Dinucleotides and their use in treatment of cancer

Abstract

Disclosed herein are dinucleotide compounds that target telomeres in cancer cells and methods for using the dinucleotide compounds to treat cancer alone or prior to administration of a checkpoint inhibitor.

Description

[0001] Priority claim

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 388,688, filed on July 13, 2022, entitled “Dinucleotides and Their Use in Treating Cancer.” Technical Field

[0003] The present invention belongs to the field of cancer treatment, and in particular, uses dinucleotide compounds that target telomeres in cancer cells for cancer treatment. Background Art

[0004] Telomeres are found at both ends of eukaryotic chromosomes. These DNA-protein structures protect the genome from nucleic acid degradation, unwanted recombination, intrachromosomal fusion and repair (Shammas, M., (2011), Curr Opin, Clin. Nutr Metab Care, 14 (1): 28-34). Due to terminal replication problems and the lack of telomere maintenance mechanisms, telomere length shortens with each cell division (Greider CW, (1996), Annu Rev Biochem. 65: 337-65). However, unicellular eukaryotic organisms, 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, Dec; 43(2 Pt 1): 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). Telomerase is a reverse transcriptase that extends telomeres by adding TTAGGG repeat sequences to the ends of chromosomes and is expressed in about 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 the development of anticancer therapies. Most therapies targeting telomerase focus on inhibiting telomerase (Andrews LG, Tollefsbol TO. Mol Biol. 2007; 405: 1-7); however, such inhibitors have performed poorly in clinical studies (Zhang G., Shay WS. Oncotarget, 2018, vol 9(88): 35803-35804).

[0005] Other cancer drugs are being developed that, rather than inhibiting telomerase, use telomerase to incorporate into telomeres, thereby causing DNA damage, senescence, 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 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 the tumor-killing effect of 6-thio-dG does not have a long lag phase. In addition, it does not directly inhibit telomerase, but is preferentially recognized by telomerase over other polymerases and incorporated into telomeres, resulting in immediate DNA chain termination. Importantly, its effect is independent of initial telomere length because it creates unstable telomeres by hijacking tumor telomerase (Mender et al., (2015) Oncoscience, 2(8): 693-695).

[0006] Disclosed below are novel telomere-targeted compounds useful for treating cancer. Summary of the invention

[0007] Disclosed herein are dinucleotide compounds or pharmaceutically acceptable salts thereof, which are telomere-targeted compounds useful for treating cancer. In some embodiments, the dinucleotide compounds are telomerase-mediated telomere-targeted compounds useful for treating cancer.

[0008] In some embodiments, the dinucleotide compounds disclosed herein have the structure of Formula I

[0009]

[0010] wherein R is independently H or OH, and X is O or S. In some embodiments, X is O. In other embodiments, X is S.

[0011] When X is O, the dinucleotide compound of Formula I may have the following structure:

[0012] (also known as MAIA-2022-12),

[0013]

[0014]

[0015] When X is S, the dinucleotide compound of Formula I may have the following structure:

[0016]

[0017]

[0018] In other embodiments, the dinucleotide compound may have one or more of the following structures:

[0019]

[0020]

[0021]

[0022] Also disclosed herein are enantiomers of Compound 11 having the following stereochemistry:

[0023]

[0024] In another embodiment are enantiomers of compound 12 having the following stereochemistry:

[0025]

[0026] In some aspects, the dinucleotide compound has the structure of Formula II:

[0027]

[0028] In the case where R1 and R2 are independently H or OH;

[0029] X is O or S;

[0030] Y is O; and

[0031] R3 is selected from the group consisting of a phosphate group, a thiophosphate group, palmitic acid, tocopherol and a cholesterol group.

[0032] In some embodiments of Formula II, R1 and R2 are independently H or OH, X is O, Y is O, and R3 is a cholesterol group. In some embodiments, the dinucleotide compound of Formula II has the following structure,

[0033]

[0034] In some embodiments of Formula II, R1 and R2 are independently H or OH, X and Y are O, and R3 is a phosphate group. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0035]

[0036] In other embodiments of Formula II, R1 and R2 are independently H or OH, X and Y are independently O, and R3 is a thiophosphate group. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0037]

[0038] In yet other embodiments of Formula II, R1 and R2

[0039] is independently H or OH; X and Y are independently O, and R3 is palmitic acid. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0040]

[0041] In still other embodiments of Formula II, R1 and R2 are independently H or OH, X and Y are independently O, and R3 is tocopherol. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0042]

[0043]

[0044] In other embodiments of Formula II, R1 and R2 are independently H or OH, X is S, Y is O, and R3 is a cholesterol group. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0045]

[0046]

[0047] In other embodiments of Formula II, R1 and R2 are independently H or OH, X is S, Y is O, and R3 is a phosphate group. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0048]

[0049] In some embodiments of Formula II, R1 and R2 are independently H or OH, X is S, Y is O, and R3 is a thiophosphate group. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0050]

[0051] In some embodiments of Formula II, R1 and R2 are independently H or OH, X is S, Y is O, and R3 is palmitic acid. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0052]

[0053]

[0054] In other embodiments of Formula II, R1 and R2 are independently H or OH, X is S, Y is O, and R3 is tocopherol. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0055]

[0056]

[0057] In other embodiments are dinucleotide compounds comprising the structure of Formula III:

[0058]

[0059] wherein X is O or S, and R' is independently H or OH. In some embodiments, the dinucleotide compound of formula III has the following structure:

[0060]

[0061]

[0062]

[0063] In other embodiments are dinucleotide compounds comprising the structure of Formula IV.

[0064]

[0065] wherein X is O or S, and R' is independently H or OH.

[0066] In some embodiments, the dinucleotide compound of Formula IV has the following structure:

[0067]

[0068]

[0069]

[0070] In other embodiments are dinucleotide compounds comprising the structure of Formula V, wherein R' is independently H or OH.

[0071]

[0072] In some embodiments, the dinucleotide compound of Formula V has the following structure:

[0073]

[0074]

[0075] In yet other embodiments are dinucleotide compounds comprising the structure of Formula VI, wherein R′ is independently H or OH

[0076]

[0077] In some embodiments, the dinucleotide compound of Formula VI has the following structure:

[0078]

[0079]

[0080]

[0081] In other embodiments are dinucleotide compounds comprising the structure of Formula VII, wherein R′ is independently H or OH and X is S or O.

[0082]

[0083] In some embodiments, the dinucleotide compound of Formula VII has the following structure, wherein X is O and R1 and R2 are H or OH:

[0084]

[0085]

[0086] In some embodiments, the dinucleotide compound of Formula VII has the following structure, wherein X is S and R1 and R2 are H or OH:

[0087]

[0088]

[0089] Another aspect of the present disclosure is a pharmaceutical salt of the dinucleotide compounds disclosed herein.

[0090] In yet other embodiments, disclosed herein are pharmaceutical compositions comprising a dinucleotide compound as described herein. In some embodiments, the pharmaceutical formulation may comprise at least one pharmaceutically acceptable excipient.

[0091] Another embodiment disclosed herein is a method for preparing a pharmaceutical formulation comprising combining a dinucleotide compound discussed herein with at least one pharmaceutically acceptable excipient.

[0092] In still other embodiments, disclosed herein are methods of treating a subject having cancer comprising administering to the subject one or more of the dinucleotide compounds discussed herein, wherein the cancer is selected from one or more of 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, pancreatic cancer, thyroid cancer, liver cancer, 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 cancer, nasopharyngeal carcinoma, and peripheral neuroepithelioid tumor.

[0093] In certain embodiments, the dinucleotide compounds disclosed herein can be administered in combination with other anticancer agents or cancer therapies. The compounds can be administered before, at the same time as, or after other cancer agents or therapies.

[0094] In some embodiments, the administration of one or more of the disclosed nucleotide compounds is followed by treatment with an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, and / or a CTLA-4 inhibitor. In other embodiments, the immune checkpoint inhibitor is administered in combination with one or more CTLA-4 inhibitors and one or more PD-1 inhibitors, or the immune checkpoint inhibitor is administered in combination with one or more CTLA-4 inhibitors and one or more PD-L1 inhibitors.

[0095] In some embodiments, the compounds disclosed herein are administered from about 1 to about 5 days per treatment cycle.

[0096] In still other embodiments, the checkpoint inhibitor is administered for about 1 to about 3 days per treatment cycle.

[0097] Also disclosed herein are methods in which the dinucleotide compounds and checkpoint inhibitors disclosed herein are administered in combination with chemotherapeutic agents, hormone therapy, toxin therapy, surgery, or a combination thereof.

[0098] In some embodiments, a dinucleotide disclosed herein is administered prior to administration of a checkpoint inhibitor in combination with a chemotherapeutic agent, hormone therapy, toxin therapy, surgery, or a combination thereof.

