demonstrating tolerability.FIG. 8 illustrates plasma and tumor concentration-time profiles of an EB inhibitor following oral administration.FIG. 9 illustrates tumor-to-plasma exposure ratios of an EB inhibitor following oral administration.15108531467.1Attorney Docket No. 134675-875134

[0027] It should be understood that the various aspects are not limited to the arrangements and instrumentality shown in the drawings.DETAILED DESCRIPTION OF THE DISCLOSURE

[0028] The present disclosure addresses an ongoing need for therapeutic modalities that more effectively disrupt exosome mediated communication within the tumor microenvironment and between tumors and distant tissues. The compounds, compositions, and methods described herein are useful for inhibiting exosome production or secretion and for providing tumor selectivity by exploiting hypoxic conditions or other features of the tumor microenvironment. In certain embodiments, exosome biogenesis is inhibited by administering an exosome biogenesis inhibitor, for example a small molecule drug or a hypoxia activated prodrug (HAP), which can suppress the release of tumor derived exosomes (TDEs), and reduce exosome driven immune suppression and tumor protection, and improving treatment outcomes for subjects with cancer.

[0029] Cancer treatment has advanced with the advent of checkpoint inhibitors, which block direct cell contact communication within the tumor micro environment (TME). This mechanism prevents tumors from suppressing immune responses, enabling immune cells to remain active at tumor sites. In cancers such as late-stage melanoma, checkpoint therapy has dramatically increased the 5-year survival rate from 5% to over 50% (1). Despite these successes, checkpoint inhibitors have limitations. These therapies often cause systemic immune activation, leading to adverse effects like long-term neuropathy. Additionally, not all tumors express checkpoint targets, leaving only 20-30% of patients eligible for treatment, and response rates are even lower, at approximately 15-20% (2-4). This underscores the critical need for novel therapeutic options for patients ineligible for checkpoint therapies.

[0030] While much research focuses on targeting direct communication pathways in the TME, indirect communication via exosomes has remained underexplored. Exosomes are nanovesicles secreted by cells that carry diverse cargo— including proteins, lipids, and nucleic acids— capable of modulating cell signaling, gene expression, and cellular function. Tumor-derived exosomes (TDEs) play a pivotal role in immune suppression and tumor protection. Studies highlight the potential impact of inhibiting TDE release. For example, blocking exosome16108531467.1Attorney Docket No. 134675-875134production has been shown to decrease primary tumor growth in metastatic carcinoma (5). Furthermore, circulating exosomes carrying miR-105 correlate with metastasis in breast cancer patients, suggesting their role as drivers of cancer progression (6). Designing therapeutics to disrupt this indirect communication could significantly reduce tumor burden and metastasis, addressing a critical gap in cancer treatment.

[0031] Exosome biogenesis begins in the endosomal system, where early endosomes mature into multivesicular bodies (MVBs). During this maturation process, the MVB membrane invaginates, forming intraluminal vesicles (ILVs) that later become exosomes upon MVB fusion with the plasma membrane (7). Lipid dynamics, particularly the role of ceramides, are central to ILV formation. Ceramide promotes membrane curvature and invagination, critical steps in the biogenesis process (8). Sphingomyelinases, enzymes that convert sphingolipids into ceramides, are essential regulators of this pathway (9). By targeting sphingomyelinases, it may be possible to effectively block exosome biogenesis, thereby disrupting the release of TDEs in the TME.

[0032] Developing an exosome biogenesis inhibitor offers a novel therapeutic strategy to complement existing cancer treatments. In various embodiments, the exosome biogenesis inhibitor is a hypoxia-activated prodrug (HAP), taking advantage of the hypoxic environments characteristic of solid tumors to achieve selective activation and minimize systemic toxicity. Alternatively, in certain embodiments, coupling the inhibitor with an antibody-drug conjugate (ADC) leverages the targeted delivery capabilities of antibodies, ensuring precise therapeutic action at tumor sites. Such approaches enhance the inhibitor’s efficacy and safety, offering a transformative solution for patients with limited treatment options.

[0033] A challenge in developing effective exosome inhibitors is achieving tumor specificity without causing off-target toxicity. Hypoxia-activated prodrugs provide a solution by selectively releasing their active form in the hypoxic conditions that characterize the tumor microenvironment (TME).

[0034] Hypoxia Activated prodrugs (HAPs) have two components: a protective group that is released in a hypoxic environment and an active drug. The protective group targets specific reductases present in the tumor microenvironment. The active17108531467.1Attorney Docket No. 134675-875134drug inhibits exosome biogenesis, a key factor in the aggressive and resistant nature of hypoxic tumors (10,11). Overall, the HAP is believed to suppress the mechanisms through which tumor cells communicate, thereby ‘isolating’ the cancer cells and making them more vulnerable to an active immune system.

[0035] Historically, designing protective groups that reliably release at the tumor site has been difficult. For example, while HAPs like the compound designated “TH-302” reached the clinic, their efficacy was insufficient for approval, primarily due to inadequate drug release at the tumor site (12). This failure was not only linked to the drug design but also to improper clinical strategies. In 2017, a trial using TH-302 to treat pancreatic ductal adenocarcinoma (PDAC) failed, likely due to a lack of proper patient screening for tumor hypoxia. Contrary to the common assumption that all PDAC cases are uniformly hypoxic, research showed significant heterogeneity in hypoxic tumor fractions, with some PDAC tumors showing as little as 0-26% hypoxia (13). Patients with low hypoxic tumor fractions were unlikely to respond to HAPs like TH-302 because the drug's protective group was not optimized to release in such environments (5). With the recent success of antibody-drug conjugates (14), targeted drug delivery is the goal of future of cancer treatment.

[0036] To investigate the potential role of oxidoreductases in a hypoxic tumor microenvironment, a comprehensive analysis of gene expression data was conducted on malignant pleural mesothelioma (MPM) clinical data. Transcriptomic profiles of MPM tumors were obtained from The Cancer Genome Atlas (TCGA) repository. Using a curated human oxido reductase database from UniProt, the dataset was filtered to include only genes encoding oxidoreductase enzymes. Spearman correlation analysis was then performed to identify oxidoreductases exhibiting significant co-expression patterns with HIF1A, a key regulator of cellular responses to hypoxia, in MPM. To expand the scope of this investigation, this analysis was repeated on transcriptomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). This approach allowed for pinpointing of candidate oxidoreductases that would enable the HAP reaction (Table 1).18108531467.1Attorney Docket No. 134675-875134Table iTop 10 candidate oxidoreductases co-expressed with HIF1A in MPM identified through Spearmen correlation of TCGA transcriptomic and CPTAC proteomic data

[0037] Further, the design of protective groups that can be reduced by the most upregulated oxidoreductases in the MPM microenvironment is contemplated. This strategy can be expanded to other solid tumors, wherein protective groups are designed to be reduced by the most upregulated oxidoreductases in the microenvironment of a specific tumor type. Accordingly, the methods and compositions described herein target hypoxia-driven mechanisms and tumor exosome biogenesis— key elements that contribute to treatment resistance and tumor progression in MPM and other cancers.

