Dinucleotides and their use in the treatment of cancer

Abstract

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

Description

Claim of Priority

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 388,688, entitled Dinucleotides and Their Use in Treating Cancer, filed on July 13, 2022. Field of the Disclosure

[0002] The present invention is in the field of cancer treatment. In particular, cancer treatment using dinucleotide compounds that target telomeres in cancer cells. Background of the Disclosure

[0003] Telomeres are found at both ends of eukaryotic chromosomes. These DNA-protein structures protect the genome from nuclease degradation, unwanted recombination, intrachromosomal fusion, and repair (Shammas, M., (2011), Curr Opin, Clin. Nutr Metab Care, 14(1):28-34). Telomere length shortens with each cell division due to the end replication problem and the absence of telomere maintenance mechanisms (Greider C.W, (1996), Annu Rev Biochem. 65:337-65). However, single-celled eukaryotes, germ cells, and immortalized 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 elongates telomeres by adding TTAGGG repeats to the ends of chromosomes and is expressed in approximately 90% of human tumors but not in most normal cells (Jafri, M.A., et al. Genome Medicine, 2016, 8:69). Therefore, telomerase is an attractive target for developing anticancer therapies. Most therapies targeting telomerase focus on inhibiting telomerase (Andrews LG, Tollefsbol TO. Mol Biol. 2007;405:1-7). However, such inhibitors have not been successful in clinical trials (Zhang G., Shay WS. Oncotarget, 2018, vol 9(88):35803-35804).

[0004] Instead of inhibiting telomerase, other cancer drugs have been developed that incorporate telomerase into telomeres and are used to cause DNA damage, senescence, or crisis in telomerase-positive cancer cells. One such compound is the nucleoside analogue, 6-thio-2'-deoxyguanosine (6-thio-dG). Its incorporation into de novo synthesized telomeres 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 significant tumor shrinkage or growth arrest with minimal side effects in tumor xenograft models (Mender et al., (2018), Neoplasia, 20(8):826-837, Sengupta et al., (2018), Mol Cancer Ther., Jul; (17(7):1504-1514). The most important advantage of this telomere-targeted therapy over direct telomerase inhibitors is that 6-thio-dG does not have a long lag period for tumoricidal effects. Furthermore, while it does not directly inhibit telomerase, it is preferentially recognized by telomerase over other polymerases and incorporated into telomeres, resulting in immediate DNA strand termination. Importantly, its effect is independent of the initial telomere length by hijacking tumor telomerase to create unstable telomeres (Mender et al., (2015) Oncoscience, 2(8):693-695).

[0005] New telomere-targeted compounds useful for treating cancer are disclosed below. Summary of Embodiments

[0006] Disclosed herein are dinucleotide compounds or pharmaceutically acceptable salts thereof that are telomere-targeted compounds useful for treating cancer. In some embodiments, the dinucleotide compounds are telomerase-mediated telomere-targeted compounds useful for treating cancer.

[0007] In some embodiments, the dinucleotide compounds disclosed herein have the structure of Formula I,

Chem.

[0008] When X is O, the dinucleotide compound of Formula I can have the following structure:

Chem.

Chem.

[0009] When X is S, the dinucleotide compound of Formula I can have the following structure:

Chem.

Chem.

[0010] In other embodiments, the dinucleotide compound can have one or more of the following structures:

Chem.

Chem.

Chem.

[0011] Also disclosed herein are enantiomers of Compound 11 having the following stereochemistry:

Chem.

[0012] In another embodiment, it is an enantiomer of compound 12 having the following stereochemistry:

Chem.

[0013] In some embodiments, the dinucleotide compound has the structure of formula II:

Chem.

[0014] 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:

Chem.

[0015] 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:

Chem.

[0016] 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: [Chemistry]

[0017] In yet other embodiments of Formula II, R1 and R2 are 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: [Chemistry]

[0018] 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: [Chemistry] [Chemistry]

[0019] 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: [Chemistry]

[0020] 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: [Chemistry]

[0021] 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:

Chemical Structure

[0022] 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:

Chemical Structure

[0023] 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:

Chemical Structure

Chemical Structure

[0024] In other embodiments, it is a dinucleotide compound containing the structure of Formula III:

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

[0025] In other embodiments, it is a dinucleotide compound containing the structure of formula IV.

Chem.

[0026] In some embodiments, the dinucleotide compound of formula IV has the following structure:

Chem.

Chem.

Chem.

[0027] In other embodiments, it is a dinucleotide compound containing the structure of formula V, wherein R' is independently H or OH

Chem.

[0028] In some embodiments, the dinucleotide compound of formula V has the following structure:

Chem.

Chem.

[0029] In still other embodiments, it is a dinucleotide compound containing the structure of formula VI, wherein R' is independently H or OH.

Chem.

[0030] In some embodiments, the dinucleotide compound of Formula VI has the following structure:

Chem.

Chem.

[0031] In other embodiments, it is a dinucleotide compound comprising the structure of Formula VII, wherein R’ is independently H or OH, and X is S or O.

Chem.

[0032] 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:

Chem.

[0033] 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:

Chem.

[0034] Another aspect of the present disclosure is a pharmaceutical salt of the dinucleotide compound disclosed herein.

[0035] In still other embodiments, a pharmaceutical composition comprising the dinucleotide compound described herein is disclosed herein. In some embodiments, the pharmaceutical formulation may comprise at least one pharmaceutically acceptable excipient.

[0036] Another embodiment disclosed herein is a process for preparing a pharmaceutical formulation comprising combining a dinucleotide compound as contemplated herein with at least one pharmaceutically acceptable excipient.

[0037] In yet other embodiments, a method of treating a subject having cancer is disclosed herein, the method comprising administering to the subject one or more of the dinucleotide compounds contemplated herein, wherein the cancer is selected from one or more of breast cancer, prostate cancer, colon cancer, gastric cancer, esophagus, liver, 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, bladder cancer, hepatoma, colorectal cancer, rectal cancer, uterine cancer, cervical cancer, endometrial cancer, salivary gland cancer, mesothelioma, kidney cancer, vulvar cancer, pancreatic cancer, thyroid cancer, hepatic carcinoma, testicular cancer, skin cancer, melanoma, brain cancer, neuroblastoma, multiple myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Merkel cell carcinoma, Ewing sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelial tumors.

[0038] In certain embodiments, the dinucleotide compounds disclosed herein can be administered in combination with other anti-cancer agents or cancer therapies. The compounds may be administered before, simultaneously with, or following other cancer agents or therapies.

[0039] In some embodiments, following administration of one or more of the disclosed nucleotide compounds, treatment with an immune checkpoint inhibitor is performed. 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.

[0040] In some embodiments, the compounds disclosed herein are administered for about 1 to about 5 days per treatment cycle.

[0041] In yet other embodiments, the checkpoint inhibitor is administered for about 1 to about 3 days per treatment cycle.

[0042] Also disclosed herein are methods of administering the dinucleotide compounds and checkpoint inhibitors disclosed herein in combination with a chemotherapeutic agent, hormone therapy, toxin therapy, surgery, or combinations thereof.

[0043] In some embodiments, the dinucleotides disclosed herein are administered before the checkpoint inhibitor is administered in combination with a chemotherapeutic agent, hormone therapy, toxin therapy, surgery, or combinations thereof.

[0044] Also disclosed herein is a method of treating a subject having cancer, the method 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, esophagus, liver, 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, 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 cancer, Ewing sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelial tumors in a human having cancer.

[0045] Also disclosed herein is a method of treating a subject having cancer, the method comprising administering to the subject one or more of the dinucleotide compounds disclosed herein, followed by treatment with radiation therapy, and administering to the subject a compound according to any one of claims 1 to 96, wherein the cancer is selected from one or more of breast cancer, prostate cancer, colon cancer, gastric cancer, esophagus, liver, 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, 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 cancer, Ewing sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelial tumors.

