Multifunctional cyclic dinucleotides and uses thereof

Abstract

This invention relates to multifunctional cyclic dinucleotide compounds of formula (X) and their derivatives, which can be used as prodrugs for inducing apoptosis or antiviral activity, and can also modulate immune pathways to generate therapeutically beneficial immune responses. This disclosure further relates to pharmaceutical compositions and combinations comprising the cyclic dinucleotide compounds of this invention, methods for synthesizing them, and their medical uses.

Description

Technical Field

[0001] This invention relates to multifunctional cyclic dinucleotide compounds and their derivatives, which can function as prodrugs to induce apoptosis in tumor cells or as antiviral agents, and as immunomodulators to regulate immune pathways to generate therapeutically beneficial immune responses, particularly activating the STING-mediated immune pathway. This disclosure further relates to pharmaceutical compositions and combinations comprising the cyclic dinucleotide compounds of this invention, methods for synthesizing them, and their medical uses. Background Technology

[0002] Cancer is a malignant disease characterized by abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness, and metastasis. It has become one of the leading causes of death in humans, and its incidence continues to rise worldwide. Meanwhile, viral infections have also caused the deaths of millions of people worldwide.

[0003] In anticancer / tumor and antiviral treatment strategies, antimetabolites are one of the main therapeutic approaches. Specifically, antimetabolites do not act directly; they must be converted into triphosphorylated forms by various cellular kinases in vivo. These triphosphorylated forms then act as pseudometabolites, serving as substrates for polymerases. They are then embedded in DNA or RNA via nucleic acid biosynthesis, inhibiting DNA or RNA modification and elongation, or inhibiting reverse transcriptases related to DNA or RNA synthesis. This induces apoptosis in tumor cells or inhibits viral replication, exhibiting cytotoxicity and making them suitable for treating cancer / tumor or viral infections. Simultaneously, the cellular debris generated by the cytotoxicity of nucleoside drugs triggers an immune response in the host cells, further inhibiting the growth and reproduction of tumor cells or viruses.

[0004] The following are examples of anticancer nucleosides currently used in clinical practice:

[0005]

[0006]

[0007] However, nucleoside analogs have multiple active groups such as hydroxyl and amino groups in their molecular structure, resulting in low membrane permeability, poor stability, and poor pharmacokinetic properties. This often necessitates special or frequent administration, causing considerable inconvenience to patients. Therefore, finding new and improved nucleoside analog solutions has always been an active area of ​​drug development.

[0008] Endogenous cyclic dinucleotide (CDN) cGAMP is a crucial component of the innate immune system—specifically, the cGAS-STING (cyclic GMP-AMP synthase-interferon gene stimulator) signaling pathway. Specifically, cGAS interacts with DNA from tumor cells, dying cells, viruses, bacteria, or mitochondria, catalyzing the synthesis of cyclic dinucleotide (CDN) cGAMP from ATP and GTP. The generated endogenous cGAMP further binds to STING on the endoplasmic reticulum (ER). The cGAMP-bound STING is activated, undergoes a conformational change, translocates to Golgi, and induces the activation of key transcription factors IRF-3 and NF-κB. The latter enter the nucleus and induce the expression of type I interferon and pro-inflammatory cytokines such as IL-6, TNF-α, and IFN-γ (Jiang et al., cGAS-STING, an important pathway in cancer immunotherapy, Journal of Hematology & Oncology, 2020, 13:81; Xiangling Cui et al., STING modulators: Predictive significance in drug discovery, European Journal of Medicinal Chemistry 182(2019)111591).

[0009] As is well known in the field, type I interferon not only exhibits antiviral activity, but also directly inhibits the proliferation of human tumor cells, significantly enhances antitumor immune responses by inducing the activation of adaptive and innate immune cells, and inhibits tumor invasion by regulating the expression of enzymes related to tissue remodeling. Therefore, it can be used as an anticancer agent.

[0010] Given that endogenous cyclic dinucleotide (CDN) cGAMP is a key mediator in the innate immune system's response to viruses and tumors, ultimately promoting the production of interferon or pro-inflammatory cytokines and thus achieving therapeutic benefits, a series of CDNSTING agonists have been synthesized and their laboratory activity validated, examples of which are described in WO2014 / 189805, WO2017 / 027645, and WO2018 / 060323. However, existing CDN-based therapies still lack sufficient clinical efficacy, and therefore improved CDN-based STING agonists are still needed to provide safer and more potent antiviral or antitumor effects.

[0011] According to the consensus in this field, the STING pathway can be activated by exogenous DNA (such as tumors or viruses). Without the mediation of protein neoantigens, the downstream pro-inflammatory factors driven by interferon and other factors lack targeting, resulting in poor tolerance to autoimmune responses and a narrow therapeutic window. First-generation single STING agonists combined with PD-1 antibodies still focus on the activation of systemic immunity, failing to address the selectivity issues of adaptive immunity and the induction of responses in the tumor microenvironment.

[0012] The compounds of this invention, on the one hand, act as known highly active STING agonists, activating signaling pathways, releasing interferon and other inflammatory factors, and activating the immune system; secondly, their cytotoxic function is subsequently activated, selectively killing tumors and releasing large amounts of tumor neoantigens and tumor DNA to establish the recognition function of adaptive immunity and train the immune system to target specific targets; thirdly, tumor neoantigens and tumor DNA continue to activate the STING pathway and other immune systems, killing tumor cells; finally, the released tumor neoantigens are recognized by DC cells, interact with T cells, form immune memory, and achieve long-term control of remote tumors and cancer cell migration.

[0013] Specifically, in the process of searching for new and improved cytotoxic nucleoside analogs, this application innovatively introduces cytotoxic nucleoside analogs as building blocks into the molecular structure of CDN, that is, introducing hidden cytotoxic pharmacophores at the molecular level of CDN. The resulting novel CDN drug molecule can first activate STING, thereby inducing the production of type I interferon, and then achieving antiviral or antitumor immunotherapy effects. More innovatively, the product formed by the in vivo decomposition of the molecule of this invention, namely the nucleoside cytotoxic drug, can specifically interfere with nucleic acid metabolism, prevent cell division and proliferation, cause tumor cell death or inhibit viral replication, release tumor DNA to continuously activate STING, and release tumor neoantigens to establish the recognition function of adaptive immunity, training the immune system to target specific targets. This overcomes the shortcomings of individual nucleoside analogs, such as low membrane permeability, lack of stability, poor pharmacokinetic properties, and the need for special or frequent administration. It also overcomes the problem of the source of exogenous DNA (such as tumor DNA) required for continuous activation of the STING pathway. More importantly, the CDN structural backbone of the molecule disclosed in this application can activate STING, thereby inducing the production of type I interferon and achieving antiviral or antitumor immunotherapy effects. On the other hand, the hidden cytotoxic pharmacophore in the novel CDN molecule of this invention will be released in a timely manner to generate apoptotic fragments, providing the immune system with antigens against tumors or viruses, and generating antibody-antigen responses in synergy with immune leukocyte subsets, thereby providing the ability of "immune memory" or durable immunity against the encountered antigens.

[0014] Therefore, this invention provides a new class of CDN compounds that, through drug combination at the submolecular level, can simultaneously achieve anti-metabolic therapy and immunosuppression for viral infections or tumors, providing enhanced or even synergistic effects compared to single cytotoxic drugs or simple CDN STING agonists.

[0015] It should be noted that the above discussion of the background of the invention is provided only to help readers understand the present invention, and does not acknowledge the prior art described or constituting the present invention. Invention Overview

[0017] The purpose of this invention is to provide a new set of antiviral or antitumor compounds based on cyclic dinucleotide structures.

[0018] On the one hand, the present invention provides a group of cyclic dinucleotide compounds having the following general formula, their stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts or solvates,

[0019] For example

[0020] Where X 1 and X 2 Each nucleoside is independently selected from -OH and -SH; and at least one of the two nucleotides in the cyclic nucleotides possesses cytotoxic or antiviral activity. Compounds with this structure, on the one hand, upon in vivo degradation, produce individual nucleoside cytotoxic or antiviral compounds, which, after activation by nucleoside kinase triphosphorylation in the cell, specifically interfere with nucleic acid metabolism, preventing cell division and proliferation, thereby inhibiting tumor cell proliferation or viral replication. On the other hand, the molecules of this invention retain the immune-activating function of the cyclic dinucleotide, namely, activating the target STING and ultimately inducing type I interferon production through the STING signaling cascade, thereby generating tumor immune activity, inhibiting tumor growth and metastasis, or exerting antiviral activity; moreover, the tissue debris from apoptosis provides the immune system with antigens not naturally expressed in the host, generating an antigen-antibody response, thereby providing the ability to "memory" or durable immunity against encountered antigens.

[0021] Specifically, the present invention provides a cyclic dinucleotide compound of formula (Y) in this respect.

[0022]

[0023] Where X 1 X 2 B1, B2, R 1 R 1 '、R 2 R 2'As defined herein; its stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts, prodrugs, or solvates.'

[0024] More specifically, the present invention provides cyclic dinucleotide compounds of formulas (I), (II), (III) and (IV) and their respective sub-formulas in this respect; more specifically, the present invention provides cyclic dinucleotide compounds of formulas (I), (II), (III) and (IV) in this respect; and their respective specific embodiments as described herein.

[0025] In another aspect, the present invention provides a method for preparing the compounds of the present invention described herein, and also provides the compounds of the present invention described herein that can be obtained by the methods described herein.

[0026] In another aspect, the present invention provides a pharmaceutical composition comprising the compound of the present invention described herein, its stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts, prodrugs or solvates, and one or more pharmaceutically acceptable excipients.

[0027] In another aspect, the present invention provides the compounds of the present invention described herein or the pharmaceutical compositions described herein as active agents for the treatment or prevention of diseases related to or mediated by immune responses, specifically for the treatment or prevention of diseases related to or mediated by STING, more specifically for the treatment or prevention of inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, especially for antiviral or antitumor purposes, or as vaccine adjuvants.

[0028] In another aspect, the present invention provides the compounds or pharmaceutical compositions described herein for use as cytotoxic agents, particularly antitumor agents, for the treatment or prevention of hyperproliferative diseases.

[0029] In another aspect, the present invention provides the compounds or pharmaceutical compositions described herein for use as cytotoxic agents for the treatment or prevention of viral infections.

[0030] In another aspect, the present invention provides the use of the compounds of the present invention or the pharmaceutical compositions described herein for the treatment or prevention of diseases related to or mediated by an immune response, such as as STING agonists, specifically for the treatment or prevention of diseases related to or mediated by STING, more specifically for the treatment or prevention of inflammation, allergic or autoimmune diseases, infectious diseases or cancer, especially tumors or viral infections; or for use as vaccine adjuvants.

[0031] In another aspect, the present invention provides the use of the compounds or pharmaceutical compositions described herein as cytotoxic agents in the treatment or prevention of proliferative diseases, particularly tumors; or as cytotoxic agents in the treatment or prevention of viral infections.

[0032] In another aspect, the present invention provides a method for treating or preventing in subjects diseases related to or mediated by an immune response, specifically diseases related to or mediated by STING, more specifically inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, especially tumors or viral infections, the method comprising administering to a human or animal the compounds of the present invention described herein or the pharmaceutical compositions described herein.

[0033] In another aspect, the present invention provides a method for treating or preventing hyperproliferative diseases, particularly tumors, in subjects, the method comprising administering to a human or animal the compounds of the present invention described herein or the pharmaceutical compositions described herein.

[0034] In another aspect, the present invention provides a method for treating or preventing viral infection in a subject, the method comprising administering to a human or animal the compounds of the present invention described herein or the pharmaceutical compositions described herein.

[0035] In another aspect, the present invention provides the use of the compounds of the present invention or the pharmaceutical compositions described herein in the preparation of a medicament for the treatment or prevention of diseases related to or mediated by an immune response, specifically diseases related to or mediated by STING, more specifically inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, especially tumors or viral infections, or as a vaccine adjuvant.

[0036] In another aspect, the present invention provides the use of the compounds or pharmaceutical compositions described herein in the preparation of medicaments for the treatment or prevention of hyperproliferative diseases, particularly tumors.

[0037] In another aspect, the present invention provides the use of the compounds or pharmaceutical compositions described herein in the preparation of medicaments for treating or preventing viral infections.

[0038] On the other hand, the present invention provides compounds or pharmaceutical compositions described herein as multifunctional active agents that possess both immunotherapeutic and cytotoxic therapeutic activities, including the ability to activate the immune system to exert antitumor and antiviral replication functions by stimulating the STING signaling pathway, to cause tumor cell death or inhibit viral replication by releasing cytotoxic agents, to continuously activate STING to kill tumor cells by releasing tumor DNA, and to provide “immune memory” or durable immunity against tumors by releasing tumor neoantigens to generate antibody-antigen responses. In this regard, the present invention also provides the use of the compounds of the present invention or the pharmaceutical compositions described herein for achieving the above-described multiple functions, for example, specifically for treating or preventing viral infections or tumors; methods for treating or preventing diseases related to or mediated by immune responses, specifically diseases related to or mediated by STING, more specifically inflammation, allergic or autoimmune diseases, infectious diseases or cancer, especially tumors or viral infections, in subjects through the above-described multiple functions, the method comprising administering the compounds of the present invention or the pharmaceutical compositions described herein to humans or animals; and the use of the compounds of the present invention or the pharmaceutical compositions described herein in the preparation of medicaments for achieving the above-described multiple functions, for example, specifically for treating or preventing viral infections or tumors.

