Tumor redox-activated 6-thiopurine containing dimer compounds

Abstract

Disclosed herein are 6-thiopurine containing dimer compounds of formula I: or a pharmaceutically acceptable salt, ester, prodrug, hydrate, or tautomer thereof, wherein both R1s are concurrently H, or (PO3)2-, and both R2s are concurrently H or OH. Methods for preparing the compounds of formula I from the corresponding 6-thiopurine containing monomer compounds are also disclosed. Also disclosed are methods for converting the dimer compounds of Formula I to the corresponding 6-thiopurine containing monomer compounds. The compounds are useful for treating, ameliorating, or preventing various types of cancer, particularly those that are drug resistant. The compounds of Formula I can also be used in conjunction with a checkpoint inhibitor and / or radiation in different ways for treating various types of cancer.

Description

TUMOR REDOX-ACTIVATED 6-THIOPURINE CONTAINING DIMER COMPOUNDS FIELD OF THE DISCLOSURE

[0001] The present disclosure is in the general field of hyper-proliferative diseases including oncology, with a focus on new compounds and methods for making them, pharmaceutical compositions containing the new compounds, and methods for treating various types of conditions including cancers using the same. BACKGROUND OF THE DISCLOSURE

[0002] Telomeres are found at both ends of eucaryotic chromosomes. These DNA-protein structures protect the genome from nucleolytic degradation, unneeded recombination, intrachromosomal fusion and repair (Shammas, M., (2011), Curr. Opin, Clin. Nutr. Metab. Care, 14(1):28-34). Telomere length shortens with each cell division due to the end replication problem and the absence of a telomere maintenance mechanism (Greider C.W, (1996), Annu. Rev. Biochem.65:337-65). However, unicellular eukaryotes, germline cells and immortal cancer cells maintain their telomeres at a constant length almost always by activating the enzyme telomerase (Greider and Blackburn, (1985) Cell, vol. 43:405-13; McEachern and Blackburn, (1996), Genes & Dev.1996;10:1822–1834; Morin GB., (1989), Cell, 59:521-529; Nakamura et al., (1997), Science, 15:277(5328); Singer and Gottschling, (1994) Science, 266:404-409; Yu et al., (1990), Nature, 344:126-132).

[0003] Telomerase is a reverse transcriptase enzyme that elongates telomeres by adding TTAGGG repeats to the ends of chromosomes and is expressed in about 90% of human tumors, but not in most normal cells (Jafri, M. A., et al. Genome Medicine, 2016, 8:69). Thus, telomerase is an attractive target to develop anti-cancer therapies. Most therapies targeting telomerase have focused on inhibiting telomerase (Andrews LG, Tollefsbol TO. Mol Biol.2007;405:1-7); however, such inhibitors have not fared well in clinical studies (Zhang G., Shay WS. Oncotarget, 2018, vol 9(88):35803-35804).

[0004] Other cancer drugs are being developed which instead of inhibiting telomerase use telomerase for incorporation of such drugs into telomeres resulting in DNA damage, senescence or crisis in telomerase positive cancer cells. One such compound is the nucleoside analogue, 6- thio-2′-deoxyguanosine (6-thio-dG). Its incorporation into de novo synthesized telomeres bytelomerase is known to induce damage on telomeric DNA (Mender et al., (2015), Cancer Discov., 5(1):82-95; Mender et al., (2015), Oncoscience, 2(8):693-695). This results in rapid tumor shrinkage or growth arrest in many tumor-derived xenograft models with minimal side effects (Mender et al., (2018), Neoplasia, 20(8):826-837); Sengupta et al., (2018), Mol Cancer Ther., Jul;(17(7):1504-1514). The most important advantage of this telomere-targeted therapy over direct telomerase inhibitors is that 6-thio-dG does not have a long-lag period for tumor killing effects. Additionally, it does not directly inhibit telomerase but is preferentially recognized by telomerase over other polymerases and incorporated into the telomeres resulting in an immediate DNA chain termination. Importantly, its effect is independent of initial telomere length by hijacking tumor telomerase to make unstable telomeres (Mender et al., (2015) Oncoscience, 2(8):693-695).

[0005] Disclosed herein are new telomere-targeting compounds useful for treating cancer. SUMMARY

[0006] One aspect of the disclosure includes 6-thiopurine dimers of Formula I:or a pharmaceutically acceptable salt, ester, prodrug, hydrate, or tautomer thereof. In some embodiments both R1s are concurrently H, or PO32-and both R2s are concurrently H or OH.

[0007] The 6-thiopurine dimer can be a 6-thiodeoxyguanosine dimer of Formula II, wherein both R1s are concurrently H, or PO32-:R1O OHor a 6-thioguanosine dimer of Formula III:wherein both R1s are concurrently H or PO32-.

[0008] In some embodiments, the dimer is compound MAIA-2021-029 with the following structure:

[0009] the followingstructure:

[0010] In some embodiments, the dimer is compound MAIA-2022-08 a ribo-thio dimer with the following structure:

[0011] In some embodiments, the dimer is compound MAIA-2021-09 a phosphorylated ribo-thio dimer with the following structure:

[0012] In another aspect, described herein are methods for producing the 6-thiopurine containing dimer of Formula I. The method comprises adding a solution of I2 / NaI to a solution of the 6-thiopurine containing monomer of Formula IV: Formula IV; and isolating the 6-of Formula I thus formed.

[0013] In another aspect, described herein are methods for forming a 6-thiopurine containing monomer. The method comprises contacting the dimer of Formula I with an organic thiol of formula R-SH, and forming the 6-thiopurine containing monomer of Formula IV,wherein R1and R2are the same as described previously.

[0014] In yet another aspect of the disclosure is a formulation comprising a compound of Formula I, or a pharmaceutically acceptable salt, ester, prodrug, hydrate, or tautomer thereof, wherein R1and R2are as described previously.

[0015] In still yet another aspect, a method for treating cancer in a subject is disclosed. The method comprises administering a pharmaceutical formulation comprising a compound of Formula I to a subject in need of treatment wherein the pharmaceutical formulation is administered in a therapeutically amount effective to treat the cancer.FIGURES

[0016] Figure 1 shows comparative dose response plots and EC50 of Lewis lung carcinoma cells measured over a period of 4 days following treatment in vitro with the 6-thiopurine dimer containing compound MAIA-2021-029 and the 8-deuterio form of 6-thio-dG designated as F1.

