Tumor redox-activated 6-thiopurine-containing dimer compound

Abstract

In this specification, the 6-thiopurine-containing dimer compound of formula I: [Formula 1] JPEG2026520230000048.jpg70159(In the formula, both R 1 H or (PO3) 2- And both R 2 Disclosed are compounds (which are simultaneously H or OH) or pharmaceutically acceptable salts, esters, prodrugs, hydrates, or tautomers thereof. Methods for preparing compounds of formula I from corresponding 6-thiopurine-containing monomer compounds are also disclosed. Methods for converting dimer compounds of formula I to corresponding 6-thiopurine-containing monomer compounds are also disclosed. These compounds are useful for treating, improving, or preventing various types of cancer, particularly drug-resistant cancers. Compounds of formula I can also be used in different ways in combination with checkpoint inhibitors and / or radiation to treat various types of cancer.

Description

[Technical Field]

[0001] This disclosure focuses on novel compounds and methods for producing the same, pharmaceutical compositions containing the novel compounds, and methods for using the same to treat various types of conditions, including cancer, in the general field of hyperproliferative diseases, including oncology. [Background technology]

[0002] Telomeres are found at both ends of eukaryotic chromosomes. These DNA protein structures protect the genome from nucleolysis, unwanted 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 problems with end replication and the lack of telomere maintenance mechanisms (Greider CW, (1996), Annu. Rev. Biochem. 65:337-65). However, single-celled eukaryotes, germline cells, and immortal cancer cells almost always maintain their telomeres at a constant length 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 that elongates telomeres by adding TTAGGG repeats to the ends of chromosomes. It is expressed in approximately 90% of human tumors but not in most normal cells (Jafri, MA, et al. Genome Medicine, 2016, 8:69). Therefore, telomerase is an attractive target for developing anti-cancer therapies. Most telomerase-targeting therapies focus on inhibiting telomerase (Andrews LG, Tollefsbol TO. Mol Biol. 2007;405:1-7). However, such inhibitors have not been successful in clinical trials (Zhang G., Shay WS. Oncotarget, 2018, vol 9(88):35803-35804).

[0004] Instead of inhibiting telomerase, other cancer drugs are being developed that incorporate these drugs into telomeres to induce DNA damage, aging, or crisis in telomerase-positive cancer cells. One such compound is the nucleoside analog, 6-thio-2'-deoxyguanosine (6-thio-dG). Its incorporation into de novo-synthesized telomeres by telomerase is known to induce damage to telomere 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 reduction or growth arrest with minimal side effects in many tumor-derived xenograft models (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 its tumor-killing effect. Furthermore, although it does not directly inhibit telomerase, it is preferentially recognized by telomerase over other polymerases, incorporated into telomeres, and leads to immediate DNA strand termination. Importantly, its effect is independent of initial telomere length by hijacking tumor telomerase and creating unstable telomeres (Mender et al., (2015) Oncoscience, 2(8):693-695).

[0005] Novel telomere-targeting compounds useful for treating cancer are disclosed herein. [Overview of the project]

[0006] One aspect of this disclosure is the 6-thiopurine dimer of formula I: [ka] or a pharmaceutically acceptable salt, ester, prodrug, hydrate, or tautomer thereof. In some embodiments, both R 1 are simultaneously H or PO3 2- and both R 2 are simultaneously H or OH .

[0007] The 6-thioguanine dimer is a 6-thiodeoxyguanosine dimer of formula II (where both R 1 are simultaneously H or PO3 2- ):

Chemical formula

Chemical formula

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

Chemical formula

[0009] In some embodiments, the dimer is the compound MAIA-2022-07 having the following structure:

Chemical formula

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

Chemical formula

[0011] In some embodiments, the dimer is compound MAIA-2021-09 (phosphorylated ribo-thio dimer) having the following structure: [ka]

[0012] In another embodiment, a method for producing a 6-thiopurine-containing dimer of formula I is described herein. The method involves adding a solution of I2 / NaI to a solution of a 6-thiopurine-containing monomer of formula IV: [ka] This also includes isolating the 6-thiopurine-containing dimer product of formula I thus formed.

[0013] In another embodiment, a method for forming a 6-thiopurine-containing monomer is described herein. The method comprises contacting a dimer of formula I with an organic thiol of formula R-SH to form a 6-thiopurine-containing monomer of formula IV. [ka] (In the formula, R 1 and R 2 (This is the same as the one mentioned above.)

[0014] In yet another aspect of this disclosure, a compound of formula I (wherein R 1 and R 2 Formulations comprising (as described above), or pharmaceutically acceptable salts, esters, prodrugs, hydrates, or tautomers thereof are disclosed.

[0015] In yet another embodiment, a method for treating cancer in a subject is disclosed. The method comprises administering a pharmaceutical formulation containing a compound of formula I to a subject in need of treatment, wherein the pharmaceutical formulation is administered in a therapeutically effective amount for treating cancer. [Brief explanation of the drawing]