[0039] In another aspect, the present invention provides pharmaceutical combinations comprising the compounds of the present invention described herein, their stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts or solvates, and at least one other therapeutic agent.

[0040] In another aspect, the present invention provides pharmaceutical compositions comprising the compounds of the present invention described herein, their stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts or solvates, at least one other therapeutic agent, and one or more pharmaceutically acceptable excipients.

[0041] In another aspect, the present invention provides pharmaceutical combinations comprising the compounds of the present invention and at least one other therapeutic agent, for the treatment or prevention of hyperproliferative diseases, viral infections or diseases related to or mediated by STING, more specifically inflammation, allergic or autoimmune diseases, infectious diseases or cancer, especially tumors or viral infections.

[0042] On the other hand, the present invention provides the use of pharmaceutical combinations comprising the compounds of the present invention and at least one other therapeutic agent, as described herein, for the treatment or prevention of hyperproliferative diseases, viral infections or diseases associated with or mediated by STING, more specifically inflammation, allergic or autoimmune diseases, infectious diseases or cancer, especially tumors or viral infections.

[0043] In another aspect, the present invention provides a method for treating or preventing hyperproliferative disorders, viral infections or diseases related to or mediated by STING, more specifically inflammation, allergic or autoimmune diseases, infectious diseases or cancer, especially tumors or viral infections, in subjects, the method comprising administering to a human or animal a pharmaceutical combination comprising the compounds of the present invention and at least one other therapeutic agent as described herein. Attached Figure Description

[0044] Appendix Figure 1 This demonstrates the interferon-stimulating activity of the representative compounds of the present invention in THP-1 cells.

[0045] Figure 2 shows the tumor growth inhibitory activity of the representative compounds of this invention in a mouse model of transplanted CT26 colon cancer. 2A: Tumor volume change after treatment; 2B: Tumor volume change after untreated tumor; 2C: Body weight change in mice; 2D: Tumor volume change in mice with and without immunization.

[0046] Figure 3 shows the immune memory of representative compounds of the present invention in mice with and without immunization. 3A: Immune memory in immunocompetent mice; 3B: Immune memory in non-immunized mice.

[0047] Figure 4 shows the hepatocyte metabolic properties of representative compounds of the present invention. 4A: Hepatocyte metabolic stability; 4B: Identification of hepatocyte metabolites.

[0048] Appendix Figure 5 : Schematic diagram of cGAS-STING signal path. Summary of the Invention

[0049] definition

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0051] Unless otherwise stated, the nomenclature used in this application is based on the IUPAC systematic nomenclature. IUPAC chemical names were generated using OpenEyeLexichem version 1.2.0, PerkinElmer E-notebook for Chemistry, or Insight for Excel 2017R2.

[0052] Unless otherwise stated, any open valence appearing on carbon, oxygen, sulfur, or nitrogen atoms in the structures described herein indicates the presence of a hydrogen atom.

[0053] The term “immune system” has its common meaning as understood by those skilled in the art, referring to the whole or any one or more components of molecules, substances (such as body fluids), anatomical structures (such as cells, tissues or organs) and physiological processes that are associated with preventing infection in the body, protecting the body during infection or disease, and / or helping the body recover health after infection or disease.

[0054] The term "disease related to or mediated by an immune response" refers to a disease related to or mediated by the body's immune system's defensive response to foreign or mutated self-components. For the purposes of this invention, "disease related to or mediated by an immune response" specifically refers to a state in a person or animal where the function of the immune system is weakened, inactivated, or otherwise impaired, or the function of one or more immune components is weakened, inactivated, or otherwise impaired, or a disease state caused by such a state, particularly a disease that can be alleviated by inducing an immune response through the STING pathway.

[0055] The term "STING" is an abbreviation for stimulator of interferon genes. STING is a transmembrane protein receptor in humans. Activation of STING by cyclic dinucleotides (CDNs) leads to activation of the IRF3 and NF-κB pathways, thereby inducing type I interferon and pro-inflammatory cytokines, respectively. The term "STING agonist" refers to any substance that activates STING in vitro or in vivo to elicit a physiological response.

[0056] The term "diseases associated with or mediated by STING" refers to diseases in which an immune response induced by the STING pathway can be alleviated, i.e., diseases in which activating STING will reduce the incidence of the disease, reduce or eliminate disease symptoms, including but not limited to inflammation, allergic or autoimmune diseases, infectious diseases, or cancer. For the purposes of this invention, "diseases associated with or mediated by STING" are preferably selected from tumors or cancer.

[0057] The terms “hyperproliferative disorder,” “tumor,” or “cancer” refer to a physiological condition in a subject characterized by uncontrolled or disordered cell growth or death, including solid tumors and hematogenous tumors, whether malignant or benign, including but not limited to brain cancer, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, leukemia, lung cancer, and bone cancer. Examples of the aforementioned cancer types include neuroblastoma, colorectal cancer such as rectal cancer, colon cancer, familial adenomatous polyposis carcinoma and hereditary non-lymphatic colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, nasopharyngeal cancer, oral cancer, salivary gland cancer, peritoneal cancer, soft tissue sarcoma, urethral epithelial carcinoma, sweat gland cancer, gastric cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, pancreatic cancer, prostate cancer, testicular cancer, breast cancer (including HER2-negative breast cancer), urinary tract cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma, and peripheral neuroectodermal tumors. Tumors, including Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CLL) and lymphocytic carcinoma, acute myeloid leukemia (AML), myeloid leukemia (chronic myeloid leukemia (CML), adult T-cell lymphoma, diffuse lymphoma (DLBCL), hepatocellular carcinoma, multiple myeloma, seminoma, osteosarcoma, chondrosarcoma, anal canal cancer, adrenocortical carcinoma, chordoma, fallopian tube cancer, gastrointestinal stromal tumor, myeloproliferative disorders, mesothelioma, biliary tract cancer, Ewing sarcoma and other rare tumor types.

[0058] The term "therapeutic agent" refers to one or more substances administered to humans or animals to achieve a therapeutic effect, including substances that prevent, cure, or mitigate the effects of disease or improve health. The therapeutic agents of this invention include not only the provided CDN compound itself, but also therapeutic agents that can be used in combination with the provided CDN compound, including but not limited to chemotherapeutic agents, immunotherapeutic agents (especially immuno-oncology agents), vaccines, adjuvants, and radiotherapy.

[0059] The term "chemotherapeutic agent" refers to one or more chemical substances that are administered to humans or animals to kill tumors, or slow or stop the growth of tumors, and / or slow or stop the division of cancer cells, and / or prevent or slow metastasis.

[0060] The term "immunizing agent" refers to any endogenous or exogenous substance that can interact with any one or more components of the immune system, including antibodies, antigens, vaccines and their components, nucleic acids, synthetic drugs, natural or synthetic organic compounds, cytokines, natural or modified cells, their synthetic analogs and / or fragments thereof.

[0061] The term "immunotherapy" refers to any medical treatment in which one or more components of the immune system of a human or animal are intentionally modulated to obtain a therapeutic benefit, either directly or indirectly, including systemic and / or local effects as well as preventative and / or therapeutic effects. Immunotherapy can be administered to human or animal subjects by any route, such as oral, intravenous, skin, injection, inhalation, or in any combination of these methods, either systemically, locally, or in combination.

[0062] The term "vaccine" refers to a biological agent administered to a human or animal to elicit or enhance a specific immune response and / or to provide protection against one or more antigens in that human or animal.

[0063] The term "adjuvant" refers to a secondary therapeutic substance administered in any order with the primary therapeutic substance to achieve a complementary, synergistic, or other beneficial effect that cannot be achieved by using the primary therapeutic substance alone. Adjuvants may be used with vaccines, chemotherapy, or other therapeutic substances to enhance the efficacy of the primary therapeutic substance, reduce its toxic side effects, or provide some protection to the subject receiving the primary therapeutic substance, such as, but not limited to, improving the function of the immune system.

[0064] As used herein, the terms "cytotoxic agent" or "apoptosis inducer" or similar expressions refer to active agents used to treat abnormal and uncontrolled growth of cells. For the purposes of this invention, "cytotoxic agent" specifically refers to nucleoside antimetabolite cytotoxic agents or antiviral agents, including but not limited to cytarabine, azacitidine, fluorouracil, deoxyuridine, enoxabin, deoxyfluorouracil, pentostatin, fludarabine, cladribine, gemcitabine, capecitabine, clofarabine, nelarabine, trifluorothymidine, 8-chloroadenosine, trichomoniasis, furofibrine, 5-fluorodeoxycytidine, ribavirin, or acalidicin.

[0065] As used herein, the term "multifunctional active agent" refers to the compound molecules of the present invention, which, based on their unique structural design, can perform multiple functions in the body of a subject, possessing both immunotherapeutic and cytotoxic therapeutic activities. These include, but are not limited to, activating the immune system by stimulating the STING signaling pathway to exert anti-tumor and antiviral replication functions, causing tumor cell death or inhibiting viral replication by releasing cytotoxic agents, continuously activating STING to kill tumor cells by releasing tumor DNA, and providing the ability to provide "immune memory" or durable immunity against tumors by releasing tumor neoantigens to generate antibody-antigen responses.

[0066] The term “treatment” for a disease includes suppressing the disease state, that is, preventing the development of the disease state or its clinical symptoms, or alleviating the disease state, that is, causing the temporary or permanent resolution of the disease state or its clinical symptoms.

[0067] The term “prevention” or “avoidance” of disease means preventing the development of clinical symptoms of the disease state in subjects who may be exposed to or susceptible to the disease state but have not yet experienced or exhibited symptoms of the disease state.

[0068] The term "therapeutic effective amount" refers to the amount of a compound or molecule of the present invention that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, improves, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. Therapeutic effective amounts will vary depending on the compound, the state of the disease being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors.

[0069] As used herein, the terms “subject,” “individual,” or “patient” refer to a vertebrate. In some embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cattle), sport animals, pets (such as guinea pigs, cats, dogs, rabbits, and horses), primates, mice, and rats. In a preferred embodiment, the mammal is a human.

[0070] The terms “pharmaceutical composition” and “pharmaceutical formulation” (or “formulation”) are used interchangeably and refer to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient and pharmaceutically acceptable excipients intended for administration to a mammal (e.g., a human) in need.

[0071] The term "pharmaceutical combination" refers to the combination of the compounds of the present invention with other active agents to achieve the objectives of the present invention. These other active agents may be one or more other compounds of the present invention, or may be a second or additional (e.g., a third) compound that is compatible with the compounds of the present invention, i.e., does not adversely affect each other, or has complementary activity. Such active agents are suitably combined in an effective amount to achieve the intended purpose. These other active agents may be administered co-administered with the compounds of the present invention in a single pharmaceutical composition, or separately administered with the compounds of the present invention in different discrete units, and when administered separately, they may be administered simultaneously or sequentially.

[0072] The term "pharmaceutically acceptable" refers to the properties of materials that can be used to prepare pharmaceutical compositions, which are generally safe, non-toxic, and not biologically or otherwise undesirable, and are acceptable for veterinary and human pharmaceutical use.

[0073] The terms “pharmaceutical-acceptable excipient,” “pharmaceutical-acceptable carrier,” and “therapeutic-inert excipient” are used interchangeably and refer to any pharmaceutically acceptable component in a pharmaceutical composition that is not therapeutically active and is non-toxic to the subject to administration, such as disintegrants, binders, fillers, solvents, buffers, tensioning agents, stabilizers, antioxidants, surfactants, carriers, diluents, or lubricants used in the formulation of pharmaceutical products.

[0074] As used herein, the term "pharmaceutically acceptable salt" means a salt of the compound of the present invention that is pharmaceutically acceptable and possesses the pharmacological activity required by the parent compound. Specifically, such salts are non-toxic and may be inorganic acid addition salts, organic acid addition salts, or base addition salts, including but not limited to: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed with organic acids such as acetic acid, propionic acid, hexanoic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, etc. Methanol, ethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucohepanoic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, mucoconic acid, etc.; or (2) salts formed when acidic protons present in the parent compound are replaced by metal ions such as alkali metal ions, alkaline earth metal ions or aluminum ions, or when coordinated with organic bases such as ethanolamine, diethanolamine, triethanolamine, N-methylglucosamine, etc. Those skilled in the art understand the general principles and techniques for preparing pharmaceutical salts, such as those described in Berge et al., Pharm ScL, 66, 1-19. (1977).

[0075] As used herein, the term "pharmaceutical-acceptable prodrug" refers to a compound having a cleavable group and which, through solvent decomposition or under physiological conditions, becomes a pharmaceutically active compound of the present invention. Specifically, prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce an active compound, including derivatives of the compounds of the present invention. Various forms of prodrugs are well known in the art, and suitable prodrug components are described, for example, in "Prodrugs and Targeted Delivery," J. Rautico, Ed., John Wiley & Sons, 2011.