[0017] Figure 2A-2B- 2A shows comparative dose response plots and EC50 of Murine Colon Adenocarcinoma (MC38) cells measured over a period of 4 days following treatment in vitro with compounds MAIA-2021-29 (referred to as MA-029) or the 8-deuterio form of 6-thio- dG referred to as F1; 2B shows a comparative plot of the average number of TIFs (Telomere dysfunction induced foci) per cell measured from treatment of MC38 cells, measured over a period of 4 days following treatment in vitro with compound MAIA-2021-29 referred to as Comp-29 and the 8-deuterio form of 6-thio-dG designated as F1.

[0018] Figure 3A-3B- 3A shows plots of % cell viability of Hep55.1C cells (cells derived from carcinogen-induced liver tumors of C57BL / 6 mice) measured over a period of 4 days following treatment in vitro with THIO; 3B shows plots of % cell viability of Hep55.1C cells (cells derived from carcinogen-induced liver tumors of C57BL / 6 mice) measured over a period of 4 days following treatment in vitro with compound MAIA-2021-29 referred to as Compound 29.

[0019] Figure 4 shows a plot of tumor volume with 5 million Hep55-1C cells, measured over a period of 4 days of treatment in vitro with compound MAIA-2021-29 (referred to as Compound 29), followed by being re-challenged with 5 million Hep55-1C cells as compared to control.

[0020] Figures 5A – 5D- shows comparative dose response plots for LP-1 cells, JHH-6 cells, CoC1 cells, and OCI-LY-19 cells, respectively, measured over a period of 4 days, following treatment in vitro with compound XX (referred to as 6S-dG-dimer) as compared to cells treated with cis-platin;

[0021] Figures 5E – 5H show comparative dose response plots for NCI-H1581 cells, DMS- 53 cells, HT-29 cells, and COLO 205 cells respectively, measured over a period of 4 days, following treatment in vitro with compound XX (referred to as 6S-dG-dimer) as compared to cells treated with cis-platin;

[0022] Figures 5I – 5L show comparative dose response plots for OCI-LY-19 cells, HCC1937 cells, AU565 cells, and H1836 cells, respectively, measured over a period of 4 days,following treatment in vitro with compound XX (referred to as 6S-dG-dimer) as compared to cells treated with cis-platin;

[0023] Figures 5M – 5O show comparative dose response plots of COLO DMS 114 cells, Capan-2 cells, and Capan-1 cells, respectively, measured over a period of 4 days, following treatment in vitro with compound XX (referred to as 6S-dG-dimer) and cis-platin.

[0024] Figure 6 shows comparative dose response plots of OCI-LY-19 cells measured over a period of 4 days, following treatment in vitro with the compounds XXVI (designated MAIA- 2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cis- platin.

[0025] Figure 7 shows a comparative plot of % cell viability of CoC1 / DDP cells (Pt- resistant ovarian cancer cells) measured over a period of 4 days, following treatment in vitro with compound XX (designated as 6S-dG-dimer) and cis-platin.

[0026] Figures 8A – 8D show comparative dose response plots of LL / 2 (LLC1) cells, A20 cells, MC-38 cells, and Hepa 1-6 cells, respectively, measured over a period of 4 days, following treatment in vitro with compound XX (6S-dG-dimer) and cis-platin;

[0027] Figures 8E – 8H show comparative dose response plots of B16-BL6 cells, Pan02 cells, RM-1 cells, and B16-F10 cells, respectively, measured over a period of 4 days, following treatment in vitro with compound XX (6S-dG-dimer or MAIA-2021-29) and cis-platin;

[0028] Figures 8I – 8L show comparative dose response plots of Renca cells, EMT6 cells, CT26.WT cells, and H22 cells, respectively, measured over a period of 4 days, following treatment in vitro with compound XX (6S-dG-dimer) and cis-platin.

[0029] Figures 9A – 9D show comparative dose response plots of LP-1 cells, JHH-6 cells, NCI-H1581 cells, and CoC1 / DDP cells, each measured over a period of 4 days, following treatment in vitro with the compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022- 09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cis-platin;

[0030] Figures 9E – 9H show comparative dose response of HT-29 cells, COLO 205 cells, AU565 cells, and DMS 53 cells, each measured over a period of 4 days, following treatment in vitro with the compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cis-platin;

[0031] Figures 9I – 9L show comparative dose response plots of CoC1 cells, NCI-H1836 cells, Capan-1 cells, and Capan-2 cells, each measured over a period of 4 days, following treatmentin vitro with the compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cis-platin.

[0032] Figures 10A and 10B show comparative plots of % cell viability of BJ human normal fibroblast cells (from BJ cell line) and Huh7 cells (human liver cancer cells), respectively, measured over a period of 4 days following treatment in vitro with compounds XX (Compound 29) and XVI (designated THIO).

[0033] Figures 11A – 11C show comparative plots of % cell viability of the mouse liver cancer cells, RIL175, Hep55-1C, and HCC53N, respectively, measured over a period of 4 days following treatment in vitro with compounds XX (Compound 29) and THIO.

[0034] Figure 12 shows comparative plots of tumor volume with 5 million Hep55-1C cells, measured over an extended period of more than 200 days from treatment in vitro with compounds XX (Compound 29) and THIO.

[0035] Figure 13 shows comparative plots of tumor volume with 5 million Hep55-1C cells, measured over an extended period of 125 days from treatment in vitro with compounds XX (Compound 29) and compound XVI (6-thio-dG), followed by tumor-free mice being re-challenged with 5 million Hep55-1C cells; and followed by tumor-free mice being re-challenged with 1 million RIL 175 cells after more than 200 days from the initial treatment.

[0036] Figures 14A – 14C show comparative plots of % cell viability of BJ human normal fibroblast cells (from BJ cell line), Huh7 human liver cancer cells, and HCC53N murine liver cancer cells, respectively, measured over a period of 4 days following treatment in vitro with compounds XX (Compound 29) and compound XVI (THIO).

[0037] Figures 15A – 15C show comparative plots of % cell viability of Hep55-1C human liver cancer cells, RIL175 murine liver cancer cells, and SB28 murine glioblastoma cancer cells, respectively, measured over a period of 4 days following treatment in vitro with compounds XX (Compound 29) and compound XVI (THIO or also designated as 6-thio-dG).

