Curcumin-conjugated Anti-cancer agent

Abstract

A curcumin-conjugated compound with a curcumin that is covalently conjugated to a chemotherapeutic agent R1. The linker is enzymatically able to be lysed to deliver the chemotherapeutic agent R1 and the curcumin.

Description

CURCUMIN-CONJUGATED ANTI-CANCER AGENT CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and is a non-provisional of, U.S. Patent Application 63 / 588,068 (filed October 5, 2023), the entirety of which is incorporated herein by reference. STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under grant number 1 R15 CA253069-01A1 awarded by the National Institute of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] The subject matter disclosed herein relates generally to cancer treatments and more specifically to cancer treatments using curcumin-conjugated compounds.

[0004] The current world of cancer therapy is energized by the popularization of immunotherapy which, at its core, involves elimination of cancer cell-mediated inhibition of cytotoxic T (Tc) cells. This is achieved by interfering with the binding of a cancer cell ligand PD-L1 to the Tc cell receptor PD-1. Two antibody-based drugs have received approval from the U.S. Food and Drug Administration (FDA) for human use: (a) OPDIVO®, which blocks the PD-1 receptor, and (b) KEYTRUDA®, which blocks the PD-L1 ligand. Whereas long-term remission of cancer, especially melanoma, has been reported for some patients using immunotherapy, side effects of autoimmune attacks of normal tissue have also been reported in other patients.

[0005] Anti-metabolites (i.e. chemotherapeutic agents) may also be used to treat cancer. Each compound that received approval for human use by the FDA ushered inimmense hope for cancer patients. Optimism eventually dissipated when patients faced side effects of chemo- and radiation therapies. Mainly three phenomena contributed to the side effects and reappearance of cancer: (a) cancer cells become resistant to chemo- and radiation therapy; (b) this chemoresistance prompts use of higher doses of the chemotherapeutic agents; (c) higher doses of the chemotherapeutic agents also kill proliferating immune cells thus causing immune suppression and thereby precipitating serious infections that often kill the patient.

[0006] As discussed above, there is a serious unmet need for a safe strategy that can selectively kill cancer cells without killing normal cells, vanquish the commonly encountered “chemo-resistance” and, finally, prime the immune system against cancer cells and cancer stem cells. In the midst of such mixed success of conventional compounds, an improved cancer therapy is therefore desired.

[0007] The discussion above is merely provided for general background information and is not intended to be used as an aid in determining the scope of the claimed subject matter. SUMMARY

[0008] This disclosure provides a curcumin-conjugated compound with a curcumin that is covalently conjugated to a chemotherapeutic agent R1. The linker is enzymatically able to be lysed to deliver the chemotherapeutic agent R1and the curcumin.

[0009] In a first embodiment, a composition of matter is provided. The composition comprising a curcumin conjugated compound with a structure of Formula A, or a pharmaceutically acceptable salt thereof:A wherein he chemotherapeutic agent to the structure.

[0010] In a second embodiment, a composition of matter is provided. The composition comprising a curcumin conjugated compound with a structure of Formula A, or a pharmaceutically acceptable salt thereof: A3- hydroxy-carboplatin, an irinotecan, a capecitabine, a temozolomide and a pegfilgrastim; L is a linker selected from the group consisting , and n is an integer from 2 to 50.

[0011] In a third embodiment, a composition of matter is provided. The composition comprising a curcumin conjugated compound with a structure of Formula B, or a pharmaceutically acceptable salt thereof: B

[0012] This brief description of the invention is intended only to provide a brief overview of subject matter disclosed herein according to one or more illustrative embodiments, and does not serve as a guide to interpreting the claims or to define or limit the scope of the invention, which is defined only by the appended claims. This brief description is provided to introduce an illustrative selection of concepts in a simplified form that are further described below in the detailed description. This brief description is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. The claimed subject matter is not limited to implementations that solve any or all disadvantages noted in the background. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] So that the manner in which the features of the invention can be understood, a detailed description of the invention may be had by reference to certain embodiments, some of which are illustrated in the accompanying drawings. It is to be noted, however, that the drawings illustrate only certain embodiments of this invention and are therefore not to be considered limiting of its scope, for the scope of the invention encompasses other equally effective embodiments. The drawings are not necessarily to scale, emphasis generally being placed upon illustrating the features of certain embodiments of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views. Thus, for further understanding of the invention, reference can be made to the following detailed description, read in connection with the drawings in which:

[0015] FIG.1 depicts a curcumin and a curcumin-conjugated compound.

[0016] FIG.2A and FIG.2B depict various examples of chemotherapeutic agents.

[0017] FIG.3A depicts one curcumin-conjugated compound (STO-1).

[0018] FIG.3B depicts another curcumin-conjugated compound (STO-4).

[0019] FIG.3C depicts another curcumin-conjugated compound (STO-3).

[0020] FIG.3D depicts another curcumin-conjugated compound (STO-2).

[0021] FIG.4A depicts an IC50 graph of paclitaxel while FIG.4B depicts an IC50 graph of STO-1.

[0022] FIG.4C, FIG.4D, FIG.4E and FIG.4F show graphs depicting the results of a pharmacokinetic analysis using high performance liquid chromatography (HPLC) of brain extracts at various times after STO-1 was injected into a tail-vein of a mouse.

[0023] FIG.5A depicts an IC50 graph of gemcitabine while FIG.5B depicts an IC50 graph of STO-2.

[0024] FIG.5C and FIG.5D show graphs depicting the results of a pharmacokinetic analysis using high performance liquid chromatography (HPLC) of brain extracts at various times after STO-2 was injected into a tail-vein of a mouse.

[0025] FIG.6A depicts an IC50 graph of methotrexate while FIG.6B depicts an IC50 graph of STO-4.

[0026] FIG.6C, FIG.6D and FIG.6E show graphs depicting the results of a pharmacokinetic analysis using high performance liquid chromatography (HPLC) of brain extracts at various times after STO-4 was injected into a tail-vein of a mouse.

[0027] FIG.7A is a Kaplan Meier graph of mice treated with STO-1 or Vehicle.

[0028] FIG.7B shows magnetic resonance images (MRIs) of four vehicle-treated mouse brains.

[0029] FIG.7C shows MRIs of four STO-1-treated mouse brains.

[0030] FIG.8A is a timeline of a treatment regimen for a mouse experiment.

[0031] FIG.8B are brightfield images of the mouse brains after the treatment regimen of FIG.8A.

[0032] FIG.8C is a CD68Ab-Dylight (near-IR) staining image of the mouse brains.

[0033] FIG.9A and FIG.9B are graphs summarizing the flow cytometry results of integrated fluorescence with respect to iNOS (FIG.9A), and Arg-1 (FIG.9B).

[0034] FIG.10A and FIG.10B are graphs showing quantification of integrated fluorescence values from each treatment group.

[0035] FIG.11A shows fluorescence images of brain tumor sections from mice that were treated with PBS (vehicle), paclitaxel and STO-1 in accordance with FIG.8A.