[0016] [Figure 1] Comparative dose-response plots and EC50 of Lewis lung cancer cells measured over 4 days after in vitro treatment with the 6-thiopurine dimer-containing compound MAIA-2021-029 and the 8-deuterio form of 6-thio-dG designated as F1 are shown. [Figure 2A] Figure 2A shows comparative dose-response plots and EC50 of mouse colon adenocarcinoma (MC38) cells measured over 4 days after in vitro treatment with the compound MAIA-2021-29 (referred to as MA-029) or F1, in the 8-duterio form. [Figure 2B] Figure 2B shows a comparative plot of the average number of TIFs (telomere dysfunction-induced lesions) per cell measured from MC38 cells treated with the compound MAIA-2021-29, designated as Comp-29, and the 8-duterio form of 6-thio-dG, designated as F1, over a 4-day period after in vitro treatment. [Figure 3A] Figure 3A shows a plot of the cell viability % of Hep55.1C cells (cells derived from carcinogen-induced liver tumors in C57BL / 6 mice) measured over 4 days after in vitro treatment with THIO. [Figure 3B] Figure 3B shows a plot of the cell viability % of Hep55.1C cells (cells derived from carcinogen-induced liver tumors in C57BL / 6 mice) measured over 4 days after in vitro treatment with compound MAIA-2021-29, referred to as compound 29. [Figure 4] Five million Hep55-1C cells were measured over four days of in vitro treatment with compound MAIA-2021-29 (referred to as compound 29), and the tumor volume after rechallenge with five million Hep55-1C cells is plotted against the control. [Figure 5A] Comparative dose-response plots of LP-1 cells, JHH-6 cells, CoC1 cells, and OCI-LY-19 cells, measured over 4 days after in vitro treatment with compound XX (referred to as 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5B] Comparative dose-response plots of LP-1 cells, JHH-6 cells, CoC1 cells, and OCI-LY-19 cells, measured over 4 days after in vitro treatment with compound XX (referred to as 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5C] Comparative dose-response plots of LP-1 cells, JHH-6 cells, CoC1 cells, and OCI-LY-19 cells, measured over 4 days after in vitro treatment with compound XX (referred to as 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5D] Comparative dose-response plots of LP-1 cells, JHH-6 cells, CoC1 cells, and OCI-LY-19 cells, measured over 4 days after in vitro treatment with compound XX (referred to as 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5E] Comparative dose-response plots for NCI-H1581 cells, DMS-53 cells, HT-29 cells, and COLO 205 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5F] Comparative dose-response plots for NCI-H1581 cells, DMS-53 cells, HT-29 cells, and COLO 205 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5G] Comparative dose-response plots for NCI-H1581 cells, DMS-53 cells, HT-29 cells, and COLO 205 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5H]Comparative dose-response plots for NCI-H1581 cells, DMS-53 cells, HT-29 cells, and COLO 205 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5I] Comparative dose-response plots for OCI-LY-19 cells, HCC1937 cells, AU565 cells, and H1836 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5J] Comparative dose-response plots for OCI-LY-19 cells, HCC1937 cells, AU565 cells, and H1836 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5K] Comparative dose-response plots for OCI-LY-19 cells, HCC1937 cells, AU565 cells, and H1836 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5L] Comparative dose-response plots for OCI-LY-19 cells, HCC1937 cells, AU565 cells, and H1836 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer), are shown, compared to cells treated with cisplatin. [Figure 5M] The comparative dose-response plots for COLO DMS 114 cells, Capan-2 cells, and Capan-1 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer) and cisplatin, are shown. [Figure 5N] The comparative dose-response plots for COLO DMS 114 cells, Capan-2 cells, and Capan-1 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer) and cisplatin, are shown. [Figure 5O] The comparative dose-response plots for COLO DMS 114 cells, Capan-2 cells, and Capan-1 cells, measured over 4 days after in vitro treatment with compound XX (referred to as a 6S-dG-dimer) and cisplatin, are shown. [Figure 6] Comparative dose-response plots of OCI-LY-19 cells measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin are shown. [Figure 7] This plot compares the % cell viability of CoC1 / DDP cells (Pt-resistant ovarian cancer cells) measured over 4 days after in vitro treatment with compound XX (specified as a 6S-dG-dimer) and cisplatin. [Figure 8A] The comparative dose-response plots for LL / 2 (LLC1) cells, A20 cells, MC-38 cells, and Hepa 1-6 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer) and cisplatin, are shown. [Figure 8B] The comparative dose-response plots for LL / 2 (LLC1) cells, A20 cells, MC-38 cells, and Hepa 1-6 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer) and cisplatin, are shown. [Figure 8C] The comparative dose-response plots for LL / 2 (LLC1) cells, A20 cells, MC-38 cells, and Hepa 1-6 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer) and cisplatin, are shown. [Figure 8D] The comparative dose-response plots for LL / 2 (LLC1) cells, A20 cells, MC-38 cells, and Hepa 1-6 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer) and cisplatin, are shown. [Figure 8E]The comparative dose-response plots for B16-BL6 cells, Pan02 cells, RM-1 cells, and B16-F10 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer or MAIA-2021-29) and cisplatin, are shown. [Figure 8F] The comparative dose-response plots for B16-BL6 cells, Pan02 cells, RM-1 cells, and B16-F10 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer or MAIA-2021-29) and cisplatin, are shown. [Figure 8G] The comparative dose-response plots for B16-BL6 cells, Pan02 cells, RM-1 cells, and B16-F10 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer or MAIA-2021-29) and cisplatin, are shown. [Figure 8H] The comparative dose-response plots for B16-BL6 cells, Pan02 cells, RM-1 cells, and B16-F10 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer or MAIA-2021-29) and cisplatin, are shown. [Figure 8I] The comparative dose-response plots for Renca cells, EMT6 cells, CT26.WT cells, and H22 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer) and cisplatin, are shown. [Figure 8J] The comparative dose-response plots for Renca cells, EMT6 cells, CT26.WT cells, and H22 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer) and cisplatin, are shown. [Figure 8K] The comparative dose-response plots for Renca cells, EMT6 cells, CT26.WT cells, and H22 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer) and cisplatin, are shown. [Figure 8L]The comparative dose-response plots for Renca cells, EMT6 cells, CT26.WT cells, and H22 cells, measured over 4 days after in vitro treatment with compound XX (6S-dG-dimer) and cisplatin, are shown. [Figure 9A] Comparative dose-response plots of LP-1 cells, JHH-6 cells, NCI-H1581 cells, and CoC1 / DDP cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9B] Comparative dose-response plots of LP-1 cells, JHH-6 cells, NCI-H1581 cells, and CoC1 / DDP cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9C] Comparative dose-response plots of LP-1 cells, JHH-6 cells, NCI-H1581 cells, and CoC1 / DDP cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9D] Comparative dose-response plots of LP-1 cells, JHH-6 cells, NCI-H1581 cells, and CoC1 / DDP cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9E]The comparative dose-response data for HT-29 cells, COLO 205 cells, AU565 cells, and DMS 53 cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9F] The comparative dose-response data for HT-29 cells, COLO 205 cells, AU565 cells, and DMS 53 cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9G] The comparative dose-response data for HT-29 cells, COLO 205 cells, AU565 cells, and DMS 53 cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9H] The comparative dose-response data for HT-29 cells, COLO 205 cells, AU565 cells, and DMS 53 cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9I] Comparative dose-response plots of CoC1 cells, NCI-H1836 cells, Capan-1 cells, and Capan-2 cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9J]Comparative dose-response plots of CoC1 cells, NCI-H1836 cells, Capan-1 cells, and Capan-2 cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9K] Comparative dose-response plots of CoC1 cells, NCI-H1836 cells, Capan-1 cells, and Capan-2 cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 9L] Comparative dose-response plots of CoC1 cells, NCI-H1836 cells, Capan-1 cells, and Capan-2 cells, measured over 4 days after in vitro treatment with compounds XXVI (designated MAIA-2021-001), XXIII (MAIA-2022-09), XXII (MAIA-2022-08), XXI (MAIA-2022-07), and cisplatin, are shown. [Figure 10A] This plot compares the cell viability percentages of BJ human normal fibroblasts (derived from the BJ cell line) and Huh7 cells (human liver cancer cells) measured over 4 days after in vitro treatment with compound XX (compound 29) and XVI (designated as THIO). [Figure 10B] This plot compares the cell viability percentages of BJ human normal fibroblasts (derived from the BJ cell line) and Huh7 cells (human liver cancer cells) measured over 4 days after in vitro treatment with compound XX (compound 29) and XVI (designated as THIO). [Figure 11A] This plot shows a comparison of the % cell viability of mouse liver cancer cells, RIL175, Hep55-1C, and HCC53N, measured over 4 days after in vitro treatment with compound XX (compound 29) and THIO. [Figure 11B] This plot shows a comparison of the % cell viability of mouse liver cancer cells, RIL175, Hep55-1C, and HCC53N, measured over 4 days after in vitro treatment with compound XX (compound 29) and THIO. [Figure 11C] This plot shows a comparison of the % cell viability of mouse liver cancer cells, RIL175, Hep55-1C, and HCC53N, measured over 4 days after in vitro treatment with compound XX (compound 29) and THIO. [Figure 12] This shows a comparative plot of tumor volume when 5 million Hep55-1C cells were measured over an extended period of more than 200 days following in vitro treatment with compound XX (compound 29) and THIO. [Figure 13] Five million Hep55-1C cells were measured over an extended period of 125 days following in vitro treatment with compound XX (compound 29) and compound XVI (6-thio-dG). Subsequently, tumor-free mice were rechallenged with five million Hep55-1C cells, and then a comparative plot of tumor volume is shown when tumor-free mice were rechallenged with one million RIL 175 cells more than 200 days after the initial treatment. [Figure 14A] This plot compares the cell viability percentages of BJ human normal fibroblasts (derived from the BJ cell line), Huh7 human hepatocellular carcinoma cells, and HCC53N mouse hepatocellular carcinoma cells, measured over 4 days after in vitro treatment with compound XX (compound 29) and compound XVI (THIO). [Figure 14B] This plot compares the cell viability percentages of BJ human normal fibroblasts (derived from the BJ cell line), Huh7 human hepatocellular carcinoma cells, and HCC53N mouse hepatocellular carcinoma cells, measured over 4 days after in vitro treatment with compound XX (compound 29) and compound XVI (THIO). [Figure 14C]This plot compares the cell viability percentages of BJ human normal fibroblasts (derived from the BJ cell line), Huh7 human hepatocellular carcinoma cells, and HCC53N mouse hepatocellular carcinoma cells, measured over 4 days after in vitro treatment with compound XX (compound 29) and compound XVI (THIO). [Figure 15A] This plot shows a comparison of the % cell viability of Hep55-1C human liver cancer cells, RIL175 mouse liver cancer cells, and SB28 mouse gliablastoma cancer cells, measured over 4 days after in vitro treatment with compound XX (compound 29) and compound XVI (also designated as THIO or 6-thio-dG). [Figure 15B] This plot shows a comparison of the % cell viability of Hep55-1C human liver cancer cells, RIL175 mouse liver cancer cells, and SB28 mouse gliablastoma cancer cells, measured over 4 days after in vitro treatment with compound XX (compound 29) and compound XVI (also designated as THIO or 6-thio-dG). [Figure 15C] This plot shows a comparison of the % cell viability of Hep55-1C human liver cancer cells, RIL175 mouse liver cancer cells, and SB28 mouse gliablastoma cancer cells, measured over 4 days after in vitro treatment with compound XX (compound 29) and compound XVI (also designated as THIO or 6-thio-dG). [Figure 16A] This plot compares the cell viability percentages of H1993 human NSCLC cells and H2081 SCLC cells, measured over 4 days after in vitro treatment with compound XX (compound 29) and compound XVI (THIO, also designated as 6-thio-dG). [Figure 16B] This plot compares the cell viability percentages of H1993 human NSCLC cells and H2081 SCLC cells, measured over 4 days after in vitro treatment with compound XX (compound 29) and compound XVI (THIO, also designated as 6-thio-dG). [Figure 17A]This plot compares the cell viability % of H1693 human NSCLC cells (THIO-inherent resistance) and H2087 clone human NSCLC cells (THIO-acquired resistance), measured over 4 days after in vitro treatment with compound XX (compound 29) and compound XVI (THIO). [Figure 17B] This shows a comparative plot of the cell viability % of H1693 human NSCLC cells (THIO-inherent resistance) and H2087 clone human NSCLC cells (THIO-acquired resistance), measured over 4 days after in vitro treatment with compound XX (compound 29) and compound XVI (THIO). Further details of this disclosure are provided below. [Modes for carrying out the invention]