[0046] In other embodiments, disclosed herein is a method of treating cancer in a subject, the method 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, esophagus, liver, 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, 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 cancer, Ewing sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelial tumors.

[0047] In other embodiments, methods of treating cancer in a subject are disclosed herein, the methods comprising administering to the subject one or more of the dinucleotide compounds disclosed herein after treatment with radiation, 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, esophagus, liver, 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, 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, nasopharyngeal, and peripheral neuroepithelial tumors.

Brief Description of the Drawings

[0048] The following drawings form a 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 combination with the detailed description of the specific embodiments presented herein.

[0049]

Figure 1

Figure 2

Figure 3

Figure 4

Figures 5A - B

Figures 6A - E

Figures 7A - B

Figures 8A - C

Figures 9A - C

Figures 10A - C

Figures 11A - E

[0050] This specification discloses dinucleotide compounds that target and modify the telomeric structure of cancer cells. All of the compounds are linear, acyclic dinucleotides.

[0051] In aspects of the present disclosure, the compounds disclosed herein are dinucleotide molecules containing modified nucleosides that are converted in cells to human telomerase substrates, such as nucleoside-5'-triphosphates. The nucleotides may be linked via a phosphodiester group (a relatively rapidly cleavable bond, Rp) or via a phosphorothioate group (a relatively slowly cleavable bond, Sp). In certain embodiments of this aspect, the general structure of the disclosed compounds having a free 5'-hydroxyl group molecule is a linear, acyclic, dinucleotide. The 5'-3' internucleoside linkage that links the nucleosides is either a phosphodiester (wherein X = O) or a phosphorothioate (wherein X = S). The nucleosides that make up the dinucleotide consist of 6-thio-2'-deoxyguanosine, 6-thio-guanosine, 4-thio-thymidine, 5-fluorouridine. When X = S, it is either a mixture of two Rp isomers and Sp isomers, or either an individual single Rp isomer or Sp isomer (Figure 1).

[0052] In some embodiments of the above aspect, the dinucleotide compound comprises a structure of Formula I or a pharmaceutically acceptable salt thereof.

Chemical formula

[0053] When X is O, the dinucleotide compound of Formula I comprises the following structure or a pharmaceutically acceptable salt thereof:

Chemical formula

[0054] When X is S, the dinucleotide compound of Formula I comprises the following structure or a pharmaceutically acceptable salt thereof:

Chemical formula

[0055] In still other embodiments, the dinucleotide compound comprises the following structure or a pharmaceutically acceptable salt thereof.

Chem.

[0056] Also disclosed herein are enantiomers of Compound 11 having the following stereochemistry:

Chem.

[0057] In another embodiment, it is an enantiomer of Compound 12 having the following stereochemistry:

Chem.

[0058] In another aspect, disclosed herein are dinucleotide compounds of Formula II having derivatized 5'-hydroxyl groups, such as, but not limited to, 5'-conjugated lipids, 5'-phosphate, or 5'-thio-phosphate groups, or pharmaceutically acceptable salts thereof (Figure 2).

Chem.

[0059] 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:

Chem.

[0060] 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:

Chemical formula

[0061] 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:

Chemical formula

[0062] In still other embodiments of Formula II, R1 and R2 are 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:

Chemical formula

[0063] 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:

Chemical formula

Chemical formula

[0064] 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:

Chemical Formula

[0065] 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:

Chemical Formula

[0066] 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:

Chemical Formula

[0067] 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:

Chemical Formula

[0068] 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:

Chemical Formula

[0069] In certain embodiments, the structure of the disclosed dinucleotide compounds has a free 3'-hydroxyl group. All molecules are linear, acyclic dinucleotides. The 5'-5' internucleoside linkage connecting the nucleosides is either a phosphodiester (where X = O) or a phosphorothioate (where X = S). The nucleosides that make up the dinucleotide consist of 6-thio-2'-deoxyguanosine, 6-thioguanosine, and 5-fluorouridine. When X = S, it can be either a mixture of the two Rp isomers and Sp isomers, or an individual single Rp isomer or Sp isomer (Figure 3).

[0070] In an embodiment of this aspect, it is a dinucleotide compound having the structure of Formula III or a pharmaceutically acceptable salt thereof: [Chemical formula] 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: [Chemical formula] [Chemical formula] [Chemical formula]

[0071] In other embodiments, it is a dinucleotide compound having the structure of Formula IV. [Chemical formula] Wherein X is O or S, and R' is independently H or OH.

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

[0073] In other embodiments, the general structure of the disclosed dinucleotide compound has a free 5'-hydroxyl group. All molecules are linear, acyclic dinucleotides. The 3'-3' internucleoside linkage connecting the nucleosides is either a phosphodiester (where X = O) or a phosphorothioate (where X = S). The nucleosides constituting the dinucleotide are 6-thio-2'-deoxyguanosine, 6-thio-guanosine, and 5-fluorouridine. When X = S, it can be either a mixture of two Rp isomers and Sp isomers, or an individual single Rp isomer or Sp isomer (Figure 4).

[0074] The compound representing Figure 4 is a dinucleotide compound having the structure of Formula V or a pharmaceutically acceptable salt thereof, wherein R' is independently H or OH: [Chemical Formula]

[0075] In still other embodiments representing Figure 4, it is a dinucleotide compound having the structure of Formula VI or a pharmaceutically acceptable salt thereof, wherein R' is independently H or OH: [Chemical Formula]

[0076] In other embodiments, it is a dinucleotide compound having the structure of Formula VII, wherein R1 or R2 is independently H or OH, and X is S or O. [Chemistry]

[0077] In some embodiments, the dinucleotide compound of Formula VII has the following structure, where X is O, and R1 and R2 are H or OH: [Chemistry]

[0078] In some embodiments, the dinucleotide compound of Formula VII has the following structure, where X is S, and R1 and R2 are H or OH: [Chemistry]

[0079] One aspect of the present disclosure includes a method for treating cancer, including resistant, refractory, and / or metastatic cancer, the method comprising administering to a subject a first amount or dose of one or more of the 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 an immune response, reduce cancer progression, extend the lifespan of the subject, or combinations thereof.

[0080] In embodiments of this aspect, the dinucleotide compounds disclosed herein are administered to a subject in combination with one or more other cancer therapies, the combination being effective to shorten telomere length, reduce tumor size, reduce tumor growth rate, reduce the incidence of metastasis, promote an immune response, reduce cancer progression, extend the lifespan of the subject, or combinations thereof.

[0081] As used herein, the term "subject" refers to any of a human or non-human, such as a primate, mammal, and vertebrate. According to some embodiments, the subject is human.

[0082] As used herein, the term "subject in need of or requiring treatment" refers to (i) a patient suffering from cancer, and (ii) a subject to whom a dinucleotide compound or dinucleotide compounds disclosed herein are administered.

[0083] As used herein, the term "therapeutically effective dose" refers to the dose (i.e., the administered dose and the dosing frequency) that eliminates, reduces, or prevents the progression of a particular disease symptom in a proportion of a population. Examples of commonly used therapeutic components are ED 50 which is a specific administered dose that is therapeutically effective against a particular disease symptom in 50% of the population and describes the dose.

[0084] As used herein, the term "therapeutic effect" refers to the treatment result, and the result is determined to be desirable and beneficial. The therapeutic effect can directly or indirectly include the cessation, reduction, or elimination of disease symptoms. The therapeutic effect can also directly or indirectly include the cessation, reduction, or elimination of the progression of disease symptoms.

[0085] 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 the active agent that can be used according to the present invention described can generally range from about 0.01 mg per kg of body weight to about 100 g per kg of body weight. However, the dosing level is based on various factors including the type of injury, the age, weight, sex, medical condition, severity of the condition, route of administration, and the particular active agent used. Accordingly, the dosing regimen can vary widely but can be routinely determined by a physician using standard methods.