[0038] Therapies addressing cell-to-cell communication focus on blocking direct immune checkpoint pathways, overlooking the crucial role of indirect cell communication within the TME. Tumor-derived exosomes (TDEs) play a pivotal role in this indirect communication, facilitating immune suppression and shielding tumors from treatment (15,16). Despite the recognition of their involvement in immune evasion, therapeutic strategies targeting exosome biogenesis are notably lacking.

[0039] In various embodiments, the methods, compounds, and compositions described herein, inhibit exosome biogenesis using small-molecule drugs which target acid sphingomyelinase (aSMase, SMPD1), a key enzyme in the formation of exosomes. This approach effectively reduces exosome production and secretion,19108531467.1Attorney Docket No. 134675-875134which has been shown to contribute to tumor growth, metastasis, and treatment resistance (9). Disruption of the immune-suppressive environment within the TME is accomplished by impeding TDE release, making tumors more vulnerable to immunotherapies and traditional treatments such as chemotherapy. This strategy is a first-of-its-kind attempt to halt tumor exosome biogenesis and represents a significant shift in cancer therapy by focusing on the indirect cellular communication that supports tumor survival.

[0040] In further embodiments, the methods, compounds, and compositions described herein combine exosome inhibition with hypoxia-activated drug delivery, an approach that transforms the treatment paradigm for solid and hypoxic tumors. Current treatments often focus on immune checkpoint inhibitors or direct tumor targeting, but they overlook the significant role of exosome-mediated immune suppression and metastasis. By addressing this underexplored aspect of the TME, the \methods, compounds, and compositions described herein substantially improve treatment efficacy and increase survival rates for patients with few current options. Furthermore, as hypoxic tumors account for nearly 40% of all cancer cases, the broader implications of the presently disclosed technology offer a scalable and adaptable solution for multiple tumor types.

[0041] Nonlimiting examples of cancers that are the tumor type include malignant pleural mesothelioma (MPM); lung cancers such as non-small cell lung cancer (NSCLC) and small cell lung cancer; head and neck cancers, including squamous cell carcinoma; gastrointestinal cancers such as esophageal, gastric, colorectal, pancreatic, and hepatobiliary cancers like hepatocellular carcinoma (HCC) and cholangiocarcinoma; genitourinary cancers including renal cell carcinoma (RCC), bladder cancer, and prostate cancer; gynecologic cancers such as ovarian, endometrial, and cervical cancers; breast cancer, including triple-negative breast cancer (TNBC); skin cancers, including melanoma and non-melanoma types such as basal cell carcinoma and squamous cell carcinoma; sarcomas, including osteosarcoma and soft tissue sarcomas; central nervous system cancers such as glioblastoma and other brain tumors; hematologic malignancies such as leukemia, lymphoma (Hodgkin and non-Hodgkin), and myeloma; and rare cancers such as thyroid carcinoma and other uncommon malignancies.20108531467.1Attorney Docket No. 134675-875134

[0042] In one aspect, the present disclosure relates compounds of Formula (A) and Formula (B), and pharmaceutically acceptable salts thereof; and to methods for inhibiting, ameliorating, or treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds of Formula (A) and / or Formula (B), or pharmaceutically acceptable salts thereof:wherein:Ri and R2 are interconnected to form an optionally substituted C4-C5 heterocyclyl;R3is selected from: -C(O)-NR4R5, -C(O)-NH-R4, -C(O)-NH-NR4R5, -C(O)- NH-NH-R4, -C(O)-NH-OH, -C(O)-NH-S(O)2-R4, -C(O)-R6, -NH- C(O)-R4, -P(O)(OH)-OR5, -P(0)(0H)2, -NR5-C(O)-R4, -C(S)-NH-R4, -C(S)-NH-NR4R5, and -C(S)-NH-NH-R4;R4is an optionally substituted Ci-Ce alkyl;Re is an optionally substituted thiadiazolyl.

[0043] The substituents of the optionally substituted Ci-Ce alkyl (R and the optionally substituted thiadiazolyl (Re) when substituted are independently preferably selected from halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and21108531467.1Attorney Docket No. 134675-875134optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Preferred substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.

[0044] The substituents are more preferably selected from C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 alkenyl, C1-C4 substituted alkenyl, C1-C4 alkynyl, C1-C4 substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, C1-C4 alkoxy, substituted C1-C4 alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, C1-C4 alkylthio, substituted C1-C4 alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, and substituted phosphonyl.

[0045] When substituted, the Ci-Ce alkyl (R4) preferably includes 1, 2, or 3 substitutions, preferably 1 substitution. When substituted, the thiadiazolyl (Re), preferably includes 1 or 2 substitutions, preferably 1 substitution.

[0046] In a preferred embodiment Re is selected from:22108531467.1Attorney Docket No. 134675-875134

[0047] The optionally substituted C4-C5 heterocyclyl is preferably an optionally substituted oxazolidin-2-one or an optionally substituted oxetane.

[0048] The substituents of the optionally substituted oxazolidin-2-one or an optionally substituted oxetane when substituted are independently preferably selected from halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Preferred substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, aiylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.

[0049] The substituents are more preferably selected from C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 alkenyl, C1-C4 substituted alkenyl, C1-C4 alkynyl, C1-C4 substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, C1-C4 alkoxy, substituted C1-C4 alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, C1-C4 alkylthio, substituted C1-C4 alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, and substituted phosphonyl.

[0050] Nonlimiting examples of C4-C5 heterocyclyls include the following, which may be optionally substituted:23108531467.1Attorney Docket No. 134675-875134

[0051] The substituents independently preferably selected from halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Preferred substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaiyl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.

[0052] The substituents are more preferably selected from C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 alkenyl, C1-C4 substituted alkenyl, C1-C4 alkynyl, C1-C4 substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, C1-C4 alkoxy, substituted C1-C4 alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, C1-C4 alkylthio, substituted C1-C4 alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, and substituted phosphonyl.

[0053] Nonlimiting examples of compounds of Formula (A) or Formula (B) include compounds 1-50, and pharmaceutically acceptable salts thereof.24108531467.1Attorney Docket No. 134675-87513425108531467.1Attorney Docket No. 134675-87513426108531467.1Attorney Docket No. 134675-87513427108531467.1Attorney Docket No. 134675-87513428108531467.1Attorney Docket No. 134675-87513429108531467.1Attorney Docket No. 134675-87513430108531467.1Attorney Docket No. 134675-875134

[0054] Preferred compounds of Formula (A) and Formula (B) include compounds 41 and 42, and pharmaceutically acceptable salts thereof.

[0055] Further preferred compounds of Formula (A) and Formula (B) include compounds 43-50, and pharmaceutically acceptable salts thereof.