[0017] Tumor tissue can have different microenvironments, such as acidic, enzymatic, and reducing environments (Cheng R., et al. (2015), Nano Today, 10:656-70). The reducing environment of tumor cells is characterized by glutathione and NADPH / NADP. + Controlled by the reduction and oxidation states ( Wu G., et al., (2004) J. Nutr. 134:489). Disclosed herein are dimeric compounds that, with the help of endogenous glutathione (GSH), generate 6-thio-dG or ribo-thio in situ in tumor cells with a reducing environment. Tumors, particularly drug-resistant solid malignancies, have elevated GSH levels, and therefore the dimeric compounds disclosed herein are unique anticancer agents that can utilize the high GSH levels found in cancer cells. Examples of tumors with a reducing environment include, but are not limited to, ovarian tumors, endometrial tumors, gliomas, renal tumors, gastric tumors, urothelial tumors, pancreatic tumors, melanomas, and lung tumors (Rahul Raj Singh and Katie M. Reindl, 2021, Antioxidants (Basel) 10(5):701).

[0018] In some embodiments, the thiopurine-containing dimer has the structure of formula II, where both R1 H or PO3 2- That is the case. [ka]

[0019] In other embodiments, the dimerized compound disclosed herein is the 6-thioguanosine dimer of formula III. [ka] (In the formula, both R 1 H or PO3 2- (That is.)

[0020] In some embodiments, the dimer is compound MAIA-2021-029 having the following structure: [ka]

[0021] In some embodiments, the dimer is compound MAIA-2022-07 having the following structure: [ka]

[0022] In some embodiments, the dimer is compound MAIA-2022-08 (ribo-thio dimer) having the following structure: [ka]

[0023] In some embodiments, the dimer is compound MAIA-2022-09 (phosphorylated ribo-thio dimer) having the following structure: [ka]

[0024] In another embodiment, a method for producing a 6-thiopurine-containing dimer of formula I is disclosed herein. The method involves adding a solution of I2 / NaI to a solution of a 6-thiopurine-containing monomer of formula IV to form a reaction mixture, as shown in reaction (1): [ka] (In the formula, R 1 As mentioned above, R 2 The process involves isolating the desired 6-thiopurine-containing dimer from the reaction mixture (where is H or OH). A solution of I2 / NaI is added dropwise at a rate sufficient to maintain the decolorized state of the resulting reaction mixture.

[0025] In this embodiment of the reaction, the monomer compound of formula IV is maintained at a temperature of about 60°C from near ambient temperature (defined as a temperature of about 15°C to about 25°C), or about 40°C from ambient temperature, or about 30°C from ambient temperature, or at ambient temperature. In other embodiments, a solution of the monomer compound of formula IV can be obtained using a buffer having a pH of about 7.0 to about 8.0, or about 7.0 to about 7.5, or about 7.5 to about 8.0.

[0026] In certain embodiments, the method can be used to produce 6-thiopurine-containing dimers XX to XXIII from corresponding 6-thiopurine-containing monomers XVI to XIX, respectively, as shown in Table 1 below. Compounds XX and XXI may also be called 6-thio-2'-deoxyguanosine-containing dimers, and compounds XXII and XXIII may also be called 6-thioguanosine-containing dimers.

[0027] Table 1. 6-thiopurine-containing dimers prepared from corresponding 6-thiopurine-containing monomers. [Table 1]

[0028] In another embodiment, as shown below in reaction (2), the 6-thiopurine-containing dimer of formula I is reduced upon contact with an organic thiol R-SH (wherein R is an alkyl group) to yield the corresponding 6-thiopurine-containing monomer of formula IV: [ka] (In the formula, R 1 and R 2 (as described herein). Suitable thiols capable of reducing the 6-thiopurine-containing dimer compound of formula I by reaction (2) also include glutathione as shown below: [ka]

[0029] In one embodiment, the above-described method for converting monomers to dimers can be used to prepare a 6-thio-2'-deoxyguanosine-containing monomer of formula XXIV from a 6-thio-2'-deoxyguanosine-containing dimer of formula II: [ka] (In the formula, R 1 (As mentioned above).