[0038] Figures 16A and 16B show comparative plots of % cell viability of H1993 human NSCLC cells and H2081 SCLC cells, respectively, measured over a period of 4 days following treatment in vitro with compounds XX (Compound 29) and compound XVI (THIO also designated as 6-thio-dG).

[0039] Figures 17A and 17B show comparative plots of % cell viability of H1693 human NSCLC cells (THIO intrinsically resistant) and H2087 clone human NSCLC cells (THIO acquiredresistance), respectively, measured over a period of 4 days following treatment in vitro with compounds XX (Compound 29) and compound XVI (THIO). DETAILED DESCRIPTION OF THE DISCLOSURE

[0040] Tumor tissues can have different microenvironments, such as acidic environments, enzyme environments, and reducing environments (Cheng R., et al. (2015), Nano Today, 10:656- 70). The reducing environment of tumor cells is controlled by the reduction and oxidation states of NADPH / NADP+together with glutathione (Wu G., et al., (2004) J. Nutr.134:489). Disclosed herein are dimer compounds that generate 6-Thio-dG or Ribo-thio in situ, in tumor cells with a reducing environment, with the help of endogenous glutathione (GSH). Tumors, especially drug resistant solid malignancies, have elevated GSH levels, thus making the dimer compounds disclosed herein unique anticancer agents that are able to take advantage of the high GSH levels found in cancer cells. Examples of tumors with reducing environments include, without limitation, ovarian, endometrial, glioma, renal, stomach, urothelial, pancreatic, melanoma and lung (Rahul Raj Singh and Katie M. Reindl, 2021, Antioxidants (Basel) 10(5):701)

[0041] In some embodiments, the thiopurine containing dimer has the structure of Formula II, wherein both R1s are concurrently H or PO32-: R1OH

[0042] In other embodiments, the dimer compounds disclosed herein are a 6-thioguanosine dimer of Formula III,wherein both R1s are concurrently H or PO32-.

[0043] In some embodiments, the dimer is compound MAIA-2021-029 with the following structure:

[0044] the following structure:

[0045] In some embodiments, the dimer is compound MAIA-2022-08 a ribo-thio dimer with the following structure:

[0046] In some embodiments, the dimer is compound MAIA-2022-09 a phosphorylated ribo-thio dimer with the following structure:

[0047] In another embodiment, disclosed herein are methods for producing a 6-thiopurine containing dimer of Formula I. The method comprises adding a solution of I2 / NaI to a solution of a 6-thiopurine containing monomer of Formula IV, as shown in Reaction (1):Reaction (1); wherein R1is as described previously, and R2is H or OH, to form a reaction mixture from which the desired 6-thiopurine containing dimer is isolated. The solution of I2 / NaI is added dropwise to at a rate sufficient to maintain the resulting reaction mixture decolorized.

[0048] In embodiments of this reaction, the monomer compound of Formula IV is maintained at a temperature from about ambient temperature (defined as between a temperaturebetween about 15°C to about 25°C) to about 60oC, or from ambient temperature to about 40oC, or from ambient temperature to about 30oC, or at ambient temperature. In other embodiments, the solution of the monomer compound of Formula IV can be obtained using a buffer having a pH of from about 7.0 to about 8.0, or from about 7.0 to about 7.5, or from about 7.5 to about 8.0.

[0049] In particular embodiments, the method can be used for producing the 6-thiopurine containing dimers XX – XXIII from the corresponding 6-thiopurine containing monomers XVI – XIX, respectively, as shown in Table 1 below. The compounds XX and XXI can also be termed as 6-thio-2’-deoxyguanosine containing dimers, and the compounds XXII and XXIII can also be termed as 6-thioguanosine containing dimers.

[0050] Table 1. 6-Thiopurine containing dimers prepared from the corresponding 6- thiopurine containing monomers. 6-thiopurine containing R1R26-thiopurine containing Product Sample monomer reactant dimer product reference number

[0051] In another embodiment, the 6-thiopurine containing dimers of Formula I undergo reduction when contacted with an organic thiol, R-SH, wherein R is an alkyl group, to provide thecorresponding 6-thiopurine containing monomers of Formula IV, as shown below in Reaction (2): Reaction (2), wherein R1and R2are as described herein. Suitable thiols that can reduce the 6-thiopurine containing dimer compounds of Formula I per Reaction (2) also include glutathione, as shown below: .

[0052] In an embodiment, the monomer to dimer conversion method discussed previously can be used for preparing 6-thio-2’-deoxyguanosine containing monomers of Formulas XXIV from the 6-thio-2’-deoxyguanosine containing dimers of Formula II: ,wherein R1is as described previously.

[0053] In another embodiment, the method can be used for preparing 6-thio-2’-guanosine containing monomers of Formula XXV from the 6-thio-2’-guanosine containing dimers of Formulas XXV:, wherein R1is as described previously. In another embodiment, the method can be used for preparing the 6-thio-2’-guanosine of Formula XXVI: .

[0054] In somethe use of one or more 6-thiopurine containing dimer compounds of Formula I for the preparation of a medicament for the treatment of the conditions recited herein.

[0055] The compounds disclosed herein may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The active compounds and compositions, for example, may be administered orally, rectally, parenterally, intraperitoneally, or topically (e.g., intranasal or ophthalmic).

[0056] Other carrier materials and modes of administration known in the pharmaceutical art may also be used. Pharmaceutical compositions disclosed herein may be prepared by any of the well-known techniques of pharmacy, such as effective formulation and administration procedures. The above considerations regarding effective formulations and administration procedures are well known in the art and are described in standard textbooks. Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

[0057] The compounds disclosed herein can be used, alone or in combination with other therapeutic agents, in the treatment of various conditions or disease states. The compound(s) disclosed herein and other therapeutic agent(s) may be administered simultaneously (either in the same dosage form or in separate dosage forms) or sequentially.

[0058] The administration of two or more compounds “in combination” means that the two compounds are administered closely enough in time that the presence of one alters the biological effects of the other. The two or more compounds may be administered simultaneously, concurrently or sequentially. Additionally, simultaneous administration may be carried out by mixing the compounds prior to administration or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration.

[0059] The phrases “concurrent administration,” “co-administration,” “simultaneous administration,” and “administered simultaneously” mean that the compounds are administered in combination.

[0060] Treatment Indications: In an embodiment, the 6-thiopurine containing dimer compounds disclosed herein are useful for treating, ameliorating, or preventing cancer.