[0036] FIG.11B and 11C are graphs that quantify the fluorescence images of FIG. 11A.

[0037] FIG.12A shows fluorescence images of brain tumor sections from mice that were treated with PBS (vehicle), paclitaxel and STO-1 in accordance with FIG.8A.

[0038] FIG.12B is a graph quantifying the fluorescence data of FIG.12A.

[0039] FIG.13 is an example of a synthetic scheme for producing curcumin- conjugated compound linked to a chemotherapeutic agent with an amine linking point.

[0040] FIG.14A and FIG.14B provide an example of a synthetic scheme for producing STO-1.

[0041] FIG.15A and FIG.15B provide an example of a synthetic scheme for producing STO-2.

[0042] FIG.16 is an example of a synthetic scheme for producing STO-3.

[0043] FIG.17A and FIG.17B provide an example of a synthetic scheme for producing STO-4. DETAILED DESCRIPTION OF THE INVENTION

[0044] This disclosure provides compounds that link a chemotherapeutic agent through cleavable bonds to a curcumin - a dietary anticancer agent. The compounds reduce or eliminate chemo-resistance and also primes the innate immune system to kill cancer cells and cancer stem cells.

[0045] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

[0046] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g. a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and / or a clone.

[0047] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to astated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0048] In Vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g. in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0049] In Vivo: As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell- based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0050] Patient: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition may be administered, e.g. for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g. mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. A human includes pre and post-natal forms.

[0051] Pharmaceutically acceptable: The term “pharmaceutically acceptable” as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. For example, a composition can include a pharmaceutically acceptable ester or amide of a compound herein. In certain implementations, a composition includes a pharmaceutically acceptable salt of a compound herein. Non-limiting examples of pharmaceutically acceptable salts include carboxylate salts, amino acid addition salts and zwitterionic forms thereof, which areknown to those skilled in the art as suitable for use with humans and animals. In cases where a compound is sufficiently basic or acidic to form a stable nontoxic acid or base salt, a composition includes a pharmaceutically acceptable salt of the compound. Non- limiting examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, non-limiting examples of which include tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts are obtained using standard procedures known in the art. For example, pharmaceutically acceptable salts may be obtained by reacting a sufficiently basic compound with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium, magnesium) salts of carboxylic acids and other anionic groups in molecules within a pharmaceutical composition also are contemplated.

[0052] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0053] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the symptom(s) of the disease, disorder, and / or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0054] Treating: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0055] As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 50 carbon atoms (“C1-50 alkyl”). In some embodiments, an alkyl group has 1 to 40 carbon atoms (“C1-40 alkyl”). In some embodiments, an alkyl group has 1 to 30 carbon atoms (“C1-30 alkyl”). In some embodiments, an alkyl group has 1 to 20 carbon atoms (“C1-20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”). Examples of C1-6 alkyl groups include, without limitation, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n- butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one ormore substituents. In certain embodiments, the alkyl group is an unsubstituted C1-50 alkyl. In certain embodiments, the alkyl group is a substituted C1-50 alkyl.

[0056] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example “C1-6 alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3- 4, C4-6, C4-5, and C5-6 alkyl.

[0057] FIG.1 depicts a curcumin 100. In one embodiment, curcumin is covalently conjugated to a chemotherapeutic agent R1using a linker L, thereby producing curcumin- conjugated compound 102. The curcumin-conjugated compound 102 may be present as the compound itself or as a pharmaceutically acceptable salt thereof.

[0058] Various chemotherapeutic agents R1are disclosed elsewhere in this specification. The disclosed methodology may be applied to any antimetabolite. For example, and with reference to FIG.2A, a paclitaxel 200, a methotrexate (MTX) 202, a doxorubicin 204 or a gemcitabine 206 is conjugated to the curcumin 100 in certain embodiments. In other embodiments, a chemotherapeutic antibody is used as the chemotherapeutic agents R1that is conjugated to the curcumin 100. Referring to FIG. 2B, in still other embodiments, the chemotherapeutic agents R1is a 3-hydroxy- carboplatin 208 (e.g. PARAPLATIN®), an irinotecan 210, a capecitabine 212, a temozolomide 214 or a pegfilgrastim 216 is the chemotherapeutic agents R1.

[0059] Referring to FIG.3A, the paclitaxel 200 is shown conjugated to the curcumin 100 by a linker L, thereby producing a curcumin-conjugated compound 300. In one embodiment, the linker L is an optionally branched alkyl with terminal diketones such as:wherein n is an integer from 1 to 50, from 1 to 25, from 1 to 15, from 1 to 10, from 1 to 5, is 2, is 3 or is 4. In other embodiments, n is from 2 to 50, from 2 to 25, from 2 to 15, from 2 to 10 or from 2 to 5. In one such embodiment, n is 3 (i.e. a glutarate) and the curcumin-conjugated compound is compound STO-1 (see FIG.3A). In the embodiment of FIG.3A, the linker is conjugated to both the chemotherapeutic agent R1and the curcumin 100 through respective esters. Such linkers are useful in connecting to chemotherapeutic agents R1with at least one hydroxyl.

[0060] Referring to FIG.3B, the MTX 202 is shown conjugated to the curcumin 100 by a linker L, thereby producing a curcumin-conjugated compound 302. In one embodiment, the linker L is an optionally branched alkyl with terminal diketones, as described elsewhere in this specification. In one such embodiment, n is 3 (i.e. a glutarate) and the curcumin-conjugated compound is compound STO-4 (see FIG.3B). In the embodiment of FIG.3B, the linker is conjugated to both the chemotherapeutic agent R1and the curcumin 100 through respective esters.

[0061] In the curcumin-conjugated compound 302 the linker L is an optionally branched alkyl with a terminal ketone such as:wherein n is an integer from 1 to 50, from 1 to 25, from 1 to 15, from 1 to 10, from 1 to 5, 2, or 3. In the embodiment of STO-4, n is 11. In the embodiment of FIG.3B, the linker is conjugated to both the chemotherapeutic agent R1and the curcumin 100 through respective esters. Such linkers are useful in connecting to chemotherapeutic agents R1with at least one carboxylic acid.

[0062] In one embodiment, R2of FIG.3B is H, a C1-C50 alkyl or :.

[0063] In one embodiment, R is a C1-C3 alkyl (e.g. methyl, ethyl, n-propyl, iso- propyl). R2is connected to the curcumin-conjugated compound 302 via an ester that is hydrolyzed under physiological conditions (e.g. within a human body), thereby liberating R2and forming the corresponding carboxylic acid of the curcumin-conjugated compound 302.

[0064] Referring to FIG.3C, the doxorubicin 204 is shown conjugated to the curcumin 100 by a linker L, thereby producing a curcumin-conjugated compound 304. In one embodiment, the linker L is an optionally branched alkyl with terminal diketones, as described elsewhere in this specification. In one such embodiment, n is 3 (i.e. a glutarate) and the curcumin-conjugated compound is compound STO-3 (see FIG.3C). In the embodiment of FIG.3C, the linker is conjugated to both the chemotherapeutic agent R1and the curcumin 100 through respective esters.