[0030] In another embodiment, the method may be used to prepare a 6-thio-2'-guanosine-containing monomer of formula XXV from a 6-thio-2'-guanosine-containing dimer of formula XXV: [ka] (In the formula, R 1 (As described above). In another embodiment, this method can be used to prepare 6-thio-2'-guanosine of formula XXVI: [ka]

[0031] In some embodiments, the use of one or more 6-thiopurine-containing dimer compounds of formula I for the preparation of pharmaceuticals for the treatment of conditions listed herein is disclosed herein.

[0032] The compounds disclosed herein may be administered by any preferred route, preferably in the form of a pharmaceutical composition adapted to such route, and in a dose effective for the intended treatment. The active compounds and compositions may be administered, for example, orally, rectally, parenterally, intraperitoneally, or topically (e.g., intranasally or intraocularly).

[0033] Other carrier materials and administration methods known in the pharmaceutical art may also be used. The pharmaceutical compositions disclosed herein may be prepared by any of the well-known pharmaceutical techniques, such as effective formulations and administration procedures. The above considerations regarding effective formulations and administration procedures are well-known in the art and are described in standard textbooks. The formulation of pharmaceuticals is discussed, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

[0034] The compounds disclosed herein may be used alone or in combination with other therapeutic agents in the treatment of various conditions or medical conditions. The compounds(s) disclosed herein and other therapeutic agents(s) may be administered simultaneously (in the same dosage form or different dosage forms) or sequentially.

[0035] The administration of a "combination" of two or more compounds means that the two compounds are administered at a time close enough that the presence of one alters the biological effect of the other. Two or more compounds may be administered simultaneously, in parallel, or sequentially. Furthermore, simultaneous administration may be achieved by mixing the compounds before administration, or by administering the compounds at the same time but at different anatomical sites or using different routes of administration.

[0036] The terms "concurrent administration," "co-administration," "simultaneous administration," and "administered simultaneously" all refer to the administration of compounds in combination.

[0037] Therapeutic indications: In one embodiment, the 6-thiopurine-containing dimer compounds disclosed herein are useful for the treatment, improvement, or prevention of cancer.

[0038] In another embodiment, a method for treating a target cancer further includes treating the target with an immune checkpoint inhibitor. Non-limiting examples of checkpoint inhibitors include PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

[0039] In another embodiment, a method for treating the target cancer further includes treating the target with chemotherapy agents, hormone therapy, toxin therapy, radiation therapy, surgery, or a combination thereof.

[0040] Different types of cancer that can be treated with 6-thiopurine-containing dimer compounds include, but are not limited to, one or more of the following: breast cancer, prostate cancer, colon cancer, gastric cancer, esophageal cancer, liver cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, biliary tract cancer, bladder cancer, hepatoma, colorectal cancer, rectal cancer, uterine cancer, cervical cancer, endometrial cancer, salivary gland cancer, mesothelioma, kidney cancer, vulvar cancer in cancer patients, pancreatic cancer, thyroid cancer, hepatic carcinoma, testicular cancer, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancers, acute lymphoblastic leukemia, acute myeloid leukemia, Merkel cell carcinoma, Ewing's sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelioma.

[0041] In some embodiments, the compounds described herein are administered in combination with other compounds, biologics, and other treatments known in the art and used for the treatment, improvement, and prevention of the conditions and diseases considered herein.

[0042] For the treatment of the conditions mentioned above, the compounds disclosed herein can be administered as compounds themselves.

[0043] Alternatively, pharmaceutically acceptable salts are more water-soluble than the parent compound and are therefore suitable for medical applications.

[0044] In another embodiment, the disclosure includes pharmaceutical formulations or compositions. Such formulations include compounds disclosed herein, presented together with a pharmaceutically acceptable carrier. The carrier may be solid, liquid, or both, and may be formulated with the compound as a unit dose composition, for example, a tablet containing 0.05% to 95% by weight of the active compound. The compounds disclosed herein may be bound to polymers suitable as targetable drug carriers. Other pharmacologically active substances may also exist.

[0045] Formulation: In another embodiment, the disclosure includes the use of one or more compounds disclosed herein for the preparation of a medicament for the treatment of any of the conditions listed herein.

[0046] The compounds disclosed herein may be administered orally. Oral administration may involve swallowing so that the compounds enter the gastrointestinal tract, or oral or sublingual administration may be employed so that the compounds enter the bloodstream directly from the mouth.

[0047] Oral administration in solid dosage forms may be presented as individual units, such as hard or soft capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the Disclosure. In another embodiment, oral administration may be in the form of powder or granules. In another embodiment, the oral dosage form is sublingual, such as lozenges. In such solid dosage forms, the compounds of the Disclosure are typically combined with one or more adjuvants. Such capsules or tablets may contain sustained-release formulations. In the case of capsules, tablets, and pills, the dosage forms may also contain buffers or be prepared with enteric coatings.

[0048] In another embodiment, oral administration may be in liquid form. Examples of liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art (e.g., water). Such compositions may also contain adjuvants such as wetting agents, emulsifiers, suspending agents, flavoring agents (e.g., sweeteners), and / or fragrances.

[0049] In another embodiment, the compounds of this disclosure may also be administered directly into the bloodstream, intramuscularly, or viscerally. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, subarachnoid, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Devices suitable for parenteral administration include needle (including microneedle) syringes, needle-free syringes, and injection techniques.

[0050] In another embodiment, the disclosure includes parenteral dosage forms. Parenteral administration includes, for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intracisional injection, and infusion. Injectable formulations (i.e., sterile aqueous or oily suspensions for injection) may be formulated according to known techniques using appropriate dispersants, wetting agents, and / or suspending agents, and may include depot formulations.

[0051] In another embodiment, the compounds disclosed herein may also be formulated as topical administration forms such that topical administration to the skin or mucous membrane (i.e., cutaneous or transdermal) results in systemic absorption of the compound. "Topical administration" includes transdermal administration, such as via a transdermal patch or iontophoresis device, intraocular administration, or intranasal or inhalation administration. Compositions for topical administration also include, for example, topical gels, sprays, ointments, and creams. Topical formulations may contain compounds that enhance the 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 is achieved using patches of either reservoir and porous membrane type or solid matrix variety. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, 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 also be incorporated (see, for example, Finnin and Morgan, J. Pharm. Sci., 88 (10), 955-958 (1999)).

[0052] Formulations suitable for topical administration to the eye include, for example, eye drops in which the compounds of this disclosure are dissolved or suspended in a suitable carrier. Typical formulations suitable for intraocular or ocular administration may be in the form of eye drops of a micronized suspension or solution in isotonic, pH-adjusted sterile saline. Other formulations suitable for intraocular and ocular administration include ointments, biodegradable (e.g., absorbent gel sponge, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and particulate or vesicle systems such as niosomes or liposomes. Polymers such as cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, cellulosic polymers such as hydroxypropyl methylcellulose, hydroxyethylcellulose, or methylcellulose, or heteropolysaccharide polymers such as gellan gum may be incorporated together with preservatives such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

[0053] For intranasal or inhalation administration, the active compounds of this disclosure are conveniently delivered in the form of a solution or suspension from a pump spray container squeezed or pumped in by the patient using a suitable propellant, or as an aerosol spray presentation from a pressurized container or nebulizer. Formulations suitable for intranasal administration are typically delivered from a dry powder inhaler in the form of a dry powder (either alone, as a mixture, e.g., a dry blend with lactose, or as mixed component particles mixed with phospholipids such as phosphatidylcholine), 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, or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer that uses electrohydrodynamics to produce a misty atomization), or nebulizer. For intranasal use, the powder may also contain a bioadhesive, e.g., chitosan or cyclodextrin.