[0056] In various embodiments, the exosome biogenesis inhibitor comprises a hypoxia-activated prodrug (HAP), for example a HAP that includes a protective group configured to be released under hypoxic conditions. Nonlimiting examples of protective groups include Compound A and Compound B:Compound A Compound B

[0057] Preferably, the HAP is inert in normal oxygen conditions but activates in hypoxic environments.

[0058] In various embodiments, the protective group targets reductases, such as sphingomyelinase (aSMase, SMPD1), present in the tumor microenvironment.31108531467.1Attorney Docket No. 134675-875134

[0059] Nonlimiting examples of exosome biogenesis inhibitors comprising a hypoxia-activated prodrug include compounds 11-40 and pharmaceutically acceptable salts thereof .

[0060] In another aspect, the present disclosure relates to exosome biogenesis inhibitors selected from compounds of Formula (I) and Formula (II), and pharmaceutically acceptable salt thereof; and to methods for inhibiting, ameliorating, or treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds of Formula (I) and Formula (II), or pharmaceutically acceptable salts thereof:Ri and R2are interconnected to form an optionally substituted C4-C5heterocyclyl;R3is selected from: -C(O)-NH-OH, -C(O)-NH-S(O)2-R4, -(CH2)nC(0)-NH- OH, -(CH2)nC(O)-NHS(O)2-R4, and -C(O)-R6;Ri is hydrogen or an optionally substituted Ci-Ce alkyl;n is o, 1, or 2; andRe is an optionally substituted heterocycle.

[0061] The substituents of the optionally substituted C4-Cs heterocyclyl (Ri and R2), the optionally substituted Ci-Ce alkyl (R , and optionally substituted heterocycle (Re) are independently preferably selected from halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-1432108531467.1Attorney Docket No. 134675-875134carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats.Preferred substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaiyl, substituted polyaiyl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.

[0062] The substituents are more preferably selected from C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 alkenyl, C1-C4 substituted alkenyl, C1-C4 alkynyl, C1-C4 substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, C1-C4 alkoxy, substituted C1-C4 alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, C1-C4 alkylthio, substituted C1-C4 alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, and substituted phosphonyl.

[0063] In certain embodiments, in Formula (I), when R3is -C(O)-NH-OH, Ri and R2are not :

[0064] In further embodiments, the compounds of Formula (I) are preferably selected from compounds of Formula (I-A) and Formula (I-B), or pharmaceutically acceptable salts thereof:33108531467.1Attorney Docket No. 134675-875134(I-A) (I-B) wherein:R3is -C(O)-NH-OH, -C(O)-NH-S(O)2-R4, or -C(O)-R6; andR4is hydrogen or an optionally substituted Ci-Co alkyl.

[0065] R.3 is preferably -C(O)-NH-OH.

[0066] The substituents of the optionally substituted Ci-Ce alkyl (R are independently preferably selected from halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Preferred substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.

[0067] The substituents are more preferably selected from Ci-C4alkyl, C1-C4 substituted alkyl, C1-C4 alkenyl, C1-C4 substituted alkenyl, C1-C4 alkynyl, Ci-C434108531467.1Attorney Docket No. 134675-875134substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, C1-C4 alkoxy, substituted C1-C4 alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, C1-C4 alkylthio, substituted C1-C4 alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, and substituted phosphonyl.

[0068] In a further embodiment, the compounds of Formula (I) are selected from compounds of Formula (I-C):(I-C)wherein R3is -C(O)-NH-OH, -C(O)-NH-S(O) R4, or -C(O)-R6, where R6is as defined above as an optionally substituted heterocycle, and wherein R3is preferably -C(O)-NH-OH.

[0069] In another embodiment, the compounds of Formula (II) are preferably selected from compounds of Formula (II-A) and pharmaceutically acceptable salts thereof:35108531467.1Attorney Docket No. 134675-875134(II-A)wherein R3is -C(O)-NH-OH, -C(O)-NHS(O)2R4, or -C(O)-R.6, where Re is as defined above as an optionally substituted heterocycle, and wherein R3is preferably -C(O)-NH-OH.

[0070] In certain embodiments, R6 is an optionally substituted thiadiazolyl. Nonlimiting examples include:

[0071] The substituents of the optionally substituted thiadiazolyl (Re) when substituted are independently preferably selected from halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats.Preferred substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted36108531467.1Attorney Docket No. 134675-875134sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaiyl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.

[0072] The substituents are more preferably selected from C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 alkenyl, C1-C4 substituted alkenyl, Ci-C4alkynyl, Ci-C4substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, Ci-C4alkoxy, substituted C1-C4 alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, C1-C4 alkylthio, substituted Ci-C4alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, and substituted phosphonyl.

[0073] The EB inhibitors are preferably administered to subject, preferably a human subject, in a pharmaceutical composition containing the one or more EB inhibitors and one or more a pharmaceutically acceptable carriers. Moreover, the EB inhibitors may be administered to the subject in conjunction with one or more additional anti-cancer therapies, for example selected from immunotherapy, chemotherapy, radiation therapy, and surgery.

[0074] In further embodiments, the compounds of Formulae (A), (B), (I), (I-A), (I-B), (I-C), (II), and (II-A) and pharmaceutically acceptable salts thereof, are modified with a protective group configured to be released under hypoxic conditions, i.e., the compounds are HAPs. Nonlimiting examples of protective groups include Compound A and Compound B:Compound A Compound B.

[0075] For example, the protective group (e.g., Compounds A and B) can be attached through an amine group on the compounds of Formulae (A), (B), (I), (I-A), (I-B), (I-C), (II), and (II-A). This is shown, for example, by Compounds 1-10, which37108531467.1Attorney Docket No. 134675-875134become compounds 12-40 by addition of protective groups (protective groups represented by Compounds A and B). All compounds of Formulae (A), (B), (I), (I-A), (I-B), (I-C), (II), and (II-A), including Compounds 41-50 can similarly be modified with one or more protective groups.

[0076] The exome biogenesis inhibitor described herein may be formulated and administered to a subject in a composition, in particular, a pharmaceutical or therapeutic composition. The pharmaceutical compositions preferably include one or more exosome biogenesis inhibitors and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier is "pharmaceutically acceptable" in that it is compatible with the other ingredients of the formulation. It is also suitable for contact with any tissue, organ, or portion of the body that it can encounter, meaning that, ideally it will not carry a significant risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0077] In some embodiments, the methods described herein include administering a pharmaceutical composition to a subject. The pharmaceutical, or therapeutic, compositions can be administered by any suitable route of administration. A route of administration can refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch), or vaginal. "Transdermal" administration can be accomplished using a topical cream or ointment or by means of a transdermal patch. "Parenteral" refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intracranial, intraventricular, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, sublingual, transmucosal, or transtracheal.38108531467.1Attorney Docket No. 134675-875134

[0078] The pharmaceutical, or therapeutic, compositions described herein can be administered by any suitable route of administration. A route of administration can refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch), or vaginal. "Transdermal" administration can be accomplished using a topical cream or ointment or by means of a transdermal patch. "Parenteral" refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intracranial, intraventricular, intrauterine, intravenous, subarachnoid, subcapsular, sublingual, subcutaneous, transmucosal, or transtracheal. In some embodiments, the composition can be delivered intraoperatively as a local administration during an intervention or resection.Preferably, the pharmaceutical composition is administered to the subject orally

[0079] In some embodiments, the present disclosure provides methods for the inhibition of exosome biogenesis in cancer therapy. For example, a method for inhibition of exosome biogenesis comprising administering an exosome biogenesis inhibitor to a subject, preferably a subject in need thereof.