[0099] Also disclosed herein are methods of treating a subject having cancer comprising administering to the subject one or more of the dinucleotide compounds disclosed herein after treatment with radiation therapy, wherein the cancer is selected from one or more of 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, pancreatic cancer, thyroid cancer, liver cancer, 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 cancer, nasopharyngeal carcinoma, and peripheral neuroepithelioid tumor.

[0100] Also disclosed herein are methods of treating a subject having cancer comprising administering to the subject one or more of the dinucleotide compounds disclosed herein followed by treatment with radiation therapy, comprising administering to the subject a compound of any one of claims 1-96, wherein the cancer is selected from 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, One or more of 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, liver cancer, 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 cancer, nasopharyngeal carcinoma, and peripheral neuroepithelioid tumor.

[0101] In other embodiments, disclosed herein are methods of treating cancer in a subject comprising administering to the subject one or more of the dinucleotide compounds disclosed herein, 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, 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, pancreatic cancer, thyroid cancer, liver cancer, 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 cancer, nasopharyngeal carcinoma, and peripheral neuroepithelioid tumor.

[0102] In other embodiments, disclosed herein is a method of treating cancer in a subject comprising administering to the subject one or more of the dinucleotide compounds disclosed herein after radiation therapy, and subsequently treating with an immune checkpoint inhibitor, wherein the cancer is selected from one or more of 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, pancreatic cancer, thyroid cancer, liver cancer, 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 cancer, nasopharyngeal carcinoma and peripheral neuroepithelioid tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] The following drawings form part of this specification and are included to further demonstrate certain aspects or embodiments of the present disclosure. The present disclosure may be better understood by referring to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.

[0104] Figure 1 The general structure of the claimed compounds with a free 5'-hydroxyl group and X being O or S is shown.

[0105] Figure 2 The general structure of the claimed compounds with a derivatized 5'-hydroxy group is shown.

[0106] Figure 3 The general structure of the claimed compounds with a free 3'-hydroxyl group is shown. All molecules are linear non-cyclic dinucleotides.

[0107] Figure 4 The general structure of the claimed compounds with a free 5'-hydroxyl group is shown.

[0108] Figure 5A-5B -5A shows compound 11 and its two enantiomers, compound 11-Rp and compound 11-Sp; 5B shows compound 12 and its two enantiomers, compound 12-Rp and compound 12-Sp.

[0109] Figures 6A-6E The effects of compounds 1-17 on the cell viability of LLC and MC38 cells are shown. 6A shows the effects of compounds 1-4 on the cell viability of LLC and MC38 cells. 6B shows the effects of compounds 5-8 on the cell viability of LLC and MC38 cells. 6C shows the effects of compounds 9-12 on the cell viability of LLC and MC38 cells. 6D shows the effects of compounds 9-12 on the cell viability of LLC and MC38 cells. 6E shows the effects of compound 17 on the cell viability of LLC and MC38 cells.

[0110] Figure 7A-7B - shows the specificity of the tested dinucleotides for telomerase negative normal non-cancerous human BJ fibroblasts. 7A shows the effect of compounds 1-2 and 5-6 on cell viability of non-cancerous / telomerase negative BJ cells. 7B shows the effect of compound 13 on cell viability of non-cancerous / telomerase negative BJ cells.

[0111] Figure 8 A-8C-H22 hepatocellular carcinoma murine model (syngeneic mouse model); Figure 8 A shows tumor volume (mm) of BALB / c mice inoculated with H22 tumor cells and treated with vehicle, MAIA-2022-12, MAIA-2021-20, anti-PD1, MAIA-2022-12 + anti-PD-1, or MAIA-2021-20 + anti-PD-1. 3 ). Figure 8 B shows the body weight changes of treated mice. Figure 8 C shows individual data from the H22 experiment.

[0112] Fig. 9 A-9C-M38 colorectal cancer murine model (syngeneic mouse model); Fig. 9A shows the tumor volume (mm) of C57Bl / 6N mice inoculated with MC38 tumor cells and treated with vehicle, MAIA-2022-12, MAIA-2021-20, anti-PD1, MAIA-2022-12 + anti-PD-1, or MAIA-2021-20 + anti-PD-1. 3 ). Fig. 9 B shows the body weight changes of treated mice. Fig. 9 C shows individual data from the M38 experiment.

[0113] Fig.10 A-10C-YUMM1.7 melanoma murine model (syngeneic mouse model). Fig.10 A shows tumor volume of c57BL / 6N mice inoculated with YUMM1.7 tumor cells and treated with vehicle, MAIA-2022-12, or MAIA-2021-20; Fig.10 B shows the body weight changes of mice treated with vehicle, MAIA-2022-12 or MAIA-2021-20; Fig.10 (c) shows individual data from the YUMM1.7 experiment.

[0114] Fig.11 A-11E-YUMM1.7 rechallenge study. Fig.11 A shows tumor volume in mice after re-challenge with control, MAIA-2022-12, or MAIA-2021-020. Fig.11 B shows body weight after rechallenge with control, MAIA-2022-12, or MAIA-2021-020; Panels CE show individual data from control, MAIA-2022-12, and MAIA-2021-20 tumor volume experiments. DETAILED DESCRIPTION

[0115] Disclosed herein are dinucleotide compounds that target and modify the telomere structure of cancer cells. The compounds are all linear non-cyclic dinucleotides.

[0116] In aspects of the present disclosure, the compounds disclosed herein are dinucleotide molecules containing modified nucleosides, which are converted into human telomerase substrates in cells, such as nucleoside-5'-triphosphates. The nucleotides can be connected via phosphodiester groups (relatively fast dissociable bonds, Rp) or via thiophosphate groups (relatively slow dissociable bonds, Sp). In certain embodiments of this aspect, the general structure of the disclosed compounds with free 5'-hydroxyl group molecules is a straight-chain non-cyclic dinucleotide. The 5'-3' internucleoside bond connecting the nucleosides is a phosphodiester (where X=O) or a thiophosphate (where X=S). The nucleosides that make up the dinucleotide are composed of 6-thio-2'-deoxyguanosine, 6-thioguanosine, 4-thio-thymidine, and 5-fluorouridine. When X=S, it is either a mixture of two Rp- and Sp-isomers, or a single Rp- or Sp-isomer ( Figure 1 ).

[0117] In some embodiments of the above aspects, the dinucleotide compound comprises the structure of Formula I or a pharmaceutically acceptable salt thereof.

[0118]

[0119] wherein R is independently H or OH, and X is O or S. In some embodiments, X is O. In other embodiments, X is S.

[0120] When X is O, the dinucleotide compound of Formula I comprises the following structure or a pharmaceutically acceptable salt thereof:

[0121]

[0122] When X is S, the dinucleotide compound of Formula I comprises the following structure or a pharmaceutically acceptable salt thereof:

[0123]

[0124] In yet other embodiments, the dinucleotide compound comprises the following structure or a pharmaceutically acceptable salt thereof

[0125]

[0126]

[0127] Also disclosed herein are enantiomers of Compound 11 having the following stereochemistry:

[0128]

[0129] In another embodiment are enantiomers of compound 12 having the following stereochemistry:

[0130]

[0131] In another aspect, disclosed herein are dinucleotide compounds of Formula II or pharmaceutically acceptable salts thereof having a derivatized 5′-hydroxyl group, such as, but not limited to, a 5′-conjugated lipid, a 5′-phosphate, or a 5′-thio-phosphate group ( Figure 2 ).

[0132]

[0133] Wherein R1 and R2 are independently H or OH;

[0134] X is O or S;

[0135] Y is O; and

[0136] R3 is selected from the group consisting of a phosphate group, a thiophosphate group, palmitic acid, tocopherol and a cholesterol group.

[0137] In some embodiments of Formula II, R1 and R2 are independently H or OH, X is O, Y is O, and R3 is a cholesterol group. In some embodiments, the dinucleotide compound of Formula II has the following structure,

[0138]

[0139] In some embodiments of Formula II, R1 and R2 are independently H or OH, X and Y are O, and R3 is a phosphate group. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0140]

[0141]

[0142] In other embodiments of Formula II, R1 and R2 are independently H or OH, X and Y are independently O, and R3 is a thiophosphate group. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0143]

[0144] In yet other embodiments of Formula II, R1 and R2

[0145] is independently H or OH; X and Y are independently O, and R3 is palmitic acid. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0146]

[0147]

[0148] In yet other embodiments of Formula II, R1 and R2 are independently H or OH, X and Y are independently O, and R3 is tocopherol. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0149]

[0150]

[0151]

[0152] In other embodiments of Formula II, R1 and R2 are independently H or OH, X is S, Y is O, and R3 is a cholesterol group. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0153]

[0154]

[0155] In other embodiments of Formula II, R1 and R2 are independently H or OH, X is S, Y is O, and R3 is a phosphate group. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0156]

[0157] In some embodiments of Formula II, R1 and R2 are independently H or OH, X is S, Y is O, and R3 is a thiophosphate group. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0158]

[0159] In some embodiments of Formula II, R1 and R2 are independently H or OH, X is S, Y is O, and R3 is palmitic acid. In some embodiments, the dinucleotide compound of Formula II has the following structure:

[0160]

[0161]

[0162] In other embodiments of Formula II, R1 and R2 are independently H or OH, X is S, Y is O, and R3 is tocopherol. In some embodiments, the compound of Formula II has the following structure:

[0163]

[0164]

[0165] In certain aspects, the structures of the disclosed dinucleotide compounds have a free 3′-hydroxyl group. All molecules are linear non-cyclic dinucleotides. The 5′-5′ internucleoside bond connecting the nucleosides is a phosphodiester (where X=O) or a thiophosphate (where X=S). The nucleosides that make up the dinucleotide are composed of 6-thio-2′-deoxyguanosine, 6-thioguanosine, and 5-fluorouridine. When X=S, it is either a mixture of two Rp- and Sp-isomers, or a single Rp- or Sp-isomer ( Figure 3 ).

