Anthracycline derivatives and their therapeutic applications

Abstract

The invention relates generally to the treatment of cancer, and chemoresistant cancer types in particular.

Description

ANTHRACYCLINE DERIVATIVES AND THEIR THERAPEUTIC APPLICATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U. S. C. § 119(e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 733,277 filed December 12, 2024, the disclosure of which is herein incorporated by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Number GM103430 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The invention relates generally to the treatment of cancer, and chemoresistant cancer types in particular.BACKGROUND OF THE INVENTION

[0004] Anthracyclines, such as doxorubicin, have been used in the clinic to treat many types of cancers. Despite the potency of these drugs, their clinical utility is limited by efflux pump-mediated multidrug resistance (MDR).

[0005] It would be an advancement in the art to create anthracyclines which are potent and are not subject to MDR. Such compounds, as well as other inventions, are described herein.SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides a method of treating cancer comprising: administering to a subject not otherwise in need of treatment thereof a therapeutically effective amount of a compound having a structure according to Formula Ior a pharmaceutically acceptable salt, hydrate, or solvate thereof, thereby treating the(“doxorubamine”), the doxorubamine is not chelated to platinum when administered to the subject.

[0007] In another aspect, the invention provides a compound having a structure according to Formula IOMe O NH2NH2Me (I),or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the

[0008] In another aspect, the invention provides a pharmaceutical composition comprising: (a) a compound having a structure according to Formula IO OH O.OMe O OH O. NH2y NH2Me (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein theOMe O OH O.NH2compound is notMe; and (b) a pharmaceutically acceptable excipient.

[0009] In another aspect, the invention provides a pharmaceutical composition comprising: (a) doxorubamine, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and (b) a pharmaceutically acceptable excipient, wherein the pharmaceutical composition does not comprise platinum.

[0010] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG 1. Dose-response curves showing the effect of doxorubicin (Dox) and doxorubamine (DoxNH2NH2) on the viability of doxorubicin-sensitive MES-SA human uterine sarcoma cells.

[0012] FIG 2. Dose-response curves showing the effect of doxorubicin (Dox) and doxorubamine (DoxNH2NH2) on the viability of doxorubicin-resistant MES-SA / Dx5 human uterine sarcoma cells.

[0013] FIG 3. Dose-response curves showing the effect of doxorubicin (Dox) and doxorubamine (DoxNH2NH2) on the viability of doxorubicin-sensitive MES-SAhuman uterine sarcoma cells co-treated with verapamil, an efflux pump (P-gp) inhibitor.

[0014] FIG 4. Dose-response curves showing the effect of doxorubicin (Dox) and doxorubamine (DoxNH2NH2) on the viability of doxorubicin-resistant MES-SA / Dx5 human uterine sarcoma cells co-treated with verapamil.

[0015] FIG 5. (A) Dose-response curves (n = 3 biological replicates) for Dox with mouse T-cell lymphoma EL4 cells (dashed blue line, without verapamil, and solid blue line, with 10 pM verapamil) and the doxorubicin-resistant variant EL4-DoxR (dashed red line, without verapamil, and solid red line, with 10 pM verapamil). (B) Dose-response curves (n = 3 biological replicates) for DoxNH2NH2with mouse T-cell lymphoma EL4 cells (dashed blue line, without verapamil, and solid blue line, with 10 pM verapamil) and the doxorubicin-resistant variant EL4-DoxR (dashed red line, without verapamil, and solid red line, with 10 pM verapamil). Error bars represent standard deviation.