[0080] In some embodiments, the exosome biogenesis inhibitor comprises a small molecule drug.

[0081] In some embodiments, the exosome biogenesis inhibitor comprises a hypoxia-activated prodrug (HAP).

[0082] In some embodiments, the HAP comprises a protective group that is released in a hypoxic environment. In some embodiments, the exosome biogenesis inhibitor can suppress release of tumor-derived exosomes (TDEs). Nonlimiting examples of protective groups include Compounds A and B.

[0083] In some embodiments, the HAP is inert in normal oxygen conditions but will activate in hypoxic environments.

[0084] In some embodiments, the protective group is designed to target specific reductases present in the tumor microenvironment. Thus, in some embodiments, the HAP targets reductases present in the tumor microenvironment. In a preferred39108531467.1Attorney Docket No. 134675-875134embodiment, the exosome biogenesis inhibitor or HAP targets acid sphingomyelinase (aSMase, SMPD1).

[0085] In some embodiments, the method further comprises administration of any combination of immunotherapy, chemotherapy, radiation therapy, and surgery.

[0086] In some embodiments, the cancer is malignant pleural mesothelioma (MPM).

[0087] In some embodiments, by reducing TDE biogenesis, the exosome biogenesis inhibitor inhibits TDE contribution to immune suppression and tumor protection.EXAMPLES

[0088] Embodiments of the present disclosure are further defined in the following Examples. It should be understood that these Examples are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the disclosure to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the disclosure, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosure of each reference set forth herein is incorporated herein by reference in its entirety, and for the disclosure referenced herein.Example 1Identification of aSMase-Targeted Exosome Biogenesis Inhibitors

[0089] Using computational modeling, a library of 2.6 million compounds was screened, including small molecules, PROTACs (PROteolysis TArgeting Chimera), molecular glues, and fragment -based entities, against key enzymes in the exosome biogenesis pathway. The initial focus was on compounds targeting acid sphingomyelinase (aSMase), an essential enzyme in lipid metabolism linked to exosome formation. Computational chemistry enabled refinement of the selection to 172 compounds based on optimal target binding modes, energetics, and structureactivity relationships (SAR).40108531467.1Attorney Docket No. 134675-875134

[0090] To validate these computational hits, a cell-free assay was employed, screening all 172 compounds alongside ARC39, a known small-molecule inhibitor of acid sphingomyelinase (aSMase), for anti-aSMase activity using a modified assay kit (Invitrogen®, A12220). Among these, Compound 41 and Compound 42 emerged as the top inhibitors.

[0091] Both were experimentally confirmed to have superior binding affinity to aSMase compared to the other candidates, and compared to ARC39, the known aSMase inhibitor, as shown in Figure 1.

[0092] Further data showed that inhibition of aSMase in vitro with Compound 41 suppresses exosome release. This was validated by immunoblot of a total exosome pellet precipitated from conditioned serum-free media of MSTO-211H cells treated for 6 hours using an exosome precipitation solution (System Biosciences®, EXOTC5OA-1). ARC39 was used as a positive control. As shown by Figure 2, the expression of CD63 (Cell Signaling Technologies, 50290S) (Fig. 2(a)) and TSG101 (Cell Signaling Technologies, 72312S) (Fig. 2(b)), both markers of exosomes, is dramatically reduced in conditioned media from the mesothelioma cell line MSTO-211H after 6-hour treatment with Compound 41 compared to vehicle control (DMSO).

[0093] Suppression of exosome release was also assessed by flow cytometry. Precipitated exosomes from a separate 6-hour treatment of MSTO-211H cells were purified with anti-CD63-conjugated magnetic beads (System Biosciences®, EXOFLOW2OOA-1) and visualized by flow cytometry using a general exosome marker conjugated to FITC. As shown in Figure 3, Compound 41, like ARC39, suppressed exosome release.41108531467.1Attorney Docket No. 134675-875134

[0094] Figures 2 and 3 show that Compound 41 produces a reduction in exosome markers and suppresses exosome release comparable to that observed with ARC39. Similar results were observed in NCI-H2452 and SH-SY5Y cell lines (data not shown).Example 2Biological Effect on Exosome Release In Vitro

[0095] The ability of exosome biogenesis (EB) inhibitors to block exosome biogenesis in human Triple-Negative Breast Cancer (TNBC) tumor cell lines (HCC1806 and MDA-MB-231) was assessed. Building on preliminary data showing that aSMase inhibitors such as ARC39 significantly reduced exosome secretion in mesothelioma cells (Example 1), this study extends those findings to another indication.

[0096] HCC1806 and MDA-MB-231 cell lines were treated with EB inhibitors for 6 hours, with control groups receiving vehicle (DMSO) or ARC39 as a positive control. At each time point, conditioned media were collected and clarified for exosome analysis.

[0097] Exosome inhibition was evaluated using nanoparticle tracking analysis (NTA).

[0098] For nanoparticle tracking analysis (NTA), exosomes were isolated from conditioned media using the ExoQuickTC reagent (System Biosciences, Cat. # EXOTCioA Extracellular vesicles were analyzed by NTA using a ZetaView (Particle Metrix) to assess both concentration (number per pL) and size distribution of exosomes. EB inhibitor treatment reduced exosome particle counts relative to DMSO controls, with levels of inhibition comparable to or greater than those observed with ARC39, as shown in Figure 4 & 5. Treatment of HCC1806 (Fig. 4(a)) andMDA-MB-231 cells (Fig. 4(b)) with Compound 42, resulted in a marked decrease in exosome production compared to vehicle control, exceeding the decrease shown by ARC39, as shown in Figure 4 analysis. Figure 5 represents both the concentration (number per pL) and size distribution of exosomes for HCC1806 (Fig. 5(a)) and MDA-MB-231 (Fig. 5(b)).42108531467.1Attorney Docket No. 134675-875134Example 3Biological Effect In Vivo

[0099] The tolerability, pharmacokinetics (PK), tissue distribution, tumor penetration, and anti-tumor efficacy of Compound 42 was evaluated in mice.

[0100] Prior studies demonstrated that Compound 42 was well tolerated in mice when administered intraperitoneally (i.p.) at doses up to 75 mg / kg once daily for 7 consecutive days. Animals exhibited less than 1% body weight loss and no observable clinical signs of distress. To evaluate maximum tolerated dose (MTD), mice received vehicle or Compound 42 by i.p. administration at 10 mg / kg, 20 mg / kg, 50 mg / kg, 75 mg / kg, too mg / kg, or 200 mg / kg once daily for 7 days. Animals exhibiting severe clinical signs or approximately 20% body weight loss were euthanized early. Animals exhibiting approximately 10% body weight loss with minimal distress were placed on a drug holiday prior to study completion (data now shown).