[0166] In an embodiment of this aspect is a dinucleotide compound comprising the structure of Formula III or a pharmaceutically acceptable salt thereof:

[0167]

[0168] wherein X is O or S, and R' is independently H or OH. In some embodiments, the dinucleotide compound of formula III has the following structure:

[0169]

[0170]

[0171]

[0172] In other embodiments are dinucleotide compounds comprising the structure of Formula IV.

[0173]

[0174] wherein X is O or S, and R' is independently H or OH.

[0175] In some embodiments, the dinucleotide compound of Formula IV has the following structure:

[0176]

[0177]

[0178]

[0179] In other aspects, the general structure of the disclosed dinucleotide compounds has a free 5'-hydroxyl group. All molecules are linear non-cyclic dinucleotides. The 3'-3' internucleoside bond connecting the nucleosides is a phosphodiester (where X=O) or a thiophosphate (where X=S). The nucleosides that make up the dinucleotide are composed of 6-thio-2'-deoxyguanosine, 6-thioguanosine and 5-fluorouridine. When X=S, it is either a mixture of two Rp- and Sp-isomers, or a single Rp- or Sp-isomer ( Figure 4 ).

[0180] Figure 4 A representative compound is a dinucleotide compound comprising a structure of Formula V or a pharmaceutically acceptable salt thereof, wherein R′ is independently H or OH:

[0181]

[0182] In yet other embodiments, Figure 4 A representative compound is a dinucleotide compound comprising the structure of Formula VI or a pharmaceutically acceptable salt thereof, wherein R′ is independently H or OH:

[0183]

[0184] In other embodiments are dinucleotide compounds comprising the structure of Formula VII, wherein R1 or R2 are independently H or OH and X is S or O.

[0185]

[0186] In some embodiments, the dinucleotide compound of Formula VII has the following structure, wherein X is O and R1 and R2 are H or OH:

[0187]

[0188]

[0189] In some embodiments, the dinucleotide compound of Formula VII has the following structure, wherein X is S and R1 and R2 are H or OH:

[0190]

[0191]

[0192] One aspect of the present disclosure includes a method for treating cancer, including drug-resistant, refractory and / or metastatic cancers, comprising administering to the subject a first amount or dose of one or more dinucleotide compounds disclosed herein in a therapeutically effective amount effective to shorten telomere length; reduce tumor size; reduce tumor growth rate; reduce the incidence of metastasis; eliminate metastasis, promote immune response; slow cancer progression; extend the lifespan of the subject; or a combination thereof.

[0193] In embodiments of this aspect, the dinucleotide compounds disclosed herein are administered to a subject in need thereof in combination with one or more other cancer therapies, wherein the combination is effective to shorten telomere length; reduce tumor size; reduce tumor growth rate; reduce the incidence of metastasis; promote immune response; slow cancer progression; extend the lifespan of the subject; or a combination thereof.

[0194] As used herein, the term "subject" refers to a human or non-human, such as a primate, a mammal, and a vertebrate. According to some embodiments, the subject is a human.

[0195] As used herein, the term "a subject in need of such treatment or in need thereof" refers to (i) a patient suffering from cancer; and (ii) a subject to whom a dinucleotide compound or dinucleotide compounds as disclosed herein will be administered.

[0196] As used herein, the term "therapeutically effective dose" refers to a dose (i.e., dosage and frequency of administration) that eliminates, reduces, or prevents the progression of a particular disease manifestation in a certain percentage of a population. An example of a commonly used therapeutic component is ED 50 , which describes a dose at a particular dosage that is effective in treating a particular disease manifestation in 50% of a population.

[0197] As used herein, the term "therapeutic effect" refers to the result of treatment, which is judged to be desirable and beneficial. Therapeutic effect may include directly or indirectly preventing, reducing or eliminating disease manifestations. Therapeutic effect may also include directly or indirectly preventing, reducing or eliminating the progression of disease manifestations.

[0198] The terms "therapeutically effective amount," "effective amount," or "pharmaceutically effective amount" of an active agent are used interchangeably to refer to an amount sufficient to provide the intended benefit of treatment. The effective amount of an active agent that can be employed in accordance with the described invention is generally in the range of about 0.01 mg / kg body weight to about 100 g / kg body weight. However, dosage levels are based on a variety of factors, including the type of injury, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific active agent employed. Thus, dosage regimens can vary widely, but can be routinely determined by a physician using standard methods.

[0199] The terms "treat" or "treating" include eliminating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating the clinical symptoms of a condition, substantially preventing the appearance of clinical symptoms of a disease, condition or disorder, and preventing harmful or bothersome symptoms. Treating further refers to achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of symptoms characteristic of the one or more disorders being treated; (c) limiting the worsening of symptoms characteristic of the one or more disorders being treated; (d) limiting the recurrence of the one or more disorders in a patient previously suffering from the one or more disorders; and (e) limiting the recurrence of symptoms in a patient previously asymptomatic for the one or more disorders.

[0200] According to some embodiments, the cancer treated using the dinucleotide compounds disclosed herein is, but is not limited to, carcinoma, sarcoma, leukemia, lymphoma / myeloma, or brain / spinal cord cancer. According to some embodiments, the cancer comprises a solid tumor comprising tumor cells, a metastatic cancer comprising metastatic tumor cells, or a combination thereof.

[0201] In certain embodiments, cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus. Tumors may include malignant or benign growths.

[0202] Cancer may specifically be of the following histological types, but is not limited to these: malignant neoplasms; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocellular carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial colon polyposis; solid carcinoma; malignant carcinoid tumor; bronchioalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and Follicular adenocarcinoma; nonenclosed sclerosing carcinoma; adrenal cortical cell carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; cerumenic adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary cystadenocarcinoma, severe; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; malignant testicular cell tumor; sertolicell cell carcinoma; malignant Leydig cell carcinoma cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephric carcinoma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma sarcoma); malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondrosarcoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma;protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease disease); Hodgkin's; paragranuloma; small lymphocytic malignant lymphoma; large cell diffuse malignant lymphoma; follicular malignant lymphoma; myeloid granuloma; other specified non-Hodgkin's lymphomas; malignant histiocytic disease; multiple myeloma; mast cell sarcoma; immunoproliferative enteropathy; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. ;

[0203] The anticancer activity of dinucleotides (formulated and administered in PBS solution) was evaluated in vitro in various cancer cell lines according to the commonly used compound cytotoxicity assessment procedure, as described in the following publications: Mender, I., Gryaznov, S., Dikmen, ZG, Wright, WE and Shay, JW (2015). Induction of telomere dysfunctionmediated by the telomerase substrate precursor 6-thio-2′-deoxyguanosine. Cancer Discovery, 5, 82-95; Mender et al., (2020). Telomere Stress Potentiates STING-Dependent Anti-tumor Immunity. Cancer Cell, 38, 400-411.

[0204] Representative dose-dependent anticancer activity data of some dinucleotide compounds disclosed herein are shown in FIG6 and FIG7 .

[0205] Table 1 shows the IC values ​​of compounds 1, 5, 6, 11 and 12 and isomers (Sp and Rp) of compound 11 and isomers (Sp and Rp) of compound 12 tested in human cell lines HEK293 (kidney), Hep3B (liver; childhood HCC), HepG2 (liver; hepatoblastoma), NCI-H23 (lung; adenocarcinoma), H2081 (lung; SCLC) relative to untreated cells. 50 .

[0206] Table 1 - IC values ​​of various dinucleotides disclosed herein in different human cancer cell lines 50 Determination of.

[0207]

[0208]

[0209] Cells were seeded at a density of 10.000 cells per well in 96-well plates, treated with the indicated doses of dinucleotides and incubated for 96 hours prior to analysis.

[0210] FIG6 shows the anticancer activities of some of the disclosed dinucleotides and the IC values ​​reported in Table 1 determined therefrom. 50 Representative dose-response curves of .