[0076] The prodrugs of the CDN compounds described in this article generally increase the activity, bioavailability, or stability of the compound. Typically, alkylation, acylation, or other lipophilic modifications of the phosphate moiety, or the use of other analogues of nucleotides, will help increase the stability of the nucleotide.

[0077] As used herein, the term "solvent" refers to a solvation form containing a stoichiometric or non-stoichiometric solvent, including, for example, solvates with water, such as hydrates, or solvates with organic solvents, such as methanol, ethanol, or acetonitrile, i.e., as methanolides, ethanolides, or acetonitrileides, respectively; or in any polymorphic form. It should be understood that such solvates of the compounds of the present invention also include solvates of pharmaceutically acceptable salts of the compounds of the present invention.

[0078] As used herein, the term "isotope variant" refers to a compound in which one or more atoms constituting the compound are replaced by atoms having a different atomic mass or mass number than those commonly found in nature. Examples of isotopes that can be incorporated into one or more atoms of the compounds of the present invention include, for example... 2 H, 3 H, 13 C 14 C 15 N、 17 O、 18 O、 31 P, 32 P, 35 S and 18 F, thereby forming isotopic variations of the compounds of the present invention, whether or not they are radioactive, are intended to be covered within the scope of the present invention. In some embodiments, the incorporated isotope is 2H (deuterium); in other embodiments, the incorporated isotope is 3H (tritium).

[0079] As used herein, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., 1, 2, 3, or 4) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Similarly, the compounds of the present invention can exist as mixtures of two or more different structural forms in rapid equilibrium (commonly referred to as tautomers). It should be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0080] The compounds of this invention may have one or more asymmetric centers, and therefore can be prepared as (R)- or (S)- stereoisomers, or mixtures thereof. The structural formulas or structural fragments of the compounds used herein... The configuration of the asymmetric center, i.e., the chiral center, is indicated by R and S in the nomenclature of the compounds or intermediates provided in this invention. Those skilled in the art will recognize that the thiophosphate bonds in the compounds of this invention are inherently chiral and can each exist in either an R or S configuration; therefore, it is possible for the two thiophosphate bonds to be in the forms of R,R,S,S,S,R, and R,S. This invention covers the compounds of this invention in substantially pure form or in mixtures thereof, and specific embodiments thereof. Compounds containing two thiophosphate bonds are preferably in substantially pure forms of R,R,S,S,S,R, and R,S stereoisomers, particularly preferably substantially pure R,R stereoisomers, i.e., both phosphorus atoms have an R configuration. Their absolute configuration assignments can be made according to the literature method (Zhao et al. Nucleosides, Nucleotides and Nucleic Acids 2009, 289, 352-378; Knouse et al. Science 2018, 361, 1234). It should be noted that errors in the specification of configuration due to methodological mistakes in the literature do not affect the actual configuration of the compounds of this invention.

[0081] The term "substantially pure" as used herein with respect to CDN means that a certain stereoconfiguration is at least 75% pure relative to other possible stereochemical configurations at the chiral centers indicated above. In preferred embodiments, substantially pure CDN is at least 85% pure, at least 90% pure, at least 95% pure, at least 97% pure, and at least 99% pure. The substantially pure CDN formulation of the present invention is "stereochemically pure," meaning that all CDNs within the formulation have a specific stereochemical configuration at these chiral centers, and is not intended to indicate that all CDNs within the formulation having a specific stereochemical configuration at these chiral centers are otherwise identical. For example, a substantially pure CDN R,RcGAMP thiophosphate formulation may comprise a combination of R,Rc-di-GMP thiophosphate and R,Rc-di-AMP thiophosphate, and is still a substantially pure cyclic purine dinucleotide formulation.

[0082] Throughout this document, the term "protecting group" refers to a group that selectively blocks a reactive site in a multifunctional compound, allowing the chemical reaction to proceed selectively at another unprotected reactive site in the sense commonly associated with it in synthetic chemistry. Protecting groups can be removed at appropriate points in time. Exemplary protecting groups include amino protecting groups, including but not limited to TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), Bz (benzoyl), i-BuCO (isobutyryl), benzyl, benzyloxycarbonyl (carbonylbenzyloxy, CBZ), Fmoc (9-fluorenylmethoxycarbonyl), p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, tert-butoxycarbonyl (BOC), and trifluoroacetyl; and hydroxyl protecting groups, including but not limited to groups that form esters and ethers, particularly tetrahydropyranoxy, Bz (benzoyl). i- BuCO (isobutyryl), DMTr (bis(4-methoxyphenyl)benzyl), acetoxy, carbamoyloxy, benzyl, and silyl ethers such as TBS (tert-butyldimethylsilyl) and TBDPS (tert-butyldiphenylsilyl). Other examples of these groups can be found in TWGreene and PGMWuts, “Greene’s protective groups in organic synthesis,” 5th ed. John Wiley & Sons., Inc., Hoboken, New Jersey, 2014.

[0083] The term "deprotection" or "deprotection" refers to the process of removing the protecting group after a selective reaction has been completed. Deprotecting agents include acids, bases, or hydrogen, particularly potassium carbonate or sodium carbonate, alcoholic solutions of lithium hydroxide, methanolic solutions of zinc, acetic acid, trifluoroacetic acid, palladium catalysts, or boron tribromide.

[0084] As used herein, the term "alkyl" refers to a straight-chain or branched aliphatic hydrocarbon group having a specified number of carbon atoms. Specifically, an alkyl group may have 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. Examples of suitable C1-14 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, dimethylmethyl, dipropylmethyl, ethylbutylmethyl, diethylmethyl, methylethylmethyl, ethylpropylmethyl, diethylethyl, diethylpropyl, dipropylethyl, etc. Certain alkyl groups have 1 to 7 carbon atoms, for example, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.

[0085] As used in this article, the terms "halogenated" or "halogen" refer to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). A specific halogenation is fluorine or chlorine.

[0086] For clarity, the term "guanine" is used in this article. It can also be expressed as The thiophosphate groups used in the structural formula of the compounds of this invention can be drawn as follows:

[0087] Compounds of the present invention

[0088] Throughout this application, the terms “compound described herein,” “compound of the invention,” and “compound of the invention,” unless otherwise indicated, encompass compounds of formulas (X), (Y), and specific embodiments (I), (II), (III), and (IV) as defined herein, their respective sub-formula embodiments and their specific or preferred embodiments, their stereoisomers, tautomers, racemates, stable isotopic variants, pharmaceutically acceptable salts or solvates, and pharmaceutically acceptable prodrugs, each as described in the definitions section above. The compounds of the invention can be isolated as mixtures of isomers or as individual isomers, the individual isomers being prepared by, for example, resolution of racemates by chromatography or fractional crystallization, or by synthesis from optically active raw materials. Similarly, the term “intermediate,” whether or not it is claimed for protection, is intended to encompass its free form and the aforementioned derivatives, if the context permits.

[0089] Preferably, the compound of the present invention is in its free form or a pharmaceutically acceptable salt or solvate thereof; most preferably, it is in its free form or a pharmaceutically acceptable salt thereof.

[0090] Some compounds of the present invention may exist in polymorphic or amorphous forms, and these also fall within the scope of the present invention. When in solid crystalline form, the compounds of the present invention may be in eutectic form with another chemical entity, and this specification includes all such eutectics.

[0091] Specifically, on the one hand, the present invention provides cyclic dinucleotide compounds of the following formula, their stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts, prodrugs, or solvates:

[0092] For example

[0093] Where X 1 and X 2Each nucleotide is independently selected from -OH and -SH; and at least one of the two nucleosides of the two cyclic nucleotides possesses cytotoxic or antiviral activity, preferably antimetabolite anticancer drugs, i.e., nucleoside anticancer drugs or their derivatives, including but not limited to clinically used enocitabine, fludarabine, cytarabine, nelarabine, doxifluridine, capecitabine, azacitidine, pentostatin, cladribine, gemcitabine, and clofarabine, more preferably cladribine, gemcitabine, and clofarabine or their derivatives. The nucleoside anticancer drug derivatives mentioned herein refer to compounds that retain anticancer toxicity by having hydrogen atoms or groups of atoms in their structure replaced by other atoms or groups of atoms.

[0094] In a specific embodiment, the present invention provides a cyclic dinucleotide compound of formula (Y),

[0095]

[0096] Where X 1 and X 2 Each is independently selected from -OH and -SH;

[0097] B1 is adenine replaced by X. Where X is selected from Cl, F or -NHC 1-6 Alkyl; or optionally R a Substituted cytosine Where R a Selected from H or -C(O)-C 1-14 alkyl;

[0098] R1 and R1' are each independently selected from H, F or -OH;

[0099] B2 is selected from adenine that is arbitrarily replaced by X. Where X is selected from H, F, or Cl; R is arbitrarily selected. a Substituted cytosine Where R a Selected from H or -C(O)-C 1-14 Alkyl; or guanine The OH group can be optionally converted by C. 1-6 Alkyl substitution;

[0100] This indicates that the phosphate bond can be attached to either the 2' or 3' position of the pentose sugar, where the site not cyclized with the phosphate is replaced by R2 and R2'; and

[0101] R2 and R2' are each independently selected from H, -OH, or F;

[0102] The condition is that either B1 or B2 is arbitrarily selected by R. a When cytosine is substituted, the carbon atom adjacent to it on the pentose ring is replaced by two F atoms;

[0103] Its stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts, prodrugs, or solvates.

[0104] In a specific implementation, the compound of formula (Y) can be

[0105]

[0106] B1, B2, R1, R1', R2, and R2' have the meanings defined above for compound (Y).

[0107] In specific embodiments, the two thiophosphate bonds in the compounds of the present invention (when present) exist in the form of R,R,S,S,S,R or R,S configurations or mixtures thereof. In preferred embodiments, the two thiophosphate bonds in the compounds of the present invention (when present) exist in substantially pure forms of R,R,S,S,S,R or R,S configurations, particularly preferably in substantially pure forms of R,R configurations.

[0108] In specific embodiments, the single thiophosphate bond present in the compound of the present invention may exist in the R or S configuration, preferably in the R configuration.

[0109] In an embodiment of a compound of formula (I), (II), (III) or (IV), B1 is an adenine substituted with X. Where X is selected from Cl or F, preferably Cl, or optionally R a Substituted cytosine Where R a Selected from H or -C(O)-C 1-14 alkyl.

[0110] In an embodiment of a compound of formula (I), (II), (III) or (IV), B1 is an adenine substituted with X. Where X is -NHC 1-6 Alkyl groups, such as -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, preferably -NHCH3.

[0111] In an embodiment of a compound of formula (I), (II), (III) or (IV), B1 is optionally subjected to R a Substituted cytosine Where R a Selected from H or -C(O)-C 1-14 alkyl.

[0112] In an embodiment of a compound of formula (I), (II), (III) or (IV), R1 and R1' are both H, or one of them is H and the other is F, or both are F.

[0113] In an embodiment of a compound of formula (I), (II), (III), or (IV), B2 is an adenine optionally substituted with X. X is selected from H, F or Cl, with H or Cl being preferred.

[0114] In embodiments of a compound of formula (I), (II), (III), or (IV), B2 is optionally reacted with R. a Substituted cytosine Where R a Selected from H or -C(O)-C 1-14 alkyl.

[0115] In an embodiment of a compound of formula (I), (II), (III), or (IV), B2 is guanine.

[0116] In an embodiment of a compound of formula (I), (II), (III) or (IV), R2 and R2' are both H, or one of them is H and the other is F, or one of them is H and the other is OH, or both are F.

[0117] In an embodiment of a compound of formula (I), (II), (III) or (IV), R a -C(O)C 1-14 Alkyl groups can be, for example, -C(O)C 1-10 Alkyl, -C(O)C 1-9 Alkyl, -C(O)C 1-8 Alkyl, -C(O)C 1-7 Alkyl, -C(O)CH(C) 1-4 Alkyl)2、-C(O)CH(C 1-3 Alkyl)2、-C(O)CH2CH(C 1-4 Alkyl)2、-C(O)CH2CH(C 1-3 Alkyl)2.

[0118] The compounds of formulas (I), (II), (III) or (IV) of the present invention also cover any combination between the above-described embodiments and their preferred or exemplary embodiments.

[0119] In a further specific embodiment, the present invention provides a cyclic dinucleotide compound having the following general formula (Y),

[0120]

[0121]

[0122] B1, B2, R1, R1', R2, and R2' have the meanings defined above for compounds of formulas (I), (II), (III), or (IV) and their respective specific embodiments.

[0123] In an embodiment of a compound of formula (II-a) or (II-b), especially a compound of formula (II-a), B1 is... And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, one of R1 and R1' is H and the other is F. Further, in each of the specific embodiments, both R2 and R2' are H, or one is H and the other is F, or one is H and the other is OH.

[0124] In an embodiment of a compound of formula (II-a) or (II-b), especially a compound of formula (II-a), B1 and B2 are both optional components. a Substituted cytosine In this specific implementation, R1, R1', R2, and R2' are all F.

[0125] In an embodiment of a compound of formula (II-a) or formula (II-b), especially a compound of formula (II-b), B1 is... And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, one of R1 and R1' is H and the other is F. Further, in each of these embodiments, both R2 and R2' are H, or one is H and the other is F, or one is H and the other is OH, preferably one is H and the other is OH.