[0101] Female C57BL / 6 mice were inoculated subcutaneously with ixioA6 MC38 cells. After tumors reached an average volume of approximately 150 mmA3 (about 8 days post -inoculation), animals were randomized into treatment groups. Mice received Compound 42 by daily i.p. injection for 21 days. Animals were monitored three times per week for body weight, clinical signs of distress, and tumor growth. In an MC38 syngeneic study (n=i2 per group), Compound 42 treatment produced approximately 44% tumor growth inhibition compared to vehicle by day 16, when vehicle-treated tumors reached approximately 2000 mmA3 in volume (FIG. 6).

[0102] No meaningful body weight loss or observable clinical signs of distress were observed during Compound 42 treatment, including at doses of 20 mg / kg and 75 mg / kg, consistent with low toxicity under the tested conditions (FIG. 7).Example 4Tumor Penetration Following Oral Administration

[0103] To evaluate tumor penetration via oral administration, mice bearing established MC38 tumors (about 7 days post -inoculation) received Compound 42 at 20 mg / kg by oral gavage, with vehicle as control. Plasma and tumor samples were collected at o hours, 1 hour, 2 hours, and 4 hours post-dose and analyzed by mass spectrometry. Concentration-time profiles demonstrated measurable Compound 42 levels in both plasma and tumor (FIG.8) after oral dosing.43108531467.1Attorney Docket No. 134675-875134

[0104] An AUC tumor-to-plasma ratio of approximately 1.52 was observed, indicating greater overall exposure in tumor compared to plasma. The average maximum concentration (C_ max) of Compound 42 in tumor was approximately 82.6 ng / g, compared to approximately 63.0 ng / mL in plasma, consistent with preferential accumulation in tumor tissue (FIG. 9).Example 5The Effects of Blocking Exosome Biogenesis in Immune Cells

[0105] Exosomes mediate intercellular communication and can modulate immune responses. The ability of exosome biogenesis (EB) inhibitors, including small -molecule inhibitors of acid sphingomyelinase (aSMase), to alter immune cell activation and function in tumor-immune co-cultures is evaluated using human peripheral blood mononuclear cells (PBMCs) and tumor cells. To confirm that observed effects arise from inhibition of aSMase-mediated exosome biogenesis, an aSMase-knockout A549 cell line is used as a specificity control in parallel with wildtype A549 cells.

[0106] PBMCs from healthy human donors are co-cultured with wild-type A549 cells treated with an EB inhibitor and with corresponding vehicle-treated A549 cells. Co-cultures are maintained for approximately 24 hours. In parallel, PBMCs are cocultured with the aSMase-knockout A549 cell line under otherwise similar conditions. Following co-culture, immune activation and functional responses are assessed in PBMC populations by measuring immune activation markers, maturation and antigen presentation markers, proliferation, and cytokine production.

[0107] T cells and natural killer (NK) cells are analyzed by flow cytometry for activation markers including CD25 and CD69.

[0108] Dendritic cells and monocytic cells are analyzed for maturation and antigen presentation markers including CD80, CD86, and MHC class II (HLA-DR). Increased expression of CD80, CD86, and / or HLA-DR relative to vehicle control is indicative of enhanced immune-stimulatory function and antigen presentation capacity.

[0109] PBMC proliferation is assessed by labeling PBMCs with a proliferation dye and analyzing dye dilution by flow cytometry. PBMCs are stained with CellTrace™ Violet (CTV) and proliferation is quantified in one or more immune subsets.44108531467.1Attorney Docket No. 134675-875134[ono] Cytokine production is assessed from co-culture supernatants using a multiplex cytokine assay. Cytokine profiles are measured using a panel assay capable of detecting a plurality of human immune cytokines, including the Legendplex™ Human Essential Immune Response Panel (or an equivalent multiplex immunoassay).

[0111] The ability of EB inhibitors to reverse immune suppression induced by tumor-derived exosomes (TDEs) is evaluated by contacting PBMCs with TDEs for approximately 24 hours or approximately 48 hours to induce an immunosuppressed phenotype. Following suppression, PBMC cultures are contacted with an EB inhibitor for an additional period of approximately 24 hours.

[0112] After EB inhibitor treatment in the presence of TDE-induced suppression, changes in surface marker expression and cytokine profiles are assessed as described above.

[0113] EB inhibitor treatment is expected to increase activation marker expression on T cells and NK cells, increase dendritic / monocyte maturation and antigen presentation marker expression, and shift cytokine production toward an immune-stimulatory profile relative to vehicle-treated controls. The aSMase-knockout A549 control is expected to reduce or eliminate effects attributable to aSMase-dependent exosome biogenesis, thereby supporting target-specific activity of the EB inhibitor.45108531467.1Attorney Docket No. 134675-875134Example 6(Synthesis of Compound 41 and Compound 42)

[0114] Compound 41 is prepared as follows. (4-(hydroxycarbamoyl)phenyl)boronic acid 1 is reacted with 2,4-dichloropyrimidine 2 in the presence of tetrakis(triphenylphosphine)palladium catalyst resulting in the formation of 4-(2-chloropyrimidin-4-yl)-N-hydroxybenzamide 3.

[0115] Next, 4-(2-chloropyrimidin-4-yl)-N-hydroxybenzamide 3 is reacted with 2-oxa-7-azaspiro[3.5]nonane 4 in the presence of palladium(II) acetate and BINAP, to form Compound 41. Compound 41 is characterized byNMR and LC-MS.3Compound 41

[0116] Compound 42 is prepared as follows. (4-(hydroxycarbamoyl)phenyl)boronic acid 1 is reacted with 2,4-dichloropyrimidine 2 in the presence of tetrakis(triphenylphosphine)palladium catalyst resulting in the formation of 4-(2-chloropyrimidin-4-yl)-N-hydroxybenzamide 3.46108531467.1Attorney Docket No. 134675-875134

[0117] Next, 4-(2-chloropyrimidin-4-yl)-N-hydroxybenzamide 3 is reacted with i-oxa-3,8-diazaspiro[4.5]decan-2-one 5 in the presence of palladium(II) acetate and BINAP, resulting in the formation of Compound 42. Compound 42 is characterized by iH NMRand LC-MS.Example 7(Synthesis of Compound 41)

[0118] Compound 41 was prepared as follows. The synthesis can be carried out at laboratory scale or larger scale. 1 g of (4-(methoxycarbamoyl)phenyl)boronic acid 6 was reacted with 1 eq of 2,4-dichloropyrimidine 2 in the presence of 0.05 eq of [i,i'-bis(diphenylphosphino)-ferrocene]palladium(II) dichloride and 1.2 eq. of sodium carbonate in a mixed dioxane / water (10:1) solvent system. The reaction mixture was heated to 90 °C for 4 hours, resulting in the formation of methyl 4- (2-chloropyrimidin-4-yl)benzoate 7. LC-MS showed 76.3% of the peak with the desired mass.Dioxane:H20 = 10:1 (10V)90 °C, 4 h