[0016] FIG 6. Quantifications of images of AKPS-zsG and AKPS-tdT-DoxR 1:1 cocultures in the form of dose-response curves. (A) Dose-response curves (n = 3 biological replicates) for Dox with AKPS-zsG colon cancer organoids (dashed blue line, without verapamil, and solid blue line, with 10 pM verapamil) and the doxorubicin-resistant variant AKPS-tdT-DoxR (dashed red line, without verapamil, and solid red line, with 10 pM verapamil). (B) Dose-response curves (n = 3 biological replicates) for DoxNH2NH2with AKPS-zsG colon cancer organoids (dashed blue line, without verapamil, and solid blue line, with 10 pM verapamil) and the doxorubicin-resistant variant AKPS-tdT-DoxR (dashed red line, without verapamil, and solid red line, with 10 pM verapamil). Error bars represent standard deviationDETAILED DESCRIPTION OF THE INVENTION

[0017] Compounds, compositions thereof, preparative methods thereof, and related therapeutic methods are disclosed herein. In certain embodiments, the compounds disclosed herein, and related compositions thereof, can be used for treating a disease in a subject in need thereof. For example, in some cases, the compounds disclosed herein, and related compositions thereof, can be used for treating cancer (e.g., ovarian cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, osteosarcoma,lymphoma, and / or leukemia), as well as other diseases caused by abnormal cell proliferation and growth in a subject in need thereof.

[0018] As used herein, when this symbol, connects two atoms, it indicates that allstereoisomeric options are encompassed. As an example, this structure,encompassesI. Compounds

[0019] In one aspect, the invention provides a compound of Formula I:Meor a pharmaceutically acceptable salt, solvate, or hydrate thereof. In an exemplary embodiment, the compound is of Formula I, or a pharmaceutically acceptable salt,In an exemplary embodiment, the compound is of Formula II:or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In an exemplary embodiment, the compound is of Formula II, or a pharmaceutically acceptable salt,O OHOMe O OH O,solvate, or hydrate thereof, wherein the compound is not•OHOMe O OH 0.In an exemplary embodiment, the compound is Me (also known as “epi-DoxNf FN’l F" or “epirubamine”), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In an exemplary embodiment, the compound is0 OH Os.. OHOMe 0 OH 0,(also known as “3’,4’-Di-epi-DoxNH2NH2” or “di-epi- epirubamine”), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Inan exemplary embodiment, the compoundis Me (also known as “3’- epi-DoxNI [2NII2" or “3’-epi-doxorubamine”), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In an exemplary embodiment, the compound isO OH °<OMe O OH O.. NH2Me (also known as “DoxNH2NH2” or “doxorubamine”), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In an exemplary embodiment, the invention is an enantiomer of doxarubamine, or a pharmaceutically acceptable salt, hydrate, or solvate thereof. In an exemplary embodiment, the invention is a diastereomer of doxarubamine, or a pharmaceutically acceptable salt,hydrate, or solvate thereof. In an exemplary embodiment, for any of the compounds in Section I, the compound is not chelated to platinum. In an exemplary embodiment, for any of the compounds in Section I, the compound is not chelated to a complexed platinum.II. Pharmaceutical Compositions

[0020] Certain aspects relate to pharmaceutical compositions. As used herein, a pharmaceutical composition comprises a compound described herein (e.g., a compound described in “I. Compounds”) and an excipient (e.g., a pharmaceutically acceptable excipient). Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

[0021] In an exemplary embodiment, the invention is a pharmaceutical composition comprising (a) the compound described herein, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and (b) a pharmaceutically acceptable excipient. In an exemplary embodiment, the compound is of Formula I:O OH. OHOMe O OH O.or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein theOMe O OH O.NH2compound is notMe. In an exemplary embodiment, the invention is a pharmaceutical composition comprising (a) doxorubamine, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and (b) a pharmaceutically acceptable excipient. In an exemplary embodiment, for any of the pharmaceutical compositions in Section II, the pharmaceutical composition does not comprise platinum. In an exemplary embodiment, for any of the pharmaceuticalcompositions in Section II, the pharmaceutical composition does not comprise a complexed platinum.III. Methods

[0022] Certain aspects relate to methods of treating a disease in a subject in need of treatment thereof. In certain embodiments, the compounds disclosed herein, and pharmaceutical compositions thereof, can be used for treating a disease in a subject not otherwise in need of treatment thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound and / or pharmaceutical composition disclosed herein. According to some embodiments, administering can be accomplished by implanting, absorbing, ingesting, injecting, or inhaling the compound and / or composition.