[0065] Referring to FIG.3D, the gemcitabine 206 is shown conjugated to the curcumin 100 by a linker L, thereby producing a curcumin-conjugated compound 306. In one embodiment, the linker L is an optionally branched alkyl with terminal diketones, as described elsewhere in this specification. In one such embodiment, n is 3 (i.e. a glutarate) and the curcumin-conjugated compound is compound STO-2 (see FIG.3D). In the embodiment of FIG.3D, the linker is conjugated to both the chemotherapeutic agent R1and the curcumin 100 through respective esters.

[0066] FIG.4A and FIG.4B provide IC50 graphs for paclitaxel 200 (FIG.4A) and STO-1 (FIG.4B) against glioblastoma cell line GL261. Viability of GL261 cells was determined after a 96-h treatment in serum-free EPMI1640 supplemented with ITSfollowed by WST-1 assays. The graphs comparing IC50 for paclitaxel vs STO-1 (46.82 nM vs 20.51 nM) demonstrate that STO-1 twice as potent as paclitaxel in killing GL261 cells.

[0067] FIGS 4C-4F show the results of a HPLC experiment which demonstrates STO-1 is detected in brain extracts of mice within 15 minutes of tail-vein injection. The figures depict different time intervals after injection (i.v.) into mice wherein FIG.4C is t=0, FIG.4D is t=15 min, FIG.4E is t=30 min and FIG.4F is t=60 min. Brain volume was 0.5 mL. Mean STO-1 concentration at 15 minutes was 0.4 ^M (0.52 mg per mL).

[0068] FIG.5A and FIG.5B provide IC50 graphs for gemcitabine 206 (FIG.5A) and STO-2 (FIB.5B) against pancreatic cancer cell line Pan02. Viability of Pan02 cells was determined after a 96-h treatment in serum-free RPMI1640 supplemented with ITS followed by WST-1 assays. The graphs IC50 of Gem vs STO-2 (57.78 nM vs 25.43 nM) demonstrating STO-2 is twice as potent against the Pan02 cells.

[0069] FIG.5C and FIG.5D show the results of a HPLC experiment which demonstrates STO-2 is detected in brain extracts of mice within 60 minutes of tail-vein injection. STO-2 peaks at about 30 minutes. Brain volume was 0.5 mL. Mean STO-2 concentration at 60 minutes was 0.64 ^M (0.47 mg per mL).

[0070] The IC50 for STO-2 is less than half of the IC50 for gemcitabine for the pancreatic cancer cells Panc02. Gemcitabine is part of the standard of care for pancreatic cancer. Thus, the reversible attachment of curcumin to gemcitabine does not alter its cancer cell-type selectivity, rather this curcumin tag slightly increases the potency of gemcitabine in killing the pancreatic Pac02 cells directly. Additionally, the data from STO-1 and also prior studies in a heterotopic mouse model of cervical cancer (Cancer Immunology, Immunotherapy (2018) 67:761–774) suggest that STO-2 will be able to enhance the anticancer activity of gemcitabine by repolarizing the tumor associated microphages (TAMs) in the pancreatic tumor.

[0071] FIG.6A and FIG.6B provide IC50 graphs for MTX 202 (FIG.6A) and STO- 4 (FIB.5B) against glioblastoma (GBM) GL261 cells in culture. Viability of GL261 cells was determined after a 96-h treatment in serum-free RPMI1640 supplemented with ITS followed by WST-1 assays. The graphs show a comparable IC50 (28.74 nM vs 80.98 nM) demonstrating STO-4 maintains biological activity.

[0072] FIG.6C-6E show the results of HPLC analysis which demonstrates STO-4 is detected in brain extracts of mice within 15 minutes of tail-vein injection. The figures depict different time intervals wherein FIG.6C is t=15, FIG.6D is t=30 min and FIG.6E is t=60 min. Brain volume was 0.5 mL. Mean STO-4 concentration at 30 minutes was 0.91 ^M (1.28 mg / mL).

[0073] Theory of operation

[0074] Without wishing to be bound any particular theory, once a curcumin conjugated compound is introduced to a cancer cell, intracellular esterases are believed to cleave the ester linkages of the side arm, thereby releasing both curcumin and the chemotherapeutic agent R1(e.g. paclitaxel, etc.). An amide-linked chemotherapeutic agents R1, in a similar manner, releases the chemotherapeutic agent R1due to the presence of cytosolic fatty acid amide hydrolases. In this manner, the disclosed compounds provide a curcumin moiety in a releasable form.

[0075] Importantly, curcumin per se is not effective in vivo because of its low bioavailability (rapid degradation and low solubility). Accordingly, the disclosed curcumin conjugated compounds provide a mechanism to simultaneously deliver bioavailable curcumin and a chemotherapeutic agent R1.

[0076] Cancer

[0077] Due to the presence of the curcumin moiety in a releasable form, the disclosed curcumin-conjugated compounds are more effective than the anti-metabolite alone in eliminating cancer cells and cancer stem cells in tumors in vivo.

[0078] The disclosed curcumin conjugated compounds are useful on glioblastoma (GBM) as well as most other cancers including pancreatic cancer, neuroblastoma, medulloblastoma, human papillomavirus (HPV) positive and negative cervical cancers, HPV positive and negative head and neck cancers (squamous cell carcinoma), ovarian / endometrial cancer, prostate cancer, breast cancer, liver cancer, colon cancer, melanoma, adenocarcinoma, and leukemia. In some embodiments, the disclosed curcumin conjugated compounds confer blood-brain-barrier permeability to a chemotherapeutic agent R1which, in isolation, lacks such permeability.

[0079] The presence of curcumin serves to eliminate cancer stem cells and proteins that confer chemoresistance on cancer cells. Thus sensitized, these treated cancer cells become vulnerable to significantly lower concentrations of the chemotherapeutic agent R1, which in turn eliminates a wide range of side effects commonly observed during chemotherapy. Furthermore, conjugation of curcumin to the chemotherapeutic agent R1confers an increased level of hydrophobicity on the chemotherapeutic agent R1, which increases blood-brain-barrier permeability. Although the antimetabolite dose from the chemotherapeutic agent R1may be too low to kill the innate immune cells (such as macrophages, natural killer (NK) cells and T lymphocytes) the released curcumin molecules are, in some embodiments, sufficient to cause repolarization of tumor- associated immune cell milieu into tumoricidal M1-type cells, which decimates the cancer cells and cancer stem cells.

[0080] STO-1 is active against GBM

[0081] Adult C57BL / 6 male mice (4-5 months old) were used to evaluate the efficacy of STO-1 in eliminating GBM. Each mouse received orthotopic implantation of 2 x 104GL261 (mouse GBM) cells on Day 1. Starting from Day 5, one group of six mice (randomly-chosen) received intravenous lipid vesicles in PBS (vehicle) treatment every 72 hours, while a second group of six mice received 0.46 mmole (0.6 mg) per mouse of STO-^^LQWUDYHQRXVO\^LQ^^^^^^ / ^RI^OLSLG^YHVLFOHV^LQ^3%6^HYHU\^^^^KRXUV^^

[0082] The Kaplan Meier graph of FIG.7A shows this STO-1 treatment resulted in long-term remission of GBM in 67% (four out of six) of the GL261-implanted mice, whereas all the vehicle-treated mice succumbed to GBM within 42 days.