[0061] In another embodiment, the method for treating cancer in a subject further comprises treating the subject with an immune checkpoint inhibitor. Non-limiting examples of checkpoint inhibitors include a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

[0062] In another embodiment, the method for treating cancer in a subject further comprises treating the subject with a chemotherapeutic agent, a hormonal therapy, a toxin therapy, radiation therapy, surgery, or combinations thereof.

[0063] Different types of cancer that can be treated using the 6-thiopurine containing dimer compounds include, without limitation, one or more of breast cancer, prostate cancer, colon cancer, stomach cancer, esophagus, liver, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, biliary tract, bladder cancer, hepatoma, colorectal cancer, rectal cancer, uterine cancer, cervical cancer, endometrial carcinoma, salivary gland carcinoma, mesothelioma, kidney cancer, vulval cancer who has cancer, pancreatic cancer, thyroid cancer, hepatic carcinoma, testicular cancer, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Merkel cell carcinoma, Ewing sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelioma.

[0064] In some embodiments, the compounds described herein are given in combination with other compounds, biologics, and other treatments known in the art and used in the treatment, amelioration, and prevention of the conditions and diseases discussed herein.

[0065] For the treatment of the conditions referred to above, the compounds disclosed herein can be administered as the compounds per se.

[0066] Alternatively, pharmaceutically acceptable salts are suitable for medical applications because of their greater aqueous solubility relative to the parent compound.

[0067] In another embodiment, the present disclosure comprises pharmaceutical formulations or compositions. Such formulations comprise a compound disclosed herein presented with a pharmaceutically acceptable carrier. The carrier can be a solid, a liquid, or both, and may be formulated with the compound as a unit-dose composition, for example, a tablet, which can contain from 0.05% to 95% by weight of the active compounds. A compound disclosed herein may be coupled with suitable polymers as targetable drug carriers. Other pharmacologically active substances can also be present.

[0068] Formulations: In another embodiment, the present disclosure comprises the use of one or more compounds disclosed herein for the preparation of a medicament for the treatment of the conditions recited herein.

[0069] The compounds disclosed herein may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed, by which the compound enters the blood stream directly from the mouth.

[0070] Oral administration of a solid dose form may be, for example, presented in discrete units, such as hard or soft capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the present disclosure. In another embodiment, the oral administration may be in a powder or granule form. In another embodiment, the oral dose form is sub-lingual, such as, for example, a lozenge. In such solid dosage forms, the compounds of the present disclosure are ordinarily combined with one or more adjuvants. Such capsules or tablets may contain a controlled-release formulation. In the case of capsules, tablets, and pills, the dosage forms also may comprise buffering agents or may be prepared with enteric coatings.

[0071] In another embodiment, oral administration may be in a liquid dose form. Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art (e.g., water). Such compositions also may comprise adjuvants, such as wetting, emulsifying, suspending, flavoring (e.g., sweetening), and / or perfuming agents.

[0072] In another embodiment, the compounds of the disclosure may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

[0073] In another embodiment, the present disclosure comprises a parenteral dose form. “Parenteral administration” includes, for example, subcutaneous injections, intravenous injections, intraperitoneal injections, intramuscular injections, intracisternal injections, and infusion. Injectable preparations (i.e., sterile injectable aqueous or oleaginous suspensions) may beformulated according to the known art using suitable dispersing, wetting, and / or suspending agents, and include depot formulations.

[0074] In another embodiment, the compounds disclosed herein may also be formulated as a topical dose form such that administration topically to the skin or mucosa (i.e., dermally or transdermally) leads to systemic absorption of the compound. “Topical administration” includes, for example, transdermal administration, such as via transdermal patches or iontophoresis devices, intraocular administration, or intranasal or inhalation administration. Compositions for topical administration also include, for example, topical gels, sprays, ointments, and creams. A topical formulation may include a compound that enhances absorption or penetration of the active ingredient through the skin or other affected areas. When the compounds of this disclosure are administered by a transdermal device, administration will be accomplished using a patch either of the reservoir and porous membrane type or of a solid matrix variety. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated—see, for example, Finnin and Morgan, J. Pharm. Sci., 88 (10), 955-958 (1999).

[0075] Formulations suitable for topical administration to the eye include, for example, eye drops wherein the compound of this disclosure is dissolved or suspended in a suitable carrier. A typical formulation suitable for ocular or aural administration may be in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

[0076] For intranasal administration or administration by inhalation, the active compounds of the disclosure are conveniently delivered in the form of a solution or suspension from a pumpspray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant. Formulations suitable for intranasal administration are typically administered in the form of a dry powder (either alone; as a mixture, for example, in a dry blend with lactose; or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.

[0077] In another embodiment, the present disclosure comprises a rectal dose form. Such rectal dose form may be in the form of, for example, a suppository. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.

[0078] In another embodiment, the compounds of the disclosure may be formulated such that administration vaginally leads to systemic absorption of the compound.

[0079] The dosage regimen for the compounds and / or compositions containing the compounds is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed. Thus, the dosage regimen may vary widely. Dosage levels of the order from about 0.01 mg to about 100 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions. In one embodiment, the total daily dose of a compound disclosed herein (administered in single or divided doses) is typically from about 0.01 to about 100 mg / kg. In another embodiment, the total daily dose of a compound disclosed herein is from about 0.1 to about 50 mg / kg, and in another embodiment, from about 0.5 to about 30 mg / kg (i.e., mg compound of the disclosure per kg body weight). In one embodiment, dosing is from 0.01 to 10 mg / kg / day. In another embodiment, dosing is from 0.1 to 1.0 mg / kg / day. Dosage unit compositions may contain such amounts or submultiples thereof to make up the daily dose. In many instances, the administration of the compound will be repeated a plurality of times in a day (typically no greater than 4 times). Multiple doses per day typically may be used to increase the total daily dose, if desired.

[0080] For oral administration, the compositions may be provided in the form of tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 75.0, 100, 125, 150, 175, 200,250 and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, or in another embodiment, from about 1 mg to about 100 mg of active ingredient. Intravenously, doses may range from about 0.1 to about 10 mg / kg / minute during a constant rate infusion.

[0081] Oral administration of a solid dose form may be, for example, presented in discrete units, such as hard or soft capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the present disclosure. In another embodiment, the oral administration may be in a powder or granule form. In another embodiment, the oral dose form is sub-lingual, such as, for example, a lozenge. In such solid dosage forms, the compounds of the present disclosure are ordinarily combined with one or more adjuvants. Such capsules or tablets may contain a controlled-release formulation. In the case of capsules, tablets, and pills, the dosage forms also may comprise buffering agents or may be prepared with enteric coatings.