[0086] The terms "treating" or "treatment" include inhibiting, substantially inhibiting, delaying or reversing the progression of a disease, condition or disorder, substantially improving the clinical symptoms of a condition, substantially preventing the appearance of clinical symptoms of a disease, condition or disorder, and protecting from adverse or bothersome symptoms. Treating further refers to achieving one or more of the following: (a) reducing the severity of a disorder, (b) limiting the manifestation of symptoms characteristic of the disorder being treated, (c) limiting the worsening of symptoms characteristic of the disorder being treated, (d) limiting the recurrence of a disorder in a patient who has previously had the disorder, and (e) limiting the recurrence of symptoms in a patient who was previously asymptomatic for the disorder.

[0087] 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 includes solid tumors containing tumor cells, metastatic cancers containing metastatic tumor cells, or combinations thereof.

[0088] In certain embodiments, the cancer can occur in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, rectum, anus, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. The tumor can include malignant or benign growths.

[0089] Cancer may specifically be, but is not limited to, the following histological types: neoplasm, malignant; cancer tumor; cancer tumor, undifferentiated; giant cell cancer and spindle cell cancer; small cell cancer; papillary cancer; squamous cell cancer; lymphoepithelial cancer; basal cell cancer; pilomatrix carcinoma; transitional cell cancer; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyp; adenocarcinoma, familial polyposis coli; solid cancer; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobic cancer; acidophil carcinoma; oxyphilic adenocarcinoma, basophil carcinoma; clear cell adenocarcinoma; granular cell cancer; follicular adenocarcinoma; papillary and follicular adenocarcinoma, non-encapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrial carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma, papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucous gland carcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; lobular cell carcinoma; adeno-squamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; capsular cell tumor, malignant; granulosa cell tumor, malignant; male germ cell tumor, malignant; Sertoli cell tumor; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; malignant glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epitheloid cell melanoma; blue nevus malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;Mixed tumors, malignant; Müllerian duct mixed tumors; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumors, malignant; Brenner tumors, malignant; phyllodes tumors, malignant; synovial sarcoma; mesothelioma, malignant; undifferentiated embryonal cell tumors; embryonic stage cancer; teratomas, malignant; ovarian teratomas, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; vascular endothelioma, malignant; Kaposi sarcoma, perivascular cell tumor, malignant; lymphangiosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma, chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumors, malignant; ameloblastic odontogenic sarcoma, ameloblastic tumor, malignant; ameloblastic fibrosarcoma, pinealoma, malignant; chordoma; glioma, malignant; epithelioma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma; astroblastoma; glioblastoma, oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory nerve tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin; lateral granuloma; malignant lymphoma, small lymphocyte type; malignant lymphoma, large cell type, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia, plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia, myelogenous leukemia; basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia; megakaryoblastoid leukemia, myelosarcoma; and hairy cell leukemia.;

[0090] The anti-cancer activity of dinucleotides (formulated and administered in PBS solution) was evaluated in vitro in various cancer cell lines according to procedures commonly used for the assessment of the cytotoxicity of compounds, as outlined in the following publications: Mender, I., Gryaznov, S., Dikmen, Z.G., Wright, W.E., & Shay, J.W. (2015). Induction of telomere dysfunction mediated 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.

[0091] Some representative dose-dependent anti-cancer activity data of the dinucleotide compounds disclosed herein are shown in FIGS. 6 and 7.

[0092] Table 1 shows the IC of compounds 1, 5, 6, 11, and 12 and the enantiomers of compounds 11 (Sp and Rp) and 12 (Sp and Rp) tested in human cell lines HEK293 (kidney), Hep3B (liver, pediatric HCC), HepG2 (liver, hepatoblastoma), NCI-H23 (lung, adenocarcinoma), H2081 (lung, SCLC) against untreated cells 50 values.

Table 1

[0093] FIG. 6 shows the anti-cancer activity of some of the disclosed dinucleotides and representative dose-response curves for which the IC 50 values were determined.

[0094] The specificity of the dinucleotides tested against telomerase-positive cancer cells versus telomerase-negative normal non-cancer cells was demonstrated by testing these molecules in normal human BJ fibroblasts - Figure 7. Importantly, these data show that in normal cells, the dinucleotides tested did not show significant cytotoxicity even at concentrations 100-fold higher than the EC 50 values observed in cancer cells, demonstrating specificity for telomerase-positive cancer cells.

[0095] Dinucleotide compounds were synthesized using the solid-phase supported phosphoramidite method and appropriately sugar- and base-protected nucleoside 3'-phosphoramidite components (all commercially available from Glen Research). The product was cleaved from the universal solid support (Glen Research), fully deprotected with aqueous ammonia, partially concentrated in vacuo (to remove ammonia), and then purified by RP HPLC. The molecular weight and purity of the final dinucleotide product were confirmed by LC MS.

[0096] Pharmaceutical formulations and routes of administration When clinical use is contemplated, the pharmaceutical composition is prepared in a form suitable for the intended use. Generally, this necessarily involves preparing a composition that is essentially pyrogen-free and contains no other impurities that may be harmful to humans or animals.

[0097] Generally, it is desirable to use appropriate salts and buffers to stabilize the drug and enable uptake by target cells. The aqueous compositions of the present disclosure comprise an effective amount of a drug dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes solvents, buffers, solutions, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are acceptable for use in formulating pharmaceutical articles such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of such in the therapeutic compositions is contemplated, except in cases where any of the conventional media or agents are incompatible with the active ingredient of the present disclosure. Supplementary active ingredients can also be incorporated into the compositions, provided that they do not inactivate the agents of the compositions.

[0098] The active compositions of the present disclosure can include classical pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any general route as long as the target tissue is available via that 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 intratumoral or locally to the tumor, for example to the tumor vasculature. Such compositions will usually be administered as pharmaceutically acceptable compositions as described above.

[0099] The active compound may also be administered parenterally or intraperitoneally. By way of example, solutions of the active compound as the free base or as a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations will usually contain a preservative to prevent the growth of microorganisms.

[0100] Pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to an extent that permits easy injection. The preparations are stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Suitable solvents or dispersion media can contain, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycols, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of injectable compositions can be brought about by using in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0101] Sterile injectable solutions can be prepared by incorporating the appropriate amount of the active compound in the solvent with any other desired ingredients (for example, those enumerated above) and subsequently filtering the solution sterilize. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the desired other ingredients, such as those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying techniques to obtain a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0102] The compositions of the present disclosure can generally be formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide), or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0103] Upon formulation, the solution is preferably administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations can be readily administered in various dosage forms such as injection solutions, drug release capsules, etc. For parenteral administration in an aqueous solution, for example, the solution is generally suitably buffered and the liquid diluent is first made isotonic, for example, with sufficient saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, especially in view of the present disclosure, a sterile aqueous medium is used as is known to those skilled in the art. By way of illustration, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of a subcutaneous injection solution or injected at the proposed injection site (see, for example, "Remington’s Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will determine the appropriate dosage for each individual subject in any case. Further, for administration to humans, the preparation needs to meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biologics criteria.

[0104] The actual dosage of the composition according to a particular embodiment of the present disclosure administered to a subject can be determined by physical and physiological factors such as the particular compound used, age, general health of the subject, diet, weight, severity of the condition, type of disease being treated, previous or current therapeutic interventions, idiopathic diseases of the patient, absorption rate, distribution rate, inactivation rate, excretion rate, administration time, administration route, etc., and by the judgment of the person supervising the administration. Depending on the dosage and administration route, the preferred dosage, and / or the number of administrations of an effective amount can vary according to the response of the subject. The active ingredient may be administered once, or may be divided into several smaller dosages administered at various time intervals. Thus, for any particular subject, a specific dosing regimen can be adjusted over time according to the individual needs and the professional judgment of the person performing or supervising the administration of the composition, and it is understood that the concentration ranges described herein are merely illustrative and are not intended to limit the scope or practice of the composition recited in the claims.