[0083] FIG.7B shows magnetic resonance imaging (MRI) scans of brains from three vehicle-treated mice (vehicle-1, vehicle-2, vehicle-3) at experimental endpoint showed large tumors. A vehicle-presymptomatic brain shows a smaller GBM tumor in a separate vehicle-treated mouse before symptoms arose (loss of body weight, hunching, impairment of mobility, and lack of feeding- this is considered as the experimental endpoint when the mice are euthanized).

[0084] Referring to FIG.7C, in contrast, brains from four STO-1-treated and asymptomatic mice (STO-1-1, STO-1-2, STO-1-3, and STO-1-4) showed no discernible tumor after 85 days.

[0085] FIG.8A depicts a treatment schedule. After orthotopic implantation of 105GL261 cells, 4-5-month-old mice were randomly divided into three groups and treated on day 12 with PBS (vehicle), or 0.46 mmole of paclitaxel (Pac), or 0.46 mmole of STO-1 per day per mouse for five days (i.v.).

[0086] FIG.8B shows brightfield images of the mouse brains (three mice per group) illustrating that STO-1 has a stronger effect on tumors than paclitaxel. The outline of hemorrhage is clearly observed around the deformed tumor mass in the brains from vehicle- or paclitaxel-treated mice, but only in one STO-1-treated mouse. A physical mixture (1:1 mole ratio) of paclitaxel and curcumin (CC) was not effective in reducing the tumor load.

[0087] For STO-1, a total of four mice were tested (the three shown in FIG.8B plus one). For better detection of tumor in this group, CD68Ab-Dylight (near-IR) staining was conducted (see FIG.8C), followed by near-IR scanning. Due to hemorrhagic leakage, such near-IR staining does not yield quantitative information in larger GBMtumors. The CD68-AB-Dylight staining more clearly depicts the tumors when they are too small to be detected in brightfield images.

[0088] Table 1 summarizes the results of flow cytometry (FC) of cells dissociated from mouse GBM tumors shown in FIG.8B: STO-1 treatment causes an inhibition of Iba1, Arg-1+,+ cells (associated with the M2-type TAMs that cause tumor survival and growth) and an induction of Iba1, iNOS+,+ cells (associated with the M1-type TAMs that are tumoricidal) relative to vehicle-treated mice. Table 1: Flow cytometry. . ith respect to iNOS (FIG.9A), and Arg-1 (FIG.9B). FIG.9A shows an increase in iNOS production relative to paclitaxel and vehicle control groups. FIG.9B shows a decrease in ARG-1 production relative to paclitaxel and vehicle control groups.

[0090] FIG.10A and FIG.10B are graphs showing comparison of quantified integrated fluorescence values among the three groups of mice with three mice in each group. These data demonstrate STO-1-treatment evokes a significant decrease in Arg-1and a significant increase in iNOS among the Iba1+ lymphocytes. Integrated fluorescence = average fluorescence per cell x total number of cells in the iNOS, Iba1+,+ or Arg-1, Iba1+,+ lymphocytes. (n = 3; dispersed GBM tumor cells from three brains used). Arg-1: Pac vs vehicle, p = 0.059; STO-1 vs vehicle, p = 0.033. iNOS: Pac vs vehicle, p = 0.066; iNOS vs vehicle, 0.047.

[0091] Table 2 shows Flow Cytometry (FC): STO-1 (Pac-curcumin) treatment as shown in FIG 8A causes a dramatic increase in Iba1, STAT-1+,+ TAMs among the dissociated GBM cells. Table 2 atwere treated with PBS (vehicle), paclitaxel and STO-1 in accordance with FIG.8A. These images were obtained confocal microscopy. Fluorescence staining was performed for Arg-1, Iba1, HOESCHST and iNOS. The lefthand “Merged” column depicts the merged fluorescence data of Arg-1, Iba1 and HOECHST33342. The right hand “Merged” column depicts the merged fluorescence data of iNOS, Iba1 and HOECHST. These data show STO-1 treatment causes repolarization of Iba1+ TAMs from Arg-1high, iNOSlowM2 state to the Arg-1low, iNOShighM1 state. FIG.11B and 11C are graphs that quantify the fluorescence images of FIG.11A for three mice in each treatment group. These data show STO-1 treatment lowers Arg-1 and iNOS expression. *p < 0.03 (vs vehicle); **p <0.002 (vs vehicle); ***p <0.0005 (vs vehicle).

[0093] FIG.12A shows fluorescence images of GBM tumor sections from mice that were treated with PBS (vehicle), paclitaxel and STO-1 in accordance with FIG.8A. These images were obtained using confocal microscopy after five days of the treatment depicted in FIG.8A. These data show recruitment of activated, tumoricidal (NKp46+) NK cells into the GBM tumor. FIG.12B is a graph quantifying the fluorescence data of FIG.12A. Vehicle-treated mice (n = 3 per treatment). p < 0.001 (STO-1 vs Vehicle); p = 0.0120 (paclitaxel vs Vehicle).

[0094] Thus, STO-1 is a single, natural product-derived compound, which can replace multiple clinically-used drugs o selectively regulate cells in the innate and adaptive immune systems within the GBM tumor micro-environment (TME). RagGTPase-independent mTORC1 has been suggested to stimulate TAMs to outcompete cancer cells (Nature, Vol 619, 20 July 2023, p616). Similarly, poly (I:C) treatment has been shown to stimulate anti-cancer activity of microglia (Neuro-Oncology 14(1):64–78, 2012). Additionally, Arginase-1 inhibition has been shown stimulate CD8+ T-cell function against tumors (Clinical Cancer Research 2011 Vol.17 Pages 4296-4308). The stimulation of CD8+ T-cells has been at the center of immunotherapy including the use of check point inhibitors like KEYTRUDA® and OPDIVO®, as mentioned elsewhere in this disclosure. However, these therapies have worked only for a limited number of melanoma patients because, reportedly, repeated activation of CD8+ T-cells causes exhaustion of these cells (similar to a state of senescence). In contrast, STO-1 is one natural product-derived compound that is pleiotropic, acting through the TAMs which, in turn, causes recruitment of tumoridical NK cells and also CD8+ T-cells to launch a combined attack on GBM cells and GBM stem cells, thereby rescuing 67% of the GBM- harboring mice.

[0095] Ectopic Pregnancies

[0096] STO-4 is believed to be useful in the treatment of conditions such as ectopic pregnancy and arthritis while precipitating minimal side effects. Ectopic pregnancies pose a major health risk in women. Gynecologists commonly treat this condition byadministering (intramuscular) a chemotherapeutic agent such as methotrexate to eliminate the ectopically located embryo. This causes major side effects such as prolonged bleeding, extreme abdominal discomfort during movement, searing pain while passing stool, etc.