[0082] In some embodiments administration of one or more of the disclosed 6-thiopurine containing dimer compounds are followed by treatment with an immune checkpoint inhibitor. In some embodiments the immune checkpoint inhibitor is a PD-1 inhibitor, PD-L1 inhibitor, and / or a CTLA-4 inhibitor. In other embodiments, the immune checkpoint inhibitor is given in combination with one or more CTLA-4 inhibitors and one or more PD-1 inhibitors, or the immune checkpoint inhibitor is given in combination with one or more CTLA-4 inhibitors and one or more PD-L1 inhibitors.

[0083] In some embodiments the compounds disclosed herein are administered for about 1 to about 5 days per therapeutic cycle.

[0084] In yet other embodiments, the checkpoint inhibitor is administered for about 1 to about 3 days per therapeutic cycle.

[0085] In another embodiment, the 6-thiopurine containing dimer compounds and the checkpoint inhibitor are administered in combination with a chemotherapeutic agent, a hormonal therapy, a toxin therapy, surgery or combinations thereof.

[0086] In some embodiments, the 6-thiopurine containing dimer compounds are administered before the checkpoint inhibitor is administered in combination with a chemotherapeutic agent, a hormonal therapy, a toxin therapy, surgery or combinations thereof.

[0087] In other embodiments, the method of treating cancer in a subject comprises administering to said subject one or more of the 6-thiopurine containing dimer compounds, followed by treatment with an immune checkpoint inhibitor and radiation therapy, wherein the cancer is selected from one or more of breast cancer, prostate cancer, colon cancer, stomach cancer, esophagus, liver, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, biliary tract, bladder cancer, hepatoma, colorectal cancer, rectal cancer, uterine cancer, cervical cancer, endometrial carcinoma, salivary gland carcinoma, mesothelioma, kidney cancer, vulval cancer, pancreatic cancer, thyroid cancer, hepatic carcinoma, testicular cancer, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Merkel cell carcinoma, Ewing sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelioma

[0088] Suitable subjects / patients according to the present disclosure include mammalian subjects. Mammals according to the present disclosure include, but are not limited to, canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, and the like, and encompass mammals in utero. In one embodiment, humans are suitable subjects. Human subjects may be of either gender, and at any stage of development.

[0089] As used throughout this application, including the claims, the following terms have the meanings defined below, unless specifically indicated otherwise. The plural and singular should be treated as interchangeable, other than the indication of number.

[0090] The embodiments disclosed herein are also meant to encompass all pharmaceutically acceptable compounds of Formula (I), including isotopically labeled compounds in which one or more atoms can be replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as2H,3H,13C,14C,13N,15N,15O,17O,18O,31P,32P,35S,18F,36C1,123I, and125I. These radiolabeled compounds could be useful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action. Certain isotopically labeled compounds of Formula (I), such as for example, those incorporating a radioactive isotope, may be useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.,3H, and carbon-14, i.e.,14C, mayparticularly be useful for this purpose in view of their ease of incorporation and ready means of detection.

[0091] Substitution with heavier isotopes such as deuterium, i.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability. For example, in vivo half-life may increase, or dosage requirements may be reduced. Thus, heavier isotopes may be preferred in some circumstances.

[0092] Substitution with positron emitting isotopes, such as C, F, O and N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically labeled compounds of Formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Examples as set out below using an appropriate isotopically labeled reagent in place of the non- labeled reagent previously employed.

[0093] The methods, compositions, kits and articles of manufacture provided herein use or include compounds (e.g., compounds of Formula (I), or pharmaceutically acceptable salts, prodrugs, or solvates thereof, in which from 1 to n hydrogen atoms attached to a carbon atom may be replaced by a deuterium atom or D, in which n is the number of hydrogen atoms in the molecule. As known in the art, the deuterium atom is a non-radioactive isotope of the hydrogen atom. Such compounds may increase resistance to metabolism, and thus may be useful for increasing the half- life of compounds or pharmaceutically acceptable salts, prodrugs, or solvates thereof, when administered to a mammal. See, e.g., Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism”, Trends Pharmacol. Sci., 5(12):524-527 (1984). Such compounds are synthesized by means well known in the art, for example, by employing starting materials in which one or more hydrogen atoms have been replaced by deuterium.

[0094] The embodiments disclosed herein are also meant to encompass the in vivo metabolic products of the disclosed compounds. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the embodiments disclosed herein include compounds produced by a process comprising administering a compound according to the embodiments disclosed herein to a mammal for a period of time sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled compound according to the embodiments disclosed herein in a detectable dose to an animal, such as rat,mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples. “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. “Mammal” includes humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like. “Optional” or “optionally” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.

[0095] “Pharmaceutically acceptable excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0096] Examples of “pharmaceutically acceptable salts” of the compounds disclosed herein include salts derived from an appropriate base, such as an alkali metal (for example, sodium), an alkaline earth metal (for example, magnesium), ammonium and NX4+(wherein X is C1-C4 alkyl). Pharmaceutically acceptable salts of a nitrogen atom or an amino group include for example salts of organic carboxylic acids such as acetic, benzoic, lactic, fumaric, tartaric, maleic, malonic, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids; and inorganic acids, such as hydrochloric, hydrobromic, sulfuric, phosphoric and sulfamic acids. Pharmaceutically acceptable salts of a compound of a hydroxy group include the anion of said compound in combination with a suitable cation such as Na+and NX4+(wherein X is independently selected from H or a C1-C4 alkyl group).

[0097] For therapeutic use, salts of active ingredients of the compounds disclosed herein will typically be pharmaceutically acceptable, i.e., they will be salts derived from a physiologically acceptable acid or base. However, salts of acids or bases which are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of a compound of Formulas (I) as disclosed herein. All salts, including those derived from a physiologically acceptable acid or base, are within the scope of the embodiments disclosed herein.

[0098] Metal salts typically are prepared by reacting the metal hydroxide with a compound according to the embodiments disclosed herein. Examples of metal salts which are prepared in this way are salts containing Li+, Na+, and K+. A less soluble metal salt can be precipitated from the solution of a more soluble salt by addition of the suitable metal compound.