[0054] In another embodiment, the disclosure includes a rectal administration form. Such a rectal administration form may be, for example, a suppository. Cocoa butter is a conventional suppository base, but various alternatives may be used as needed.

[0055] In another embodiment, the compounds of the present disclosure may be formulated such that intravaginal administration leads to systemic absorption of the compound.

[0056] The administration regimen for compounds and / or compositions containing compounds is based on various factors including the patient's type, age, weight, sex, and medical condition, the severity of the condition, the route of administration, and the activity of the specific compound used. Therefore, the administration regimen can vary considerably. Dosage levels equivalent to about 0.01 mg to about 100 mg per kilogram of body weight per day are useful for treating the above conditions. In one embodiment, the total daily dose of the compounds disclosed herein (administered as a single dose or in divided doses) is typically about 0.01 to about 100 mg / kg. In another embodiment, the total daily dose of the compounds disclosed herein is about 0.1 to about 50 mg / kg, and in yet another embodiment, about 0.5 to about 30 mg / kg (i.e., mg of the compounds disclosed herein per kg of body weight). In one embodiment, the dose is 0.01 to 10 mg / kg / day. In another embodiment, the dose is 0.1 to 1.0 mg / kg / day. A dose unit composition may contain such an amount or a fraction thereof to constitute a daily dose. In many cases, the compound is administered multiple times a day (typically four times or less). Multiple daily doses can typically be used to increase the total daily dose as needed.

[0057] For oral administration, the composition 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 symptomatic dose adjustment to patients. The pharmacopoeia typically contains about 0.01 mg to about 500 mg of the active ingredient, or in another embodiment, about 1 mg to about 100 mg of the active ingredient. Intravenously, the dose may range from about 0.1 to about 10 mg / kg / min during constant-rate infusion.

[0058] Oral administration in solid dosage forms may be presented as individual units, such as hard or soft capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the Disclosure. In another embodiment, oral administration may be in the form of powder or granules. In another embodiment, the oral dosage form is sublingual, such as lozenges. In such solid dosage forms, the compounds of the Disclosure are typically combined with one or more adjuvants. Such capsules or tablets may contain sustained-release formulations. In the case of capsules, tablets, and pills, the dosage forms may also contain buffers or be prepared with enteric coatings.

[0059] In some embodiments, treatment with an immune checkpoint inhibitor is performed following administration of one or more of the disclosed 6-thiopurine-containing dimer compounds. In some embodiments, the immune checkpoint inhibitors are PD-1 inhibitors, PD-L1 inhibitors, and / or CTLA-4 inhibitors. In other embodiments, the immune checkpoint inhibitors are administered in combination with one or more CTLA-4 inhibitors and one or more PD-1 inhibitors, or in combination with one or more CTLA-4 inhibitors and one or more PD-L1 inhibitors.

[0060] In some embodiments, the compounds disclosed herein are administered for about 1 to about 5 days per treatment cycle.

[0061] In yet another embodiment, the checkpoint inhibitor is administered for approximately 1 to 3 days in each treatment cycle.

[0062] In another embodiment, the 6-thiopurine-containing dimer compound and the checkpoint inhibitor are administered in combination with chemotherapeutic agents, hormone therapy, toxin therapy, surgery, or a combination thereof.

[0063] In some embodiments, the 6-thiopurine-containing dimer compound is administered before the checkpoint inhibitor is administered in combination with chemotherapeutic agents, hormone therapy, toxin therapy, surgery, or a combination thereof.

[0064] In other embodiments, a method for treating cancer in a subject includes administering one or more 6-thiopurine-containing dimer compounds to the subject, followed by treatment with an immune checkpoint inhibitor and radiotherapy, wherein the cancer is breast cancer, prostate cancer, colon cancer, gastric cancer, esophageal cancer, liver cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, biliary tract cancer, bladder cancer, hepatitis B One or more of the following are selected: myeloma, colorectal cancer, rectal cancer, uterine cancer, cervical cancer, endometrial cancer, salivary gland cancer, mesothelioma, kidney cancer, vulvar cancer, pancreatic cancer, thyroid cancer, liver cancer, testicular cancer, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancers, acute lymphoblastic leukemia, acute myeloid leukemia, Merkel cell carcinoma, Ewing's sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelioma.

[0065] Preferred subjects / patients as provided in this disclosure include mammalian subjects. Mammals as provided in this disclosure include, but are not limited to, dogs, cats, cattle, goats, horses, sheep, pigs, rodents, lagomorphs, primates, and mammals in utero. In one embodiment, humans are preferred subjects. Human subjects may be of either sex and at any developmental stage.

[0066] When used throughout this application, including the claims, the following terms have the meanings defined below unless otherwise indicated. The plural and singular forms should be treated as interchangeable except for their numerical representation.

[0067] Embodiments disclosed herein are also intended to encompass all pharmaceutically acceptable compounds of formula (I), including isotope-labeled compounds in which one or more atoms may be substituted with atoms having different atomic masses or mass numbers. Examples of isotopes that may be incorporated into the compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, 2 H, 3 H, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 C1, 123 I, and 125 I is one example. These radiolabeled compounds may be useful in determining or measuring the efficacy of compounds by characterizing, for example, the site or mode of action, or the binding affinity to pharmacologically important sites of action. Certain isotope-labeled compounds of formula (I), such as those incorporating radioisotopes, may be useful in studying the tissue distribution of drugs and / or substrates. Radioisotope tritium, i.e., 3 H, and carbon-14, that is, 14 C may be particularly useful for this purpose in terms of their ease of integration and rapid detection means.

[0068] Deuterium, that is, 2 Substitution with heavier isotopes, such as 1H, can offer certain therapeutic benefits stemming from greater metabolic stability. For example, the in vivo half-life may increase, or the required dose may decrease. Therefore, in some situations, heavier isotopes may be preferable.

[0069] Substitution with positron-emitting isotopes such as C, F, O, and N may be useful in positron emission topography (PET) studies to investigate substrate acceptor occupancy. The isotope-labeled compound of formula (I) can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the examples below, using appropriate isotope-labeling reagents instead of previously employed unlabeled reagents.

[0070] The methods, compositions, kits, and products provided herein use or contain compounds, for example, compounds of formula (I), or pharmaceutically acceptable salts, prodrugs, or solvates thereof, where 1 to n hydrogen atoms bonded to a carbon atom may be substituted with deuterium atoms or D, and n is the number of hydrogen atoms in the molecule. As is known in the art, deuterium is a non-radioactive isotope of hydrogen. Such compounds can increase resistance to metabolism and may therefore be useful in increasing the half-life of the compound or its pharmaceutically acceptable salts, prodrugs, or solvates when administered to mammals. See, for example, Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism”, Trends Pharmacol. Sci., 5(12):524-527 (1984). Such compounds are synthesized by means known in the art, for example, by employing starting materials in which one or more hydrogen atoms are substituted with deuterium.