[0119] Next, methyl 4-(2-chloropyrimidin-4-yl)benzoate 7 was reacted with 2 eq. of lithium hydroxide in a mixed THF / water (2:1) solvent system at 20 °C for 16 hours, resulting in the formation of 4-(2-chloropyrimidin-4-yl)benzoic acid 8 (74.6% purity). LC-MS showed 69.6% of the peak with the desired mass.47108531467.1Attorney Docket No. 134675-875134,

[0120] 4-(2-chloropyrimidin-4-yl)benzoic acid 8 was subsequently reacted with 1.5 eq of propylphosphonic anhydride in the presence of 2 eq. of N,N- diisopropylethylamine (DIEA) in dichloromethane. The reaction mixture was heated to 45 °C for 16 hours, resulting in the formation of 4-(2-chloropyrimidin- 4-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide 9.45 C, 16 h

[0121] 4-(2-chloropyrimidin-4-yl)-N-((tetrahydro-2H-pyran-2- yl)oxy)benzamide 9 was reacted with 2-oxa-7-azaspiro[3.5]nonane 4 in the presence of palladium(II) acetate and BINAP resulting in the formation of 4-(2- (2-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-N-((tetrahydro-2H-pyran-2- yl)oxy)benzamide 10.

[0122] 4-(2-(2-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-N-((tetrahydro- 2H-pyran-2-yl)oxy)benzamide 10 was subsequently treated with trifluoroacetic acid in dichloromethane (1:1) at 20 °C for 16 hours to remove the protecting group and form Compound 41. The identity of Compound 41 was confirmed by 'H NMR spectroscopy, and purity is assessed by LC-MS. The purity was >95%.48108531467.1Attorney Docket No. 134675-87513410 Compound 41Example 8(Synthesis of Compound 42)

[0123] Compound 42 was prepared as follows. The synthesis can be conducted at both laboratory scale and larger scale.1 g of (4- (methoxycarbamoyl)phenyl)boronic acid 6 is reacted with 1 eq of 2,4- dichloropyrimidine 2 in the presence of 0.05 eq of [1,1'- bis(diphenylphosphino)ferrocene]palladium(II) dichloride and 1.2 eq. of sodium carbonate in a mixed dioxane / water (10:1) solvent system. The reaction mixture was heated to 90 °C for 4 hours, resulting in the formation of methyl 4-(2- chloropyrimidin-4-yl)benzoate 7. LC-MS showed 76.3% of the peak with the desired mass.>Dioxane: H2O = 10:1 (10V) 90 °C, 4 h

[0124] Next, methyl 4-(2-chloropyrimidin-4-yl)benzoate 7 was reacted with 2 eq. of lithium hydroxide in a mixed THF / water (2:1) solvent system at 20 °C for 16 hours, resulting in the formation of 4-(2-chloropyrimidin-4-yl)benzoic acid 8 (74.6% purity). LC-MS showed 69.6% of the peak with the desired mass.49108531467.1Attorney Docket No. 134675-875134

[0125] 4-(2-chloropyrimidin-4-yl)benzoic acid 8 was subsequently reacted with 1.5 eq of propylphosphonic anhydride in the presence of 2 eq. of N,N- diisopropylethylamine (DIEA) in dichloromethane. The reaction mixture was heated to 45 °C for 16 hours, resulting in the formation of 4-(2-chloropyrimidin- 4-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide 9.

[0126] 4-(2-chloropyrimidin-4-yl)-N-((tetrahydro-2H-pyran-2- yl)oxy)benzamide 9 was reacted with i-oxa-3,8-diazaspiro[4.5]decan-2-one 5 in the presence of palladium(II) acetate and BINAP resulting in the formation of 4- (2-(2-oxo-i-oxa-3,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-yl)-N-((tetrahydro- 2H-pyran-2-yl)oxy)benzamide 11." "

[0127] 4-(2-(2-oxo-i-oxa-3,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-yl)-N- ((tetrahydro-2H-pyran-2-yl)oxy)benzamide 11 was subsequently treated with trifluoroacetic acid in di chloromethane (1:1) at 20 °C for 16 hours to remove the protecting group and form Compound 42. The identity of Compound 42 was confirmed byXH NMR spectroscopy, and purity is assessed by LC-MS. The purity was >95%.11 Compound 42Example 950108531467.1Attorney Docket No. 134675-875134(Generic Synthesis)

[0128] Compound (e) is prepared as follows. Compound (a) is reacted with 2,4-dichloropyrimidine (b) in the presence of tetrakis(triphenylphosphine)palladium catalyst resulting in the formation of compound (c).Pd(pph3)4

[0129] Next, compound (c) is reacted with compound (d) in the presence of palladium(II) acetate and BINAP, to form compound (e).

[0130] Compound (i) is prepared as follows. Compound (f) is reacted with 2,4-dichloropyrimidine (b) in the presence of tetrakis(triphenylphosphine)palladium catalyst resulting in compound (g).Pd(pph3)4

[0131] Next, compound (g) is reacted with compound (d) in the presence of palladium(II) acetate and BINAP, resulting in the formation of compound (i).51108531467.1Attorney Docket No. 134675-875134Terms

[0132] All terms have their ordinary and customary meaning as understood by one of ordinary skill in the art, in view of the present disclosure.

[0133] As used herein, “exosome” has its plain and ordinary meaning as understood in light of the specification and refers to nanovesicles released by cells during the process of exosome biogenesis.

[0134] As used herein, “exosome biogenesis inhibitor” has its plain and ordinary meaning as understood in light of the specification and refers to a compound, such as a small molecule that suppresses exosome biogenesis. In some embodiments, the exosome biogenesis inhibitor targets one or more enzymes in the exosome biogenesis pathway.

[0135] As used herein, “hypoxia activated prodrug (HAP)” has its plain and ordinary meaning as understood in light of the specification and refers to drugs that are inert in normal oxygen conditions but activate in hypoxic environments.

[0136] As used herein, “tumor-derived exosome (TDE)” has its plain and ordinary meaning as understood in light of the specification and refers to exosomes released by a tumor.

[0137] The term “treating” or “treatment” of a condition can refer to preventing the condition, slowing the onset and / or rate of development of the condition, reducing the risk of developing the condition, preventing and / or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof. The term “prevent” does not require the absolute prohibition of the disorder or disease. For example, the term “prevent” may be prevention of the worsening of the disorder.

[0138] A “therapeutically effective amount” or a “therapeutically effective dose” is an amount that produces a desired therapeutic effect in a subject, such as preventing, treating a target condition, delaying the onset of the disorder and / or symptoms, and / or alleviating symptoms associated with the condition. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, biodistribution and bioavailability), the physiological condition52108531467.1Attorney Docket No. 134675-875134of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and / or the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for example by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly, given the present disclosure. For additional guidance, see Remington: The Science and Practice of Pharmacy 21stEdition, Univ, of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0139] The terms "subject," "patient," and "individual" interchangeably refer to an entity that is being examined and / or treated. The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys), humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, or guinea pigs. The terms “subject,” “patient,” and “mammal” include a human, a preferred subject, patient, and mammal.