[0023] In an aspect, the invention provides a method of treating a proliferative disease comprising: administering to a subject not otherwise in need of treatment thereof a therapeutically effective amount of a compound having a structure according to Formula Ior a pharmaceutically acceptable salt, hydrate, or solvate thereof, thereby treating the proliferative disease. In an exemplary embodiment, the compound is not chelated to platinum when administered to the subject. In an exemplary embodiment, the compound is not chelated to complexed platinum when administered to the subject. In an exemplary embodiment, the compound is not chelated to platinum in the subject. In an exemplary embodiment, the compound is not chelated to complexed platinum in the subject. In an exemplary embodiment, the proliferative disease is described herein. In an exemplary embodiment, the proliferative disease is cancer. In another exemplary embodiment, when the compound has the structureO OH 9x / OH(;c^OMe O OH, XNH2°"r *N H2Me (“doxorubamine”), the doxorubamine is not chelated to platinum when administered to the subject. In another exemplary embodiment, when the compound is doxorubamine, the doxorubamine is not chelated to complexed platinum when administered to the subject. In an exemplary embodiment, when the compound is doxorubamine, the doxorubamine is not chelated to platinum in the subject. In an exemplary embodiment, when the compound is doxorubamine, the doxorubamine is not chelated to complexed platinum in the subject.

[0024] Examples of possible subjects include, but are not limited to, humans (i.e., a male or female human of any age group, e.g., a pediatric subject (e.g, infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and / or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cats, and / or dogs. In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal. In another exemplary embodiment, the subject is a human.

[0025] A "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent. In some embodiments, a therapeutically effective amount refers to an amount sufficient to treat a disease.

[0026] The terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatmentmay be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0027] In certain embodiments, the proliferative disease is cancer, an inflammatory diseases, or an autoimmune disease. Exemplary cancers include, but are not limited to, acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, women's gynecological cancers, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barnet's adenocarcinoma), Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), blood cancers, hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and nonHodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g.,mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, ymphoplasmacytic lymphoma (i.e., " Waldenstrom's macroglobulinemia"), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer ( e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type I or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer ( e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissuesarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer ( e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer, cervical cancer, breast cancer, testicular cancer, lung cancer, and vulvar cancer ( e.g., Paget' s disease of the vulva). For example, in some embodiments, the cancer is ovarian cancer and / or colorectal cancer. In an exemplary embodiment, the cancer is selected from the group consisting of uterine cancer, ovarian cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, osteosarcoma, kidney cancer, bladder cancer, liver cancer, lymphoma, and leukemia. In an exemplary embodiment, the cancer is a uterine cancer which is uterine sarcoma.

[0028] In accordance with some embodiments, compounds and / or compositions disclosed herein have numerous applications and benefits. For example, in some cases, the compounds and / or compositions disclosed herein are more potent than other drugs. In certain instances, compounds and / or compositions disclosed herein can be used to treat diseases which are resistant to other drugs. In certain embodiments, cells uptake a higher amount of compounds and / or compositions disclosed herein compared to other drugs. In an exemplary embodiment, the proliferative disease is resistant to doxorubicin. In an exemplary embodiment, the cancer is a doxorubicin-resistant cancer. In an exemplary embodiment, the cancer is an efflux pump-mediated multidrug resistant cancer. In an exemplary embodiment, the cancer is an ATP-binding cassette transporter-mediated multidrug resistant cancer. In an exemplary embodiment, the cancer is resistant to anthracyclines and / or other chemotherapuetics, including vinca alkaloids, camptotheca drugs, podophyllum drugs, and taxanes.