[0126] In an embodiment of a compound of formula (II-a) or formula (II-b), especially a compound of formula (II-b), B1 is optionally subjected to R a Substituted cytosine And B2 is In this specific embodiment, both R1 and R1' are F. Further, in each of these embodiments, both R2 and R2' are H, or one is H and the other is F, or one is H and the other is OH, preferably one is H and the other is OH.

[0127] In an embodiment of a compound of formula (II-a) or (II-b), R a For H, or for -C(O)C 1-10 Alkyl groups, such as -C(O)C 1-9 Alkyl, -C(O)C 1-8 Alkyl, -C(O)C 1-7 Alkyl, -C(O)CH(C) 1-4 Alkyl)2、 -C(O)CH(C 1-3 Alkyl)2、-C(O)CH2CH(C 1-4 Alkyl)2、-C(O)CH2CH(C 1-3 Alkyl)2.

[0128] In an embodiment of a compound of formula (II-a) or (II-b), B1 is Both R1 and R1' are H.

[0129] In an embodiment of a compound of formula (II-a) or (II-b), B1 is One of R1 and R1' is H, and the other is F; in this specific embodiment, F and B1 are located on the same side of the ribose.

[0130] In an embodiment of a compound of formula (II-a) or formula (II-b), especially a compound of formula (II-b), B1 is... Preferred Both R1 and R1' are H, or one of R1 and R1' is H and the other is F, preferably one of R1 and R1' is H and the other is F.

[0131] In an embodiment of a compound of formula (II-a) or (II-b), B1 is optionally subjected to R a Substituted cytosine Where R a Selected from H, R1 and R1' are both F.

[0132] In an embodiment of a compound of formula (II-a) or (II-b), B1 is optionally subjected to R a Substituted cytosine Where R a -C(O)C 1-10 Alkyl group, preferably -C(O)CH(C) 1-4 Alkyl)2, R1 and R1' are both F.

[0133] In an exemplary embodiment, R a is -C(O)CH(CH3)2, -C(O)CH(CH2CH3)2, - C(O)CH(CH3)(CH2CH3), -C(O)CH(CH2CH2CH3)2, - C(O)CH(CH2CH3)(CH2CH2CH3), -C(O)CH2CH(CH2CH3)(CH2CH2CH3), C(O)CH2CH(CH2CH2CH3)2, preferably R a It is C(O)CH(CH2CH2CH3)2.

[0134] In an embodiment of a compound of formula (II-a) or (II-b), B2 is guanine. or adenine Furthermore, one of R2 and R2' is H, and the other is selected from -OH or F.

[0135] In one embodiment of a compound of formula (II-a), B2 is guanine. One of R2 and R2' is H, and the other is selected from -OH or F.

[0136] In one embodiment of a compound of formula (II-a), B2 is adenine. One of R2 and R2' is H, and the other is selected from -OH or F.

[0137] In one embodiment of a compound of formula (II-a), B2 is an adenine substituted with halogen X. Where X is Cl; one of R2 and R2' is H and the other is F, preferably F and B2 are located on the same side of the ribose.

[0138] In one embodiment of a compound of formula (II-a), B2 is an adenine substituted with halogen X. Where X is C1; R2 and R2' are both H.

[0139] In one embodiment of a compound of formula (II-a), B2 is optionally replaced by R. a Substituted cytosine Where R a Selected from H, R1 and R1' are both F.

[0140] In one embodiment of a compound of formula (II-a), B2 is R a Substituted cytosine Where R a -C(O)C 1-10 Alkyl group, preferably -C(O)CH(C) 1-4 Alkyl)2, R1 and R1' are both F.

[0141] In an exemplary embodiment, R a is -C(O)C(CH3)2, -C(O)CH(CH2CH3)2, - C(O)CH(CH3)(CH2CH3), -C(O)CH(CH2CH2CH3)2, - C(O)CH(CH2CH3)(CH2CH2CH3), -C(O)CH2CH(CH2CH3)(CH2CH2CH3), C(O)CH2CH(CH2CH2CH3)2, preferably R a It is C(O)CH(CH2CH2CH3)2.

[0142] In one embodiment of a compound of formula (II-b), B2 is guanine. or adenine One of R2 and R2' is H, and the other is -OH.

[0143] In the compounds of formula (II-a) or (II-b) and their respective embodiments, the two thiophosphate bonds exist in the form of R,R,S,S,S,R or R,S configurations or mixtures thereof; preferably in substantially pure form in the R,R,S,S,S,R or R,S configurations, and particularly preferably in substantially pure form in the R,R configuration.

[0144] The specific embodiments and preferred or exemplary methods given above for compounds of formula (II-a) or (II-b) are also applicable to compounds of formula (Ia) or (Ib), formula (III-a) or (III-b), and formula (IV-a) or (IV-b), respectively. That is, the present invention also covers compounds of formula (Ia) or (Ib), formula (III-a) or (III-b), and formula (IV-a) or (IV-b), wherein each specific substituent takes the specific definition given above for compounds of formula (II-a) or (II-b) or their specific embodiments.

[0145] For example, in an embodiment of a compound of formula (Ia) or (Ib), a compound of formula (III-a) or (III-b), or a compound of formula (IV-a) or (IV-b), especially a compound of formula (Ib), formula (III-b), or formula (IV-b), B1 is... And B2 is Preferred In one specific embodiment, one of R1 and R1' is H and the other is F. Further, in each of these embodiments, one of R2 and R2' is H and the other is OH.

[0146] The compounds of formula (Ia) / (Ib), (II-a) / (II-b), (III-a) / (III-b) or (IV-a) / (IV-b) of the present invention also cover any combination between the above-described embodiments and their preferred or exemplary embodiments.

[0147] In a further specific embodiment, the present invention provides cyclic dinucleotide compounds of the following general formula:

[0148]

[0149]

[0150] Wherein B1, B2, R1, R1', R2, R2' have the meanings defined above for compounds of formula (I), (II), (III) or (IV) or their respective specific embodiments; more specifically, they have the meanings defined above for compounds of formula (Ia) / (Ib), (II-a) / (II-b), (III-a) / (III-b) or (IV-a) / (IV-b) or their respective specific embodiments.

[0151] In an embodiment of a compound of formula (II-a') or formula (II-b'), especially a compound of formula (II-a'), B1 is And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, R1 is F and R1' is H, meaning F and B1 are on the same side of the ribose. Further, in each of these specific embodiments, both R2 and R2' are H, or R2 is H and R2' is F, or R2 is F and R2' is H, or R2 is OH and R2' is H.

[0152] In an embodiment of a compound of formula (II-a') or formula (II-b'), especially a compound of formula (II-a'), B1 and B2 are both optional components. a Substituted cytosine In this specific implementation, R1, R1', R2, and R2' are all F.

[0153] In an embodiment of a compound of formula (II-a') or formula (II-b'), especially a compound of formula (II-b'), B1 is And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, R1 is F and R1' is H, meaning F and B1 are on the same side of the ribose. Further, in each of these embodiments, both R2 and R2' are H, or R2 is H and R2' is F, or R2 is F and R2' is H, or R2 is OH and R2' is H; preferably, R2 is OH and R2' is H.

[0154] In an embodiment of a compound of formula (II-a') or formula (II-b'), especially a compound of formula (II-b'), B1 is optionally subjected to R a Substituted cytosine And B2 is In this specific embodiment, both R1 and R1' are F. Further, in each of these embodiments, both R2 and R2' are H, or R2 is H and R2' is F, or R2 is F and R2' is H, or R2 is OH and R2' is H; preferably R2 is OH and R2' is H.

[0155] In an embodiment of a compound of formula (II-a') or formula (II-b'), R a For H, or -C(O)C 1-10 Alkyl groups, such as -C(O)C 1-9 Alkyl, -C(O)C 1-8 Alkyl, -C(O)C 1-7 Alkyl, -C(O)CH(C) 1-4 Alkyl)2、-C(O)CH(C 1-3 Alkyl)2、-C(O)CH2CH(C 1-4 Alkyl)2、-C(O)CH2CH(C 1-3 Alkyl)2.

[0156] In an embodiment of a compound of formula (II-a') or formula (II-b'), B1 is... Both R1 and R1' are H, thus B1, together with the ribose it is attached to, forms the nucleoside antitumor drug cladribine.

[0157] In an embodiment of a compound of formula (II-a') or formula (II-b'), B1 is... R1 is F and R1' is H, meaning that F and B1 are located on the same side of the ribose. Thus, B1, together with the ribose it is attached to, forms the nucleoside antitumor drug clofarabine.

[0158] In an embodiment of a compound of formula (II-a') or formula (II-b'), especially a compound of formula (II-b'), B1 is Preferred Both R1 and R1' are H, or R1 is F and R1' is H, preferably R1 is F and R1' is H.

[0159] In an embodiment of a compound of formula (II-a') or (II-b'), B1 is optionally subjected to R a Substituted cytosine Where R a Selected from H, with R1 and R1' both being F, this B1, together with the ribose it is attached to, forms the nucleoside antitumor drug gemcitabine.

[0160] In an embodiment of a compound of formula (II-a') or (II-b'), B1 is optionally subjected to R a Substituted cytosine Where R a -C(O)C 1-10 Alkyl group, preferably -C(O)CH(C) 1-4 Alkyl group B1, R1, and R1' are both F, thereby forming an alkylacylated derivative of the nucleoside antitumor drug gemcitabine together with the ribose it is attached to. In an exemplary embodiment, R1... a is -C(O)CH(CH3)2, -C(O)CH(CH2CH3)2, - C(O)CH(CH3)(CH2CH3), -C(O)CH(CH2CH2CH3)2, -C(O)CH(CH2CH3)(CH2CH2CH3), -C(O)CH2CH(CH2CH3)(CH2CH2CH3), C(O)CH2CH(CH2CH2CH3)2, preferably R a It is C(O)CH(CH2CH2CH3)2.

[0161] In one embodiment of a compound of formula (II-a'), B2 is guanine. R2' is H, and R2 is selected from -OH or F.

[0162] In one embodiment of a compound of formula (II-a'), B2 is adenine. R2' is H, and R2 is selected from -OH or F.

[0163] In one embodiment of a compound of formula (II-a'), B2 is an adenine substituted with halogen X. Where X is Cl, R2 is H, and R2' is F, and F and B2 are located on the same side of the ribose, thus B2 together with the ribose it is attached to forms the nucleoside antitumor drug clofarabine.

[0164] In one embodiment of a compound of formula (II-a'), B2 is an adenine substituted with halogen X. Where X is Cl, and R2 and R2' are both H, thus B2 together with the ribose it is attached to forms the nucleoside antitumor drug cladribine.

[0165] In one embodiment of a compound of formula (II-a'), B2 is optionally replaced by R. a Substituted cytosine Where R a Selected from H, with R1 and R1' both being F, this B2, together with the ribose it is attached to, forms the nucleoside antitumor drug gemcitabine.

[0166] In one embodiment of a compound of formula (II-a'), B2 is R a Substituted cytosine Where R a -C(O)C 1-10 Alkyl group, preferably -C(O)CH(C) 1-4 Alkyl group B2, where R1 and R1' are both F, thereby forming an alkylacylated derivative of the nucleoside antitumor drug gemcitabine together with the ribose it is attached to. In an exemplary embodiment, R... a is -C(O)CH(CH3)2, -C(O)CH(CH2CH3)2, - C(O)CH(CH3)(CH2CH3), -C(O)CH(CH2CH2CH3)2, - C(O)CH(CH2CH3)(CH2CH2CH3), -C(O)CH2CH(CH2CH3)(CH2CH2CH3), C(O)CH2CH(CH2CH2CH3)2, preferably R a It is C(O)CH(CH2CH2CH3)2.

[0167] In one embodiment of a compound of formula (II-b'), B2 is guanine. or adenine R2' is H, and R2 is -OH.

[0168] The specific embodiments and preferred or exemplary methods given above for compounds of formula (II-a') or (II-b') are also applicable to compounds of formula (I-a') or (I-b'), formula (III-a') or (III-b'), and formula (IV-a') or (IV-b'), respectively. That is, the present invention also covers compounds of formula (I-a') or (I-b'), formula (III-a') or (III-b'), and formula (IV-a') or (IV-b'), wherein each specific substituent takes the specific definition given above for compounds of formula (II-a') or (II-b') or their specific embodiments.

[0169] For example, in an embodiment of a compound of formula (I-a') or (I-b'), a compound of formula (III-a') or (III-b'), or a compound of formula (IV-a') or (IV-b'), especially a compound of formula (I-b'), formula (III-b') or (IV-b'), B1 is And B2 is Preferred In one specific embodiment, R1 is F and R1' is H. Further, in each of these embodiments, R2 is OH and R2' is H.

[0170] It should be noted that the compounds of the present invention cover the various specific embodiments described above, as well as embodiments consisting of any combination or sub-combination of the various specific embodiments described above, and embodiments consisting of any combination of any preferred or exemplary embodiments described above.

[0171] Preferred embodiments of the compounds of the present invention include the following compounds, their stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts or solvates.