[0097] STO-4 was synthesized (see FIG.17). One of the two carboxyl groups retains a rearranged form of dicyclohexylurea (DCU) while the other carboxyl group forms an ester with the linker L. Following esterase hydrolysis inside cells, the released 12-hydroxy-dodecanoic acid is metabolized in vivo like all fatty acids and is, therefore, biocompatible. Next, DCU is another biocompatible, and beneficial, agent that has been shown to lower systolic blood pressure in hypertensive rats (Basic Clin Pharmacol Toxicol, 2008.102(5): p.453-8). Therefore, STO-4 may offer added benefits.

[0098] Due to the presence of the curcumin moiety in a releasable form, STO-4 is more effective than methotrexate in eliminating the rapidly dividing cells in the ectopic embryo and therefore requires a greatly reduced dose of methotrexate to treat the condition. This, in turn, reduces or eliminates the side effects described above.

[0099] Referring to FIG.13, the linker L may also include an amide coupling wherein the nitrogen from the amide comes from the chemotherapeutic agent R1. In FIG. 13, a curcumin derivative 1300 is converted to a succinimide 1302 using conventional methods. For exmple, one may use N-hydroxysuccinimide (NHS) followed by treatment with dicyclohexylcarbodiimide (DCC). An amine-containing chemotherapeutic agent R1may then be covalently linked to the curcumin, thereby producing a curcumin-conjugated compound 1304. Examples of suitable chemotherapeutic agents R1include antibodies (e.g. lysine amino acids therein) and other proteins. In one embodiment, R1is a monoclonal antibody (e.g. IgG). Antibodies used in immunotherapy may be used such as ipilimumab, pembrolizumab, nivolumab and bevacizumab. Examples of non-proteins that may be linked using such a strategy include temozolomide 214 (see FIG.2B).

[0100] Delivery

[0101] The disclosed compositions may be administered in a variety of forms. The compositions may be delivered in a liquid form (e.g. a dilute solution) with or without a buffer (e.g. PBS). The compositions may be administered along with an emulsifier, in nanoparticles with biocompatible vesicles, as a phytosome complex with phosphatidylcholine. such nanoparticles may have appropriate chemical groups on their surface to target them to either cancer cells or the immune cells or both.

[0102] Synthesis of STO-1

[0103] FIG.14A and FIG.14B provide one method for the coupling of paclitaxel monocarboxylic acid to curcumin via EDC-mediated esterification to obtain Paclitaxel- curcumin (STO-1). 5-(((1S,2R)-1-benzamido-3-(((2aR,4S,4aS,6R,9S,12S,12bS)-6,12b- diacetoxy-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo- 2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11- methanocyclodeca[3,4]benzo[1,2-b]oxet-9-yl)oxy)-3-oxo-1-phenylpropan-2-yl)oxy)-5- oxopentanoic acid (s2): Reaction conditions were adapted from a previously published paper (Lim et al., Organic Letters 2008, 10(2), 201-204). To a solution of protected Pacitaxol 200 (1 g, 4 mmol) and glutaric anhydride (4 g, 0.04 mol) in 100 mL of CH2Cl2was added TEA (2 mL, 0.02 mol). Stir the reaction mixture at room temperature overnight. This mixture was then washed with NaHCO3 (10%, 2 × 30 mL), water (2 × 30 mL) and brine (30 mL). The organic phase was then dried (Na2SO4), filtered and concentrated under reduced pressure. The crude was then purified by combi-flash chromatography (DCM / Methanol). Yield: 1.6 g (60%), white solid 1400.

[0104] (1S,2R)-1-benzamido-3-(((2aR,4S,4aS,6R,9S,12S,12bS)-6,12b-diacetoxy-12- (benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo- 2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11- methanocyclodeca[3,4]benzo[1,2-b]oxet-9-yl)oxy)-3-oxo-1-phenylpropan-2-yl (4- ((1E,6E)-7-(4-hydroxy-3-methoxyphenyl)-3,5-dioxohepta-1,6-dien-1-yl)-2-methoxyphenyl) glutarate (STO-1): To a solution of compound 1400 (350 mg, 362 ^PRO^^DQG^FRPSRXQG^100 ^^^^^PJ^^^^^^^PRO^^LQ^^^^P / ^PHWK\OHQH^FKORULGH^ZDV^DGGHG^ EDC (398 mg, 2.08 mmol) and DMAP (33.8 mg, ^^^^^PRO^^DW^DPELHQW^WHPSHUDWXUH^^WKH^ reaction was allowed to stir for 12 h The reaction mixture was concentrated under vacuum and purified via preparative HPLC using a H2O-CH3CN gradient (99:1 to5:95, v / v, 0.1%FA). A fraction containing the target molecule was lyophilized to give a yellow solid. (100 mg, 21%).1H NMR (600 MHz, CDCl3^^į^^^^^^– 8.14 (m, 2H), 7.74 – 7.72 (m, 2H), 7.64 – 7.59 (m, 3H), 7.52 (d, J = 7.9 Hz, 2H), 7.48 – 7.45 (m, 1H), 7.43 – 7.37 (m, 4H), 7.33 (d, J = 8.0 Hz, 3H), 7.14 (ddd, J = 12.4, 8.2, 1.9 Hz, 2H), 7.07 (dd, J = 9.3, 1.9 Hz, 3H), 7.02 (d, J = 8.2 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.55 (d, J = 15.8 Hz, 1H), 6.50 (d, J = 15.8 Hz, 1H), 6.31 (s, 1H), 6.30 – 6.26 (m, 1H), 6.02 (dd, J = 9.2, 3.0 Hz, 1H), 5.89 (s, 1H), 5.83 (s, 1H), 5.69 (d, J = 7.1 Hz, 1H), 5.54 (d, J = 3.0 Hz, 1H), 5.00 – 4.98 (m, 1H), 4.46 (td, J = 6.6, 3.3 Hz, 1H), 4.32 (d, J = 8.5 Hz, 1H), 4.22 – 4.20 (m, 1H), 3.95 (s, 3H), 3.83 (d, J = 7.0 Hz, 1H), 3.77 (s, 3H), 2.74 – 2.55 (m, 7H), 2.49 (d, J = 4.0 Hz, 1H), 2.46 (s, 3H), 2.41 – 2.37 (m, 1H), 2.23 (s, 3H), 2.21 – 2.17 (m, 1H), 2.08 (d, J = 7.1 Hz, 2H), 1.97 (d, J = 1.5 Hz, 3H), 1.92 – 1.87 (m, 1H), 1.69 (s, 3H), 1.25 (s, 3H), 1.14 (s, 3H).13C NMR (151 MHz, CDCl3^^į^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^170.8, 169.8, 168.0, 167.1, 167.0, 151.2, 148.0, 146.8, 142.8, 141.2, 141.0, 139.2, 136.9, 134.2, 133.7, 133.5, 132.8, 131.9, 130.2, 129.1, 129.0, 128.7, 128.6, 128.4, 127.5, 127.1, 126.4, 124.4, 123.1, 123.0, 121.7, 120.9, 114.8, 111.4, 109.6, 101.5, 84.4, 81.1, 79.2, 75.6, 75.1, 74.1, 72.1, 71.8, 58.5, 55.9, 55.8, 52.6, 45.5, 43.2, 35.5, 35.5, 32.6, 32.5, 29.7, 26.8, 22.7, 22.1, 20.8, 20.2, 14.8, 9.6. MS: m / z calculated for C73H76NO22 [M+H]+: 1318.49, found: 1318.40, Purity (HPLC-UV 254 nm): >95%. (tR= 3.791min).