[0099] In addition, salts may be formed from acid addition of certain organic and inorganic acids, e.g., HC1, HBr, H2SO4, H3PO4or organic sulfonic acids, to basic centers, typically amines. Finally, it is to be understood that the compositions herein comprise compounds disclosed herein in their un-ionized, as well as zwitterionic form, and combinations with stoichiometric amounts of water as in hydrates.

[0100] Often crystallizations produce a solvate of a compound of the embodiments disclosed herein. As used herein, the term “solvate” refers to an aggregate that comprises one or more molecules of a compound of the embodiments disclosed herein with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the embodiments disclosed herein may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. The compounds of the embodiments disclosed herein may be true solvates, while in other cases, a compound of the embodiments disclosed herein may merely retain adventitious water or be a mixture of water plus some adventitious solvent.

[0101] Also, within the scope of the present disclosure are so-called “prodrugs” of the compounds disclosed herein. Thus, certain derivatives of the compounds disclosed herein that may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into the compounds of the disclosure having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as “prodrugs.” Further information on the use of prodrugs may be found in “Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and “Bioreversible Carriers in Drug Design,” Pergamon Press, 1987 (ed. E. B. Roche, American Pharmaceutical Association). Prodrugs in accordance with the disclosure can, for example, be produced by replacing appropriate functionalities present in the compounds of the present disclosure with certain moieties known to those skilled in the art as “pro- moieties” as described, for example, in “Design of Prodrugs” by H. Bundgaard (Elsevier, 1985).

[0102] A “pharmaceutical composition” refers to a formulation of a compound of the embodiments disclosed herein and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable excipients. “Effective amount” or “therapeutically effective amount” refers to an amount of a compound according to the embodiments disclosed herein, which when administered to a patient in need thereof, is sufficient to effect treatment for disease-states, conditions, or disorders for which the compounds have utility. Such an amount would be sufficient to elicit the biological or medical response of a tissue system, or patient that is sought by a researcher or clinician. The amount of a compound according to the embodiments disclosed herein which constitutes a therapeutically effective amount will vary depending on such factors as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of the treatment, the type of disease-state or disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds of the embodiments disclosed herein, and the age, body weight, general health, sex and diet of the patient. Such a therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge, the state of the art, and this disclosure.

[0103] “Effective amount” or “therapeutically effective amount” refers to an amount of a compound according to the embodiments disclosed herein, which when administered to a patient in need thereof, is sufficient to effect treatment for disease-states, conditions, or disorders for which the compounds have utility. Such an amount would be sufficient to elicit the biological or medical response of a tissue system, or patient that is sought by a researcher or clinician. The amount of a compound according to the embodiments disclosed herein which constitutes a therapeutically effective amount will vary depending on such factors as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of the treatment, the type of disease-state or disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds of the embodiments disclosed herein, and the age, body weight, general health, sex and diet of the patient. Such a therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge, the state of the art, and this disclosure.

[0104] The term “treatment” as used herein is intended to mean the administration of a compound or composition according to the present embodiments disclosed herein to alleviate or eliminate symptoms of the conditions described herein.

[0105] “EC50” as used herein means the dose or concentration of a drug that results in the death of 50% of the cancer cells being treated.

[0106] The compounds disclosed herein and pharmaceutically acceptable salts thereof may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, such as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (−), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0107] A “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0108] A “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any said compounds.

[0109] The 6-thiopurine containing dimer compounds disclosed herein which have a deoxyribose monosaccharide unit are sometimes referred to as “diTHIO”, and those having a ribose monosaccharide unit are referred to as “diRIBOTHIO”. The 6-thio-2’-deoxyguanosine containing monomer compounds disclosed herein also referred to as “THIO”, and the 6- thioguanosine containing monomer compounds are referred to as “RIBOTHIO”. These notations are used in Table 1.EXAMPLES

[0110] Example 1. Synthesis of 6-thiopurine containing dimer compounds of Formula I: This is illustrated by the representative synthesis of Compound XX (MAIA-2021-029): The preparation of compound XX from compound XVI is shown schematically in reaction (3):Reaction (3).

[0111] The compound of formula XVI (30 mg, 105.89 μmol) was dissolved in pH=7.6 buffer (7.5 mL) maintained at 45oC, and the solution was cooled to 30 °C. To the solution was added 0.5N I2 / NaI solution (105.89 μmol, 0.3 mL) dropwise with stirring at such a rate that the color appearing in the reaction mixture with each drop was dispersed. A white precipitate was formed, which was recovered by filtration, and the filtered solid was washed with water (0.2*2 mL), then EtOH (0.2*2 ml), and dried to give the product of Formula XX (16.7 mg, 28.99 μmol, 27.37% yield).1H-NMR (600 MHz, DMSO-d6):δ =8.27 (s, 2H), 6.61 (s, 4H), 6.80 (s, 2H), 6.22 (t, 2H, J = 6.6 Hz), 5.30 (s, 2H), 4.95 (s, 2H), 4.37 (s, 2H), 3.28~3.50 (m, 4H), 2.64~ 2.60 (m, 2H), 2.25~ 2.24 (m, 2H). ESI-LCMS: m / z 565.1[M+H]+.

[0112] Using the above procedure, the other 6-thiopurine containing dimers XXI – XXIII were prepared from the corresponding 6-thiopurine containing monomers XVII – XIX, respectively, as shown in Table 1.

[0113] Cell viability assay for determination of EC50 was measured by screening a given type of cancer cells with a drug compound with a dilution series in 9 different points in 96-well plates. Cells were plated prior to the addition of drug, incubated for 4-5 days, and assayed using CellTiter-Glo Cell Viability Assay according to the manufacturer’s instructions (Promega). Dose response curves were generated and EC50 values were calculated using Graphpad Prism. All samples were analyzed in triplicate and standard deviations are from 2-3 independent experiments.

[0114] Telomere dysfunction induced foci (TIF) was measured by seeding a given type of cancer cells onto a previously sterilized slide and placing the slide in a 10 cm Petri disk (Falcon). After 24 h, the cells were treated with the drug compound at EC50 concentration for 48 h. Then, the slides were rinsed in PBS 1X for 10 minutes on a shaking platform, then fixed in 4% formaldehyde (Thermo Fisher) for 10 minutes on ice, and then washed twice in PBS 1X for 5 minutes. Subsequently, the slides were permeabilized with 0.5% Triton X-100 for 10 minutes on ice and then blocked with BSA / PBS 1X for 30 minutes at RT.