[0071] The embodiments disclosed herein are also intended to encompass in vivo metabolites of the disclosed compounds. Such products may arise, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily due to enzymatic processes. Accordingly, the embodiments disclosed herein include compounds produced by processes comprising administering the compounds according to the embodiments disclosed herein to mammals for a period of time sufficient to produce their metabolites. Such products are typically identified by administering a detectable dose of the radiolabeled compound according to the embodiments disclosed herein to animals such as rats, mice, guinea pigs, monkeys, or humans, allowing sufficient time for metabolism to occur, and then isolating the conversion products from urine, blood, or other biological samples. “Stable compound” and “stable structure” mean a compound that is robust enough to withstand isolation from a reaction mixture to a useful purity and formulation into an effective therapeutic agent. “Mammal” includes humans, as well as domestic animals such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domesticated animals such as wild animals. "Optional" or "optionally" means that the events of the situation described thereafter may or may not occur, and that the description includes both cases in which the event or situation occurs and cases in which it does not.

[0072] "Pharmacologically acceptable excipients" include, but are not limited to, any adjuvants, carriers, excipients, flow enhancers, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers approved by the U.S. Food and Drug Administration as acceptable for use in humans or livestock.

[0073] Examples of "pharmaceutically acceptable salts" of the compounds disclosed herein include alkali metals (e.g., sodium), alkaline earth metals (e.g., magnesium), ammonium, and NX4 +Examples of pharmaceutically acceptable salts of nitrogen atoms or amino groups include salts derived from suitable bases such as (wherein X is a C1-C4 alkyl group). Examples of pharmaceutically acceptable salts of nitrogen atoms or amino groups include organic carboxylic acids such as acetic acid, benzoic acid, lactic acid, fumaric acid, tartaric acid, maleic acid, malonic acid, malic acid, isethionic acid, lactobionic acid, and succinic acid, organic sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid, and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and sulfamic acid. Examples of pharmaceutically acceptable salts of compounds with hydroxyl groups include Na + and NX 4+ Examples include anions of the compound combined with suitable cations such as (wherein X is independently selected from H or C1-C4 alkyl groups).

[0074] For therapeutic applications, salts of the active ingredients of the compounds disclosed herein are typically pharmaceutically acceptable, i.e., salts derived from physiologically acceptable acids or bases. However, salts of pharmaceutically unacceptable acids or bases may also be used, for example, in the preparation or purification of the compounds of formula (I) disclosed herein. All salts, including those derived from physiologically acceptable acids or bases, are within the scope of the embodiments disclosed herein.

[0075] Metal salts are typically prepared by reacting a metal hydroxide with a compound according to the embodiments disclosed herein. An example of a metal salt prepared in this way is Li + kaNa + , and K + It is a salt containing [a certain substance]. Less soluble metal salts can be precipitated from solutions of more soluble salts by adding a suitable metal compound.

[0076] Furthermore, the salt may be formed from the acid addition of certain organic and inorganic acids, such as HC1, HBr, H2SO4, H3PO4, or organic sulfonic acids, to a basic center, typically an amine. Finally, it should be understood that the compositions herein include their non-ionized forms, as well as combinations of the compounds disclosed herein in zwitterionic form with stoichiometric amounts of water, as seen in hydrates.

[0077] Crystallization often produces solvates of the compounds of the embodiments disclosed herein. As used herein, the term “solvate” refers to an aggregate containing one or more molecules of the compounds of the embodiments disclosed herein, having one or more solvent molecules. 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 hydrates including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, and the corresponding solvated forms. The compounds of the embodiments disclosed herein may be true solvates, while in other cases, the compounds of the embodiments disclosed herein may simply retain exogenous water or be a mixture of water and some exogenous solvent.

[0078] Furthermore, so-called “prodrugs” of the compounds disclosed herein are within the scope of this disclosure. Therefore, certain derivatives of the compounds disclosed herein, which may themselves have little or no pharmacological activity, can be converted to the compounds of this disclosure having the desired activity, for example, by hydrolysis, when administered in or onto the body. Such derivatives are called “prodrugs.” Further information on the use of prodrugs can 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. EB Roche, American Pharmaceutical Association). Prodrugs according to this disclosure may be produced, for example, by replacing appropriate functional groups present in the compounds of this disclosure with specific parts known to those skilled in the art as “pro-moieties,” as described, for example, in “Design of Prodrugs” by H. Bundgaard (Elsevier, 1985).

[0079] "Pharmaceutical composition" refers to a combination of the compounds of the embodiments disclosed herein with a medium generally accepted in the art for the delivery of the biologically active compounds to a mammal, such as a human. Such a medium includes all pharmaceutically acceptable excipients. "Effective dose" or "therapeutic dose" refers to the amount of the compound of the embodiments disclosed herein that, when administered to a patient in need, is sufficient to produce a therapeutic effect against a disease, condition, or disorder for which the compound is useful. Such a dose would be sufficient to induce the tissue system or the patient's biological or medical response desired by the researcher or clinician. The amount of the compound of the embodiments disclosed herein that constitutes a therapeutic dose varies depending on 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 treatment, the type and severity of the disease or disorder being treated, any drugs used in combination with or concurrently with the compound of the embodiments disclosed herein, and factors such as the patient's age, weight, general health, sex, and diet. Such a therapeutic dose can usually be determined by a person skilled in the art, taking into account their own knowledge, the latest technology, and the present disclosure.

[0080] "Effective dose" or "therapeutic dose" means the amount of a compound according to the embodiments disclosed herein that, when administered to a patient in need, is sufficient to exert a therapeutic effect on a disease, condition, or disorder for which the compound is useful. Such a dose would be sufficient to induce the tissue system or the patient's biological or medical response desired by the researcher or clinician. The amount of a compound according to the embodiments disclosed herein that constitutes a therapeutic dose varies depending on 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 treatment, the type and severity of the disease or disorder being treated, any agents used in combination with or concurrently with the compound according to the embodiments disclosed herein, and factors such as the patient's age, weight, general health, sex, and diet. Such a therapeutic dose can usually be determined by a person skilled in the art, taking into account their own knowledge, advanced technology, and this disclosure.

[0081] As used herein, the term “treatment” is intended to mean the administration of a compound or composition according to this embodiment to alleviate or eliminate the symptoms of any condition described herein.

[0082] When used herein, "EC 50 " " refers to the dose or concentration of a drug that kills 50% of the cancer cells being treated.

[0083] The compounds disclosed herein and their pharmaceutically acceptable salts may contain one or more chiral centers, and thus may result in enantiomers, diastereomers, and other stereoisomers, which can be defined in terms of the absolute configuration of amino acids, such as (R)- or (S)-, or (D)- or (L)-. This disclosure includes 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 may be decomposed using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or decomposition of racemics (or racemics of salts or derivatives) using, for example, chiral high-pressure liquid chromatography (HPLC). Where a compound described herein contains an olefinic double bond or other geometrically asymmetric center, unless otherwise specified, the compound is intended to include both E and Z geometric isomers. Similarly, all tautomers are also intended to be included.