[0105] Synthesis of STO-2

[0106] FIG.15A and FIG.15B provide one method for the Boc-protection of gemcitabine, attaching a glutarate side arm, coupling to curcumin via EDC-mediated ester bond formation, and removal of Boc protection to obtain gemcitabine-curcumin (STO-2).

[0107] tert-butyl(1-(4-((tert-butoxycarbonyl)oxy)-3,3-difluoro-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (1500): Reaction conditions were adapted from a previously published paper (Fan et al., European Journal of Pharmaceutical Sciences, 2015, 79, 20-26). To a solution of gemcitabine 206 (3g, 0.01 mol) in 30 mL of DMF was added DBDC(0.02 kg , 0.1 mol).The reaction mixture was maintained at 37oC for 4h. The crude compound product which was purified by combi-flash chromatography (PE EA 1:1 to EA) to yield the compound 1500 as white solid (3 g, 61%).

[0108] 5-((5-(4-((tert-butoxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-3-((tert- butoxycarbonyl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methoxy)-5-oxopentanoic acid (1502): Reaction conditions were adapted from a previously published paper (Karampelas et al., Bioconjugate Chem.2014, 25(4) 813-823). To a solution of protected gemcitabine (2 g, 4 mmol) and glutaric anhydride (4 g, 0.04 mol) in 100 mL of CH2Cl2 was added TEA (2 mL, 0.02 mol). Stir the reaction mixture at room temperature overnight. This mixture was then washed with NaHCO3 (10%, 2 × 30 mL), water (2 × 30 mL) and brine (30 mL). The organic phase was then dried (Na2SO4), filtered and concentrated under reduced pressure. The crude was then purified by combi-flash chromatography (DCM / Methanol). Yield: 1.6 g (60%), white solid 1502.

[0109] (5-(4-((tert-butoxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-3-((tert- butoxycarbonyl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methyl(4-((1E,6E)-7-(4-hydroxy- 3-methoxyphenyl)-3,5-dioxohepta-1,6-dien-1-yl)-2-methoxyphenyl) glutarate (1504): To a solution of compound 100 in (800 mg, 1.39 mmol), EDC (398 mg, 2.08 mmol) and DMAP (33.8 mg, ^^^^^PRO^^LQ^^^^PO^PHWK\OHQH^FKORULGH^ZDV^DGGHG^&XUFXPLQ^ compound 4 (510 mg, 1.39 mmol) in 20 ml methylene chloride dropwise at ambient temperature, the reaction was allowed to stir for 12 h. The reaction mixture was concentrated under vacuum and purified via preparative HPLC using a H2O-CH3CN gradient (99:1 to5:95, v / v, 0.1%FA). A fraction containing the target molecule was lyophilized to give a yellow solid. (500 mg, 38.9%).1H NMR (600 MHz, CDCl3^^į^^^^^^(dd, J = 7.7, 1.7 Hz, 1H), 7.60 (dd, J = 15.8, 9.8 Hz, 2H), 7.25 (d, J = 7.7 Hz, 1H), 7.13 (ddd, J = 18.9, 9.5, 1.9 Hz, 3H), 7.07 – 7.04 (m, 2H), 6.94 (d, J = 8.2 Hz, 1H), 6.52 (dd, J = 29.8, 15.8 Hz, 2H), 6.44 (dd, J = 11.5, 5.6 Hz, 1H), 5.83 (s, 1H), 5.08 (dd, J = 14.5, 5.6 Hz, 1H), 4.47 (qd, J = 12.5, 4.0 Hz, 2H), 4.37 (td, J = 5.1, 3.1 Hz, 1H), 3.95 (s, 3H), 3.86 (s, 3H), 2.70 (t, J = 7.1 Hz, 2H), 2.58 (td, J = 7.3, 1.5 Hz, 2H), 2.12 (p, J = 7.2 Hz, 2H), 1.51 (s, 9H), 1.51(s, 3H).13C NMR (151 MHz, CDCl3^^į^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ 163.0, 154.5, 151.5, 151.3, 150.8, 148.0, 146.8, 144.0, 141.1, 141.10, 139.3, 134.1, 127.5, 124.2, 123.2, 123.0, 121.8, 121.0, 118.5, 114.8, 111.4, 109.6, 101.5, 95.5, 84.8, 83.2, 62.0, 55.9, 55.9, 32.8, 32.7, 28.0, 27.5, 20.0. MS: m / z calculated for C45H52F2N3O16 [M+H]+:928.33 found: 928..30, Purity (HPLC-UV 254 nm): >95%. (tR= 6.28 min).

[0110] (5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3- hydroxytetrahydrofuran-2-yl)methyl (4-((1E,6E)-7-(4-hydroxy-3-methoxyphenyl)-3,5- dioxohepta-1,6-dien-1-yl)-2-methoxyphenyl) glutarate (STO-2): To a solution of 1504 (370 PJ^^^^^^^PRO mmol), in 20 ml methylene chloride was added TFA (1.2 mL, in 2 mL methylene chloride) at 0oC and then slowly warm up to room temperature and allowed to stir for 12 h. The completion reaction was confirmed by LC-MS. The reaction mixture was concentrated under vacuum and purified via preparative HPLC using a H2O- CH3CN gradient (99:1 to5:95, v / v, 0.1%FA). A fraction containing the target molecule was lyophilized to give a yellow solid. (110 mg, 37.9%).1H NMR (600 MHz, DMSO) į 9.69 (s, 1H), 7.61 (d, J = 15.8 Hz, 1H), 7.60 (d, J = 15.8 Hz, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 1.4 Hz, 1H), 7.35 (d, J = 1.6 Hz, 1H), 7.32 (dd, J = 1.4, 8.3 Hz, 1H), 7.18 (dd, J = 1.7, 8.1 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 6.96 (d, J = 16.0 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 15.8 Hz, 1H), 6.19 (s, 1H), 6.14 (s, 1H), 5.80 (d, J = 7.5 Hz, 1H), 4.42 (dd, J = 2.4, 12.4 Hz, 1H), 4.33 (dd, J = 6.2, 12.4 Hz, 1H), 4.20 (s, 1H), 4.03 (ddd, J = 2.4, 6.2, 8.3 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 2.66 (t, J = 7.4 Hz, 2H), 2.53 (t, J = 7.4 Hz, 2H), 1.91 (m, J = 7.4 Hz, 2H).13&^105^^^^^^0+]^^'062^^į^^^^^^^^ 181.8, 172.7, 171.0, 166.1, 163.5, 154.9, 151.5, 150.0, 148.4, 142.0, 141.1, 139.4, 134.3, 126.6, 125.0, 123.8, 123.7, 123.1, 121.7, 121.6, 116.1, 112.4, 111.9, 101.7, 95.3, 77.8,70.6, 70.5, 63.2, 56.4, 56.1, 32.7, 32.5, 20.3. MS: m / z calculated for C35H36F2N3O12[M+H]+: 728.23 found: 728.40, Purity (HPLC-UV 254 nm): >95%. (tR= 2.476 min).