[0029] U. S. Pat. No 11,203,608 is herein incorporated by reference in its entirety for all purposes.EXAMPLES

[0030] The following Examples illustrate the synthesis of representative compounds used in the invention and the following Reference Examples illustrate the synthesis of intermediates in their preparation. These examples are not intended, nor are they to be construed, as limiting the scope of the invention. It will be clear that the inventionmay be practiced otherwise than as particularly described herein. Numerous modifications and variations of the invention are possible in view of the teachings herein and, therefore, are within the scope of the invention.EXAMPLE 1DoxNH2NH2synthesis

[0031] Doxarubamine (8S,10S)-10-(((2 / ?,4S,57?,6S)-4,5-diamino-6-methyltetrahydro- 2H-pyran-2-yl)oxy)-6, 8,1 l-trihydroxy-8-(2-hydroxyacetyl)-l -methoxy -7, 8, 9,10- tetrahydrotetracene-5, 12-dione (DoxNH2NH2), and stereoisomers of DoxNH2NH2, are synthesized as shown in this general scheme:

[0032] DoxNH2NH2was synthesized as described in U. S. Pat. No 11,203,608.

[0033] Epirubamine is synthesized as shown below:

[0034] 3’, 4 ’-Di-epi-doxorubainine is synthesized as shown below:

[0035] 3’ -epi-doxorubamine is synthesized as shown below:EXAMPLE 2 DoxNH2NH2chemoresistance testing

[0036] The ability of doxarubamine (DoxNH2NH2):to overcome chemoresistance, particularly ABC transporter-mediated drug efflux, was tested. Doxarubamine (DoxNH2NH2) was tested in comparison with doxorubicin (Dox).In vitro testing protocol

[0037] MES-SA or MES-SA / Dx5 cells were grown in media (10 mL) in 10 cm tissue culture plates. The cells were trypsinized and seeded into 96-well plates with 5×103cells per well. Cells were treated with Dox- or DoxNH₂NH₂-containing media (200 pL) at the time of plating. The cells were then incubated for 96 h following the treatment. Cell growth was measured by resazurin assay. In brief, at the end of 96 h, resazurin was added to the cells at a final concentration of 50 pg / mL. The baseline fluorescence was determined immediately. The cells were then incubated at 37 °C for 2 h. The fluorescence of the resorufin product was measured at 560 / 590 nm and baseline corrected. The cell growth at each Dox or DoxNH2NH2concentration was normalized to the untreated control.Testing results

[0038] As shown in FIGs 1-4, a striking effect was observed with the uterine sarcoma cell line MES-SA and its doxorubicin-resistant counterpart MES-SA / Dx5. As shown in FIG 1, compared to Dox, DoxNH2NH2showed more than 20-fold higher activity against the MES-SA cell line. As shown in FIG 2, despite MES-SA / Dx5 conferring more than 100-fold resistance to doxorubicin, DoxNH2NH2completely overcame this drug resistance. MES-SA and MESSA / Dx5 cells were also treated with doxorubicin in the presence of verapamil, a commonly used P-gp inhibitor. As expected, we observed a complete reversal of doxorubicin resistance in MES-SA / Dx5 cells. In contrast, verapamil did not sensitize either MES-SA (FIG 3) or MES-SA / Dx5 (FIG 4) cells to DoxNH2NH2, indicating that DoxNH2NH2is not a P-gp substrate.EXAMPLE 3DoxNH2NH2chemoresistance testing

[0039] ABSTRACT. Efflux pump-mediated multidrug resistance is a common mechanism by which cancer cells reduce the efficacy of a broad range of smallmolecule therapeutics. We discovered that substituting the 4’ -hydroxy group of doxorubicin - a known efflux pump substrate - with an e i-amino group results in a new compound, doxorubamine, which exhibits substantially improved activity against drag-sensitive and -resistant cancer cells and organoids. Mechanistic studies revealthat doxorubamine is a poor substrate of P-glycoprotein and it thus retains high potency against multidrug-resistant cancer. This synthetic modification provides a promising strategy for circumventing multidrug resistance beyond conventional approaches that rely on efflux pump inhibition.