[0172]

[0173]

[0174] Pharmacological activity and beneficial effects of the compounds of this invention

[0175] Studies have revealed that the cyclic dinucleotide compound provided by this invention possesses the following pharmacological activities and beneficial effects:

[0176] ● It effectively stimulates THP-1 cells to secrete IFN-β, which indicates that it has a high affinity for the STING receptor, can effectively activate STING, and induce the production of type I interferon.

[0177] ● It effectively inhibits the in vitro growth of the mouse colorectal cancer cell line CT26, indicating that it has a toxic effect on tumor cells and prevents tumor cell division and proliferation;

[0178] ● It showed superior antitumor activity in immunocompetent CT26 syngeneic mouse bilateral xenograft models compared to immunodeficient mouse models, indicating that it simultaneously activates STING to produce tumor immune activity and cytotoxic effects, resulting in an additive or even synergistic antitumor effect.

[0179] ● It demonstrated immune memory function in a CT26 syngeneic mouse xenograft model, effectively preventing tumor recurrence;

[0180] ● Demonstrates favorable pharmacokinetic properties in hepatocyte metabolism studies, such as a long t0.1 / 2 And a low clearance rate, which allows for longer dosing intervals and better patient compliance; and

[0181] ● Local application of medication to the lesion site allows for precise targeting, reduces the amount of medication required, and due to the high molecular polarity, it is difficult to diffuse outside the lesion, resulting in limited toxicity and good safety.

[0182] Pharmaceutical Composition

[0183] Another aspect of the present invention provides a pharmaceutical composition comprising the compound of the present invention and one or more pharmaceutically acceptable excipients, and a method for preparing the composition using the compound of the present invention.

[0184] The composition or dosage form is formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery, the method of administration, the timing of administration, and other factors known to the medical practitioner.

[0185] Typical pharmaceutical compositions or dosage forms are prepared by mixing the compounds of the present invention with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, in Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000), Lippincott, Williams & Wilkins, Philadelphia. Formulations may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, flow aids, processing aids, colorants, sweeteners, flavoring agents, diluents, and other known additives to provide an elegant appearance for the pharmaceutical product (i.e., the compound of the present invention or a pharmaceutical composition thereof) or to facilitate the preparation of the pharmaceutical product (i.e., the dosage form).

[0186] The compounds of the present invention can be administered by any suitable manner, including oral, topical (including sublingual and sublingual), rectal, vaginal, percutaneous, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal, epidural, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0187] In a preferred embodiment, a pharmaceutical composition comprising one or more compounds of the present invention is provided, wherein the pharmaceutical composition is suitable for intravenous, intratumoral, peritumoral, or subcutaneous administration. Intratumoral (direct entry into the tumor mass) or peritumoral (around the tumor mass) administration of the compounds of the present invention can directly activate locally infiltrative dendritic cells, directly promote tumor cell apoptosis, or sensitize tumor cells to cytotoxic agents.

[0188] The compounds of this invention can be administered in any convenient form of drug delivery, such as tablets, powders, capsules, sterile injectable formulations, solutions, dispersants, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain components conventional to pharmaceutical formulations, such as diluents, carriers, pH adjusters, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for altering osmotic pressure, buffers, masking agents, antioxidants, and other active agents. They may also contain other substances of therapeutic value. Various formulations can be prepared according to conventional methods in the pharmaceutical field, see, for example, Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000), Lippincott, Williams & Wilkins, Philadelphia, or national pharmacopoeias.

[0189] In a preferred embodiment, a pharmaceutical composition comprising one or more compounds of the present invention is provided, in the form of a parenteral preparation, particularly a sterile injectable preparation, such as a sterile injectable solution or suspension in a non-toxic, parenteral-acceptable diluent or solvent, or prepared as a lyophilized powder. Acceptable media or solvents may include, for example, water, 1,3-butanediol, Ringer's solution, or isotonic sodium chloride solution; furthermore, sterile non-volatile oils may conventionally be used as solvents or suspension media, for which any mild non-volatile oil may be used, including, for example, synthetic monoglycerides or diglycerides, fatty acids, etc.

[0190] The dosage of the compounds of this invention can vary over a wide range, and of course, can be adjusted according to individual needs in each specific case. Typically, the effective dose of the compounds of this invention for systemic administration is from about 0.1 μg / kg / day to about 50 mg / kg / day, for example, from 0.5 μg / kg / day to about 10 mg / kg / day, or from 1 μg / kg / day to about 1 mg / kg / day. Each dosage unit can conveniently contain from 0.001 μg to 10 mg, for example, from 0.01 μg to 1 mg, or from 50 μg to 500 μg. The effective dose can be given in one or more doses, i.e., administered once, twice, or more times, and can be administered multiple times at equal or unequal time intervals, including once or more daily, once or more weekly, or according to a regimen of several days / weeks.

[0191] Uses and methods

[0192] Given that the compounds of the present invention can activate STING, induce the expression of type I interferon and pro-inflammatory cytokines such as IL-6, TNF-α and IFN-γ, and also have cytotoxic activity, the present invention, in another aspect, provides therapeutic uses and methods for the compounds of the present invention.

[0193] On the one hand, the compounds or pharmaceutical compositions described herein can be used as therapeutic substances for the treatment or prevention of diseases related to or mediated by immune responses, specifically for the treatment or prevention of diseases related to or mediated by STING, including inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, or as vaccine adjuvants.

[0194] In a preferred embodiment, the compounds or compositions of the present invention are used as cytotoxic agents for the treatment or prevention of proliferative diseases, particularly tumors. In another preferred embodiment, the compounds or compositions of the present invention are used to treat recurrent tumors or to prevent tumor recurrence.

[0195] In a preferred embodiment, the compounds or compositions of the present invention are used as cytotoxic agents for the treatment or prevention of viral infections.

[0196] On the other hand, the present invention thereby provides methods for inducing, stimulating, or assisting an immune response in an individual, including administering the compounds or pharmaceutical compositions of the present invention to the individual. In one embodiment, the compounds of the present invention are administered to an individual as an immunotherapy to induce the production in the body of a variety of cytokines that are therapeutically useful in humans or animals, including type I interferon and pro-inflammatory cytokines such as IL-6, TNF-α, and IFN-γ, to modulate the immune system of the human or animal to achieve certain therapeutic benefits.

[0197] On the other hand, the present invention thereby provides methods for treating or preventing diseases related to or mediated by immune responses, specifically diseases related to or mediated by STING, including inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, including administering a therapeutically effective amount of the compound or pharmaceutical composition of the present invention to a subject in need.

[0198] In a preferred embodiment, the present invention provides a method for treating or preventing hyperproliferative diseases, particularly tumors, comprising administering a therapeutically effective amount of a compound or pharmaceutical composition of the present invention to a subject in need. In another preferred embodiment, the present invention provides a method for treating or preventing tumor recurrence, comprising administering a therapeutically effective amount of a compound or pharmaceutical composition of the present invention to a subject in need.

[0199] In a preferred embodiment, the present invention provides a method for treating or preventing viral infection, comprising administering a therapeutically effective amount of the compound or pharmaceutical composition of the present invention to a subject in need.

[0200] On the other hand, the present invention thereby provides the use of the compounds or pharmaceutical compositions of the present invention in the preparation of medicaments for the treatment or prevention of diseases related to or mediated by immune responses, specifically diseases related to or mediated by STING, including inflammatory, allergic or autoimmune diseases, infectious diseases or cancer.

[0201] On the other hand, the present invention also provides the use of the compounds or pharmaceutical compositions of the present invention in the preparation of vaccine adjuvants.

[0202] In a preferred embodiment, the present invention provides the use of the compounds or pharmaceutical compositions of the present invention in the preparation of a medicament for treating or preventing proliferative diseases, particularly tumors. In another preferred embodiment, the present invention provides the use of the compounds or pharmaceutical compositions of the present invention in the preparation of a medicament for treating recurrent tumors or for preventing tumor recurrence.

[0203] In a preferred embodiment, the present invention provides the use of the compounds or pharmaceutical compositions of the present invention in the preparation of a medicament for treating or preventing viral infections.

[0204] Inflammation related to the above uses and methods can be acute or chronic, involving any organ or tissue in the body, including musculoskeletal inflammation, vascular inflammation, neurological inflammation, digestive system inflammation, eye inflammation, reproductive system inflammation, or other inflammations, as well as autoimmune diseases and allergic diseases with inflammatory characteristics such as contact dermatitis, urticaria, and respiratory allergies.

[0205] Autoimmune diseases for the purposes and methods described above refer to diseases in which the body's own tissues are damaged due to an immune response to its own antigens, including but not limited to systemic lupus erythematosus, psoriasis, insulin-dependent diabetes mellitus, dermatomyositis, Sjögren's syndrome, chronic fatigue syndrome, aplastic anemia, autoimmune hepatitis, multiple sclerosis, optic neuritis, pemphigus, rheumatoid arthritis, ulcerative colitis, regional ileitis, scleroderma, scleroderma, etc.

[0206] The hyperproliferative diseases for the purposes and methods described above refer to physiological conditions in subjects characterized by uncontrolled or disordered cell growth or death, particularly tumors or cancers, including solid tumors and hematogenous tumors, including but not limited to brain cancer, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, leukemia, lung cancer, and bone cancer. Examples of the aforementioned cancer types include neuroblastoma, colorectal cancer such as rectal cancer, colon cancer, familial adenomatous polyposis carcinoma and hereditary non-lymphatic colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, nasopharyngeal cancer, oral cancer, salivary gland cancer, peritoneal cancer, soft tissue sarcoma, urothelial carcinoma, sweat gland cancer, gastric cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, pancreatic cancer, prostate cancer, testicular cancer, breast cancer (including HER2-negative breast cancer), urinary tract cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, Hodgkin's lymphoma. Lymphoma, non-Hodgkin's lymphoma, Burkitt lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CLL) and lymphocytic carcinoma, acute myeloid leukemia (AML), myeloid leukemia (chronic myeloid leukemia (CML), adult T-cell lymphoma, diffuse lymphoma (DLBCL), hepatocellular carcinoma, multiple myeloma, seminoma, osteosarcoma, chondrosarcoma, anal canal cancer, adrenocortical carcinoma, chordoma, fallopian tube cancer, gastrointestinal stromal tumor, myeloproliferative disorders, mesothelioma, biliary tract cancer, Ewing sarcoma and other rare tumor types, as well as recurrent forms of the above tumors.

[0207] In a preferred embodiment, the proliferative diseases for which the above-described uses and methods are applied are small cell lung cancer, non-small cell lung cancer, colorectal cancer, liver cancer, breast cancer, ovarian cancer, gastric cancer, prostate cancer, melanoma, renal cell carcinoma, head and neck cancer, pancreatic cancer, Hodgkin's lymphoma, leukemia, or bladder cancer.

[0208] Viral infection, as described in the above-mentioned uses and methods, refers to the process by which viruses invade the body through various pathways and multiply in susceptible host cells. The viruses involved include, but are not limited to, double-stranded DNA viruses and single-stranded DNA viruses, single-stranded positive-sense RNA viruses, single-stranded negative-sense RNA viruses and double-stranded RNA viruses, and retroviruses. Examples include hepatitis B virus, TTV virus, adenovirus, papillomavirus, herpes zoster virus, smallpox virus and vaccinia virus, influenza virus, classical swine fever virus, hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola virus, enteroviruses, and human immunodeficiency virus. The therapeutic uses and methods provided by this invention can be used for the above-mentioned viral infections and the diseases they cause.

[0209] The use of the compounds or compositions of the present invention as vaccine adjuvants and in the preparation of vaccine adjuvants refers to the use of the compounds or compositions of the present invention as adjuvants in therapeutic or preventive strategies employing vaccines, i.e., the compounds or compositions of the present invention are used with one or more vaccines selected to stimulate an immune response to one or more predetermined antigens, said vaccines comprising inactivated or attenuated bacteria or viruses, such as inactivated tumor cells expressing and secreting one or more of GM-CSF, CCL-20, CCL3, IL-12p70, FLT-3 ligands, and cytokines.

[0210] Drug combination

[0211] In view of the beneficial pharmacological activity of the compounds of the present invention, in addition to being used alone in the therapeutic uses or methods mentioned above, they can also be used in combination with at least one other therapeutic agent or therapy to provide further therapeutic benefits.

[0212] Therefore, another aspect of the present invention provides a pharmaceutical combination comprising, or consisting of, the cyclic dinucleotide compound described herein, its stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts, prodrugs or solvates or pharmaceutical compositions thereof, and at least one other therapeutic agent.

[0213] On the other hand, the present invention provides pharmaceutical compositions comprising the pharmaceutical combination described herein and one or more pharmaceutically acceptable excipients.

[0214] On the other hand, the present invention provides use of pharmaceutical combinations or pharmaceutical compositions comprising the pharmaceutical ingredients described herein for the treatment or prevention of diseases, or use in the preparation of pharmaceuticals for the treatment or prevention of diseases, including proliferative disorders, viral infections or diseases related to or mediated by STING, more specifically inflammatory, allergic or autoimmune diseases, infectious diseases or cancer. In a preferred embodiment, the disease involved in the use is a tumor or a viral infection.