[0115] Anti-mouse primary antibody ^H2AX (Millipore) was diluted 1:200 in blocking solution and cells were incubated in a humid chamber at 4 ^C O / N. Following washes with PBS 1X, cells were incubated with AlexaFluor 488 conjugated goat anti-mouse, for 45 minutes at RT. After washing with PBS 1X, TIF assay was conducted as previously described (Mender and Shay, 2015) with minor modifications.

[0116] The cancer cell slides obtained above were seeded, treated, and stained with ^H2AX as described in the ^H2AX Foci paragraph. After PBS 1X washes, cells were fixed in 4% formaldehyde in PBS for 20 min at RT. The slides were sequentially dehydrated with 70%, 90%, 100% ethanol and subsequently denatured for 3 minutes at 80°C with 20 μL of hybridization mixture contained 70% deionized formamide, 1M Tris pH 7.2, 8.56% buffer MgCl2, 5% maleic blocking reagent, and 25 μg / mL Cy3-conjugated PNA Tel-C (CCCTAA)3 probe (PANAGENE, South Korea) and incubated overnight at 4°C in a humid chamber. Slides were washed two times for 15 minutes in wash solution containing 70% formamide, 10mM Tris pH 7.2, 0.1% BSA, and washed three times for 5 minutes in a solution containing 0.1 M Tris pH7.5, 0.15 M NaCl, and 0.08% Tween-20. The slides were dehydrated by ethanol series, air- dried, and counterstained with Vectashield / DAPI (Vector Laboratories, Burlingame, CA). Images were captured at 63X magnification with an Axio Imager Z2 (Carl Zeiss) equipped with an automatic capture system (Metafer, Metasystems) and analyzed with ISIS software (Metasystems).

[0117] Testing of the 6-thiopurine containing dimer and monomer compounds: The inhibition of cancer cell viability by the 6-thiopurine containing dimer compounds were compared with the corresponding 6-thiopurine containing monomer compounds in a variety of human and mouse cancer cells. The in vitro anti-tumor activity of the 6-thiopurine containing dimer compounds against various types of tumor cells were compared with the activity of the corresponding 6-thiopurine containing monomer compound XVI and the known compound cisplatin. The results are displayed in Figures 1-17 and Tables 1-7.

[0118] Table 2. In vitro activity of 6-Thiopurine containing dimer compound XX (also called diTHIO, or THIO-dimer or 6S-dG-dimer) in other tumor cell lines. Cancer cell type B-Lymphoma Myeloma Liver SCLC NSCLC Ovarian Seeding density2

[0009] abe 3 . n vitro actvty o te 6- opurne contanng dmer compound XX (6S-dG-dimer) compared with cisplatin in other tumor cell lines. Tumor type B-NHL Breast SCLC CRCSeeding density

[0120] Table 3B. In vitro activity of the 6-Thiopurine containing dimer compound XX (6S- dG-dimer) compared with cisplatin in other tumor cell lines. Tumor type Pancreatic Colon TNBC SCLC PancreaticSeeding density

[0000] abe . n vitro actvty o te 6- o- -deoxyguanosne containing dimer compound XX compared with the 6-Thio-2’-deoxyguanosine containing monomer compound XVI. Seeding density Cll li OCILY19 JHH6 DMS 53 CC1 C C1 / DDP

[0122] Table 4B. In vitro activity of the 6-Thio-2’-deoxyguanosine containing dimer compound XX compared with the corresponding 6-Thio-2’-deoxyguanosine containing monomer compound XVI. Seeding density Cell line AU565 HCC1937 HT-29DMSCaan-2[ ] a e . n vtro ac v y o e mer compoun X compared with the corresponding monomer compound XVI. Seeding density Cll lin LP1 NCIH1581 COLO 205 NCIH1836 C n1

[0124] Table 5A. Cell line characteristics used in evaluating compounds XVI and XX. Gene Expression Copy Number (SNP)S 5 3

[0125] Table 5B. Cell line characteristics used in evaluating compounds XVI and XX. Copy Number (WES) MutationsSample TERT GSTP1 KRAS TERT GSTP1 KRAS Vontaining dimer compound XX compared with cisplatin in murine cell lines. Absolute IC50 (μM) NSCLC B cell CRClymphoma HCC Melanoma Pancreatic[ ] a e . n vtro actvty o -t o- -eoxyguanosne mer containing compound XX compared with cisplatin in murine cell lines. Absolute IC50(μM) Prostate Melanoma Renal Breast CRC HCC Cll lin RM1 B16F10 Rn EMT6 CT26WT H22 3 5

[0128] Table 7A. In vitro activities of the novel tumor RedOx activated 6-thiopurine containing dimers XX - XXIII, and the 6-thiopurine containing monomers XVI and XXVI, compared with cisplatin in various cell lines. Cell line OCI-LY- JHH-6 DMS 53 CoC1 CoC1 / DDP AU565 HCC1937 HT-29 19 C ll / ll 15000 1200 13000 15000 10000 4000 3000 5000

[0129] Table 7B. In vitro activities of the novel tumor RedOx activated 6-thiopurine containing dimers XX - XXIII, and the 6-thiopurine containing monomers XVI and XXVI, compared with cisplatin in various cell lines. Cell line DMS 114 Capan-2 LP-1 NCI-H1581 COLO 205 NCI-H1836 Capan-1 Cells / well 4000 3000 10000 12000 5000 10000 3000

[0130] Figure 1 shows that the 6-thio-2 -deoxyguanosine containing dimer XX exhibits a much lower EC50(half maximal effective concentration) of 0.7 µM when tested with NSCLC LLC (Non-Small Cell Lung Cancer Lewis Lung Carcinoma) cells over 4 days, as compared to the 6- thio-2’deoxyguanosine monomer compound XVI (2.4 µM). In other words, the concentration of XX necessary to reduce the number of NSCLC LLC cells by 50% is markedly less than the concentration of XVI needed under the same conditions. When CRC MC38 (Colorectal Cancer Murine Colon Adenocarcinoma) cells were treated with compounds XX and XVI, the dimer compound XX exhibited a markedly lower EC50(3.1µM) than XVI (1.0 µM).

[0131] The dimer compound XX is also efficient in forming TIFs (Telomere dysfunction- Induced Focii) as seen in Figure 2A. This means XX is efficient in inducing telomere dysfunctions in the cancer DNA molecules, which leads to cancer cell death, thereby leading to a beneficial therapeutic effect.