[0040] Drug resistance is a formidable barrier to effective cancer treatment. Among various mechanisms, multidrug resistance (MDR) is widely observed in vitro and is associated with the poor prognoses of different cancers. Szakacs et al. Nat. Rev. Drug Discovery 2006, 5, 219; Shaffer et al. Drug Resist. Updates 2012, 15, 62-69; Holohan et al. Nat. Rev. Cancer 2013, 13, 714-726; Szakacs, et al. Chem. Rev. 2014, 114, 5753-5774; Gottesman et al. Annu. Rev. Pharmacol. Toxicol. 2016, 56, 85-102;Wijdeven et al. Drug Resist. Updates 2016, 28, 65-81; Seluanov et al. Nat. Rev. Cancer 2018, 18, 433-441; Ward et al. Chem. Rev. 2021, 121, 3297-3351; Robey et al. Nat. Rev. Cancer 2018, 18, 452-464; Tamaki et al. Essays Biochem. 2011, 50, 209-232; Patch et al. Nature 2015, 527, 489-494; Robey et al. Drug Transporters, 2022; pp 235-256. Extensive studies have shown that typical MDR is enabled by several ATP-binding cassette (ABC) transporters, including P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and ATP-binding cassette superfamily G member 2 (ABCG2). Szakacs et al. Nat. Rev. Drug Discovery 2006, 5, 219; Shaffer et al. Drug Resist. Updates 2012, 75, 62-69; Robey et al. Nat. Rev. Cancer 2018, 18, 452-464; Patch et al. Nature 2015, 527, 489-494; Robey et al. Drug Transporters, 2022; pp 235-256. The overexpression of these proteins, either as an intrinsic or acquired phenotype, leads to the undesired removal of a broad range of structurally unrelated small-molecule therapeutics from cancer cells, which reduces the efficacy of treatment. Conventional strategies for overcoming MDR primarily rely on inhibiting ABC transporters, thereby enhancing the retention of anticancer drugs by blocking efflux activity. This approach has led to the development of several generations of P-gp inhibitors with high in vitro activity. These inhibitors, however, confer limited clinical benefit or induce severe side effects. Szakacs et al. Nat. Rev. Drug Discovery 2006, 5, 219; Robey et al. Nat. Rev. Cancer 2018, 18, 452-464; Binkhathlan et al. Curr. Cancer Drug Targets 2013, 13, 326-346; Palmeira et al. Curr. Med. Chem. 2012, 19, 1946-2025. One problem arises from the need for inhibitors and anticancer drugs to act simultaneously in a particular tumor, thus requiring each agent to have similar pharmacokinetic profiles. Because non-malignant tissues also express ABC transporters to clear endogenous and exogenous toxins, non-selective inhibition often causes adverse effects. Additional complications stem from the possible co-expression of multiple types of ABC transporters, which may require the use of several different inhibitors. These challenges underscore the need for new strategies to overcome MDR.