[0105] The dinucleotide compounds disclosed herein are contained 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 severe toxic effects to the treated patient. The preferred dosage of the dinucleotide for all conditions referred to herein ranges from about 10 ng / kg to 100 mg / kg, preferably 0.1 to 50 mg / kg per day, more generally in the range of 0.5 to about 25 mg per kilogram of the subject's body weight per day. As a non-limiting example, typical dosages can range from 50.01 to 20 weight / weight % in a suitable carrier. Similarly, the compound can be administered in any suitable unit dosage form, including but not limited to those containing less than 1 mg, 1 mg to 3000 mg, or 5 to 1000 mg of the dinucleotide compounds disclosed herein.

[0106] The composition can be administered continuously, sequentially, as needed, or 1, 2, 3, 4, 5, 6, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times, and includes but is not limited to those containing less than 1 mg.

[0107] Combination therapy In order to enhance the effectiveness of the treatment, it may be desirable to combine the composition of the present disclosure with a second treatment or pharmaceutical composition. For example, the method of use can further include the administration of a second pharmaceutical composition comprising an anti-cancer agent or other agent effective in the treatment of a proliferative disorder. The anti-cancer agent can, for example, kill cancer cells, induce apoptosis in cancer cells, reduce the growth rate of cancer cells, reduce the occurrence or number of metastases, shrink tumor size, inhibit tumor growth, reduce the blood supply to the tumor or cancer cells, promote the immune response against cancer cells or tumors, prevent or inhibit the progression of cancer, or extend the lifespan of a subject having cancer, thereby having an adverse effect on cancer in the subject. More generally, the second pharmaceutical composition can be administered in an effective amount or a combination effective amount to kill specific cells or inhibit the proliferation of specific cells.

[0108] In some embodiments, the method of treatment can include co-administration, simultaneously. This process can involve administering simultaneously or sequentially. Co-administration can be achieved by contacting the cells with a single composition or pharmaceutical formulation comprising one or more of the dinucleotide compounds disclosed herein and another anti-cancer agent, or by contacting the cells simultaneously with two separate compositions or formulations (one composition comprising one or more of the dinucleotide compounds disclosed herein and the other comprising a second agent). Similarly, the two compositions can be administered not simultaneously, but, for example, on the same day or within the same week, in temporal proximity to each other.

[0109] In other embodiments, the treatment method may include a first stage in which a pharmaceutical composition comprising one or more of the dinucleotide compounds disclosed herein is administered, and a second stage in which a second pharmaceutical composition is administered. The first stage and the second stage may be temporally consecutive, may be temporally spaced (minutes, days, weeks, or months), or may be temporally overlapping. Further, the sequential order of the treatment stages can be reversed or repeated. To ensure, any combination of treatment stages can be used. As an example, the administration of one or more of the dinucleotide compounds disclosed herein is "A", and the treatment with the second agent is "B": A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0110] In the context of the present disclosure, it is contemplated that the administration of a pharmaceutical composition comprising one or more of the dinucleotide compounds disclosed herein can be used in combination with treatment B, such as gene therapy, chemotherapy, radiation therapy, or immunotherapy intervention, in addition to other apoptosis promoters or cell cycle regulators. It is also contemplated that various standard therapies, as well as surgical interventions, can be applied in combination with the described telomere shortening and telomere dysfunction inducing therapies.

[0111] a. Chemotherapy Chemotherapy includes, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastone, and methotrexate, or analogues or derivative variants of any of the foregoing.

[0112] b. Radiation therapy Radiation therapy can cause DNA damage and includes what are commonly known as X-rays and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents, such as microwave and UV radiation, are also contemplated.

[0113] c. Immunotherapy Immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Therefore, immunotherapy can be used as part of a combination therapy in conjunction with the administration of pharmaceutical compositions containing one or more of the dinucleotide compounds disclosed herein. Immunotherapy modalities involve targeting tumor cells via some marker of the tumor cells that is suitable for targeting, i.e., that is not present in the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting with a second therapeutic modality in the context of this 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.

[0114] d.Surgery Curative surgery is a cancer treatment that can be used in combination with a pharmaceutical composition comprising one or more of the dinucleotide compounds disclosed herein. Curative surgery includes resection in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumorectomy refers to the physical removal of at least a portion of the tumor. In addition to tumorectomy, surgical treatments include laser surgery, cryosurgery, electrocautery, and microsurgically controlled surgery (Mohs surgery). It is further contemplated that the compositions of the present disclosure can be administered in conjunction with the removal of superficial cancer, precancer, or an incidental amount of normal tissue. When all portions of the cancer cells, tissue, or tumor are excised, a cavity can be formed in the body. Treatment can be achieved by administration of a pharmaceutical composition comprising one or more of the dinucleotide compounds disclosed herein.

[0115] e. Other anticancer agents Other anticancer agents are contemplated to be used in combination with including one or more of the dinucleotide compounds disclosed herein to additively or synergistically enhance the therapeutic efficacy of treatment.

[0116] These additional agents include immunomodulators, agents that affect the upregulation of cell surface receptors and GAP junctions, cell growth inhibitors and differentiating agents, cell adhesion inhibitors, or agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing substances. Immunomodulators include tumor necrosis factor, interferon alpha, beta, and gamma, IL-2 and other cytokines, F42K and other cytokine analogs, or MIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines. Upregulation of cell surface receptors or their ligands, such as Fas / Fas ligand, DR4, or DR5 / TRAIL, is further contemplated to 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 adjacent hyperproliferative cell populations. In other embodiments, cell growth inhibitors or differentiating agents can be used in combination with the present invention to improve the anti-hyperproliferative effect of the treatment. Cell adhesion inhibitors are contemplated to improve the effectiveness of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Other anti-cancer agents that increase the sensitivity of hyperproliferative cells to apoptosis, and signal transduction inhibitors such as antibody c225, can be used in combination with one or more of the dinucleotide compounds disclosed herein to improve therapeutic efficacy.

[0117] Finally, the additional agent may also include anti-cancer agents that are broadly characterized as antimetabolites, inhibitors of topoisomerase I and II, alkylating agents, and microtubule inhibitors (e.g., taxol). Anti-cancer agents for use in the present invention include, for example, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, BCG Live, bexarotene capsules, bexarotene gel, bleomycin, busulfan for intravenous injection, busulfan for oral use, calusterone, capecitabine, carboplatin, carmustine, carmustine by polifeprosan 20 implant, celecoxib, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytarabine liposome, dacarbazine, dactinomycin, actinomycin D, darbepoetin alfa, daunorubicin liposome, daunorubicin, daunomycin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, doxorubicin liposome, drostanolone propionate, Elliott's BSolution), 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 alpha-2a, interferon alpha-2b, irinotecan, letrozole, leucovorin, levamisole, lomustine (CCNU), mechlorethamine (nitrogen mustard), megestrol acetate, melphalan (L-PAM), mercaptopurine (6-MP), mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenylpropionate, nofetumomab, LOddC, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pegademase, pegaspargase, pegfilgrastim, pentostatin, pipobroman, plicamycin, mitramycin, porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, streptozocin, talc, tamoxifen, temozolomide, teniposide (VM-26), testolactone, thioguanine (6-TG), thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin (ATRA), uracil mustard, valrubicin, valcitabine (monoval LDC), vinblastine, vinorelbine, zoledronate, and mixtures thereof are specifically mentioned.

[0118] Hormone therapy may also be used in combination with the administration of a pharmaceutical composition that can be used in combination with one or more of the dinucleotide compounds disclosed herein. The use of hormones is employed in the treatment of certain cancers such as breast cancer, prostate cancer, ovarian cancer, or cervical cancer to reduce the levels of certain hormones such as testosterone or estrogen or to block their effects. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastasis.