[0084] A "stereoisomer" refers to a compound that is composed of the same atoms bonded together by the same bonds, but has different three-dimensional structures that are incompatible. This disclosure intends various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers that are mirror images of each other and whose molecules cannot be superimposed.

[0085] "Tautomerism" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. This disclosure includes tautomers of any such compound.

[0086] The 6-thiopurine-containing dimer compounds disclosed herein that have a deoxyribose monosaccharide unit may be called "diTHIO," and those that have a ribose monosaccharide unit may be called "diRIBOTHIO." The 6-thio-2'-deoxyguanosine-containing monomer compounds disclosed herein may also be called "THIO," and the 6-thioguanosine-containing monomer compounds may be called "RIBOTHIO." These notations are used in Table 1. Examples

[0087] Example 1. Synthesis of a 6-thiopurine-containing dimer compound of formula I: This is exemplified by the typical synthesis of compound XX (MAIA-2021-029): The preparation of compound XX from compound XVI is schematically shown in reaction (3): [ka]

[0088] The compound of formula XVI (30 mg, 105.89 μmol) was dissolved in a pH=7.6 buffer solution (7.5 mL) maintained at 45°C, and the solution was cooled to 30°C. A 0.5N I2 / NaI solution (105.89 μmol, 0.3 mL) was added dropwise to the solution while stirring at a rate sufficient to disperse the color appearing in the reaction mixture with each drop. A white precipitate formed, which was collected by filtration. The filtered solid was washed with water (0.2 × 2 mL) and then with EtOH (0.2 × 2 mL), dried, and the product of formula XX (16.7 mg, 28.99 μmol, yield 27.37%) was obtained. 1H-NMR (600MHz, DMSO-d6): δ=8.27(s,2H),6.61(s,4H),6.80(s,2H),6.22(t,2H,J=6.6Hz),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] + .

[0089] Using the procedure described above, other 6-thiopurine-containing dimers XXI to XXIII were prepared from the corresponding 6-thiopurine-containing monomers XVII to XIX, as shown in Table 1.

[0090] EC 50 Cell viability assays for determining the efficacy were measured by screening a given type of cancer cells with drug compounds in a dilution series at nine different points in a 96-well plate. Cells were seeded before drug addition, incubated for 4-5 days, and assayed using the CellTiter-Glo cell viability assay according to the manufacturer's instructions (Promega). Dose-response curves were constructed and analyzed using Graphpad Prism. 50 The values ​​were calculated. All samples were analyzed three times, and the standard deviations are from two to three independent experiments.

[0091] Telomere dysfunction-induced lesions (TIFs) were measured by seeding a given type of cancer cells onto a pre-sterilized slide and placing the slide in a 10 cm Petri disc (Falcon). After 24 hours, the cells were measured using EC. 50 The slides were treated with a drug compound of a specified concentration for 48 hours. The slides were then rinsed in PBS 1X for 10 minutes on a shaking platform, fixed in 4% formaldehyde (Thermo Fisher) on ice for 10 minutes, and then washed twice in PBS 1X for 5 minutes each. The slides were then permeabilized with 0.5% Triton X-100 on ice for 10 minutes, and subsequently blocked in BSA / PBS 1X for 30 minutes at room temperature.

[0092] The anti-mouse primary antibody γH2AX(Millipore) was diluted 1:200 in blocking solution, and cells were incubated over-the-air in a humid chamber at 4°C. After washing with PBS 1X, the cells were incubated with AlexaFluor 488 conjugated goat anti-mouse for 45 minutes at room temperature. After washing with PBS 1X, the TIF assay was performed with slight modifications to the method described above (Mender and Shay, 2015).

[0093] As described in the paragraph on γH2AX Foci, the slides of cancer cells obtained above were seeded, treated, and stained with γH2AX. After washing with PBS 1X, the cells were fixed in 4% formaldehyde in PBS at room temperature for 20 minutes. The slides were sequentially dehydrated with 70%, 90%, and 100% ethanol, and then denatured at 80°C for 3 minutes in 20 μL of hybridization mixture containing 70% deionized formamide, 1 M Tris pH 7.2, 8.56% buffer MgCl2, 5% maleic acid blocking reagent, and 25 μg / mL Cy3 conjugate PNA Tel-C (CCCTAA)3 probe (PANAGENE, Korea), and incubated overnight at 4°C in a humid chamber. The slides were washed twice for 15 minutes in a washing solution containing 70% formamide, 10 mM Tris pH 7.2, and 0.1% BSA, and then washed three times for 5 minutes in a solution containing 0.1 M Tris pH 7.5, 0.15 M NaCl, and 0.08% Tween-20. The slides were dehydrated with an ethanol-based solution, air-dried, and counterstained with Vectashield / DAPI (Vector Laboratories, Burlingham, California). Images were captured at 63X magnification using an Axio Imager Z2 (Carl Zeiss) equipped with an automated capture system (Metafer, Metasystems) and analyzed using ISIS software (Metasystems).

[0094] Tests of 6-thiopurine-containing dimers and monomer compounds: The inhibition of cancer cell survival by 6-thiopurine-containing dimers was compared with that of corresponding 6-thiopurine-containing monomer compounds in various human and mouse cancer cells. The in vitro antitumor activity of 6-thiopurine-containing dimers against various types of tumor cells was compared with that of corresponding 6-thiopurine-containing monomer compound XVI and the known compound cisplatin. The results are shown in Figures 1-17 and Tables 1-7.

[0095] Table 2. In vitro activity of 6-thiopurine-containing dimer compound XX (also known as diTHIO, THIO-dimer, or 6S-dG-dimer) in other tumor cell lines. [Table 2]

[0096] Table 3A. In vitro activity of 6-thiopurine-containing dimer compound XX (6S-dG-dimer) compared to cisplatin in other tumor cell lines. [Table 3A]

[0097] Table 3B. In vitro activity of 6-thiopurine-containing dimer compound XX (6S-dG-dimer) compared to cisplatin in other tumor cell lines. [Table 3B]

[0098] Table 4A. In vitro activity of 6-thio-2'-deoxyguanosine-containing dimer compound XX compared to monomer compound XVI containing 6-thio-2'-deoxyguanosine. [Table 4A]

[0099] Table 4B. In vitro activity of 6-thio-2'-deoxyguanosine-containing dimer compound XX compared to the corresponding 6-thio-2'-deoxyguanosine-containing monomer compound XVI. [Table 4B]

[0100] Table 4C. In vitro activity of dimer compound XX compared to the corresponding monomer compound XVI. [Table 4C]

[0101] Table 5A. Characteristics of cell lines used for the evaluation of compounds XVI and XX. [Table 5A]

[0102] Table 5B. Characteristics of cell lines used to evaluate compounds XVI and XX. [Table 5B]

[0103] Table 6A. In vitro activity of 6-thio-2'-deoxyguanosine-containing dimer compound XX compared to cisplatin in mouse cell lines. [Table 6A]

[0104] Table 6B. In vitro activity of 6-thio-2'-deoxyguanosine dimer-containing compound XX compared to cisplatin in mouse cell lines. [Table 6B]

[0105] Table 7A. In vitro activity of novel tumor redox-activating 6-thiopurine-containing dimers XX-XXIII and 6-thiopurine-containing monomers XVI and XXVI compared to cisplatin in various cell lines. [Table 7A]

[0106] Table 7B. In vitro activity of novel tumor redox-activating 6-thiopurine-containing dimers XX-XXIII and 6-thiopurine-containing monomers XVI and XXVI compared to cisplatin in various cell lines. [Table 7B]

[0107] Figure 1 shows that when the 6-thio-2'-deoxyguanosine-containing dimer XX was tested in NSCLC LLC (non-small cell lung cancer, Lewis lung cancer) cells for 4 days, it exhibited a significantly lower EC50 (half of the maximum effective concentration) of 0.7 μM compared to the 6-thio-2'-deoxyguanosine monomer compound XVI (2.4 μM). In other words, the concentration of XX required to reduce the number of NSCLC LLC cells by 50% is significantly lower than the concentration of XVI required under the same conditions. When CRC MC38 (colorectal cancer, mouse colon adenocarcinoma) cells were treated with compounds XX and XVI, the dimer compound XX showed a significantly lower EC50 (3.1 μM) than XVI (1.0 μM).