[0041] While studying transition metal-based agents for treating multidrug-resistant cancers (Wang et al. Nat. Commun. 2025, 16, 9263), we synthesized doxorubamine (DoxNH2NH2), the 4’ - tyri-dehy drox y ami nated derivative of doxorubicin (Dox), which is a classic efflux pump substrate. Shen et al. J. Pharmacol. Exp. Ther. 2008, 324, 95- 102; Biedler et al. Cancer Res. 1970, 30, 1174-1184. We serendipitously discovered that doxorubamine efficiently killed drug-sensitive human ovarian cancer A2780 cells and the corresponding multidrug-resistant A2780ADR cell line. Rogan et al. Science 1984, 224, 994-996. Surprisingly, compared to various strategies to enhance theactivity of anthracycline drugs (Wander et al. J. Org. Chem. 2021, 86, 5757-5770;van Gelder et al. J. Med. Chem. 2024, 67, 13802-13812; Priebe et al. Pharmacol.Ther. 1993, 60, 215-234; Perez-Soler et al. Int. J. Cancer 1997, 71, 35-41; Consoli et al. Blood 1996, 88, 633-644; Barbieri et al. Cancer Res. 1987, 47, 4001-4006;Monneret, Eur. J. Med. Chem. 2001, 36, 483-493; Mazel et al. Anti-Cancer Drugs2001, 12, 107-116; Kolar, et al. Carbohydr. Res. 1990, 208, 67-81), this relatively minor structural modification not only introduces a 10-fold increase in potencyagainst A2780 cells but also enables doxorubamine to retain activity in efflux-high A2780ADR cells, which exhibit 410-fold resistance toward doxorubicin (Table 1, Entries 1 and 2).Table 1. Comparison of the cytotoxicity of Dox and DoxNH2NH2against various cell lines.IC50± (nM) IC50+ (nM) for Fold resistance entry treatment ' for A2780 A2780ADR (IC5O. A27. SOADR-TIC5(). A27S()) 1 Dox 8.4 ± 2.2 577 ± 349 i 69 2 DoxNH2NH20.83 + 0.38 1.4 ± 0.6: 1.7 3 Dox + verapamil 7.6 + 2.1 25 + 8 ■ 3.4 4 DoxNH2NH2+ 0.93 + 0.14 0.46 + 0.27 i 0.49 verapamil: Potency increase ■ verapamil (-) 10 410 i (IC50, Dox±IC50, DoxNH2NH2) iICS0± (nM) IC\, ± (nM) for Fold resistance treatment A.r-c c * Mrc o * / n - (IC5O. MI’S-SA / DX5-TIC5<>. MI’S-tor MES-SA MES-SA / Dx5 SA,) 5 Dox 12 ± 1 185 ± 61: 156 DoxNH2NH22.0 ± 0.2 2.7 ± 0.1 1.37 Dox + verapamil 7.2 ± 0.4 42 ± 5; 5.98 DoxNH2NH2+ 2.5 ± 0.3 2.3 ± 0.3 i 0.92verapamil;Potency increase:verapamil (-) 6.0 68:(IC50, Dox±IC50, DoxNH2NH2) ’IC50± (nM) IC50± (nM) for Fold resistance for EL4 EL4-DoxR (IC.so.i;i.4 -DoxRFlC.so.iiu) 9 Dox 9.1 ± 0.9 1400 ± 50: 155 10 DoxNH2NH22.8 ± 2.1 37 ± 3 13 11 Dox + tariquidar 3.3 ± 0.4 18 ± 2 ■ 5.3 12 DoxNH2NH2+ 1 3±o i 2.6 ± 0.7 i 2.0 tariquidar; Potency increase: verapamil (−) 3.3 39(IC50, Dox±IC50, DoxNH2NH2)!

[0042] Prompted by these observations, we evaluated the anticancer activity of doxorubamine with additional drug-sensitive and -resistant cell line pairs. As shown in Table 1, compared to Dox, DoxNH2NH2was six times more potent against drug- sensitive human uterine sarcoma MES-SA cells. More importantly, althoughmultidrug-resistant MES-SA / Dx5 cells (Wesolowska et al Anticancer Res. 2005, 25,383-389; Harker et al. Cancer Res. 1985, 45, 4091-4096) exhibited 15-fold resistance toward doxorubicin, DoxNH2NH2completely abrogated drug resistance (Table 1, Entries 5 and 6). Given the broad clinical use of anthracyclines in lymphomatreatment, we generated a drug -resistant lymphoma cell line, EL4-DoxR, by exposing the parental mouse T-cell lymphoma cell line, EL4, to increasing concentrations of doxorubicin, resulting in 115-fold resistance. In contrast, although DoxNH2NH2displayed some cross-resistance toward EL4-DoxR, DoxNH2NH2maintained high activity, with 3.3- and 39-fold higher potency than Dox against EL4 and EL4-DoxR lines, respectively (Table 1, Entries 9 and 10). Overall, these results suggest that the4’-epi-dehydroxyamination of doxorubicin enhances both the anticancer capacity and the ability to circumvent efflux -pump-mediated chemoresistance.