[0119] The present disclosure provides a sequential treatment of cancer using the dinucleotide compounds disclosed herein, followed by PD-L1, PD-1, and / or CTLA-4 therapy. The duration for each treatment may vary, and short intervals between treatments are considered advantageous. For example, for the dinucleotide compounds disclosed herein, the treatment may be as short as 2 days, but may also be 3, 4 days or more, including 2 - 4 days. The interval before PD-L1, PD-1, and / or CTLA-4 treatment needs to be at least 1 day and can be up to 14 days, for example 2 - 4 days. Overlap between the dinucleotide compounds disclosed herein and PD-L1, PD-1, and / or CTLA-4 needs to be avoided as it may potentially have a harmful effect on the activated effector T cells of the dinucleotide compounds disclosed herein.

[0120] The daily dosage of the dinucleotide compounds disclosed herein is preferably intravenous or oral and is 0.5 mg / kg - 10 mg / kg. The dosages of PD-L1, PD-1, and / or CTLA-4 are in accordance with the currently approved dosing schedules.

[0121] In the context of the present disclosure, it is also contemplated that the dinucleotide compounds disclosed herein and anti-PD-L1 such as atezolizumab, or anti-PD-1 or anti-CTLA-4 such as dinucleotide and Libtayo® can be used in combination with chemotherapy or radiation therapy interventions, or other treatments. In particular, it may also be demonstrated that it is effective to combine the dinucleotide compounds, anti-PD-L1, anti-PD-1, or anti-CTLA-4 disclosed herein with other therapies targeting different aspects of cancer cell function.

[0122] In general, to kill cells, inhibit cell proliferation, 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 dinucleotide compounds disclosed herein and at least one other agent will be contacted with the "target" cells. These compositions will be provided continuously or in combination in an amount effective to kill cells or inhibit cell proliferation. This process may involve contacting the cells simultaneously with the dinucleotide / anti-PD-L1, anti-PD-1 or anti-CTLA-4 and another agent or factor. This can be achieved by contacting the cells with a single composition or pharmaceutical formulation containing both agents, or by contacting the cells simultaneously with two separate compositions or formulations (one composition containing the interferon prodrug according to the present disclosure and the other containing the other agent).

[0123] Alternatively, the dinucleotide / anti-PD-L1, anti-PD-1, or anti-CTLA-4 therapy can precede or follow other drug treatments at intervals ranging from a few minutes to a few weeks. In embodiments where the other drug and the interferon prodrug are applied separately to the cells, usually, a significant period will not pass between each delivery so that the drug and the expression construct can still advantageously exert a combined effect on the cells. In such cases, it is contemplated that both modalities contact the cells within about 12 to 24 hours of each other, more preferably within about 6 to 12 hours of each other, with a delay time of only about 12 hours being most preferred. However, depending on the situation, it may be desirable to significantly extend the period for treatment, in which case, several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between each administration.

[0124] Also, it is contemplated that more than one administration of either the interferon prodrug or the other drug may be desirable. As illustrated below, when the dinucleotide compound / anti-PD-L1, anti-PD-1, or anti-CTLA-4 therapy disclosed herein is "A" and the other therapy is "B", various combinations can be used: A / B / A B / A / B B / B / A A / A / B B / A / A A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B B / B / B / A A / A / A / B B / A / A / A A / B / A / A A / A / B / A A / B / B / B B / A / B / B B / B / A / B Other combinations are also contemplated. Again, to achieve cell killing, both drugs are delivered to the cells in a combination amount effective for cell killing.

[0125] Agents or factors suitable for cancer therapy include any compound or treatment method that induces DNA damage when applied to cells. Such agents and factors include radiation and waves that induce DNA damage, such as irradiation, microwaves, and electron emission. Various compounds, also referred to as "chemotherapeutic agents" or "genotoxic substances," may be used. This may be achieved by irradiating a local tumor site, or tumor cells may be contacted with the agent by administering a therapeutically effective amount of the pharmaceutical composition to the subject.

[0126] Various classes of chemotherapeutic agents are contemplated for use in the present disclosure. Other chemotherapeutic agents include selective estrogen receptor modulators (SERMs) such as tamoxifen, 4-hydroxytamoxifen (afimoxifene), Falsodex, raloxifene, bazedoxifene, clomifene, femarelle, lasofoxifene, ormeloxifene, and toremifene. The drugs camptothecin, actinomycin-D, and mitomycin C are commonly used chemotherapeutic agents. The present disclosure also encompasses the use of combinations of one or more DNA-damaging agents, such as the use of X-rays and cisplatin, or the use of cisplatin and etoposide, regardless of whether radiation-based or actual compounds. The agent may be prepared and used as a combination therapeutic composition.

[0127] 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.

[0128] Agents that directly crosslink DNA or form adducts are also envisioned. Agents such as cisplatin, and other DNA alkylating agents may be used. Cisplatin is widely used to treat cancer and in clinical applications, it is administered at 20 mg / m for 5 days every 3 weeks for a total of 3 courses. 2It is used at an effective dose. Since cisplatin is not absorbed orally, it needs to be delivered via intravenous, subcutaneous, intratumoral, or intraperitoneal injection.

[0129] 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. Widely used in the clinical setting for the treatment of neoplasms, these compounds are intravenously administered via bolus injection at a dose in the range of 25 - 75 mg / m 2 for doxorubicin, and for etoposide, it is intravenously administered at a dose in the range of 35 - 50 mg / m 2 or orally administered at twice the intravenous dose. Microtubule inhibitors such as taxanes are also contemplated. These molecules are diterpenes produced by plants of the Taxus genus and include paclitaxel and docetaxel.

[0130] Epidermal growth factor receptor inhibitors such as Iressa, mammalian target of rapamycin, mTOR (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, rapamycin and its analogs ("rapalogs") are contemplated for use in cancer therapy according to the present disclosure. Another EGFR inhibitor that is particularly useful here is gefitinib.

[0131] Another possible therapy is TNF - α (tumor necrosis factor alpha), which is a cytokine involved in systemic inflammation and is a member of the group of cytokines that stimulate the acute - phase response. The main role of TNF is the regulation of immune cells. TNF can also induce apoptotic cell death, trigger inflammation, and inhibit tumor formation and viral replication.

[0132] Agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits also cause DNA damage. Therefore, numerous nucleic acid precursors have been developed. Agents that are readily available and have undergone extensive testing are particularly useful. Thus, agents such as 5-fluorouracil (5-FU) are preferentially utilized by neoplastic tissues and are particularly useful for targeting neoplastic cells. 5-FU, which is highly toxic, can be applied with a wide range of carriers, including topically, but intravenous administration at doses in the range of 3 - 15 mg / kg / day is commonly used.

[0133] Other factors that cause DNA damage and are widely used include γ-rays, X-rays, and / or those commonly known as the targeted delivery of radioisotopes to tumor cells. Other forms of DNA damage factors such as microwave and UV irradiation are also contemplated. All of these factors are most likely to affect a wide range of damaged DNA in the precursors of DNA, DNA replication and repair, and chromosome assembly and maintenance. The dose range of X-rays extends from a daily dose of 50 - 200 roentgens over a long period (3 - 4 weeks) to a single dose of 2000 - 6000 roentgens. The dose range of radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the uptake by neoplastic cells.

[0134] Furthermore, it is also contemplated that separate immunotherapy, hormone therapy, toxin therapy, and / or surgery can be used.

[0135] Those skilled in the art are referred to “Remington’s Pharmaceutical Sciences” 15th Edition, Chapter 33, particularly pages 624 - 652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any case, determine the appropriate dosage for the individual subject. Furthermore, for administration to humans, the preparation needs to meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biologics.