[0215] On the other hand, the present invention also provides a treatment method in which the compound of the present invention is administered together with one or more other therapeutic agents.

[0216] Inflammation, autoimmune diseases, hyperproliferative diseases, and viral infections for the purpose of use of the pharmaceutical combinations of the present invention and pharmaceutical compositions comprising the present invention, as described above in the uses and methods of the present invention.

[0217] The compounds of the present invention can also be used in combination with surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immuno-oncology drugs, etc.

[0218] Other therapeutic agents used in combination with the present invention may be administered simultaneously, separately, or sequentially with the compounds of the present invention via the same or different routes of administration. These other therapeutic agents may be administered co-administered with the compounds of the present invention in a single pharmaceutical composition, or separately administered in different discrete units, such as combination products, preferably in the form of a pillbox, and when administered separately, may be simultaneous or sequential, with the sequential administration occurring close or spaced apart in time. They may be prepared and / or formulated by the same or different manufacturers. Furthermore, the compounds of the present invention and other therapeutic agents may (i) be sent to a physician before the combination product is delivered (e.g., in the case of a pillbox containing the compounds of the present invention and other drugs); (ii) be administered by the physician himself (or under the physician's guidance) immediately before administration; or (iii) be added by the patient himself, for example, during the sequential administration of the compounds of the present invention and other therapeutic agents, to the combination therapy.

[0219] Therefore, on the other hand, the present invention also provides a pillbox comprising two or more separate pharmaceutical compositions, at least one of which comprises the cyclic dinucleotide compound of the present invention, its stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts, prodrugs, or solvates, the remaining co-occurring pharmaceutical compositions comprising at least one other therapeutic agent, and means for separately containing said compositions, such as containers, dispensing bottles, or separate foil packages, for example, blister packs for packaging tablets, capsules, etc. The pillbox of the present invention is particularly suitable for administering different dosage forms, such as oral and parenteral dosage forms, or for administering different compositions at different dose intervals.

[0220] The other therapeutic agents may be one or more other compounds of the present invention, or may be a second or other (e.g., a third) therapeutic agent that is compatible with the compounds of the present invention, i.e., does not adversely affect each other, or has complementary or other activities.

[0221] In specific implementation schemes, other therapeutic agents that can be used in combination with the compounds of the present invention include, but are not limited to, vaccines, adjuvants, immune checkpoint inhibitors, T-cell receptor agonists, TLR agonists, therapeutic antibodies, lipids, liposomes, chemotherapeutic agents, and immunomodulatory cell lines.

[0222] In one specific embodiment, adjuvants used in combination with the compounds of the present invention can be used, due to their properties, to stimulate or otherwise utilize the immune system to respond to cancer antigens present on tumor cells, including but not limited to lipids, liposomes, fire-inducing bacteria that induce innate immunity, compounds that mediate the activation of innate immunity, etc.

[0223] In one specific embodiment, the immune checkpoint inhibitor used in combination with the compounds of the present invention is selected, for example, from CTLA-4 pathway antagonists, PD-1 pathway antagonists, Tim-3 pathway antagonists, Vista pathway antagonists, BTLA pathway antagonists, LAG-3 pathway antagonists, or TIGIT pathway antagonists.

[0224] In one specific embodiment, the T-cell receptor agonists used in combination with the compounds of the present invention include, but are not limited to, CD28 agonists, OX40 agonists, GITR agonists, CD137 agonists, CD27 agonists, or HVEM agonists.

[0225] In one specific embodiment, the TLR agonists used in combination with the compounds of the present invention include, but are not limited to, Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, polyadenosine-polyuridine (polyAU), LPS, bacterial flagellin, monophosphoryl lipid A (MPL), imiquimod, reciprocitate, loxoribin, etc.

[0226] In one specific embodiment, the chemotherapeutic agents used in combination with the compounds of the present invention include, but are not limited to, alkylated antitumor drugs, platinum-based antitumor drugs, antimetabolites, antimicrotubule agents, antimitotic agents, topoisomerase inhibitors, and antitumor antibiotics.

[0227] In one specific embodiment, the therapeutic antibodies used in combination with the compounds of the present invention include, but are not limited to, Muromonab-CD3 and Infliximab. and adalimumab Omalizumab Daclizumab Rituximab(trade ), Ibritumomab (trade ), Tositumomab Cetuximab Trastuzumab Alemtuzumab Lym-1 Ipilimumab Vitaxin, Bevacizumab Abciximab Other therapeutic antibodies that can be used in combination include prolactin receptor inhibitors, HER3 inhibitors, EGFR2 and / or EGFR4 inhibitors, M-CSF inhibitors, anti-APRIL antibodies, or anti-SIRPa or anti-CD47 antibodies.

[0228] In other embodiments, the compounds of the present invention may also be used in combination with PKC inhibitors, BCR-ABL inhibitors, HSP90 inhibitors, PI3K and / or mTOR inhibitors, FGFR inhibitors, cytochrome P450 inhibitors, HDM2 inhibitors, aromatase inhibitors, p53 and / or p53 / Mdm2 interaction inhibitors, or CSF-1R tyrosine kinase inhibitors.

[0229] Examples of the above-mentioned therapeutic agents and other therapeutic agents that can be used in combination with the compounds of the present invention can be found in WO2016 / 145102 and WO2018 / 060323, the contents of which are incorporated herein by reference.

[0230] For the compounds, pharmaceutical compositions, methods, uses, pharmaceutical combinations and kits of the present invention described above, the cyclic dinucleotide compounds of the present invention, their stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts, prodrugs or solvates or pharmaceutical compositions thereof in the various preferred embodiments described above are preferred, the compounds defined in the specific embodiments, i.e. the compounds of Examples 1-26, are more preferred, and those compounds that show excellent activity in the active embodiments are most preferred.

[0231] For the compounds, pharmaceutical compositions, methods, uses, pharmaceutical combinations and kits of the present invention described above, it is preferred to use the free form of the cyclic dinucleotide compound as defined herein or a pharmaceutically acceptable salt or prodrug, and more preferably the free form of the substantially pure cyclic dinucleotide compound as defined herein or a pharmaceutically acceptable salt or prodrug.

[0232] For the therapeutic uses and methods of the present invention described above, it is preferred that the object of use or treatment be a mammal, preferably a human.

[0233] When this article describes a dosage of a drug or its pharmaceutically acceptable salt, it should be understood that the dosage is based on the weight of the free base, excluding any of its hydrates or solvates, unless otherwise stated.

[0234] Preparation method of the compound of the present invention

[0235] General Synthesis Method

[0236] The compounds of the present invention, their stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts or solvates can be prepared by a variety of methods well known in the field of organic synthesis, including the methods given below, the methods given in the examples, or similar methods as understood by those skilled in the art.

[0237] The following examples illustrate general synthetic schemes for synthesizing the compounds of the present invention. For each reaction step, suitable reaction conditions are known to those skilled in the art or can be conventionally determined. Specifically, the method steps for synthesizing the compounds of the present invention can be carried out under reaction conditions known per se (including those specifically mentioned), in the absence or generally in the presence of solvents or diluents (including, for example, solvents or diluents that are inert to the reagents used and soluble in the reagents used), in the absence or in the presence of catalysts, condensing agents, or neutralizing agents (e.g., ion exchangers, such as cation exchangers, e.g., in the H+ form), depending on the nature of the reaction and / or the reactants, at reduced, normal, or elevated temperatures (e.g., from about -100°C to about 190°C, including, for example, from about -78°C to about 150°C, e.g., from about 0°C to about 125°C, room temperature, -20 to 40°C, or reflux temperature), at atmospheric pressure or in a closed container, under pressure when appropriate, and / or in an inert atmosphere such as oxygen or nitrogen.

[0238] The starting materials and reagents used in the preparation of these compounds are generally commercially available or can be prepared by the methods described below, similar methods, or methods known in the art. If desired, the starting materials and intermediates in the synthetic reaction process can be separated and purified using conventional techniques, including but not limited to filtration, distillation, crystallization, and chromatography. The materials can be characterized using conventional methods, including physical constants and spectroscopic data.

[0239] Unless otherwise specified in the description of the method, solvents suitable for any particular reaction include: those solvents specifically mentioned, or, for example, water; esters, such as lower fatty acid alkyl esters, such as ethyl acetate; ethers, such as aliphatic ethers, such as diethyl ether, or cyclic ethers, such as tetrahydrofuran or dioxane; liquid aromatic hydrocarbons, such as benzene or toluene; alcohols, such as methanol, ethanol, or 1- or 2-propanols, such as acetonitrile; halogenated hydrocarbons, such as dichloromethane or chloroform; amides, such as N,N-dimethylformamide or N,N-dimethylacetamide; bases, such as heterocyclic nitrogen bases, such as pyridine; carboxylic anhydrides, such as lower aliphatic chain carboxylic anhydrides, such as acetic anhydride; cyclic, straight-chain, or branched hydrocarbons, such as cyclohexane, hexane, or isopentane; or mixtures of these solvents, such as aqueous solutions. Such solvent mixtures may also be used for post-processing, such as post-processing by chromatography or partitioning.

[0240] Those skilled in the art will recognize the presence of a stereocenter in the Formula I compound. At all stages of the reaction, the mixture of isomers formed can be separated into individual isomers, such as diastereomers or enantiomers, or into any desired mixture of isomers, such as racemates or mixtures of diastereomers, see, for example, “Stereochemistry of Organic Compounds” by E.L. Leel, S.H. Wilen, and L.N. Mander (Wiley-Interscience, 1994).

[0241] The following procedure 1 illustrates a general synthetic route that can be used to prepare compounds of formula II as defined herein and their various specific embodiments. The variables in the general formulas of the following procedure have the same meaning as those for the compounds defined herein or their various specific embodiments, unless otherwise stated.

[0242] Process 1

[0243]

[0244] P1 and P2 are suitable hydroxyl protecting groups, and P3 and P4 are suitable hydroxyl or amino protecting groups, including but not limited to TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), and Bz (benzoyl). i- BuCO (isobutyryl). The deprotection process used in the synthesis of the compounds of this invention is carried out under acidic conditions (including but not limited to acetic acid / water, trifluoroacetic acid / water, etc.), alkaline conditions (including but not limited to ammonia water, ammonia / methanol solution, etc.), or in the presence of fluorine-containing anionic compounds (including but not limited to tetrabutylammonium fluoride, triethylamine trihydrofluoride, etc.).

[0245] Process 2 illustrates the synthetic routes of compound A and compound C in Process 1, as well as the synthesis of the intermediates used therein.

[0246] Process 2

[0247]

[0248] Process 3 illustrates the synthetic route when compound H is compound H-1 (gemcitabine prodrug LY2334737).

[0249] Process 3

[0250]

[0251] Procedures 4-7 illustrate the synthetic route of compound B in procedure 1, and the synthesis of the intermediates used therein.

[0252] Process 4

[0253]

[0254] Process 5

[0255]

[0256] Process 6

[0257]

[0258] Process 7

[0259]

[0260] Process 8 illustrates the synthetic route of compound D in Process 1.

[0261] Process 8

[0262]

[0263] Specifically, the present invention provides a method for preparing the above-mentioned compound, comprising:

[0264] Compound A

[0265]

[0266] Wherein B1, R1, and R1' have the meanings defined above for the compound of formula (II) of the present invention or its various specific embodiments; P1 is a suitable hydroxyl protecting group, such as, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), Bz (benzoyl), i- BuCO (isobutyryl);

[0267] The compound of formula B reacts with the compound in the presence of a base, such as DBU.

[0268]

[0269] Wherein B2, R2, and R2' have the meanings defined above for the compound of formula (II) of the present invention or its various specific embodiments; P2 is a suitable hydroxyl protecting group, and P3 and P4 are each a suitable hydroxyl or amino protecting group, such as, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), and Bz (benzoyl). i- BuCO (isobutyryl);

[0270] Or the compound of formula C

[0271]

[0272] Where B1, R1, R1' and P1 are as defined above for compound A;

[0273] The compound of formula D reacts with the compound in the presence of a base, such as DBU.

[0274]

[0275] Wherein B2, R2, R2', P2, P3, and P4 are as defined above for compound B;

[0276] Compound E was obtained.

[0277]

[0278] The above compound E is selectively deprotected, for example, under conditions of trifluoroacetic acid / water, tetrabutylammonium fluoride, or triethylamine trihydrofluoride, to obtain compound F.

[0279]

[0280] Each of the groups has the meaning defined above;

[0281] a) When R2′=-O(H) and the protecting group P3 is benzoyl, cyclization of compound F with a (-)-PSI reagent in the presence of a base, such as DBU, yields compound G of cyclic dinucleotide form.

[0282]

[0283] Then, the benzoyl group is removed and protected in ammonia or ammonia-methanol solution to obtain the cyclic dinucleotide II compound.

[0284]

[0285] Where R2′=-O(H), and the other groups have the meanings defined above for the compound of formula II or its various specific embodiments;

[0286] Alternatively, b) when R2′ = -F or -H, cyclization of compound F with a (-)-PSI reagent in the presence of a base, such as DBU, yields a cyclic dinucleotide II compound.

[0287]

[0288] Where R2′ = -F or -H, and the other groups have the meanings defined above for the compound of formula II or its various specific embodiments.