[0111] Synthesis of STO-3

[0112] FIG.16 provides one method for the coupling of curcumin monocarboxylic acid to doxorubicin via EDC coupled ester bond formation (STO-3). The amine of doxorubicin is protected as the tert-butyloxycarbonyl (Boc) protecting group. The curcumin derivative 1300 is coupled to the protected doxorubicin using coupling chemistry, such as 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The Boc protecting group is then removed.

[0113] Synthesis of STO-4

[0114] FIG.17A and FIG.17B provide one method for the coupling of curcumin to methotrexate via EDC-mediated ester bond formation.

[0115] Benzyl 12-bromododecanoate (1702): A mixture of 12-bromododecanoic acid 1700 (750 mg, 2.69 mmol), DMAP (0.54 mmol), and DCC (4.03 mmol) in CH2Cl2was stirred at 0 °C for 30 minutes. Benzyl alcohol (3.22 mmol) was then added to the reaction mixture. The mixture was allowed to warm to room temperature and stirred for 12 hours. After completion, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product obtained was purified by column chromatography using a hexane / EtOAc mixture (150:1, v / v) as an eluent to afford colorless oil 1702 (91% yield).1H NMR (600 MHz, CDCl3^^SSP^^į^^^^^^–7.31 (m, 5H, - PhH), 5.12 (s, 2H, --COOCH2), 3.40 (t, J = 6.9 Hz, 2H, -CH2Br), 2.35 (t, J = 7.5 Hz, 2H, -COCH2), 1.85 (quint, J = 7.2 Hz, 2H, -CH2CH2Br), 1.65 (quint, J = 7.3 Hz, 2H, - COCH2CH2), 1.42 (quint, J = 7.2 Hz, 2H, dodecyl -CH2), 1.45–1.39 (m, 2H, dodecyl - CH2), 1.34-1.24 (m, 12H, dodecyl -CH2);13C NMR (150 MHz, CDCl3^^SSP^^į^^173.8, 128.8, 128.6, 128.4, 128.2, 66.2, 34.6, 34.5, 34.4, 34.2, 33.0, 29.6, 29.5, 29.4, 29.3, 28.9, 28.3, 25.1.

[0116] Bis(12-(benzyloxy)-12-oxododecyl) (4-(((2,4-diaminopteridin-6- yl)methyl)(methyl)amino) benzoyl)-^-glutamate (1704): To a suspension of methotrexate 202 (114 mg, 0.25 mmol) in water (2 mL), 1 M NaHCO3 solution (0.5 mL) was added. The resulting solution was lyophilized to quantitatively yield methotrexate disodium salt as a yellow solid. To a mixture of methotrexate disodium salt (0.25 mmol) and Cs2CO3 (0.55 mmol) in DMSO (1 mL), benzyl 12-bromododecanoate 1700 (2.2 mmol) were added. The reaction mixture was stirred at 50 °C for 4 hours. After completion, the reaction mixture was poured into water (10 mL) and extracted with dichloromethane (20 mL × 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure using a rotary evaporator. The crude product was purified by column chromatography on silica gel, using a dichloromethane / methanol mixture (40:1, v / v) as the eluent to afford yellow oil 1704 (77% yield).1H NMR (600 MHz, CDCl3^^SSP^^į^^^^^^^^V^^^+^^SWHULGLQH^^-H), 7.70 (d, J = 8.8 Hz, 2H, -PhH), 7.36–7.28 (m, 10H, benzyl -PhH), 6.88 (d, J = 7.4 Hz, 1H, -CONH), 6.74 (d, J = 8.7 Hz, 2H, -PhH), 6.65 (br. s, 1H, -NH2), 5.96 (br. s, 1H, -NH2), 5.39 (br. s, 2H, -NH2), 5.09 (s, 4H, -CH2Ph), 4.76 (dt, J = 7.7 Hz, J = 4.8 Hz, 1H, -NHCH), 4.72 (s, 2H, -NCH2), 4.13 (t, J = 6.7 Hz, 2H, dodecyl -COOCH2), 4.00 (ddd, J = 6.8 Hz, J = 6.5 Hz, J = 4.0 Hz, 2H, dodecyl -COOCH2), 3.16 (s, 3H, -NCH3), 2.50–2.44 (m, 1H, - NHCHCH2), 2.43–2.36 (m, 1H, -NHCHCH2), 2.33 (t, J = 7.5 Hz, 4H, dodecyl - CH2COOBn), 2.30–2.24 (m, 1H, -NHCHCH2), 2.13–2.06 (m, 1H, -NHCHCH2), 1.65- 1.58 (m, 6H, dodecyl -CH2), 1.57–1.51 (m, 2H, dodecyl -CH2), 1.33-1.25 (m, 4H, - CH2CH3), 1.34-1.18 (m, 28H, dodecyl -CH2);13C NMR (150 MHz, CDCl3^^SSP^^į^^ 173.8, 173.4, 163.1, 162.6, 155.2, 151.5, 147.0, 136.2, 129.1, 128.9, 128.9, 128.7, 128.6, 128.6, 128.5, 128..5, 128.3, 128.1, 122.1, 121.7, 111.5, 111.4, 66.1, 65.9, 65.2, 65.0, 64.9, 55.9, 52.4, 34.5, 34.4, 34.2, 29.7, 29.5, 29.4, 29.3, 29.2, 28.6, 25.9, 25.0.