[0136] The following examples are included to demonstrate preferred embodiments of the present invention. The techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present invention and, accordingly, may be considered to constitute preferred modes for carrying out the present invention, which should be understood by those skilled in the art. However, those skilled in the art should understand that, in view of the present disclosure, many modifications can be made to the specific embodiments disclosed, and similar or analogous results can be obtained without departing from the spirit and scope of the present invention. Experiment: [Table 2]

[0137] Process Overview: The production of the disclosed dinucleotides included the following process steps: 1. Solid-phase synthesis on an H8-custom solid-phase synthesizer using universal linker-CPG as the solid support. 2. Cleavage and deprotection. 3. Purification by reverse-phase (RP) HPLC (column: Waters Xbridge C18 (19 mm × 50 mm, 14.2 mL); eluent A: 100 mM TEAA in water, eluent B: 25% ACN - 100 mM TEAA in water as the eluent system). 4. If necessary: Buffer exchange was performed by RP-HPLC (column: Waters Xbridge C18 (19 mm × 50 mm, 14.2 mL); eluent A: water, eluent B: ACN as the eluent system). 5. Freeze-drying cycle, followed by final weight measurement (final amount) after 2 cycles. Materials and General Procedures:

[0138] Components and Solid Support The phosphoramidite components and CPG-solid support used in the solid-phase synthesis are summarized in Table 3 below. [Table 3-1] [Table 3-2]

[0139] Solvents and auxiliary reagents: All solvents and auxiliary reagents used in these projects are summarized in Table 4. [Table 4]

[0140] Solid-phase synthesis of dinucleotides: Synthesis was carried out according to the conventional solid-phase oligonucleotide synthesis protocol using standard phosphoramidite chemistry on an automated solid-phase synthesizer. Solid-phase synthesis was performed using an H8-Custom synthesizer (K&A Laborgerate) controlled by the H8-COM software package.

[0141] The phosphoramidite was dissolved in dry acetonitrile to obtain a 0.1 M solution, which was then dried on molecular sieves (3 Å) for at least 2 hours and then attached to the designated amidite port of the H8-synthesizer.

[0142] As the activator, 0.5 M 5-ethylthio-1H-tetrazole (ETT) in ACN was used and dried on molecular sieves (3 Å) for at least 24 hours before the start of synthesis. As the sulfurization reagent, 3-amino-1,2,4-dithiazole-5-thione (or xanthine hydride; 100 mM in ACN-pyridine (2:3 v / v)) was freshly prepared before use.

[0143] All other auxiliary reagents for solid-phase synthesis were used as obtained from their respective commercial sources (Table 5).

[0144] During the synthesis cycle, a standard oxidation step followed by a capping step was used. All dinucleotides were synthesized in DMT-OFF mode. The synthesis using phosphate-ON reagents was performed omitting the final capping step. A detailed list of all raw materials, auxiliary reagents, solvents and their suppliers and product numbers is listed in Tables 3 and 4.

[0145] Cleavage and deprotection: After the synthesis was completed, the dinucleotides immobilized on the solid support were treated with DBU at ambient temperature for 4 hours to ensure the removal of the cyanoethyl protecting groups, especially from the 6-thio position of 6SdG or 6SG and the 4-thio position of the 4TdT nucleotide unit. Subsequently, the product-bearing CPG solid support was dried at room temperature using a vacuum pump, and the resulting dry CPG was transferred to a 15 mL Falcon tube. To each tube, a solution of AMA (pre-mixed 1:1 v / v of methylamine in water (41 wt%) + aqueous NH3 solution (30%, purest)) was added (3 mL), and the tubes were shaken at 30 °C for 3 hours. This resulted in the cleavage of the dinucleotides from the solid support, the generation of free 3'-OH by dephosphorylation of the uni-linker at the 3'-end, and the deprotection of the 2-NH2 position of 6SdG and 6SG by removal of the trifluoroacetyl and isobutyryl protecting groups. In the case of dinucleotides containing 6SG, TEA·3HF was then added to the deprotection mixture, and the mixture was shaken at 45 °C for an additional 90 minutes to effect 2'-O-TBDMS deprotection. Finally, the dinucleotides dissolved in the supernatant solution were separated from the solid support by centrifugation (using a HERAEUS Multifuge X3F by Thermo Scientific), followed by decantation. The residual CPG in each tube was washed with water to recover the remaining solution containing the dinucleotides. The combined liquid phase was concentrated in vacuo. The residue was diluted with TEAA buffer (100 mM TEAA in water) for subsequent HPLC purification.

[0146] Purification by RP-HPLC: Purification was performed on an Akta pure 150 HPLC system using a Waters RP Xbridge C18 column. The details of the process are listed below, and Table 5 summarizes the materials used in the purification and subsequent desalting steps. Akta Pure 150 HPLC system Reverse-phase chromatography: Column - X - Bridge C18 (19 mm × 50 mm, 14.2 mL); Eluent A: 100 mM TEAA in water Eluent B: 100 mM TEAA in an ACN - water mixture (25% ACN in water) [Table 5]

[0147] Desalting: For samples for in vivo studies, the counter - cation TEA was replaced from the purified material with Na by performing buffer exchange. After removing the RP - HPLC elution buffer by evaporation and subsequent lyophilization, the purified material was reconstituted in 500 mM NaCl solution and subjected to buffer exchange by RP - HPLC on an Akta pure 150 using a Waters X - Bridge C18 column (19 mm × 50 mm, 14.2 mL). The details are listed below, and the materials used are summarized in Table 5. + with + Na. After removing the RP - HPLC elution buffer by evaporation and subsequent lyophilization, the purified material was reconstituted in 500 mM NaCl solution and subjected to buffer exchange by RP - HPLC on an Akta pure 150 using a Waters X - Bridge C18 column (19 mm × 50 mm, 14.2 mL). The details are listed below, and the materials used are summarized in Table 5. Akta Pure 150 Reverse - phase chromatography: Column - X - Bridge C18 (19 × 50 mm; 14.2 mL); Eluent A: water Eluent B: 95% ACN - water Thereafter, acetonitrile - water was removed in vacuo, and the aqueous solution of dinucleotide was lyophilized.

[0148] Lyophilization and quantification: The oligonucleotide solution was dispensed into Falcon tubes and lyophilized (using a CHRIST Epsilon 1-4 LSCplus lyophilizer). After the first freeze cycle, the residual dinucleotides were redissolved in water and lyophilized again. This procedure was repeated until a constant weight was achieved. Prior to the final lyophilization step, a sufficient amount was aliquoted for quality control testing. After completion of the lyophilization step, the dried material was quantified by weighing.

[0149] QC analysis: For the QC release test, UV-based RP chromatogram traces of dinucleotides were recorded at both 260 nm and 350 nm (characteristic absorption maxima of 6SG-nucleobases; N.F. Krynetskaia, X. Cai, J.L. Nitiss, E.Y. Krynetski, M.V, Relling, FASEB J. 2000, 14, 2339). Although both values were reported, as agreed with the sponsor, the trace at 350 nm was monitored and integrated for the final purity determination. To confirm the purity and identity of the dinucleotides, an Ultimate 3000 HPLC system (Thermo Scientific) equipped with a Waters Acquity UPLC Oligonucleotide C18 column (2.1 mm × 100 mm, 0.37 mL) was run.

[0150] Data acquisition and integration of the HPLC chromatograms were performed using the software package Chromeleon from Thermo Fisher Scientific. For each sample, the peaks were integrated by vertical peak splitting. In the case of overlapping peaks, the splitting was done at the minimum between the two, and their identity was confirmed from the associated MS-Spectra.

[0151] The monoisotopic molecular weight of the sample was analyzed using ESI / MS detection. For this purpose, an Ultimate 3000 HPLC system equipped with a UV detector and a qTOF detector (compact) from Bruker Daltonics was used. The MS spectra were extracted in total ion current (TIC) using Bruker Hystar Data Analysis software. The identity of all dinucleotides was also established within the agreed specifications.