[0132] In the case of Hep55-1C cells (cell line derived from carcinogen-induced liver tumors of C57BL / 6 mice), compound XX shows a competitively low IC50 (the half maximalinhibitory concentration needed to inhibit the liver cancer DNA from proliferating) as compared to compound XVI (See Figure 3). Compound XX also exhibits outstanding therapeutic effect against Hep55-1C cells in in vitro tests even when re-challenged with the same number of the tumor cells. Figure 4 shows that the tumor cell growth inhibiting ability is fully preserved even after 4 days from the first treatment with the Hep55-1C cells. In Figure 12, it can be seen that the dimer compound XX inhibits Hep55-1C cells for a much longer period of 125 days and maintains the same potency even after subsequently challenging with a fresh load of 5 million Hep55-1C cells and exposure over more than 75 additional days of testing. In this extended test phase, the dimer compound XX actually shows superior potency for inhibition as compared to the corresponding monomer compound XVI. When the system was further challenged with 1 million RIL 175 cells, however, the potency for both compounds XVI and XX dropped significantly (See Figure 13), which perhaps indicates that compounds like XVI and XX exhibit highly selective potency for inhibiting liver cancer cells. These results perhaps also indicate induction of cellular immunity with XX, which portends a very powerful therapeutic option to treat liver cancer.

[0133] The dimer compound XVI also exhibits better efficacy than cisplatin in inhibiting tumor DNA growth for a broad variety of cancer cells, such as myeloma, ovarian, B-lymphoma, SCLC, lymphoma, breast cancer, TNBC (triple negative breast cancer), and human pancreatic ductal adenocarcinoma cells (See Figures 5A-5O). Other 6-thiopurine containing dimer and monomer compounds, including the 6-thio-2’-guanosine containing monomer XXVI (referenced as MAIA-2021-001) were also prepared and tested in vitro against different types of cancer cells and they all displayed superior inhibition of cancer cell proliferation (See Figures 9A-9L).

[0134] The 6-thio-2’-deoxyguanosine containing dimer compound XX also exhibits a far superior efficacy in inhibiting ovarian cancer cell proliferation, when compared to traditionally used cisplatin. Figure 7 shows that XVI has a far lower IC50 of 0.08 µM than cisplatin (3.54 µM). Therefore, by using the 6-thio-2’-deoxyguanosine containing dimer compounds such as XVI, it is likely that the toxicity-related side effects due to cisplatin can be avoided or minimized. The dimer compounds such as XX are also effective when tested in vitro against different types of murine cancer cells (See Figures 8A-8L).

[0135] Glutathione S-transferases (GSTs) are a family of isoenzymes that play an important role in protecting cells from cytotoxic and carcinogenic agents. GST-P1, glutathione S- transferase alpha 1, an isozyme of glutathione S-transferase, functions by catalyzing theconjugation of the toxic substances with GSH and removing it from the cell. When an anti-cancer agent, e.g., cisplatin, is administered, GST-P1 aids in removing the therapeutic agent from the cells, which leads to cancer progression and resistance to cancer therapy. In the presence of a 6- thio-2’-guanosine containing dimer, however, the activity of the GST-P1 is strongly inhibited, which results in a better therapeutic outcome. More importantly, the normal cells were generally not affected by the drug compounds in the tests.

[0136] All references cited herein are incorporated by reference.

[0137] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A 6-thiopurine containing dimer of Formula I: Formula I, or athereof, wherein both R1s are concurrently H, or (PO3)2-, and both R2s are concurrently H or OH.

2. A compound of claim 1 having the following structure: .

3. A compound of claim 1 having the following structure:.

4. A compound of claim 1 having the following structure: .

5. A compound of claim 1 having the following structure:.

6. A pharmaceutical composition comprising any one of claims 1-5.

7. A pharmaceutical composition comprising any one of claims 1-5, and at least one pharmaceutically acceptable excipient.

8. A method for treating cancer in a subject, comprising: administering a pharmaceutical composition of claim 6 or claim 7 to the subject in an amount effective to treat said cancer.

9. The method of claim 8, further comprising treating the subject with an immune checkpoint inhibitor after the administration of the pharmaceutical composition.

10. The method of claim 9, wherein the checkpoint inhibitor is one or more members selected from a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

11. The method of claim 10, further comprising treating the subject with a chemotherapeutic agent, a hormonal therapy, a toxin therapy, radiation therapy, surgery, or combinations thereof.

12. The method of claim 8, wherein the cancer is selected from one or more of breast cancer, prostate cancer, colon cancer, stomach cancer, esophagus, liver, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, biliary tract, bladder cancer, hepatoma, colorectal cancer, rectal cancer, uterine cancer, cervical cancer, endometrial carcinoma, salivary gland carcinoma, mesothelioma, kidney cancer, vulval cancer, pancreatic cancer, thyroid cancer, hepatic carcinoma, testicular cancer, skin cancer, melanoma, brain cancer, neuroblastoma,myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Merkel cell carcinoma, Ewing sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelioma.

13. A method for forming a 6-thiopurine containing monomer, the method comprising: a) contacting a 6-thiopurine containing a 6-thiopurine containing dimer with an organic thiol of formula R-SH, where R is an alkyl group, and b) forming the 6-thiopurine containing monomer, wherein the 6-thiopurine containing dimer is of Formula (I): Formula I,IV:2 or an ester thereof, [(P(OH)2(=S)] or an ester thereof, (PO3)2, or [(PO2)(=S)]2-; and R2in Formulas I and IV are H or OH.

14. The method of claim 13, wherein the 6-thiopurine containing dimer is selected from the group consisting of a 6-thiodeoxyguanosine containing dimer of Formula II:Formula II, a 6-thioguano Formula III, orwherein R1in Formulas II and III are independently H, P(=O)(OH)2 or an ester thereof, [(P(OH)2(=S)] or an ester thereof, (PO3)2, or [(PO2)(=S)]2.

15. The method of claim 13, wherein the 6-thiopurine containing monomer is a 6- thiodeoxyguanosine containing monomer of Formula XXIV: S Formula XXIV,an ester thereof, [(P(OH)2(=S)] or an ester thereof, (PO3)2, or [(PO2)(=S)]2-.

16. The method of claim 13, wherein the 6-thiopurine containing monomer is a 6- thioguanosine containing monomer of Formula XXV:Formula XXV, r an ester thereof, [(P(OH)2(=S)] or an ester thereof, (PO3)2, or [(PO2)(=S)]2-.

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