[0211] The specificity of the tested dinucleotides for telomerase-positive cancer cells relative to telomerase-negative normal non-cancer cells was confirmed by testing these molecules in normal human BJ fibroblasts - Figure 7. Importantly, these data show that in normal cells, the tested dinucleotides exhibited a higher EC than that observed in cancer cells. 50 There was no significant cytotoxicity at concentrations 100-fold higher than the above value, thus confirming the specificity for telomerase-positive cancer cells.

[0212] Dinucleotide compounds are synthesized using solid phase supported phosphoramidite methods and appropriate sugar ring protected and base protected nucleoside 3'-phosphoramidite building blocks (all commercially available from Glen Research). The cleaved product is cleaved from a universal solid support (Glen Research) and fully deprotected with ammonia, partially concentrated in a vacuum (to remove ammonia), and then purified by RP HPLC. The molecular weight and purity of the final dinucleotide product are confirmed by LC MS.

[0213] Drug formulations and routes of administration

[0214] When clinical use is contemplated, the pharmaceutical composition will be prepared in a form suitable for the intended use. Typically, this will require preparing a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0215] It is generally desirable to use appropriate salts and buffers to stabilize the drug and allow it to be taken up by the target cells. The aqueous composition of the present disclosure comprises an effective amount of a drug dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The expression "pharmaceutically or pharmacologically acceptable" refers to a molecular entity and composition that does not produce adverse reactions, allergic reactions or other unexpected reactions when applied to animals or humans. As used herein, "pharmaceutically acceptable carriers" include solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents that are acceptable for preparing drugs such as drugs suitable for application to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredients of the present disclosure, their use in therapeutic compositions is covered. Supplementary active ingredients can also be incorporated into the composition, provided that they do not inactivate the agents of the composition.

[0216] Active compositions of the present disclosure may include classical pharmaceutical preparations. Administration of these compositions according to the present disclosure may be carried out via any common route, as long as the target tissue is available via the route, but generally includes systemic administration. This includes oral, nasal or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or administration within a tumor or within a tumor region (such as in a tumor vessel). Such compositions will generally be administered with a pharmaceutically acceptable composition, as described above.

[0217] The active compound may also be administered parenterally or intraperitoneally. As an illustration, solutions of the active compound as a free base or a pharmacologically acceptable salt may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under typical storage and use conditions, these preparations typically contain preservatives to prevent the growth of microorganisms.

[0218] The pharmaceutical form suitable for injectable use includes, for example, sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and are fluids for easy injection. The preparation should be stable under manufacturing and storage conditions, and should prevent the contamination of microorganisms such as bacteria and fungi. Suitable solvents or dispersion media may contain, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof and vegetable oils. Suitable fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. The effect of microorganisms can be prevented by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents are preferably included, such as sugar or sodium chloride. The absorption of injectable compositions can be extended by using delayed absorption agents such as aluminum monostearate and gelatin in the composition.

[0219] Sterile injectable solutions can be prepared by incorporating the active compound into a solvent in an appropriate amount together with any other ingredients (e.g., as listed above), followed by filtration sterilization if necessary. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other desired ingredients, such as those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze drying techniques, which produce a powder of one or more active ingredients plus any other desired ingredients from a previously sterile filtered solution thereof.

[0220] The compositions of the present disclosure can generally be formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid, or derived from organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.) (formed with free amino groups of proteins). Salts formed with free carboxyl groups of proteins can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium or iron hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0221] When formulated, the solution is preferably administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation can be easily administered in various dosage forms, such as injectable solutions, drug release capsules, etc. For example, for parenteral administration in an aqueous solution, the solution is usually appropriately buffered, and the liquid diluent is first made isotonic with, for example, sufficient saline or glucose. Such an aqueous solution can be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, as known to those skilled in the art, particularly in view of the present disclosure, a sterile aqueous medium is used. As an illustration, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion liquid or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences" 15th edition, pages 1035-1038 and 1570-1580). Depending on the condition of the subject being treated, some changes in dosage are inevitable. In any case, the person responsible for administration will determine the appropriate dosage for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

[0222] The actual dosage of the composition according to certain embodiments of the present disclosure applied to the subject can be determined by physical and physiological factors such as the specific compound used, the age of the subject, general health, diet, body weight, severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathic diseases of the patient, absorption rate, distribution rate, inactivation rate, excretion rate, administration time, administration route, and the judgment of the personnel supervising the administration. Depending on the dosage and route of administration, the number of administrations and / or effective amount of the preferred dosage may vary according to the response of the subject. The active ingredient may be administered once, or may be divided into multiple smaller doses and administered at different time intervals. Therefore, it should be understood that for any particular subject, the specific dosage regimen may be adjusted over time according to individual needs and the professional judgment of the personnel administering the composition or supervising the administration of the composition, and the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0223] The dinucleotide compounds disclosed herein are included in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver a therapeutically effective amount for the desired indication to the patient without causing serious toxic effects in the treated patient. For all cases mentioned herein, the preferred dosage of the dinucleotide is in the range of about 10 ng / kg to 100 mg / kg, preferably 0.1 to 50 mg / kg per day, more usually 0.5 to about 25 mg / kg of the subject's body weight per day. As a non-limiting example, in a suitable carrier, a typical dose may be in the range of 50.01-20% wt / wt. Similarly, the compound may be administered in any suitable unit dosage form, including but not limited to a unit dosage form containing less than 1 mg, 1 mg to 3000 mg or 5 to 1000 mg of the dinucleotide compounds disclosed herein.

[0224] The composition can be administered continuously or continuously; as needed; or 1, 2, 3, 4, 5, 6, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more, including but not limited to one containing less than 1 mg.

[0225] Combination therapy

[0226] In order to improve the effectiveness of treatment, it may be necessary to combine the composition of the present disclosure with a second treatment or pharmaceutical composition. For example, the method of use may also include administering a second pharmaceutical composition, which comprises an anticancer agent or other agents effective in treating hyperproliferative diseases. Anticancer agents can have a negative impact on the subject's cancer, for example, by killing cancer cells, inducing apoptosis of cancer cells, reducing cancer cell growth rate, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting immune responses to cancer cells or tumors, preventing or inhibiting cancer progression, or extending the life span of cancer subjects. More generally, the second pharmaceutical composition can be administered in an effective amount or a combined effective amount to kill or inhibit the proliferation of certain cells.

[0227] In some embodiments, the method of treatment may include simultaneous co-administration. This method may involve simultaneous or sequential administration. Co-administration can be achieved by contacting the cell with a single composition or pharmaceutical formulation comprising one or more of the dinucleotide compounds disclosed herein and another anticancer agent, or by contacting the cell with two different compositions or formulations simultaneously, wherein one composition comprises one or more of the dinucleotide compounds disclosed herein and the other composition comprises a second agent. Similarly, the two compositions may be administered at different times but close to each other in time, such as on the same day or within the same week.

[0228] In other embodiments, the method of treatment may include a first phase in which a pharmaceutical composition comprising one or more of the dinucleotide compounds disclosed herein is administered and a second phase in which a second pharmaceutical composition is administered. The first phase and the second phase may be sequential in time, spaced apart in time (a few minutes, days, weeks, or months), or overlap in time. Additionally, the order of the treatment phases may be reversed or repeated.

[0229] Of course, any combination of treatment phases may be employed. For example, administration of one or more of the dinucleotide compounds disclosed herein is "A", and treatment with a second agent is "B":

[0230] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

[0231] B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A

[0232] B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0233] In the context of the present disclosure, it is contemplated that administration of a pharmaceutical composition comprising one or more of the dinucleotide compounds disclosed herein may be used in conjunction with treatment B such as gene therapy, chemotherapy, radiotherapy or immunotherapy interventions and other pro-apoptotic or cell cycle modulators. It is also contemplated that various standard therapies as well as surgical interventions may be used in conjunction with the described telomere shortening and telomere dysfunction induction therapies.

[0234] a. Chemotherapy

[0235] Chemotherapy includes, for example, cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, mithramycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, paclitaxel, gemcitabine, norvinblastine, farnesyl protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate or any analog or derivative variant of the foregoing.

[0236] b.Radiotherapy

[0237] Radiation therapy can cause DNA damage and includes what is commonly referred to as X-ray therapy and / or the targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves and UV irradiation.