[0140] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0141] As used herein, “exosome biogenesis inhibitor” or “EB inhibitor” refers to any agent or intervention that reduces, prevents, delays, or otherwise interferes with the formation, maturation, trafficking, secretion, or release of exosomes from a cell. Exosome biogenesis inhibitors may act at any stage of the exosome pathway, including but not limited to endosomal sorting, multivesicular body (MVB) formation, intraluminal vesicle generation, MVB trafficking, or fusion of MVBs with the plasma membrane. EB inhibitors may reduce the number, size, cargo, or biological activity of exosomes released by a cell. EB inhibitors include, without limitation, small molecules, biologies, peptides, nucleic acids, hypoxia-activated53108531467.1Attorney Docket No. 134675-875134prodrugs, antibodies, or combinations thereof. Preferably, the exosome biogenesis inhibitors are small molecules and prodrugs such as hypoxia-activated prodrugs.

[0142] Headings are included herein for reference and to help in locating various sections. These headings are not intended to limit the scope of the concepts described with respect thereto. Such concepts may have applicability throughout the entire specification.

[0143] The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.

[0144] In the foregoing description, specific details are given to provide a thorough understanding of the examples. However, it will be understood by one of ordinary skill in the art that the examples may be practiced without these specific details.

[0145] In at least some of the described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0146] With respect to the use of substantially any plural or singular terms herein, those having skill in the art can translate from the plural to the singular or from the singular to the plural as is appropriate to the context or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0147] The embodiments illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for54108531467.1Attorney Docket No. 134675-875134example, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of’ may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed. The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term “about” as used herein refers to a value within 10% of the underlying parameter, and use of the term “about” at the beginning of a string of values modifies each of the values. For example, a weight of “about too grams” can include weights between 90 grams and 110 grams. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%). Thus, it should be understood that although the present technology has been specifically disclosed by representative embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of the embodiments.

[0148] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0149] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will55108531467.1Attorney Docket No. 134675-875134be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0150] While preferred embodiments described herein have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the description. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the embodiments. It is intended that the following claims define the scope of embodiments provided herein and that methods and structures within the scope of these claims and their equivalents be covered thereby.REFERENCESCarlino MS, Larkin J, Long G V. Therapeutics Immune Checkpoint Inhibitors in Melanoma. Vol 398.; 2021.Das S, Johnson DB. Immune-related adverse events and anti-tumor efficacy of immune checkpoint inhibitors. J Immunother Cancer [Internet]. 2019 Dec 1 [cited 2024 Jun 17];7(I):3O6.Saad MB, Hong L, Aminu M, Vokes NI, Chen P, Salehjahromi M, et al. Predicting benefit from immune checkpoint inhibitors in patients with non-small-cell lung cancer by CT-based ensemble deep learning: a retrospective study. Lancet Digit Health [Internet]. 2023 Jul 1 [cited 2024 Jun 17] 55(7): 0404.Haslam A, Gill J, Prasad V. Estimation of the Percentage of US Patients With Cancer Who Are Eligible for Immune Checkpoint Inhibitor Drugs. JAMA Netw Open [Internet]. 2020 Mar 9 [cited 2024 Jun 17] ;3(3).Spiegelberg L, Houben R, Niemans R, et al. Hypoxia-activated prodrugs and (lack of) clinical progress: The need for hypoxia-based biomarker patient selection in phase III clinical trials. Published online 2019. doi:io.ioi6 / j.ctro.2019.01.005Zhou W, Fong MY, Min Y, et al. Cancer-Secreted miR-105 Destroys Vascular Endothelial Barriers to Promote Metastasis. Cancer Cell. 2O14;25(4):5O1-515. doi:io.ioi6 / j.ccr.20i4.03.oo7Gurung S, Perocheau D, Touramanidou L, Baruteau J. The exosome journey: from biogenesis to uptake and intracellular signalling. Cell Communication and Signaling.2O2i;i9(i):47. doi:io.n86 / si2964-O2i-oo73O-iSkryabin GO, Komelkov A V., Savelyeva EE, Tchevkina EM. Lipid Rafts in Exosome Biogenesis. Biochemistry (Moscow). 2O2O;85(2):177-191. doi:io.H34 / Sooo6297920020054Trajkovic K, Hsu C, Chiantia S, et al. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science. 2OO8;319(5867):1244-1247. doi:io.H26 / science.H53i2456108531467.1Attorney Docket No. 134675-875134Jiang H, Zhao H, Zhang M, et al. Hypoxia Induced Changes of Exosome Cargo and Subsequent Biological Effects. Front Immunol. 2O22;13:824188. doi:io.3389 / FIMMU.2022.824i88 / BIBTEXPaskeh MDA, Entezari M, Mirzaei S, et al. Emerging role of exosomes in cancer progression and tumor microenvironment remodeling. J Hematol Oncol.2O22;15(I):83. doi:io.n86 / si3045-022-oi305-4Li Y, Zhao L, Li XF. The Hypoxia-Activated Prodrug TH -302: Exploiting Hypoxia in Cancer Therapy. Front Pharmacol. 2021512. doi:io.3389 / fphar.202i.636892 Dhani NC, Serra S, Pintilie M, et al. Analysis of the intra- and intertumoral heterogeneity of hypoxia in pancreatic cancer patients receiving the nitroimidazole tracer pimonidazole. Br J Cancer. 2O15;113(6):864-871. doi:io.1038 / ^0.2015.284 Tong JTW, Harris PWR, Brimble MA, Kavianinia I. An Insight into FDA Approved Antibody-Drug Conjugates for Cancer Therapy. Molecules. 2021526(19).doi: IO.339O / MOLECULES26195847jarz W, Dominiak A, Zolnierzak A, Kubiak-Tomaszewska G, Lorenc T. Tumor- Derived Exosomes in Immunosuppression and Immunotherapy. J Immunol Res. 2020;2020(l):6272498. doi:lO.1155 / 2O2O / 6272498Mashouri L, Yousefi H, Aref AR, Ahadi AM, Molaei F, Alahari SK. Exosomes: composition, biogenesis, and mechanisms in cancer metastasis and drug resistance. Molecular Cancer 201918:1. 2O19;I8(I):I-14. doi:io.n86 / Si2943-oi9-O99i-5 Kim M, Yun HW, Park DY, Choi BH, Min BH. Three-Dimensional Spheroid Culture Increases Exosome Secretion from Mesenchymal Stem Cells. Tissue Eng Regen Med [Internet]. 2018 Aug 1 [cited 2024 Jun I6];IS(4):427.La Rocca A, De Gregorio V, Lagreca E, Vecchione R, Netti PA, Imparato G. Colorectal Cancer Bioengineered Microtissues as a Model to Replicate Tumor- ECM Crosstalk and Assess Drug Delivery Systems In vitro. Int J Mol Sci [Internet]. 2023 Mar 1 [cited 2024 Jun I ];24(6).Selby MJ, Engelhardt JJ, Johnston RJ, Lu LS, Han M, Thudium K, et al. Preclinical Development of Ipilimumab and Nivolumab Combination Immunotherapy: Mouse Tumor Models, In vitro Functional Studies, and Cynomolgus Macaque Toxicology. PLoS One [Internet]. 2016 Sep 1 [cited 2024 Jun 16];11(9).57108531467.1