[0152] Syngeneic mouse model: A) Tumor type: Mouse model of H22 hepatocellular carcinoma (syngeneic mouse model) Method: Female BALB / c mice were subcutaneously inoculated with H22 tumor cells (1×106) in the right flank to establish a syngeneic subcutaneous H22 tumor model. When the average tumor volume reached 70 - 100 mm3, the mice were randomly assigned to 6 groups based on tumor volume and body weight, with 8 mice in each group: control group (vehicle, i.v., QD on D0, D1, D2, D7, D8, D9), MAIA-2022-12 group (MAIA-2022-12, i.v., 6 mg / kg, QD on D0, D1, D2, D7, D8, D9), MAIA-2021-20 group (MAIA-2021-20, i.v., 6 mg / kg, QD on D0, D1, D2, D7, D8, D9), anti-PD-1 group (anti-PD-1, i.p., 10 mg / kg, QD on D4, D12, and D20), MAIA-2022-12 combination group (MAIA-2022-12, i.v., 6 mg / kg, QD on D0, D1, D2, D7, D8, D9, anti-PD-1, i.p., 10 mg / kg, QD on D4, D12, and D20), MAIA-2021-20 combination group (MAIA-2021-20, i.v., 6 mg / kg, QD on D0, D1, D2, D7, D8, D9, anti-PD-1, i.p., 10 mg / kg, QD on D4, D12, and D20). The dose volume was 10 μL / g. Drug efficacy was evaluated using tumor volume calculation and survival analysis, and drug safety was evaluated using body weight changes and animal deaths.

[0153] B) Tumor type: Mouse model of M38 colorectal cancer (syngeneic mouse model) Methods: Female C57BL / 6N mice were subcutaneously inoculated with MC38 tumor cells (4×104) in the right flank to establish a syngeneic subcutaneous MC38 tumor model. When the average tumor volume reached 70 - 100 mm3, the mice were randomly assigned to 6 groups based on tumor volume and body weight, with 8 mice per group: control group (vehicle, i.v., QD on D0, D1, D2, D7, D8, D9), MAIA-2022-12 group (MAIA-2022-12, i.v., 3 mg / kg, QD on D0, D1, D2, D7, D8, D9), MAIA-2021-20 group (MAIA-2021-20, i.v., 3 mg / kg, QD on D0, D1, D2, D7, D8, D9), anti-PD-1 group (anti-PD-1, i.p., 10 mg / kg, QD on D4, D12), MAIA-2022-12 combination group (MAIA-2022-12, i.v., 3 mg / kg, QD on D0, D1, D2, D7, D8, D9, anti-PD-1, i.p., 10 mg / kg, QD on D4, D12), MAIA-2021-20 combination group (MAIA-2021-20, i.v., 3 mg / kg, QD on D0, D1, D2, D7, D8, D9, anti-PD-1, i.p., 10 mg / kg, QD on D4, D12). The dose volume was 10 μL / g. Drug efficacy was evaluated using tumor volume calculation and survival analysis, and drug safety was evaluated using body weight changes and animal death.

[0154] c) Tumor type: Mouse model of YUMM1.7 melanoma (syngeneic mouse model) Methods: Female C57BL / 6N mice were subcutaneously inoculated with YUMM1.7 tumor cells (2×105) in the right flank to establish a syngeneic subcutaneous YUMM1.7 tumor model. When the average tumor volume reached 70 - 100 mm3, the mice were randomly assigned into 3 groups based on tumor volume and body weight, with 8 mice per group: control group (vehicle, i.p., QD on D0, D1, D2, D9, D10, D11), MAIA-2022-12 group (MAIA-2022-12, i.p., 6 mg / kg, QD on D0, D1, D2, D9, D10, D11), MAIA-2021-20 group (MAIA-2021-20, i.p., 6 mg / kg, QD on D0, D1, D2, D9, D10, D11). The dose volume was 10 μL / g. Drug efficacy was evaluated using tumor volume calculation and survival analysis, and drug safety was evaluated using body weight change and animal death.

[0155] d) Re-challenge of the YUMM1.7 experiment Methods: Four mice from the MAIA-2022-12 group and six mice from the MAIA-2021-020 group had no tumors from the initial experiment (see Slide 7 for treatment schedule and results). Re-challenge with YUMM1.7 was performed in these tumor-free mice to examine whether the mice obtained an immune protection effect after treatment. Forty-six days after the last treatment for tumor development, female C57BL / 6N mice (10 mice for the control group, 4 mice for MAIA-2022-12, 6 mice for MAIA-2021-20) were subcutaneously inoculated with YUMM1.7 tumor cells (2×105) in the left flank. One week after tumor inoculation, the mice were started to be measured for tumor development (2 - 3 times per week). Tumor volume calculation was used to confirm whether the mice formed tumors after inoculation, and body weight change and animal death were used to evaluate drug safety.

Claims

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

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. 【Chemical 2】

4. The compound according to Claim 2, wherein the compound has the following structure. [Chemical Formula 3]

5. The compound according to Claim 2, wherein the compound has the following structure. 【Chemical Formula 4】

6. The compound according to Claim 1, wherein X is S.

7. The compound according to Claim 6, wherein the compound has the following structure. [Chemical Formula 5]

8. The compound according to Claim 6, wherein the compound has the following structure. 【Chemical Formula 6】

9. The compound according to Claim 6, wherein the compound has the following structure. 【Chemical Formula 7】

10. A compound having the following structure. 【Chemical Formula 8】

11. A compound having the following structure. 【Chemical Formula 9】

12. A compound having the following structure. 【Chemical Formula 10】

13. A compound having the following structure. 【Chemical 11】

14. A compound having the following structure. 【Chemical Formula 12】

15. A compound having the following structure. 【Chemical 13】

16. A compound comprising the structure of Formula II or a pharmaceutically acceptable salt thereof, wherein: 【Chemical 14】 In the formula: R １ and R ２ are independently H or OH, X is O or S; Y is O; R ３ is a compound or a pharmaceutically acceptable salt thereof selected from the group consisting of a phosphate group, a thiophosphate group, palmitic acid, tocopherol, and a cholesterol group.

17. R １ and R ２ are independently H or OH, X is O, Y is O, and R ３ is a cholesterol group, the compound according to claim 16.

18. The compound according to Claim 17, having the following structure. 【Chemical Formula 15】

19. The compound according to Claim 17, having the following structure. 【Chemical 16】

20. The compound according to Claim 17, having the following structure. 【Chemical 17】

21. The compound according to Claim 17, having the following structure. 【Chemical 18】

22. R １ and R ２ are independently H or OH, X and Y are O, and R ３ is a phosphate group, the compound according to claim 16.

23. The compound according to Claim 22, having the following structure. 【Chemical 19】

24. The compound according to Claim 22, having the following structure. 【Chemical 20】

25. The compound according to Claim 22, having the following structure. 【Chemical 21】

26. The compound according to Claim 22, having the following structure. 【Chemical 22】

27. R １ and R ２ are independently H or OH, X and Y are independently O, and R ３ is a thiophosphate group, the compound according to claim 16.

28. The compound according to Claim 27, having the following structure. 【Chemical 23】

29. The compound according to Claim 27, having the following structure. 【Chemical Formula 24】

30. The compound according to Claim 27, having the following structure. 【Chemical 25】

31. The compound according to Claim 27, having the following structure. 【Chemical 26】

32. R １ and R ２ are independently H or OH, X and Y are independently O, and R ３ is palmitic acid, the compound according to claim 16.

33. The compound according to Claim 32, having the following structure. 【Chemical 27】

34. The compound according to Claim 32, having the following structure. 【Chemical Formula 28】

35. The compound according to claim 32, having the following structure. 【Chemical 29】

36. The compound according to claim 32, having the following structure. 【Chemical Formula 30】

37. R １ and R ２ are independently H or OH, X and Y are independently O, and R ３ is tocopherol, the compound according to claim 16.