[0238] c. Immunotherapy

[0239] Immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Therefore, immunotherapy can be used in combination with the administration of a pharmaceutical composition comprising one or more of the dinucleotide compounds disclosed herein as part of a combination therapy. Immunotherapy approaches involve targeting tumor cells through a marker of the tumor cells that is suitable for targeting, i.e., not present on most other cells. There are many tumor markers, and any of these markers may be suitable for targeting with a second treatment approach in the context of the present disclosure. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor-associated antigen, fetal antigen, tyrosinase (p9′7), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155

[0240] d. Surgery

[0241] Radical surgery is a cancer treatment method that can be used in combination with a pharmaceutical composition comprising one or more of the dinucleotide compounds disclosed herein. Radical surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised and / or destroyed. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatment also includes laser surgery, cryosurgery, electrosurgery, and microscope-controlled surgery (Mohs surgery). It is also envisioned that the compositions of the present disclosure can be administered in combination with the removal of superficial cancers, precancers, or incidental amounts of normal tissue. When some or all of the cancer cells, tissues, or tumors are removed, cavities may form in the body. Treatment can be accomplished by administering a pharmaceutical composition comprising one or more of the dinucleotide compounds disclosed herein.

[0242] e. Other anticancer agents

[0243] It is contemplated that other anticancer agents may be used in combination with one or more of the dinucleotide compounds disclosed herein to additively or synergistically enhance the efficacy of the treatment.

[0244] These additional agents include immunomodulators, agents that affect the upregulation of cell surface receptors and gap junctions (GAP junctions), cytostatics and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents. Immunomodulators include tumor necrosis factor; interferon α, β and γ; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1, RANTES and other chemokines. It is also envisioned that upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL will enhance the apoptosis-inducing ability of the present disclosure by establishing an autocrine or paracrine effect on hyperproliferative cells. Increasing intercellular signaling by increasing the number of gap junctions will increase the anti-hyperproliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cytostatics or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of treatment. It is envisioned that cell adhesion inhibitors are used to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are localized focal adhesion kinase (FAK) inhibitors and lovastatin. It is also contemplated that other anticancer agents and signal transduction inhibitors that will increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with one or more of the dinucleotide compounds disclosed herein to improve therapeutic efficacy.

[0245] Finally, additional agents may also include anticancer agents, which are broadly described as antimetabolites, inhibitors of topoisomerases I and II, alkylating agents, and microtubule inhibitors (eg, paclitaxel). Anticancer agents useful in the present invention include, for example, aldesleukin; alemtuzumab; alitretinoin; allopurinol; hexamethylmelamine; amifostine; anastrozole; arsenic trioxide; asparaginase; BCG Live; bexarotene capsules; bexarotene gel; bleomycin; busulfan intravenous injection; busulfan oral; carlostenolone; capecitabine; carboplatin; carmustine; carmustine with Polifeprosan 20 implant; celecoxib; chlorambucil; cisplatin; cladribine; cyclophosphamide; cytarabine; cytarabine liposome; dacarbazine; dactinomycin; actinomycin D; darbepoetin alfa; daunorubicin liposome; daunorubicin, daunorubicin; denileukin, dexrazoxane; docetaxel; doxorubicin; doxorubicin liposome; drostanolone propionate; Elliott's B solution; epirubicin; epoetin alfa estramustine; etoposide phosphate; etoposide (VP-16); exemestane; filgrastim; floxuridine (intra-arterial); fludarabine; fluorouracil (5-FU); fulvestrant; gemtuzumab ozogamicin; goserelin acetate; hydroxyurea; ibritumomab tiuxetan; idarubicin; ifosfamide; imatinib mesylate; interferon alfa-2a; interferon alfa-2b; irinotecan; letrozole; folinic acid; levamisole; lomustine (CCNU); chlorambucil (nitrogen mustard); megestrol acetate; melphalan (L-PAM); mercaptopurine (6-MP); mesna; methotrexate; methoxsalen; mitomycin C; m Totane; mitoxantrone; nandrolone phenylpropionate; nofitumomab; LOddC; oprelvekin; oxaliplatin; paclitaxel; pamidronate; pegaspargase; pegfilgrastim; pentostatin; pipobroman; plicamycin; mithramycin; porfimer sodium; procarbazine; quinacrine; rasburicase; rituximab; sargramostim; streptozotocin; tapavidine (LDT); talc; tamoxifen; temozolomide; teniposide (VM-26); testolactone; thioguanine (6-TG); thiotepa; topotecan; toremifene; tositumomab; trastuzumab; retinoic acid (ATRA); uracil mustard; valrubicin; vatocitabine (monoval LDC); vinblastine; vinorelbine; zoledronic acid; and mixtures thereof.

[0246] Hormonal therapy can also be used in conjunction with the administration of the pharmaceutical composition and can be used in conjunction with one or more of the dinucleotide compounds disclosed herein. The use of hormones can be used to treat certain cancers, such as breast cancer, prostate cancer, ovarian cancer, or cervical cancer, to reduce the level of certain hormones (such as testosterone or estrogen) or block their effects. This treatment is often used in conjunction with at least one other cancer therapy as a treatment option or to reduce the risk of metastasis.

[0247] The present disclosure provides sequential cancer treatments using dinucleotide compounds disclosed herein followed by PD-L 1, PD-1 and / or CTLA-4 therapy. The cycle of each treatment may be different, and it is expected that a short interval between treatments will be advantageous. For example, for dinucleotide compounds disclosed herein, treatment may be as short as 2 days, but may be 3 days, 4 days or more days, including 2-4 days. The interval before PD-L1, PD-1 and / or CTLA-4 treatment should be at least one day, and may be up to 14 days, such as 2-4 days. Due to the potential adverse effects of dinucleotide compounds disclosed herein on activated effector T cells, it may be necessary to avoid overlap between dinucleotide compounds disclosed herein and PD-L 1, PD-1 and / or CTLA-4.

[0248] The daily dosage of the dinucleotide compounds disclosed herein will be between 0.5 mg / kg and 10 mg / kg, preferably administered intravenously or orally. The dosage of PD-L1, PD-1 and / or CTLA-4 will be consistent with the current approved dosing schedule.

[0249] In the context of the present disclosure, it is also contemplated that the dinucleotide compounds disclosed herein and anti-PD-L 1 such as atezolizumab or the dinucleotide and anti-PD-1 such as Or anti-CTLA-4 can be used in combination with chemotherapeutic or radiotherapeutic intervention or other treatments. In particular, combining the dinucleotide compounds disclosed herein, anti-PD-L1, anti-PD-1 or anti-CTLA-4 with other therapies targeting different aspects of cancer cell function may also prove effective.

[0250] In order to kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells using the methods and compositions of the present disclosure, the "target" cells will generally be contacted with the dinucleotide compounds disclosed herein and at least one other agent. These compositions will be provided in an order or combination amount effective for killing or inhibiting cell proliferation. This method may involve contacting cells with dinucleotides / anti-PD-L 1, anti-PD-1 or anti-CTLA-4 and other one or more agents or one or more factors simultaneously. This can be achieved by contacting cells with a single composition or pharmacological formulation comprising two agents or by contacting cells with two different compositions or formulations simultaneously, wherein one composition comprises an interferon prodrug according to the present disclosure and the other composition comprises another agent.

[0251] Alternatively, dinucleotide / anti-PD-L 1, anti-PD-1 or anti-CTLA-4 therapy may be performed before or after treatment with another agent, with intervals ranging from minutes to weeks. In embodiments in which another agent and an interferon prodrug are applied to cells separately, it is generally ensured that there is no long interval between each delivery, so that the agent and the expression construct are still able to produce a favorable combination effect on the cell. In such a case, it is envisaged that the cells are contacted in two ways within about 12-24 hours of each other, more preferably within about 6-12 hours of each other, and the most preferred delay time is only about 12 hours. In some cases, it may be necessary to significantly extend the time period of treatment, however, wherein several days (2, 3, 4, 5, 6 or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8 weeks) are passed between each administration.

[0252] It is also contemplated that more than one administration of an interferon prodrug or other agent will be required. Various combinations may be employed in which the dinucleotide compound disclosed herein / anti-PD-L1, anti-PD-1 or anti-CTLA-4 therapy is "A" and the other therapy is "B", as exemplified below:

[0253] A / B / AB / A / BB / B / AA / A / BB / A / AA / B / BB / B / B / AB / B / A / B

[0254] A / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BB / B / B / A

[0255] A / A / A / BB / A / A / AA / B / A / AA / A / B / AA / B / B / BB / A / B / BB / B / A / B

[0256] Other combinations are contemplated. Likewise, to achieve cell killing, the two agents are delivered to the cells in a combined amount effective to kill the cells.

[0257] Agents or factors suitable for cancer therapy include any chemical compound or treatment method that will induce DNA damage when applied to cells. Such agents and factors include radiation and waves that induce DNA damage, such as irradiation, microwaves, electron emission, etc. Various chemical compounds that are also described as "chemotherapeutic agents" or "genotoxic agents" can be used. This can be achieved by irradiating the local tumor site; alternatively, tumor cells can be contacted with the agent by administering a therapeutically effective amount of a pharmaceutical composition to the subject.