[0289] Compounds of formula A and formula C can be prepared as follows:

[0290] Selective protection of primary alcohols by compound H in the presence of a base, such as imidazole.

[0291]

[0292] Compound C was obtained

[0293]

[0294] The above compound C is reacted with a (+)-PSI reagent in the presence of a base, such as DBU, to obtain compound A.

[0295]

[0296] B1, R1, R1', and P1 are as defined above for compound A.

[0297] Wherein, when compound H is compound H-1 (gemcitabine prodrug LY2334737),

[0298]

[0299] It is prepared by reacting a compound of formula H-1-1 with 2-propylpentanoic acid.

[0300]

[0301] Compound B can be prepared in the form of formula B-1 as follows:

[0302] The two hydroxyl groups of the compound of formula H are protected in the presence of a base, such as imidazole.

[0303]

[0304] Compound B-1-1 was obtained.

[0305]

[0306] The protecting group on the primary alcohol of compound B-1-1 is selectively removed under conditions such as trifluoroacetic acid / water to obtain compound B-1.

[0307]

[0308] B2, R2, R2', and P2 are as defined above for compound B.

[0309] Compound B can also be prepared as formula B-2 as follows:

[0310] The secondary alcohol can be further protected by compound C in the presence of a base, such as DBU / pyridine.

[0311]

[0312] Compound B-2-1 was obtained.

[0313]

[0314] The protecting group on the primary alcohol of compound B-2-1 is selectively removed under conditions such as acetic acid / water to obtain compound B-2.

[0315]

[0316] B2, R2, R2', P1, and P2 are as defined above for compounds of formula A or B.

[0317] Compound B can also be prepared as form B-3 as follows:

[0318] Compound B-3-1 selectively protects a primary alcohol and a secondary alcohol in the presence of a base, such as imidazole.

[0319]

[0320] Compound B-3-2 was obtained.

[0321]

[0322] The compound of formula B-3-2 is reacted with an unprotected secondary alcohol and an amino group on the base in the presence of a base, such as N-methylimidazolium, using a protecting group, such as benzoyl chloride, to obtain the compound of formula B-3-3.

[0323]

[0324] The protecting group on the primary alcohol of compound B-3-3 is selectively removed under conditions such as trifluoroacetic acid / water to obtain compound B-3.

[0325]

[0326] B2, R2, R2', P1, P2, and P3 are as defined above for compounds of formula A or B.

[0327] Compound B can also be prepared in the form of formula B-4 as follows:

[0328] The compound of formula B-4-1 is reacted with an unprotected secondary alcohol therein in the presence of a base, such as N-methylimidazolium, using a protecting group, such as benzoyl chloride.

[0329]

[0330] Compound B-4-2 was obtained.

[0331]

[0332] The protecting group on the primary alcohol of compound B-4-2 is selectively removed under conditions such as acetic acid / water to obtain compound B-4.

[0333]

[0334] B2, R2, R2', P1, P2, and P4 are as defined above for compounds of formula A or B.

[0335] Compound D can be prepared as follows:

[0336] Compound B is reacted with a (-)-PSI reagent in the presence of a base, such as DBU.

[0337]

[0338] Compound D was obtained

[0339]

[0340] B2, R2, R2', P2, P3, and P4 are as defined above for compound B.

[0341] The following procedure 9 illustrates a general synthetic route that can be used to prepare compounds of formulas (Ib), (II-b), (III-b), and (IV-b) as defined herein, and their various specific embodiments. The variables in the general formulas of the following procedure have the same meaning as those for the compounds defined herein or their various specific embodiments, unless otherwise stated.

[0342] Process 9

[0343]

[0344] Where X is independently either a hydroxyl group or a thiol group; R 1 R 1 B1 and B2 are each as defined above for formulas (Ib), (II-b), (III-b), (IV-b) and their respective sub-formulas and specific embodiments; P1 and P2 are suitable hydroxyl protecting groups, and P3 and P4 are suitable hydroxyl or amino protecting groups, including but not limited to TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), Bz (benzoyl), i- BuCO (isobutyryl). P5 is a suitable hydroxyl or mercapto protecting group on a phosphate / phosphate ester, including but not limited to cyanoethyl.

[0345] The deprotection process used in the above-described synthesis of the compounds of the present invention is carried out under acidic conditions (including but not limited to acetic acid / water, trifluoroacetic acid / water, etc.), alkaline conditions (including but not limited to ammonia water, ammonia / methanol solution, methylamine / ethanol solution, lithium hydroxide, etc.), or in the presence of fluorine-containing anionic compounds (including but not limited to tetrabutylammonium fluoride, triethylamine trihydrofluoride, ammonium fluoride, etc.). The oxidation conditions used in the synthesis of the compounds of the present invention are carried out in the presence of iodine, including but not limited to iodine, and the sulfidation conditions are carried out in the presence of, but not limited to, 3H-1,2-benzodisulfonyl-3-one.

[0346] Procedure 10 illustrates the synthetic route of compound J in procedure 9, and the synthesis of intermediates used therein.

[0347] Process 10

[0348]

[0349] Procedure 11 illustrates the synthetic route of compound K and the synthesis of intermediates used therein.

[0350] Process 11

[0351]

[0352] Specifically, the present invention provides a method for preparing the above-mentioned compound, comprising:

[0353] Compound J

[0354]

[0355] Wherein B1, R1, and R1' have the meanings defined above for compounds of formulas (Ib), (II-b), (III-b), and (IV-b) of the present invention or their respective specific embodiments; P3 is a suitable hydroxyl protecting group, such as, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), and Bz (benzoyl). i- BuCO (isobutyryl),

[0356] It reacts with compound K in the presence of a base (e.g., DBU).

[0357]

[0358] Wherein B2 has the meaning defined above for compounds of formulas (Ib), (II-b), (III-b), and (IV-b) of the present invention or their respective specific embodiments; P1 and P2 are suitable hydroxyl protecting groups, and P4 is a suitable hydroxyl or amino protecting group, such as, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), and Bz (benzoyl). i- BuCO (isobutyryl),

[0359] Alternatively, compound J may be reacted with compound L in the presence of tetrazolium, followed by oxidation or sulfidation. Oxidation conditions include, but are not limited to, the use of iodine or tert-butyl hydroperoxide. Sulfidation conditions include, but are not limited to, the use of N,N-dimethyl-N'-(3-thio-3H-1,2,4-dithiazo-5-yl)methylimidazolium (DDTT) or 3H-1,2-benzodisulfonyl-3-one. The introduction and removal of protecting groups shall be performed according to standard methods well known to those skilled in the art.

[0360]

[0361] B2, P1, P2, and P4 are as defined above for compound K;

[0362] Compound M was obtained.

[0363]

[0364] Where X is OH or SH, and P5 is a suitable hydroxyl or mercapto protecting group on a phosphate / phosphate ester, such as, but not limited to, cyanoethyl;

[0365] The above compound M is selectively deprotected, for example, under lithium hydroxide conditions, to obtain compound N.

[0366]

[0367] Each of the groups has the meaning defined above;

[0368] a) Reacting compound N with a (+)-PSI reagent or a (-)-PSI reagent in the presence of a base, such as DBU, yields compound O.

[0369]

[0370] Then, P1 protection is removed (e.g., DMTr) in acetic acid / aqueous solution to obtain compound P.

[0371]

[0372] By cyclizing compound P in the presence of a base, such as DBU, a cyclic dinucleotide compound Q is obtained.

[0373]

[0374] Then, under conditions such as ammonium fluoride, P4 protection is removed (e.g., by TBS) to obtain cyclic dinucleotide compounds of formula (Ib), (II-b), (III-b), or (IV-b), wherein R 2 It is OH and R 2 'For H,

[0375]

[0376] Each of the groups has the meaning defined above;

[0377] b) Alternatively, react compound N with diphenyl phosphite in the presence of a base, such as DBU, to obtain compound R.

[0378]

[0379] Then, P1 protection is removed (e.g., DMTr) in acetic acid / aqueous solution to give compound S.

[0380]

[0381] The compound of formula S is cyclized in the presence of an activating reagent, such as pivaloyl chloride, and then oxidized or sulfided to obtain the cyclic dinucleotide compound of formula Q.

[0382]

[0383] Then, under conditions such as ammonium fluoride, P4 protection is removed (e.g., by TBS) to obtain cyclic dinucleotide compounds of formula (Ib), (II-b), (III-b), or (IV-b), wherein R 2 It is OH and R 2 'For H,

[0384]

[0385] Each of the groups has the meaning defined above.

[0386] Compound J can be prepared as follows:

[0387] The secondary alcohol is protected by compound C in the presence of a base, such as N-methylimidazole.

[0388]

[0389] Compound J-1 was obtained.

[0390]

[0391] The compound of formula J-1 was selectively deprotected to obtain compound J.

[0392]

[0393] The individual groups are as defined above.

[0394] Compound K can be prepared as follows:

[0395] The compound of formula K-1 was reacted with a (+)-PSI reagent in the presence of a base, such as DBU.

[0396]

[0397] Compound K was obtained

[0398]

[0399] The individual groups are as defined above.

[0400] Unless otherwise specified, the experimental materials and reagents used in the above synthesis methods and procedures can be obtained from commercially available sources, prepared according to existing techniques, or prepared according to methods similar to those disclosed in this application. Unless otherwise specified, the synthesis conditions used in the above synthesis methods and procedures can be routinely determined by those skilled in the art.

[0401] The present invention also relates to preparation methods in which a compound that can be obtained as an intermediate in any step of the various preparation methods and processes described herein is used as a starting material and the remaining method steps are carried out, or in which the starting material is formed in situ under reaction conditions or used as a derivative, for example in a protected form or in salt form, or a compound that can be obtained according to the method of the present invention is generated under the method conditions and further treated in situ. Example

[0402] The present invention will be further described below with reference to embodiments. It should be noted that the following embodiments should not be construed as limiting the scope of protection of the present invention.

[0403] Unless there is an obvious error in the structural formula, the structural formula shall prevail when the chemical name of any compound of the present invention is inconsistent with the given structural formula.

[0404] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions for such reactions or as recommended by the manufacturer. Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available, prepared using existing methods, or prepared using methods similar to those disclosed in this application.

[0405] Unless otherwise stated, percentages and parts are weight percentages and weight parts; ratios of liquids are volume ratios; unless otherwise stated, all temperatures are given in degrees Celsius.

[0406] In the following embodiments, 1 H NMR spectrum 31 P NMR spectra are typically recorded using a Bruker 400MHz and 500MHz NMR spectrometer, with chemical shifts expressed as δ (ppm). Mass spectrometry is recorded using an Agilent 1290 HPLC system coupled with a 6120B LC-MS system. Silica gel column purification is performed using a Biotage Selekt SEL-2SV or ISO-1SV; preparative HPLC purification is performed using a Gilson 281 (column: Waters Xbridge 19x250mm, 5μm or Welch C18, 21.2x250mm, 10μm. Mobile phase: A: water (10mM NH4HCO3 or 0.05% formic acid), B: acetonitrile (or containing 0.05% formic acid). Flow rate: 20-30 mL / min. Detection wavelength: 214nm / 254nm), or as otherwise specified.

[0407] The following abbreviations are used in the synthetic embodiments below. The abbreviations not listed have the meanings commonly understood by those skilled in the art.

[0408] List of abbreviations

[0409] CDCl3 (deuterated chloroform)

[0410] DMSO-d6 deuterated dimethyl sulfoxide

[0411] MHz

[0412] MS-ESI Electrospray Mass Spectrometry

[0413] DBU 1,8-diazabicycloundec-7-ene

[0414] DMF N,N-dimethylformamide

[0415] CDI N,N'-carbonyldiimidazole

[0416] TMSCl Trimethylchlorosilane

[0417] TFA trifluoroacetic acid

[0418] THF Tetrahydrofuran

[0419] H2O water

[0420] HOAc Acetic Acid

[0421] BzCl Benzoyl chloride

[0422] IFN interferon

[0423] FBS Fetal Bovine Serum

[0424] PBS phosphate buffer solution

[0425] ELISA (Enzyme-Linked Immunosorbent Assay)

[0426] (+)-PSI reagent (2R, 3aR, 6S, 7aR)-3a-methyl-2-((perfluorophenyl)thio)-6- (propyl-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxothiophosphazenecyclopentane Alkyl-2-sulfide (CAS: 2245335-71-9)

[0427]

[0428] (-)-PSI reagent (2S, 3aS, 6R, 7aS)-3a-methyl-2-((perfluorophenyl)thio)-6- (propyl-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxothiophosphazenecyclopentane Alkyl-2-sulfide (CAS: 2245335-70-8)

[0429]

[0430] Synthesis Examples

[0431] Preparation of Intermediate 1 2-Chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine

[0432]

[0433] To a DMF solution (15 mL) of 2-chloro-2'-deoxy-2'-fluoro-beta-adenosine (2.50 g, 8.25 mmol), imidazole (1.12 g, 16.5 mmol) and tert-butyldimethylchlorosilane (1.31 g, 8.66 mmol) were added sequentially. The resulting mixture was stirred at 20–25 °C for 16 hours. The reaction mixture was poured into water (150 mL) and extracted twice with ethyl acetate (100 mL each time). The combined organic phases were washed twice with saturated brine (100 mL each time) and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate (2:3) to give a white solid (2.53 g).