[0108] The dimeric compound XX is also efficient in the formation of TIFs (telomere dysfunction-induced lesions), as shown in Figure 2A. This means that XX is efficient in inducing telomere dysfunction in cancer DNA molecules, which leads to the death of cancer cells and thereby yields a beneficial therapeutic effect.

[0109] In the case of Hep55-1C cells (a cell line derived from carcinogen-induced liver tumors in C57BL / 6 mice), compound XX competitively showed lower IC50 compared to compound XVI. 50 (This represents half the maximum inhibitory concentration required to inhibit the proliferation of liver cancer DNA) (see Figure 3). Compound XX also shows superior therapeutic efficacy against Hep55-1C cells in in vitro studies, even when re-challenged with the same number of tumor cells. Figure 4 shows that the ability to inhibit tumor cell proliferation is fully retained even 4 days after the initial treatment with Hep55-1C cells. Figure 12 shows that the dimeric compound XX inhibits Hep55-1C cells for a much longer period of 125 days, and then maintains the same potency even after further exposure of more than 75 days following a new challenge and test with 5 million Hep55-1C cells. In this extended testing phase, the dimeric compound XX actually shows superior inhibitory efficacy compared to the corresponding monomer compound XVI. However, when the system was further challenged with 1 million RIL 175 cells, the potency of both compounds XVI and XX decreased significantly (see Figure 13), which likely indicates that compounds such as XVI and XX exhibit highly selective inhibitory efficacy against liver cancer cells. These results also likely indicate induction of cellular immunity by XX, which foreshadows a very potent therapeutic option for treating liver cancer.

[0110] The dimer compound XVI also shows better efficacy than cisplatin in inhibiting tumor DNA growth in a wide range of cancer cells, including myeloma, ovarian cancer, 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 dimers and monomer compounds, including the 6-thio-2'-guanosine-containing monomer XXVI (referred to as MAIA-2021-001), were also prepared and tested in vitro against different types of cancer cells, all of which showed excellent inhibition of cancer cell proliferation (see Figures 9A-9L).

[0111] The 6-thio-2'-deoxyguanosine-containing dimer compound XX also shows far superior efficacy in inhibiting the proliferation of ovarian cancer cells compared to conventionally used cisplatin. Figure 7 shows that XVI showed a much lower IC50 at 0.08 μM than cisplatin (3.54 μM). 50 This indicates that it possesses [the characteristic]. Therefore, it is highly likely that toxicity-related side effects from cisplatin can be avoided or minimized by using 6-thio-2'-deoxyguanosine-containing dimer compounds such as XVI. Dimer compounds such as XX have also been effective when tested in vitro against different types of mouse cancer cells (see Figures 8A to 8L).

[0112] Glutathione S-transferase (GST) is a family of isoenzymes that play a crucial role in protecting cells from cytotoxic and carcinogenic substances. The isozymes GST-P1, glutathione S-transferase alpha-1, and glutathione S-transferase function by catalyzing the conjugate of toxic substances with GSH and removing them from cells. When anticancer drugs, such as cisplatin, are administered, GST-P1 assists in removing the therapeutic agent from cells, leading to cancer progression and resistance to cancer therapy. However, in the presence of a 6-thio-2'-guanosine-containing dimer, GST-P1 activity is strongly inhibited, resulting in better therapeutic outcomes. More importantly, normal cells were generally unaffected by the drug compounds in the study.

[0113] All references cited herein are incorporated by reference.

[0114] Several embodiments of this disclosure have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 6-thiopurine-containing dimer of formula I: 【Chemistry 1】 or a pharmaceutically acceptable salt, ester, prodrug, hydrate, or tautomer thereof (wherein both R １ H, or (PO ３ ) ２－ And both R ２ (It is simultaneously H or OH).

2. The compound according to claim 1 having the following structure: 【Chemistry 2】

3. The compound according to claim 1 having the following structure: 【Transformation 3】

4. The compound according to claim 1 having the following structure: 【Chemistry 4】

5. The compound according to claim 1 having the following structure: 【Transformation 5】

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

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

8. A method for treating cancer in the subject, A method comprising administering the pharmaceutical composition according to claim 6 or claim 7 to the subject in an amount effective for treating the cancer.

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

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

11. The method according to claim 10, further comprising treating the subject with a chemotherapy agent, hormone therapy, toxin therapy, radiotherapy, surgery, or a combination thereof.

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

13. A method for forming a 6-thiopurine-containing monomer, a) Contacting a 6-thiopurine containing a 6-thiopurine dimer with an organic thiol of formula R-SH (wherein R is an alkyl group), b) Forming the 6-thiopurine-containing monomer, Here, the 6-thiopurine-containing dimer is given by formula (I): 【Transformation 6】 It is, The aforementioned 6-thiopurine-containing monomer is of formula IV: 【Transformation 7】 It is, R of Formulas I and IV １ is H, P(=O)(OH) ２ or its ester, [(P(OH) ２ (=S)] or its ester, (PO3) ２ , or [(PO ２ )(=S)] ２ — and R of Formulas I and IV ２ is H or OH, a method.

14. The aforementioned 6-thiopurine-containing dimer is a 6-thiodeoxyguanosine-containing dimer of formula II: 【Transformation 8】 Dimer containing 6-thioguanosine of formula III: 【Chemistry 9】 Or selected from a group consisting of combinations thereof, R in Equations II and III １ These are independent of H, P (=O) (OH) ２ or its ester, [(P(OH) ２ (=S)] or its ester, (PO ３ ) ２ , or [(PO ２ ) (=S)] ２ The method according to claim 13.

15. The method according to claim 13, wherein the 6-thiopurine-containing monomer is a 6-thiodeoxyguanosine-containing monomer of formula XXIV: 【Chemistry 10】 (In the formula, R １ H, P (=O) (OH) ２ or its ester, [(P(OH) ２ (=S)] or its ester, (PO ３ ) ２ , or [(PO ２ ) (=S)]2-).

16. The method according to claim 13, wherein the 6-thiopurine-containing monomer is a 6-thioguanosine-containing monomer of formula XXV: 【Chemistry 11】 (In the formula, R １ H, P (=O) (OH) ２ or its ester, [(P(OH) ２ (=S)] or its ester, (PO ３ ) ２ , or [(PO ２ ) (=S)]2-).

Images