[0258] The present disclosure contemplates the use of various classes of chemotherapeutic agents. Other chemotherapeutic agents include selective estrogen receptor antagonists ("SERM"), such as tamoxifen, 4-hydroxytamoxifen (Afimoxfene), Falsodex, raloxifene, bazedoxifene, clomiphene, Femarelle, lasofoxifene, ormeloxifene and toremifene. Medicament camptothecin, actinomycin D and mitomycin C are conventional chemotherapeutic drugs. The present disclosure also encompasses the use of a combination of one or more DNA damaging agents, whether based on radiation or actual compounds, such as using X-rays together with cisplatin or using cisplatin together with etoposide. The medicament can be prepared and used as a combined therapeutic composition.

[0259] Heat shock protein 90 is a regulatory protein found in many eukaryotic cells. HSP90 inhibitors have been shown to be useful in the treatment of cancer. Such inhibitors include geldanamycin, 17-(allylamino)-17-demethoxygeldanamycin, PU-H71, and rifabutin.

[0260] Agents that directly crosslink DNA or form adducts are also contemplated. Agents such as cisplatin and other DNA alkylating agents may be used. Cisplatin has been widely used to treat cancer, with an effective dose of 20 mg / m used in clinical applications. 2 , administered for 5 days every three weeks for a total of three courses. Cisplatin cannot be absorbed orally and therefore must be delivered via intravenous, subcutaneous, intratumoral or intraperitoneal injection.

[0261] Agents that damage DNA also include compounds that interfere with DNA replication, mitosis, and chromosome segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, etc. These compounds are widely used in clinical settings to treat neoplasms. For doxorubicin, 25-75 mg / m2 is administered at 21-day intervals. 2 The dose range for etoposide is 35-50 mg / m 2 Oral administration of twice the intravenous dose. Microtubule inhibitors, such as taxanes, are also contemplated. These molecules are diterpenes produced by plants of the genus Taxus and include paclitaxel and docetaxel.

[0262] Epidermal growth factor receptor inhibitors, such as Iressa, mTOR, mammalian target of rapamycin (also known as FK506-binding protein 12-rapamycin associated protein 1 (FRAP1)), are serine / threonine protein kinases that regulate cell growth, cell proliferation, cell motility, cell survival, protein synthesis and transcription. Therefore, according to the present disclosure, rapamycin and its analogs ("rapamycin analogs") are contemplated for use in cancer treatment. Another EGFR inhibitor that is particularly useful herein is gefitinib.

[0263] Another possible therapy is TNF-α (tumor necrosis factor-α), which is a cytokine involved in systemic inflammation and a member of a group of cytokines that stimulate the acute phase response. The main effect of TNF is to regulate immune cells. TNF can also induce apoptotic cell death, induce inflammation, and inhibit tumorigenesis and viral replication.

[0264] Agents that destroy the synthesis and fidelity of nucleic acid precursors and subunits can also cause DNA damage. Therefore, many nucleic acid precursors have been developed. Particularly useful are agents that have been extensively tested and easily available. Therefore, agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissues, making this agent particularly suitable for targeting neoplastic cells. Although the toxicity is quite large, 5-FU is applicable to a wide range of carriers, including topical administration, however, it is usually administered intravenously at a dosage of 3 to 15 mg / kg / day.

[0265] Other factors that will cause DNA damage and have been widely used include those factors commonly referred to as gamma-rays, x-rays and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors, such as microwaves and UV irradiation, are also envisioned. Most likely, all of these factors cause extensive damage to DNA, DNA precursors, replication and repair of DNA, and assembly and maintenance of chromosomes. The dosage range of x-rays is in the range of a single dose of 2000 to 6000 roentgens for a long time (3 to 4 weeks) from 50 to 200 roentgens daily doses. The dosage range of radioisotopes varies greatly and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the uptake by the neoplastic cells.

[0266] Additionally, it is contemplated that various immunotherapies, hormone therapies, toxin therapies, and / or surgery may be used.

[0267] The skilled artisan will refer to Chapter 33 of "Remington's Pharmaceutical Sciences", 15th edition, especially pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dosage for the individual subject. In addition, for human administration, the preparation should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Products standards.

[0268] The following examples are included to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes may be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining the same or similar results.

[0269] experiment:

[0270] Table 2 - Abbreviations

[0271] ACN Acetonitrile CAS Chemical Abstracts Service conc. concentration Da Dalton g gram h Hour HPLC High performance liquid chromatography M Mole / L min minute mM mmol / L MW Molecular weight Na+ Sodium ion QC Quality Control RT Room temperature TEA+ triethylammonium ion TEAA Triethylammonium acetate UV UV Wrt about

[0272] Method Overview:

[0273] The production of the disclosed dinucleotides involves the following method steps:

[0274] 1. Solid phase synthesis was performed on an H8-custom solid phase synthesizer; a universal linker-CPG was used as a solid support.

[0275] 2. Dissociation and deprotection.

[0276] 3. Purification was performed by reverse phase (RP) HPLC (column: Waters Xbridge C18 (19 mm×50 mm, 14.2 mL); eluent A: 100 mM TEAA / water, eluent B: 100 mM TEAA / 25% ACN-water, as eluent system).

[0277] 4. If deemed necessary: ​​perform buffer exchange by RP-HPLC (column: Waters Xbridge C18 (19 mm x 50 mm, 14.2 mL); eluent A: water, eluent B: ACN, as eluent system).

[0278] 5. Lyophilization cycles followed by final weight measurement after 2 cycles (final amount).

[0279] Materials and general procedure:

[0280] Building blocks and solid supports

[0281] The phosphoramidite building blocks and CPG-solid supports used in the solid phase synthesis are summarized in Table 3 below.

[0282] Table 3: Building blocks and solid supports used in solid phase synthesis.

[0283]

[0284]

[0285]

[0286] Solvents and auxiliary reagents:

[0287] All solvents and auxiliary reagents used in these projects are summarized in Table 4.

[0288] Table 4: Solvents, reagents and auxiliary materials used in solid phase synthesis and cleavage / deprotection steps.

[0289]

[0290]

[0291] Solid Phase Synthesis of Dinucleotides:

[0292] The synthesis was performed on an automated solid phase synthesizer using standard phosphoramidite chemistry according to conventional solid phase oligonucleotide synthesis procedures. The H8-Custom synthesizer (K&A ) for solid phase synthesis.

[0293] The phosphoramidite was dissolved in dry acetonitrile to obtain a 0.1 M solution, which was then concentrated over molecular sieves ( ) for at least two hours and then loaded into the pre-set phosphoramidite port on the H8 synthesizer.

[0294] As activating agent, 0.5 M 5-ethylthio-1H-tetrazole (ETT) in ACN was used and passed through molecular sieves before the start of the synthesis. Dry for at least 24 hours. As the sulfurization reagent, 3-amino-1,2,4-dithiazole-5-thione (or xanthan gum hydride; 100 mM in ACN-pyridine (2:3 v / v)) was prepared freshly before use.

[0295] All other auxiliary reagents for solid phase synthesis were used as received from the corresponding commercial sources (Table 5).

[0296] During the synthesis cycle, a standard oxidation step was used, followed by an end-capping step. All dinucleotides were synthesized in DMT-OFF mode. The final end-capping step was omitted for syntheses involving phosphate-ON reagents. A detailed list of all raw materials, auxiliary reagents and solvents and their suppliers and product numbers is listed in Tables 3 and 4.

[0297] Dissociation and deprotection:

[0298] After the synthesis is completed, the dinucleotides still fixed on the solid support are treated with DBU at ambient temperature for four hours to ensure the removal of the cyanoethyl protecting group, especially from the 6-thio position of 6SdG or 6SG and the 4-thio position of the 4TdT nucleotide unit. Thereafter, the product with the CPG-solid support is subsequently dried at room temperature using a vacuum pump, and the resulting dry CPG in each case is transferred to a 15mL-Falcon tube. AMA (methylamine aqueous solution (41 wt%) + NH3 (30%, high purity) water is premixed at 1:1 v / v) solution (3mL) is added to each tube, and the tube is shaken at 30°C for three hours. This achieves the dissociation of the dinucleotide from the solid support, the dephosphorylation of the single linker at the 3'-end to produce a free 3'-OH, and the deprotection of the 2-NH2 position of 6SdG and 6SG by removing the trifluoroacetyl and isobutyryl protecting groups. In the case of a dinucleotide containing 6SG, 2'-O-TBDMS deprotection is then performed by adding TEA·3HF to the deprotection mixture, and the mixture is then shaken at 45°C for another 90 minutes. Finally, the dinucleotide dissolved in the supernatant solution is separated from the solid support by centrifugation (using ThermoScientific's HERAEUS MultifugeX3F) and then decantation. The residual CPG in each tube is washed with water to collect any remaining solution containing the dinucleotide. The combined liquid phase is concentrated in vacuo. The residue is diluted with TEAA buffer solution (100mM TEAA in water) for subsequent HPLC purification.