38. The compound according to claim 37, having the following structure. 【Chemical 31】

39. The compound according to claim 37, having the following structure. 【Chemical Formula 32】

40. The compound according to claim 37, having the following structure. 【Chemical 33】

41. The compound according to claim 37, having the following structure. 【Chemical Formula 34】

42. The compound according to claim 37, having the following structure. 【Chemical 35】

43. R １ and R ２ are independently H or OH, X is S, Y is O, and R ３ is a cholesterol group, the compound according to claim 16.

44. The compound according to claim 43, having the following structure. 【Chemical Formula 36】

45. The compound according to claim 43, having the following structure. 【Chemical 37】

46. The compound according to claim 43, having the following structure. 【Chemical Formula 38】

47. The compound according to claim 43, having the following structure. 【Chemical 39】

48. R １ and R ２ is independently H or OH, X is S, Y is O, and R ３ is a phosphate group, the compound according to claim 16.

49. The compound according to claim 48, having the following structure. 【Chemical Formula 40】

50. The compound according to claim 48, having the following structure. 【Chemical 41】

51. The compound according to claim 48, having the following structure. 【Chemical 42】

52. The compound according to claim 48, having the following structure. 【Chemical 43】

53. R １ and R ２ are independently H or OH, X is S, Y is O, and R ３ is a thiophosphate group, the compound according to claim 16.

54. The compound according to claim 53, having the following structure. 【Chemical 44】

55. The compound according to claim 53, having the following structure. 【Chemical 45】

56. The compound according to claim 53, having the following structure. 【Chemical Formula 46】

57. The compound according to claim 53, having the following structure. 【Chemical 47】

58. R １ and R ２ are independently H or OH, X is S, Y is O, and R ３ is palmitic acid, the compound according to claim 16.

59. The compound according to claim 58, having the following structure. 【Chemical 48】

60. The compound according to claim 58, having the following structure. 【Chemical 49】

61. The compound according to claim 58, having the following structure. 【Chemical Formula 50】

62. The compound according to claim 58, having the following structure. 【Chemical Formula 51】

63. R １ and R ２ are independently H or OH, X is S, Y is O, and R ３ is tocopherol, the compound according to claim 16.

64. The compound according to claim 63, having the following structure. 【Chemical 52】

65. The compound according to claim 63, having the following structure. 【Chemical 53】

66. The compound according to claim 63, having the following structure. 【Chemical 54】

67. The compound according to claim 63, having the following structure. 【Chemical 55】

68. The compound according to claim 63, having the following structure. 【Chemical Formula 56】

69. A compound comprising the structure of formula III or a pharmaceutically acceptable salt thereof, 【Chemical 57】 wherein X is O or S, and R' is independently H or OH, the compound or a pharmaceutically acceptable salt thereof.

70. The compound according to claim 69, having the following structure. 【Chemical Formula 58】

71. The compound according to claim 69, having the following structure. 【Chemical Formula 59】

72. The compound according to claim 69, having the following structure. 【Chemical Formula 60】

73. The compound according to claim 69, having the following structure. 【Chemical Formula 61】

74. The compound according to claim 69, having the following structure. 【Chemical Formula 62】

75. The compound according to claim 69, having the following structure. 【Chemical Formula 63】

76. The compound according to claim 69, having the following structure. 【Chemical Formula 64】

77. The compound according to claim 69, having the following structure. 【Chemical 65】

78. A compound or a pharmaceutically acceptable salt thereof comprising the structure of formula IV, wherein X is O or S and R' is independently H or OH. 【Chemical Formula 66】 Wherein X is O or S and R' is independently H or OH.

79. The compound according to claim 78, having the following structure. 【Chemical Formula 67】

80. The compound according to claim 78, having the following structure. 【Chemical Formula 68】

81. The compound according to claim 78, having the following structure. ​

82. The compound according to claim 78, having the following structure. 【Chemical Formula 70】

83. The compound according to claim 78, having the following structure. 【Chemical Formula 71】

84. The compound according to claim 78, having the following structure. 【Chemical 72】

85. The compound according to claim 78, having the following structure. 【Chemical 73】

86. The compound according to claim 78, having the following structure. 【Chemical 74】

88. A compound or a pharmaceutically acceptable salt thereof having the following structure and stereochemistry. 【Chemical 75】

89. A compound or a pharmaceutically acceptable salt thereof having the following structure and stereochemistry. 【Chemical 76】

91. A compound or a pharmaceutically acceptable salt thereof having the following structure and stereochemistry. 【Chemical 77】

92. A compound or a pharmaceutically acceptable salt thereof having the following structure and stereochemistry. 【Chemical 78】

93. A compound or a pharmaceutically acceptable salt thereof comprising the structure of formula V, wherein R' is independently H or OH. 【Chemical 79】

94. A compound or a pharmaceutically acceptable salt thereof comprising the structure of formula VI, wherein R' is independently H or OH. 【Chemical Formula 80】

95. A compound comprising the structure of formula VII or a pharmaceutically acceptable salt thereof, wherein X is O or S, and R １ and R ２ is H or OH, a compound or a pharmaceutically acceptable salt thereof. 【Chemical 81】

96. The compound according to claim 95, having the following structure. 【Chemical 82】

97. The compound according to claim 95, having the following structure. 【Chemical 83】

98. The compound according to claim 95, having the following structure. 【Chemical 84】

99. The compound according to claim 95, having the following structure. 【Chemical 85】

100. The compound according to claim 95, having the following structure. 【Chemical 86】

101. The compound according to claim 95, having the following structure. 【Chemical 87】

102. A pharmaceutical composition comprising the compound according to any one of claims 1 to 101.

103. A pharmaceutical preparation comprising any one of claims 1 to 101 and at least one pharmaceutically acceptable excipient.

104. A process for preparing the pharmaceutical preparation according to claim 103, comprising combining the compound according to any one of claims 1 to 99 with at least one pharmaceutically acceptable excipient.

105. A method for treating a subject having cancer, comprising administering to the subject a compound according to any one of claims 1 to 101, wherein the cancer is selected from breast cancer, prostate cancer, colon cancer, gastric cancer, esophagus, liver, 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, 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 cancer, Ewing sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelial tumors.

106. The method according to claim 105, wherein treatment with an immune checkpoint inhibitor continues after administration of any one of the compounds according to claims 1 to 101.

107. The method according to claim 106, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

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

109. The method according to claim 106, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor.

110. The method according to claim 106, further comprising administering two or more immune checkpoint inhibitors.

111. The method according to 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.

112. The method according to 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.

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

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

115. A method of treating a subject having cancer, comprising administering to the subject a compound according to any one of claims 1 to 101 after treatment by radiotherapy, wherein the cancer is selected from one or more of breast cancer, prostate cancer, colon cancer, gastric cancer, esophagus, liver, 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, 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 cancer, Ewing sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelial tumors.

116. A method of treating a subject having cancer, comprising administering to the subject a compound according to any one of claims 1 to 101, followed by treatment by radiotherapy, wherein the cancer is selected from one or more of breast cancer, prostate cancer, colon cancer, gastric cancer, esophagus, liver, 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, 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 cancer, Ewing sarcoma, myelodysplastic syndrome, myelofibrosis, oral, nasopharyngeal, and peripheral neuroepithelial tumors.

117. A method for treating cancer in a subject, comprising administering to the subject a compound according to any one of claims 1 to 101, followed by treatment with an immune checkpoint inhibitor and radiotherapy, wherein the cancer is selected from one or more of breast cancer, prostate cancer, colon cancer, gastric cancer, esophagus, liver, 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, 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, nasopharyngeal, and peripheral neuroepithelial tumors.

118. A method for treating cancer in a subject, comprising administering to the subject a compound according to any one of claims 1 to 101 after the subject has been treated 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, esophagus, liver, 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, 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, nasopharyngeal, and peripheral neuroepithelial tumors.

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