[0117] (S)-12,12'-((2-(4-(((2,4-diaminopteridin-6- yl)methyl)(methyl)amino)benzamido) pentanedioyl)bis(oxy))didodecanoic acid (1706): Palladium on carbon black (10%, w / w^^^^^^PRO^SDOODGLXP) was suspended in a solution of compound 1704 ^^^^PJ^^^^^^PRO^^in methanol (5 mL). A mixture of H2 / N2 (5%, v / v)was then bubbled into the suspension. The reaction progress was monitored by TLC until compound 1704 was completely consumed. The Pd / C catalyst was then filtered off and washed with 10 mL of methanol three times. The filtrate was combined and concentrated under reduced pressure to yield yellow oil. The crude product was further purified by column chromatography on silica gel, using a dichloromethane / methanol mixture (7:1, v / v) as the eluent to afford yellow oil 1706 (24 mg, 65% yield).1H NMR (600 MHz, DMSO-d6^^SSP^^į^^12.01 (br.s, 2H, -COOH), 8.56 (s, 1H, pteridine 7-H), 8.30 (d, J = 7.3 Hz, 1H, -CONH), 7.72 (d, J = 8.5 Hz, 2H, -PhH), 7.70 (br. s, 1H, -NH2), 7.46 (br. s, 1H, - NH2), 6.81 (d, J = 8.6 Hz, 2H, -PhH), 6.65 (br. s, 2H, -NH2), 4.78 (s, 2H, -NCH2), 4.40– 4.34 (m, 1H, -NHCH), 4.06–3.98 (m, 2H, - COOCH2), 3.97 (t, J = 6.4 Hz, 2H, - COOCH2), 3.20 (s, 3H, -NCH3), 2.40 (t, J = 7.1 Hz, 2H, -CH2COO), 2.17 (t, J = 7.3 Hz, 4H, -COOCH2CH2), 2.09–2.03 (m, 1H, -NHCHCH2), 2.01–1.94 (m, 1H, -NHCHCH2), 1.55–1.49 (m, 4H, dodecyl -CH2), 1.49–1.43 (m, 4H, dodecyl -CH2), 1.28–1.15 (m, 28H, dodecyl -CH2);13C NMR (150 MHz, DMSO-d6^^SSP^^į^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ 162.8, 162.7, 155.0, 150.1, 149.1, 146.0, 129.0, 121.4, 120.9, 111.0, 64.3, 63.9, 54.8, 51.9, 33.7, 30.2, 28.9, 28.8., 28.7, 28.6, 28.6, 28.1, 25.8, 25.4, 25.3, 24.5.

[0118] (STO-4): To a mixture of compound 1706 (0.05 mmol), DCC (0.11 mmol) and DMAP (0.01 mmol) was added 4 mL of DMF at 0 °C. After stirring for 15 minutes, curcumin (0.1 mmol) in THF (1 mL) was added, and the mixture was allowed to warm to room temperature. After 12 hours, the mixture was concentrated under reduced pressure and the crude product was directly purified by column chromatography on silica gel, using a dichloromethane / methanol mixture (10:1, v / v) as the eluent to afford an orange solid STO-4 (32% yield). Tg: 76 °C;1H NMR (600 MHz, CDCl3^^SSP^^į^^^^^^^^V^^^+^^ pteridine 7-H), 7.71 (d, J = 7.7 Hz, 2H, -benzyl -PhH), 7.63–7.53 (m, 2H, -COCH=CH), 7.17–7.07 (m, 3H, curcumin -PhH and -PhCONH), 7.06–7.00 (m, 2H, curcumin -PhH), 6.97–6.89 (m, 2H, curcumin -PhH), 6.74 (m, 2H, benzyl -PhH), 6.85–6.70 (m, 1H, - NH2), 6.54 (d, J = 14.9 Hz, 1H, -COCH=CH), 6.48 (d, J = 15.5 Hz, 1H, -COCH=CH), 6.24 (br.s, 1H, -NH2), 5.82 (s, 1H, -COCH2CO-), 5.66 (br.s, 2H, -NH2), 4.82–4.76 (m, 1H, -NHCH), 4.72 (s, 2H, -NCH2), 4.18–4.11 (m, 2H, -COOCH2), 4.07–3.97 (m, 2H, -COOCH2), 3.92 (s, 3H, -OCH3), 3.90–3.88 (m, 1H, cyclohexyl -CH), 3.86 (s, 3H, - OCH3), 3.72–3.64 (m, 1H, cyclohexyl -CH), 3.17 (s, 3H, -NCH3), 2.61–2.54 (m, 2H, dodecyl -CH2COO), 2.51–2.46 (m, 1H, glutamate -CH2COO), 2.46–2.42 (m, 1H, glutamate -CH2COO), 2.42–2.37 (m, 2H, dodecyl -CH2COO), 2.33–2.25 (m, 1H, - NHCHCH2), 2.16–2.08 (m, 1H, -NHCHCH2), 1.99–1.11 (m, 56H, dodecyl and cyclohexyl -CH2);13C NMR (150 MHz, CDCl3^^SSP^^į^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ 171.9, 167.0, 163.1, 162.5, 155.3, 154.3, 151.6, 151.5, 149.7, 148.4, 147.3, 147.2, 141.5, 141.3, 139.5, 134.1, 129.0, 127.6, 124.3, 123.4, 123.2, 122.3, 122.0, 121.8, 121.1, 115.2, 111.6, 111.5, 110.0, 101.7, 65.9, 65.1, 56.1, 56.0, 52.4, 49.9, 39.4, 36.0, 36.0, 34.2, 32.9, 31.1, 30.7, 29.6, 29.6, 29.5, 29.4, 29.4, 29.3, 29.3, 29.2, 28.7, 27.6, 26.5, 26.0, 25.9, 25.7, 25.6, 25.5, 25.1, 24.9; HRMS (ESI) calcd. for [C78H107N10O14] [M+H]+1407.7963 found 1407.7969.

[0119] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

Claims

What is claimed is:

1. A composition of matter comprising a curcumin conjugated compound with a structure of Formula A, or a pharmaceutically acceptable salt thereof: A binds thechemotherapeutic agent to the structure.

2. The composition as recited in claim 1, wherein the structure is:

3. The composition as recited in claim 2, wherein n is from 2 to 25.

4. The composition as recited in claim 2, wherein n is from 2 to 15.

5. The composition as recited in claim 2, wherein n is from 2 to 10.

6. The composition as recited in claim 2, wherein n is from 2 to 5.

7. The composition as recited in claim 2, where in R1is paclitaxel.

8. The composition as recited in claim 2, wherein n is 3 and R1is paclitaxel such that the structure is:.

9. The composition as recited in claim 2, wherein R1is selected from the group consisting of methotrexate (MTX), a doxorubicin and a gemcitabine.

10. The composition as recited in claim 2, wherein n is 3 and R1is gemcitabine such that the structure is: .

11. The composition as recited in claim 2, wherein n is 3 and R1is doxorubicin such that the structure is: .

12. The composition as recited in claim 1, wherein the structure is:

13. The composition as recited in claim 2, wherein n is 3 and R1is methotrexate (MTX) such that the structure is:wherein R is an organic moiety.

14. A composition of matter comprising a curcumin conjugated compound with a structure of Formula A, or a pharmaceutically acceptable salt thereof: Aof a 3- hydroxy-carboplatin, an irinotecan, a capecitabine, a temozolomide and a pegfilgrastim; L is a linker selected from the group consisting of: n is an integer15. The composition as recited in claim 14, wherein n is from 2 to 10.

16. The composition as recited in claim 14, wherein n is from 2 to 5.

17. A composition of matter comprising a curcumin conjugated compound with a structure of Formula B, or a pharmaceutically acceptable salt thereof: B18. The composition of matter as recited in claim 17, wherein the chemotherapeutic agent is a protein.

19. The composition of matter as recited in claim 17, wherein the chemotherapeutic agent is an antibody.

20. The composition of matter as recited in claim 17, wherein the chemotherapeutic agent is an antibody selected from the group consisting of ipilimumab, pembrolizumab, nivolumab and bevacizumab.

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