[0043] We investigated whether DoxNH2NH2kills MDR cancer cells independentlyof efflux pump activity. We first treated drug-sensitive A2780 cells with acombination of anticancer agents and verapamil (10 pM), a widely used P-gpinhibitor. Rogan et al. Science 1984, 224, 994-996; Tsuruo et al. Cancer Res. 1981,41, 1967-1972; Tsuruo et al. Cancer Res. 1982, 42, 4730-4733. As expected,verapamil did not affect the cytotoxicity of either doxorubicin or doxorubamine(Table 1, entries 1 vs. 3 and 2 vs. 4). In contrast, in drug-resistant A2780ADR cells, verapamil sensitized doxorubicin by 23-fold but showed little effect on the potency of doxorubamine, suggesting that doxorubamine is a poor P-gp substrate. Similar results were observed with the MES-SA and MES-SA / Dx5 cell line pairs - although efflux inhibition by verapamil substantially reversed resistance to doxorubicin, it exhibited negligible effects on the activity of doxorubamine. Subsequent tests on EL4 and EL4- DoxR cells revealed that verapamil alone reduced the viability of these cells,presumably because of off-target interactions, including inhibition of calciumtransport (Table 2; FIG 5). Tsuruo et al. Cancer Res. 1981, 41, 1967-1972. Wetested a more specific P-gp inhibitor, tariquidar (100 nM), in co-treatment studies.Martin et al. Br. J. Pharmacol. 1999, 728, 403-411; Roe, et al. Bioorg. Med. Chem.Lett. 1999, 9, 595-600. Under these conditions, a strong sensitization ofapproximately 80-fold was observed in EL4-DoxR cells treated with doxorubicin.Still, the resistance toward doxorubamine was reversed by only 14-fold, revealing the inefficient removal of doxorubamine by P-gp (Table 1, Entries 11 and 12).Collectively, these findings indicate that DoxNH2NH2overcomes MDR primarilybecause it is intrinsically a poor P-gp substrate.Table 2. Comparison of the cytotoxicity of Dox and DoxNH2NH2against AKPS andAKPS-DoxR cancer organoids.IC50± SD IC50± (nM)c i ].,; ' Fold resistance entry treatment (nM) lor lor AKPS- / ir,.ir,,AKPS DoxR 1 Dox 3.6 ± 2.6 4400 + 500: 1223 2 DoxNH2NH20.79 ± 0.20 20 ± 10 25 3 Dox + verapamil 12 ± 3 110 ± 20 ■ 9.0 4 DoxNH2NH2+ verapamil 0.95 ± 0.26 4.3 ± 1.9 i 4.5Potency increase: verapamil (-) 4.6 220 ■(IC50, Dox+IC50, DoxNH2NH2)!

[0044] Next, we assessed the activity of doxorubamine with genetically engineered mouse colorectal cancer organoids. Compared to conventional cell lines, these three-dimensional in vitro models are more physiologically relevant as they mimic the structure and function of in vivo tumors. Drost et al. Nat. Rev. Cancer 2018, 18, 407-418. We specifically generated a model harboring mutations commonly found in colon cancer, including APC- / “; KRASG12D; p53~ - SMAD4~7~ (AKPS) organoids. These organoids were also engineered to express either tdTomato (tdT) or zsGreen (zsG) to facilitate fluorescence microscopy-based in vitro co-culture studies. We derived the doxorubicin-resistant variants of these organoids, AKPS-tdT-DoxR, by repeatedly exposing them to increasing concentrations of doxorubicin. In co-culture experiments, although AKPS-tdT-DoxR organoids exhibited more than 1000-fold resistance to doxorubicin, it could be reversed by verapamil treatment, indicating a P-gp-driven drug resistance mechanism (Table 2, Entry 1, FIG 6). In contrast, compared to doxorubicin, doxorubamine effectively killed both drug-sensitive and -resistant organoids, showing a 5-fold and more than 200-fold enhancement in potency, respectively (Table 2, Entry 2). Moreover, DoxNH2NH2circumvented MDR in resistant organoids through a mechanism largely independent of P-gp activity, as indicated by the insignificant effect of verapamil co-treatment on efficacy (Table 2, Entry 4). These results recapitulate the observation with human cell line pairs: the high cellular retention of doxorubamine across multiple model systems appears to be a general feature of this compound.