Claims

Attorney Docket No. 134675-875134CLAIMS1. A method for inhibiting, ameliorating, or treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the compound of Formula (A) or Formula (B), or a pharmaceutically acceptable salt thereof:Ri and R2are interconnected to form an optionally substituted C4-C5hetero cyclyl;R3is selected from:-C(O)-NR4R5,-C(O)-NH-R4,-C(O)-NH-NR4R5,-C(0)-NH-NH-R4,-C(O)-NH-OH,-C(O)-NH-S(O)2-R4,-C(O)-R6,-NH-C(O)-R4,-P(O)(OH)-OR5,-P(O)(OH)2,-NR5-C(O)-R4,-C(S)-NH-R4,-C(S)-NH-NR4R5, and-C(S)-NH-NH-R4j58108531467.1Attorney Docket No. 134675-875134R4 is an optionally substituted CI-CG alkyl;Re is an optionally substituted thiadiazolyl.

2. The method of claim 1, wherein Re is selected from:

3. The method of claim 1 or 2, wherein the optionally substituted C4-C5 heterocyclyl is an optionally substituted oxazolidin- 2-one or an optionally substituted oxetane.

4. The method of claim 3, wherein the optionally substituted C4-C5 heterocyclyl is selected from:

5. The method of claim 1, wherein the compound of Formula (A) or Formula (B) or the pharmaceutically acceptable salt thereof is selected from compounds 1-50 and pharmaceutically acceptable salts thereof:59108531467.1Attorney Docket No. 134675-8751346o108531467.1Attorney Docket No. 134675-87513461108531467.1Attorney Docket No. 134675-87513462108531467.1Attorney Docket No. 134675-87513463108531467.1Attorney Docket No. 134675-87513464108531467.1Attorney Docket No. 134675-87513465108531467.1Attorney Docket No. 134675-8751346. The method of claim 5, wherein the compound of Formula (A) or (B), or the pharmaceutically acceptable salt, is compounds 41 or 42, or a pharmaceutically acceptable salt thereof:

7. A compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof:66108531467.1Attorney Docket No. 134675-875134(I) (II) wherein:Ri and R2are interconnected to form an optionally substituted C4-C5heterocyclyl;R3is selected from:-C(O)-NH-OH,-C(O)-NH-S(O)2-R4,-(CH2)nC(O)-NH-OH,-(CH2)nC(0)-NHS(0)2-R4, and-C(O)-R6;R4is hydrogen or an optionally substituted Ci-Ce alkyl;n is o, 1, or 2; andRe is an optionally substituted heterocycle.provided that in Formula (I), when R3is -C(O)-NH-OH, Ri and R2are not8. The compound or the pharmaceutically acceptable salt of claim 7, wherein the compound is a compound of Formula (I-A) or (I-B), or a pharmaceutically acceptable salt thereof:67108531467.1Attorney Docket No. 134675-875134(I-A) (I-B) wherein:R3is-C(O)-NH-OH,-C(0)-NH-S(0)2-R4, or-C(O)-Re; andR4is hydrogen or an optionally substituted Ci-Ce alkyl9. The compound or the pharmaceutically acceptable salt of claim 8, wherein R3is -C(O)-NH-OH.

10. The compound or the pharmaceutically acceptable salt of claim 7, wherein the compound is a compound of Formula (I-C) or a pharmaceutically acceptable salt thereof;(I-C)wherein R3is-C(O)-NH-OH,-C(O)-NH-S(O)2R4, or-C(O)-R6.it. The compound or the pharmaceutically acceptable salt of claim 10, wherein R3is -C(O)-NH-OH.68108531467.1Attorney Docket No. 134675-87513412. The compound or the pharmaceutically acceptable salt of claim 7, wherein the compound is a compound of Formula (II-A) or a pharmaceutically acceptable salt thereof:(II-A)wherein R3is-C(O)-NH-OH,-C(O)-NHS(O)2R4, or-C(O)-R6.

13. The compound or the pharmaceutically acceptable salt of claim 12, wherein R3is -C(O)-NH-OH.

14. The compound or the pharmaceutically acceptable salt of claim 12, wherein R3is -C(O)-NH-S(O)2R4.

15. The compound or the pharmaceutically acceptable salt of claim 12, wherein R3is -C(O)-R6.

16. The compound of claim 15, wherein Re is an optionally substituted thiadiazolyl.

17. The compound of claim 16, wherein Re is selected from:69108531467.1Attorney Docket No. 134675-87513418. The compound or the pharmaceutically acceptable salt of claim 7 selected from compounds 43-50 and pharmaceutically acceptable salts thereof:70108531467.1Attorney Docket No. 134675-87513419- A pharmaceutical composition comprising the compound of any one of the claims 7-18, or the pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

20. A method for inhibiting, ameliorating, or treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the compound of any one claims 7-18 or a pharmaceutically acceptable salt thereof, or a composition of claim 18.

21. The method of claim 20, wherein the method inhibits tumor growth or reduces tumor volume.71108531467.1Attorney Docket No. 134675-87513422. The method of claim 21, wherein the cancer is selected from malignant pleural mesothelioma (MPM); lung cancer, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC); head and neck cancer, including squamous cell carcinoma; gastrointestinal cancer, including esophageal, gastric, colorectal, pancreatic, hepatobiliary, hepatocellular carcinoma (HCC), and cholangiocarcinoma; genitourinary cancer, including renal cell carcinoma (RCC), bladder cancer, and prostate cancer; gynecologic cancer, including ovarian, endometrial, and cervical cancer; breast cancer, including triplenegative breast cancer (TNBC); skin cancer, including melanoma, basal cell carcinoma, and squamous cell carcinoma; sarcoma, including osteosarcoma and soft tissue sarcoma; central nervous system cancer, including glioblastoma; hematologic malignancy, including leukemia, lymphoma (Hodgkin and non-Hodgkin), and myeloma; thyroid carcinoma; and rare cancers.

23. The method of any one of claims 1-6 and 20-22, wherein the compound or the pharmaceutically acceptable salt is administered orally to the subject.

24. The method of any one of claims 1-6 and 20-23, wherein the subject is a human.

25. The method of any one of claims 1-6 and 20-24, further comprising administering to the subject at least one additional anti-cancer therapy selected from immunotherapy, chemotherapy, radiation therapy, and surgery.72108531467.1

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