[0299] Purification by RP-HPLC:

[0300] An RP Xbridge C18 column from Waters was used. Purification was performed on a pure 150 HPLC system. Details of the process are listed below and Table 5 summarizes the materials used for purification and subsequent desalting steps.

[0301] Pure 150HPLC System

[0302] Reversed Phase Chromatography:

[0303] Column - X-Bridge C18 (19 mm x 50 mm, 14.2 mL);

[0304] Eluent A: 100 mM TEAA in water

[0305] Eluent B: 100 mM TEAA in ACN-water mixture (25% ACN / water)

[0306] Table 5: Solvents, reagents and auxiliary materials used for RP-HPLC purification and desalting.

[0307]

[0308] Desalting:

[0309] In the case of samples intended for in vivo studies, the counter cation TEA is exchanged with + Replaced with Na from purified material + After removal of the RP-HPLC eluent buffer by evaporation and subsequent lyophilization, the purified material was reconstituted in 500 mM NaCl solution and Buffer exchange was performed by RP-HPLC using a Waters X-Bridge C18 column (19 mm x 50 mm, 14.2 mL) on a pure 150. The details are listed below and the materials used are summarized in Table 5.

[0310] Pure 150

[0311] Reversed Phase Chromatography:

[0312] Column - X-Bridge C18 (19x50mm; 14.2mL);

[0313] Eluent A: Water

[0314] Eluent B: 95% ACN-water

[0315] Thereafter, the acetonitrile-water was removed in vacuo and the aqueous dinucleotide solution was lyophilized.

[0316] Lyophilization and quantification:

[0317] Oligonucleotide solution is distributed in Falcon tube and freeze-dried (using CHRIST Epsilon 1-4LSCplus freeze dryer). After the first freeze drying cycle, the remaining dinucleotide is redissolved in water and freeze-dried again. Repeat the procedure until constant weight is reached. Before the final freeze drying step, the sufficient amount of subpackage is used for quality control testing. After completing the freeze drying step, the dried material is quantitatively weighed.

[0318] QC Analysis:

[0319] For QC release testing, UV-based RP-chromatographic traces of dinucleotides were recorded at 260nm and 350nm (characteristic absorption maxima of 6SG-nucleobases; NF Krynetskaia, X. Cai, JL Nitiss, EY Krynetski, MV, Relling, FASEB J. 2000, 14, 2339). While reporting these two values, the traces at 350nm were also monitored and integrated as agreed with the sponsor for final purity determination. To confirm the purity and identity of the dinucleotides, an Ultimate 3000 HPLC system (Thermo Scientific) equipped with an Acquity UPLC oligonucleotide C18 column (2.1mm x 100mm, 0.37mL) from Waters was performed.

[0320] Data acquisition and integration of HPLC chromatograms were performed using the software package Chromeleon from Thermo Fisher Scientific. For each sample, peaks were integrated by vertical peak segmentation. In the case of overlapping peaks, segmentation was performed at the minimum between the two and their identities were checked from the associated MS-spectra.

[0321] The monoisotopic molecular weight of the sample was analyzed using ESI / MS detection. For this purpose, the Ultimate 3000HPLC system from BrukerDaltonics was used, which was equipped with a UV detector and a qTOF detector (Compact). The MS spectrum was extracted in the total ion current (TIC) using Bruker Hystar data analysis software. The identities of all dinucleotides were also determined within the specifications reached.

[0322] Syngeneic Mouse Models:

[0323] A) Tumor Type: H22 Murine Model of Hepatocellular Carcinoma (Syngeneic Mouse Model)

[0324] Methods: H22 tumor cells (1×106) were subcutaneously inoculated in the right flank of female BALB / c mice to establish a syngeneic subcutaneous H22 tumor model. When the average tumor volume reached 70-100 mm3, the mice were randomly divided into 6 groups based on tumor volume and body weight: control group (vehicle, intravenous, once a day (QD) on days 0, 1, 2, 7, 8, and 9), MAIA-2022-12 group (MAIA-2022-12, intravenous, 6 mg / kg, once a day on days 0, 1, 2, 7, 8, and 9), MAIA-2021-20 group (MAIA-2021-20, intravenous, 6 mg / kg, once a day on days 0, 1, 2, 7, 8, and 9), anti-PD-1 group (anti-PD-1, intraperitoneal, 10 mg / kg g, once a day on days 4, 12, and 20), MAIA-2022-12 combination group (MAIA-2022-12, intravenous, 6 mg / kg, once a day on days 0, 1, 2, 7, 8, and 9; anti-PD-1, intraperitoneal, 10 mg / kg, once a day on days 4, 12, and 20), MAIA-2021-20 combination group (MAIA-2021-20, intravenous, 6 mg / kg, once a day on days 0, 1, 2, 7, 8, and 9; anti-PD-1, intraperitoneal, 10 mg / kg, once a day on days 4, 12, and 20), 8 mice in each group. The dose volume was 10 μL / g. Tumor volume calculation and survival analysis were used to evaluate drug efficacy, and body weight change and animal death were used to evaluate drug safety.

[0325] B) Tumor type: M38 murine model of colorectal carcinoma (syngeneic mouse model)

[0326] Methods: MC38 tumor cells (4×104) were subcutaneously inoculated in the right flank of female C57BL / 6N mice to establish a homologous subcutaneous MC38 tumor model. When the average tumor volume reached 70-100 mm3, the mice were randomly divided into 6 groups based on tumor volume and body weight: control group (vehicle, intravenous, once a day (QD) on days 0, 1, 2, 7, 8, and 9), MAIA-2022-12 group (MAIA-2022-12, intravenous, 3 mg / kg, once a day on days 0, 1, 2, 7, 8, and 9), MAIA-2021-20 group (MAIA-2021-20, intravenous, 3 mg / kg, once a day on days 0, 1, 2, 7, 8, and 9), anti-PD-1 group (anti-PD-1, peritoneal The mice were divided into two groups: the first group (MAIA-2022-12, intravenous, 3 mg / kg, once a day on day 0, day 1, day 2, day 7, day 8, day 9; anti-PD-1, intraperitoneal, 10 mg / kg, once a day on day 4 and day 12), the second group (MAIA-2021-20, intravenous, 3 mg / kg, once a day on day 0, day 1, day 2, day 7, day 8, day 9; anti-PD-1, intraperitoneal, 10 mg / kg, once a day on day 4 and day 12), and the third group (MAIA-2021-20, intravenous, 3 mg / kg, once a day on day 0, day 1, day 2, day 7, day 8, day 9; anti-PD-1, intraperitoneal, 10 mg / kg, once a day on day 4 and day 12), with 8 mice in each group. The dose volume was 10 μL / g. Tumor volume calculation and survival analysis were used to evaluate drug efficacy, and body weight change and animal death were used to evaluate drug safety.

[0327] c) Tumor type: YUMM1.7 murine model of melanoma (syngeneic mouse model)

[0328] Methods: YUMM1.7 tumor cells (2×105) were subcutaneously inoculated into the right flank of female C57BL / 6N mice to establish a syngeneic subcutaneous YUMM1.7 tumor model. When the average tumor volume reached 70-100 mm3, the mice were randomly divided into 3 groups based on tumor volume and body weight: control group (vehicle, intraperitoneal, once a day on day 0, day 1, day 2, day 9, day 10, day 11), MAIA-2022-12 group (MAIA-2022-12, intraperitoneal, 6 mg / kg, once a day on day 0, day 1, day 2, day 9, day 10, day 11), MAIA-2021-20 group (MAIA-2021-20, intraperitoneal, 6 mg / kg, once a day on day 0, day 1, day 2, day 9, day 10, day 11), with 8 mice in each group. The dose volume was 10 μL / g. Tumor volume calculation and survival analysis were used to evaluate drug efficacy, and body weight change and animal death were used to evaluate drug safety.

[0329] d) Re-stimulation of YUMM1.7

[0330] Methods: Four mice in the MAIA-2022-12 group and six mice in the MAIA-2021-020 group were tumor-free in the initial experiment (see Slide 7 for treatment schedule and results). Re-challenge of YUMM1.7 was performed in these tumor-free mice to see if the mice gained immune protection after treatment. 46 days after the last treatment for tumor development, YUMM1.7 tumor cells (2×105) were inoculated subcutaneously in the left flank of female C57BL / 6N mice (10 mice in the control group, 4 mice in the MAIA-2022-12 group, and 6 mice in the MAIA-2021-20 group). One week after tumor inoculation, tumor development in mice began to be measured (2-3 times a week). Tumor volume calculations were used to see if mice formed tumors after inoculation and weight changes and animal deaths were used to evaluate drug safety.

Claims

1. A compound comprising a structure of Formula I or a pharmaceutically acceptable salt thereof: wherein R is independently H or OH, and X is O or S.