[0045] Finally, because both doxorubicin and doxorubamine are intrinsically fluorescent, we were able to compare the subcellular distribution of these two compounds in MES-SA cells using fluorescence microscopy. Through the measurements, we found that both Dox and DoxNH2NH2were distributed throughout the cytoplasm and nucleus. Fluorescence intensisty quantification revealed that Dox-treated cells exhibited strong nuclear fluorescence. In contrast, DoxNH2NH2showed more fluorescence in the cytoplasm. As anthracycline compounds commonly undergo fluorescence quenching upon DNA intercalation (Mohan et al. Mol. Pharm. 2010, 7, 1959-1973; Chaires et al. Biochemistry 1983, 22, 4204-4211; Airoldi et al.Biochemistry 2014, 53, 2197-2207; Perez-Arnaiz et al. J. Phy. Chem. B 2014, 118, 1288-1295), these studies suggest that both Dox and DoxNH2NH2primarily accumulate in the nucleus. Moreover, the logarithm of the partition coefficient (log P) of Dox and DoxNH2NH2in / / -octanol-pH 7.4 phosphate buffer was -0.44±0.02 and0.12±0.08, respectively, indicating that DoxNH2NH2is only slightly more lipophilic or hydrophobic than Dox. Therefore, the substantially enhanced anticancer activity of doxorubamine may be attributable to factors other than alterations in subcellular distribution, cell permeability, lipophilicity, or hydrophobicity.

[0046] In conclusion, converting the 4’ -hydroxy group of doxorubicin into an epi-amino moiety substantially enhanced the anticancer activity of the resulting compound, doxorubamine, in both drug-sensitive and -resistant cancer models. Cotreatment experiments with P-gp inhibitors indicate that this structural modification renders doxorubamine a poor P-gp substrate, which allows doxorubamine to overcome efflux pump-mediated MDR. Fluorescence imaging studies reveal a similar subcellular distribution pattern for doxorubamine and the parent compound, doxorubicin, suggesting that the drastically varied bioactivity of doxorubamine may not be solely attributable to the difference in physical properties. Further investigations of the mode of action of doxorubamine and its mechanism of overcoming drug resistance are currently underway.

[0047] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0048] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to beunderstood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0049] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to " A and / or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0050] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." " Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0051] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0052] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4−salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0053] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi -closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

WHAT IS CLAIMED IS;1. A method of treating cancer comprising:administering to a subject not otherwise in need of treatment thereof a therapeutically effective amount of a compound having a structure according to Formula Ior a pharmaceutically acceptable salt, hydrate, or solvate thereof,thereby treating the cancer(“doxorubamine”), the doxorubamine is not chelated to platinum when administered to the subject.

2. The method of claim 1, wherein the doxorubamine is not chelated to a complexed platinum when administered to the subject.

3. The method of a previous claim, wherein the cancer is selected from the group consisting of uterine cancer, ovarian cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, osteosarcoma, kidney cancer, bladder cancer, liver cancer, lymphoma, and leukemia.

4. The method of claim 3, wherein the cancer is a uterine cancer which is uterine sarcoma.

5. The method of a preceding claim, wherein the cancer is a doxorubicin-resistant cancer.

6. The method of a preceding claim, wherein the cancer is an efflux pump-mediated multidrug resistant cancer.

7. The method of a preceding claim, wherein the cancer is an ATP-binding cassette transporter-mediated multidrug resistant cancer.

8. The method of a preceding claim, wherein the subject is a human.

9. A compound having a structure according to Formula IMe (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof,10. An enantiomer of doxarubamine, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

11. A diastereomer of doxarubamine, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

12. A compound selected from the group consisting ofand a pharmaceutically acceptable salt, hydrate, or solvate thereof.

13. A pharmaceutical composition comprising(a) the compound of claims 9-12, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and(b) a pharmaceutically acceptable excipient.

14. A pharmaceutical composition comprising(a) doxorubamine, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and(b) a pharmaceutically acceptable excipientwherein the pharmaceutical composition does not comprise platinum.

15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition does not comprise a complexed platinum.

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