2. The compound according to claim 1, wherein X is O.

3. The compound according to claim 2, wherein the compound has the following structure:

4. The compound according to claim 2, wherein the compound has the following structure:

5. The compound according to claim 2, wherein the compound has the following structure: The compound according to claim 1 , wherein X is S.

7. The compound according to claim 6, wherein the compound has the following structure:

8. The compound according to claim 6, wherein the compound has the following structure:

9. The compound according to claim 6, wherein the compound has the following structure:

10. A compound having the following structure:

11. A compound having the following structure:

12. A compound having the following structure:

13. A compound having the following structure:

14. A compound having the following structure:

15. A compound having the following structure:

16. A compound comprising a structure of Formula II or a pharmaceutically acceptable salt thereof: in, R1 and R2 are independently H or OH; X is O or S; Y is O; and R3 is selected from the group consisting of a phosphate group, a thiophosphate group, palmitic acid, tocopherol and a cholesterol group.

17. The compound according to claim 16, wherein: R1 and R2 are independently H or OH, X is O, Y is O, and R3 is a cholesterol group.

18. The compound according to claim 17, which has the following structure:

19. The compound according to claim 17, which has the following structure:

20. The compound according to claim 17, which has the following structure:

21. The compound according to claim 17, which has the following structure:

22. The compound according to claim 16, wherein: R1 and R2 are independently H or OH, X and Y are O, and R3 is a phosphate group.

23. The compound according to claim 22, which has the following structure:

24. The compound according to claim 22, which has the following structure:

25. The compound according to claim 22, which has the following structure:

26. The compound according to claim 22, which has the following structure:

27. The compound according to claim 16, wherein: R1 and R2 are independently H or OH, X and Y are independently O, and R3 is a phosphorothioate group.

28. The compound according to claim 27, which has the following structure:

29. The compound according to claim 27, which has the following structure:

30. The compound according to claim 27, which has the following structure:

31. The compound according to claim 27, which has the following structure:

32. The compound according to claim 16, wherein: R1 and R2 are independently H or OH; X and Y are independently O, and R3 is palmitic acid.

33. The compound according to claim 32, which has the following structure:

34. The compound according to claim 32, having the following structure:

35. The compound of claim 32, having the structure:

36. The compound of claim 32, having the structure:

37. The compound according to claim 16, wherein: R1 and R2 are independently H or OH, X and Y are independently O, and R3 is tocopherol.

38. The compound of claim 37, having the structure:

39. The compound according to claim 37, which has the following structure:

40. The compound of claim 37, having the structure:

41. The compound of claim 37, having the structure:

42. The compound of claim 37, having the structure:

43. The compound according to claim 16, wherein: R1 and R2 are independently H or OH, X is S, Y is O, and R3 is a cholesterol group.

44. The compound of claim 43, having the structure:

45. The compound of claim 43, having the structure:

46. ​​The compound of claim 43, having the structure:

47. The compound of claim 43, having the structure:

48. The compound according to claim 16, wherein: R1 and R2 are independently H or OH, X is S, Y is O, and R3 is a phosphate group.

49. The compound of claim 48, having the structure:

50. The compound of claim 48, having the structure:

51. The compound of claim 48, having the structure:

52. The compound of claim 48, having the structure:

53. The compound according to claim 16, wherein: R1 and R2 are independently H or OH, X is S, Y is O, and R3 is a phosphorothioate group.

54. The compound of claim 53, having the structure:

55. The compound of claim 53, having the structure:

56. The compound of claim 53, having the structure:

57. The compound of claim 53, having the structure:

58. The compound of claim 16, wherein: R1 and R2 are independently H or OH, X is S, Y is O, and R3 is palmitic acid.

59. The compound of claim 58, having the structure:

60. The compound of claim 58, having the structure:

61. The compound of claim 58, having the structure:

62. The compound of claim 58, having the structure:

63. The compound of claim 16, wherein: R1 and R2 are independently H or OH, X is S, Y is O, and R3 is tocopherol.

64. The compound of claim 63, having the structure:

65. The compound of claim 63, having the structure:

66. The compound of claim 63, having the structure:

67. The compound of claim 63, having the structure:

68. The compound of claim 63, having the structure:

69. A compound comprising a structure of Formula III or a pharmaceutically acceptable salt thereof: wherein X is O or S, and R' is independently H or OH.

70. The compound of claim 69, having the structure:

71. The compound of claim 69, having the structure:

72. The compound of claim 69, having the structure:

73. The compound of claim 69, having the structure:

74. The compound of claim 69, having the structure:

75. The compound of claim 69, having the structure:

76. The compound of claim 69, having the structure:

77. The compound of claim 69, having the structure:

78. A compound comprising a structure of Formula IV or a pharmaceutically acceptable salt thereof: wherein X is O or S, and R' is independently H or OH.

79. The compound of claim 78, having the structure:

80. The compound of claim 78, having the structure:

81. The compound of claim 78, having the structure:

82. The compound of claim 78, having the structure:

83. The compound of claim 78, having the structure:

84. The compound of claim 78, having the structure:

85. The compound of claim 78, having the structure:

86. The compound of claim 78, having the structure:

87. A compound or a pharmaceutically acceptable salt thereof having the following structure and stereochemistry:

88. A compound or a pharmaceutically acceptable salt thereof having the following structure and stereochemistry:

89. A compound or a pharmaceutically acceptable salt thereof having the following structure and stereochemistry:

90. A compound or a pharmaceutically acceptable salt thereof having the following structure and stereochemistry:

91. A compound or a pharmaceutically acceptable salt thereof comprising a structure of Formula V, wherein R' is independently H or OH, 92. A compound comprising a structure of Formula VI or a pharmaceutically acceptable salt thereof, wherein R' is independently H or OH 93. A compound comprising a structure of Formula VII, or a pharmaceutically acceptable salt thereof, wherein X is O or S, and R1 and R2 are H or OH, 94. The compound of claim 95, having the structure:

95. The compound of claim 95, having the structure:

96. The compound of claim 95, having the structure:

97. The compound of claim 95, having the structure:

98. The compound of claim 95, having the structure:

99. The compound of claim 95, having the structure:

100. A pharmaceutical composition comprising a compound according to any one of claims 1-101.

101. A pharmaceutical formulation comprising a compound according to any one of claims 1-101 and at least one pharmaceutically acceptable excipient.

102. A method for preparing a pharmaceutical formulation according to claim 103, said method comprising combining a compound according to any one of claims 1-99 with at least one pharmaceutically acceptable excipient.

103. A method of treating a subject having cancer, the method comprising administering to the subject a compound according to any one of claims 1-101, wherein the cancer is selected from one or more of 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, pancreatic cancer, thyroid cancer, liver cancer, 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 cancer, nasopharyngeal carcinoma, and peripheral neuroepithelioid tumor.

104. The method of claim 105, wherein administration of any one of the compounds of claims 1-101 is followed by treatment with an immune checkpoint inhibitor.

105. The method of claim 106, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

106. The method of claim 106, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor.

107. The method of claim 106, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor.

108. The method of claim 106, further comprising administering two or more immune checkpoint inhibitors.

109. The method of claim 110, wherein the two or more immune checkpoint inhibitors are selected from one or more CTLA-4 inhibitors and / or one or more PD-1 inhibitors.

110. The method of claim 110, wherein the two or more immune checkpoint inhibitors are selected from one or more CTLA-4 inhibitors and / or one or more PD-L1 inhibitors.

111. The method of claim 105, wherein the compound of any one of claims 1-101 is administered for about 1 to about 5 days per treatment cycle.

112. The method of claim 103, wherein the checkpoint inhibitor is administered for about 1 to about 3 days per treatment cycle.

113. A method of treating a subject having cancer, the method comprising administering to the subject a compound according to any one of claims 1-101 after treatment with radiation therapy, wherein the cancer is selected from one or more of 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, pancreatic cancer, thyroid cancer, liver cancer, 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 cancer, nasopharyngeal carcinoma, and peripheral neuroepithelioid tumor.

114. A method of treating a subject having cancer, the method comprising administering to the subject a compound according to any one of claims 1-101, followed by treatment with radiation therapy, wherein the cancer is selected from one or more of 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, pancreatic cancer, thyroid cancer, liver cancer, 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 cancer, nasopharyngeal carcinoma, and peripheral neuroepithelioid tumor.

115. A method of treating cancer in a subject, the method comprising administering to the subject a compound according to any one of claims 1-101, 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, 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, pancreatic cancer, thyroid cancer, liver cancer, 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 cancer, nasopharyngeal carcinoma and peripheral neuroepithelioid tumor.

116. A method of treating cancer in a subject, the method comprising administering to the subject a compound according to any one of claims 1-101 after treating the subject with radiation, followed by treatment with an immune checkpoint inhibitor, wherein the cancer is selected from one or more of 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, pancreatic cancer, thyroid cancer, liver cancer, 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 cancer, nasopharyngeal carcinoma and peripheral neuroepithelioid tumor.

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