[0434] 1 H NMR (400MHz, DMSO-d6) δ8.18 (s, 1H), 7.89 (brs, 2H), 6.33 (dd, J=12.0, 4.8 Hz, 1H), 6.00 (d, J=4.8Hz, 1H), 5.28 (dt, J=52, 4.8Hz, 1H), 4.50-4.30 (m, 1H), 3.98-3.73 (m, 3H), 0.89 (s, 9H), 0.07 (s, 6H).

[0435] Preparation of intermediate 2 :(2S,3aR,6S,7aR)-2-((2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-2-((tert-butyldimethylsilyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)-3a-methyl-6-(isopropyl-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxothiophosphazenecyclopentane 2-sulfide

[0436]

[0437] To a tetrahydrofuran solution (10 mL) of intermediate 1 (1.00 g, 2.40 mmol) and (+)-PSI reagent (1.39 g, 3.12 mmol) at 0 °C, 1,8-diazabicycloundec-7-ene (474 ​​mg, 3.12 mmol) was added, and the resulting mixture was stirred at 0–5 °C for 1 hour. The reaction mixture was diluted with ethyl acetate (50 mL). The organic phase was washed successively with 10% sodium dihydrogen phosphate aqueous solution (50 mL) and saturated brine (50 mL) and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate (3:7) to give a white solid (1.50 g).

[0438] 1H NMR (400MHz, CDCl3) δ8.09 (s, 1H), 6.52-6.40 (m, 1H), 6.23 (brs, 2H), 5.55-5.4 0(m, 1H), 5.30-5.06(m, 2H), 4.92(s, 1H), 4.52-4.40(m, 1H), 4.20-4.12(m, 1H), 4.08-3.90(m, 2H), 2.62-2.52(m, 1H), 2.36-2.23(m, 1H), 2.20-2.08(m, 1H), 2.02-1.80 (m, 3H), 1.80-1.62 (m, 7H), 0.93 (s, 9H), 0.12 (s, 6H). 31 P NMR (162MHz, CDCl3) δ101.9.

[0439] Preparation of intermediate 3 2-Chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine

[0440]

[0441] Intermediate 3 is obtained from 2'-deoxyadenosine via the route of intermediate 1.

[0442] 1 H NMR (400MHz, DMSO-d6) δ8.29 (s, 1H), 7.82 (brs, 2H), 6.26 (t, J=6.4Hz, 1H), 5. 37(d, J=4.0Hz, 1H), 4.45-4.32(m, 1H), 3.90-3.76(m, 1H), 3.73-3.60(m, 2H), 2.75-2.61 (m, 1H), 2.37-2.25 (m, 1H), 0.84 (s, 9H), 0.02 (s, 6H).

[0443] Preparation of intermediate 4 :(2S,3aR,6S,7aR)-2-((2R,3S,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-2-((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-3-yl)oxy)-3a-methyl-6-(propyl-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxothiophosphazenecyclopentane 2-sulfide

[0444]

[0445] Intermediate 4 is obtained by following the route of intermediate 2, from intermediate 3 and (+)-PSI reagent.

[0446] 1H NMR (400MHz, CDCl3) δ8.65 (s, 1H), 6.59-6.52 (m, 1H), 5.49-5.40 (m, 1H), 5.09 (s, 1H), 4.92 (s, 1H), 4.52-4.40 (m, 2H), 4.25-4.12 (m, 2H) , 3.96 (s, 2H), 2.90-2.81 (m, 1H), 2.69-2.56 (m, 2H), 2.20-2.08 (m, 2H), 2.02-1.80 (m, 3H), 1.80-1.62 (m, 7H), 0.91 (s, 9H), 0.14 (s, 6H). 31 P NMR (162MHz, CDCl3) δ100.6.

[0447] Preparation of intermediate 5 5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0448]

[0449] Intermediate 5 is obtained from 2'-deoxy-2',2'-difluorocytidine via the route of intermediate 1.

[0450] 1 H NMR (400MHz, DMSO-d6) δ7.63 (d, J=7.6Hz, 1H), 7.37 (brs, 2H), 6.29 (d, J=6.8H z, 1H), 6.14 (t, J=7.6Hz, 1H), 5.75 (d, J=7.6Hz, 1H), 4.15-4.02 (m, 1H), 3.98- 3.73 (m, 3H), 0.90 (s, 9H), 0.09 (s, 6H). MS-ESI[M+H] + : 378.2.

[0451] Preparation of intermediate 6 : 4-amino-1-((2R,4R,5R)-5-((tert-butyldimethylsilyl(oxy)methyl)-3,3-difluoro-4-(((2S,3aR,6S,7aR)-3a-methyl-6-(propyl-1-en-2-yl)-2-thiohexahydrobenzo[d][1,3,2]oxothiophosphazenecyclopentan-2-yl)oxy)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one

[0452]

[0453] Intermediate 6 is obtained by following the route of intermediate 2, from intermediate 5 and (+)-PSI reagent.

[0454] 1H NMR (400MHz, DMSO-d6) δ7.73 (d, J=8.0Hz, 1H), 6.34 (t, J=8.0Hz, 1H), 6.21(d, J=8.0Hz, 1H), 5.36-5.24(m, 1H), 5.04(s, 1H), 4.87(s, 1H), 4.55-4.43(m, 1H), 4.17-4.06(m, 1H), 4.05-3.86(m, 2H), 2.66-2.52(m, 1H), 2.30-2.05(m, 2H), 2.02- 1.80 (m, 3H), 1.80-1.62 (m, 7H), 0.92 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H). 31 P NMR (162MHz, CDCl3) δ103.0. MS-ESI[M+H] + :624.2.

[0455] Preparation of intermediate 7 N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine

[0456]

[0457] Trimethylchlorosilane (3.61 g, 33.4 mmol) was added dropwise to a pyridine solution (2.00 g, 6.67 mmol) of 2'-deoxy-2',2'-difluorocytidine hydrochloride (2.00 g, 6.67 mmol) at 0 °C, and the mixture was stirred at 0–5 °C for 2 hours. Simultaneously, carbonyl diimidazole (1.19 g, 7.33 mmol) was added in portions to an acetonitrile solution (20 mL) of 2-propylvaleric acid (1.06 g, 7.33 mmol), and the mixture was stirred at 25 °C for 2 hours. Then, the resulting acetonitrile mixture was added dropwise to the previously prepared pyridine mixture at 0 °C, and the mixture was stirred at 45 °C for 16 hours. Ethanol (20 mL) was added to the mixture, and the mixture was stirred at 45 °C for 0.5 hours. Water (20 mL) was then added to the mixture, and the mixture was stirred at 45 °C for 1 hour. The resulting reaction solution was evaporated to dryness and diluted with water (50 mL). The pH of the above mixture was adjusted to 2-3 with 2N hydrochloric acid aqueous solution, and extracted twice with ethyl acetate (50 mL each time). The organic phases were combined, washed twice with water (50 mL each time), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate (2:3) to give a white solid (1.20 g).

[0458] 1H NMR (400MHz, DMSO-d6) δ11.06 (brs, 1H), 8.25 (d, J=8.0Hz, 1H), 7.33 (d, J =8.0Hz, 1H), 6.32 (d, J = 8.0Hz, 1H), 6.17 (t, J = 8.0Hz, 1H), 5.32-5.25 (m, 1H), 4.25 -4.10(m, 1H), 3.97-3.75(m, 2H), 3.70-3.58(m, 1H), 2.70-2.55(m, 1H), 1.56-1.42 (m, 2H), 1.38-1.10 (m, 6H), 0.85 (t, J=7.6Hz, 6H). MS-ESI[M+H] + 390.2.

[0459] Preparation of intermediate 8 N4-(2-propylpentanoyl)-5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0460]

[0461] Intermediate 8 is obtained from intermediate 7 by following the route of intermediate 1.

[0462] 1 H NMR (400MHz, CDCl3) δ9.07 (brs, 1H), 8.27 (brs, 1H), 7.51 (d, J=8.0Hz, 1H), 6. 45-6.30 (m, 1H), 4.46-4.33 (m, 1H), 4.12-4.00 (m, 2H), 3.91 (dd, J=8.0, 2.4Hz, 1H), 2.25-2.12(m, 1H), 1.71-1.60(m, 2H), 1.56-1.42(m, 2H), 1.40-1.25(m, 4H), 1.05- 0.85(m, 15H), 0.13(s, 6H).

[0463] Preparation of intermediate 9 :N-(1-((2R,4R,5R)-5-((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluoro-4-(((2S,3aR,6S,7aR)-3a-methyl-6-(propyl-1-en-2-yl)-2-thiohexahydrobenzo[d][1,3,2]oxothiophosphazenecyclopentan-2-yl)oxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-propylpentanamide

[0464]

[0465] Intermediate 9 is obtained by following the route of intermediate 2, from intermediate 8 and (+)-PSI reagent.

[0466] 1 H NMR (400MHz, CDCl3) δ9.63 (brs, 1H), 8.29-8.15 (m, 1H), 7.60-7.48 (m, 1H), 6.4 8-6.39 (m, 1H), 5.40-5.25 (m, 1H), 5.04 (s, 1H), 4.88 (s, 1H), 4.55-4.45 (m, 1H), 4.28-4.05(m, 1H), 4.00-3.88(m, 2H), 2.65-2.55(m, 1H), 2.46-2.35(m, 1H), 2.33-2.23 (m, 1H), 2.20-1.80 (m, 5H), 1.79-1.60 (m, 8H), 1.58-1.45 (m, 2H), 1.45-1.25 (m, 4H), 0.98-0.85 (m, 15H), 0.15 (d, J=4.0Hz, 6H).

[0467] Preparation of intermediate 10 N6-bis(4-methoxyphenyl)benzyl-2-chloro-5'-O-tert-butyldimethylsilyl-3'-bis(4-methoxyphenyl)benzyl-2'-deoxy-2'-fluoro-beta-adenosine

[0468]

[0469] To a pyridine solution (10 mL) of intermediate 1 (1.53 g, 3.67 mmol), bis(4-methoxyphenyl)benzyl chloride (1.49 g, 4.40 mmol) was added, and the mixture was stirred at 25 °C for 2 hours. Then, bis(4-methoxyphenyl)benzyl chloride (497 mg, 1.47 mmol) and 1,8-diazabicycloundec-7-ene (669 mg, 4.40 mmol) were added sequentially, and the mixture was stirred at 20–25 °C for 16 hours. Then, bis(4-methoxyphenyl)benzyl chloride (1.49 g, 4.40 mmol) and 1,8-diazabicycloundec-7-ene (669 mg, 4.40 mmol) were added sequentially, and the mixture was stirred at 20–25 °C for 24 hours. The reaction mixture was concentrated and diluted with ethyl acetate (100 mL). The organic phase was washed twice with saturated brine (100 mL each time) and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate (5:1) to give a white solid (2.60 g). 1H NMR (400MHz, DMSO-d6) δ8.00 (s, 1H), 7.95 (s, 1H), 7.50-7.10 (m, 18H), 6.90-6.7 8(m, 8H), 6.33-6.20(m, 1H), 4.46-4.30(m, 2H), 4.25-4.15(m, 1H), 3.76-3.68(m, 12H), 3.55-3.49(m, 2H), 0.76(s, 9H), -0.08(s, 3H), -0.11(s, 3H).

[0470] Preparation of intermediate 11 N6-bis(4-methoxyphenyl)benzyl-2-chloro-3'-bis(4-methoxyphenyl)benzyl-2'-deoxy-2'-fluoro-beta-adenosine

[0471]

[0472] Tetrabutylammonium fluoride (1.33 g, 5.10 mmol) was added to a tetrahydrofuran solution (15 mL) of intermediate 10 (2.60 g, 2.55 mmol), and the resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was concentrated and diluted with ethyl acetate (30 mL). The organic phase was washed with water (30 mL) and dried over anhydrous sodium sulfate. The crude product obtained from the concentration was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate (2:3) to give a white solid (1.20 g).

[0473] 1 H NMR (400MHz, DMSO-d6) δ8.17 (d, J=2.4Hz, 1H), 7.97 (s, 1H), 7.50-7.10 (m, 18H ), 6.90-6.78(m, 8H), 6.30-6.18(m, 1H), 4.86(t, J=6.0Hz, 1H), 4.30-4.10(m, 3H), 3.76-3.68(m, 12H), 3.49-3.30(m, 2H).

[0474] Preparation of intermediate 12 : N6-bis(4-methoxyphenyl)benzyl-2-chloro-5'-O-tert-butyldimethylsilyl-3'-bis(4-methoxyphenyl)benzyl-2'-deoxyadenosine

[0475]

[0476] To a pyridine solution (10 mL) of intermediate 3 (1.80 g, 4.51 mmol), bis(4-methoxyphenyl)benzyl chloride (3.81 g, 11.3 mmol) and 1,8-diazabicycloundec-7-ene (743 mg, 11.3 mmol) were added sequentially. The resulting mixture was stirred at 20–25 °C for 16 hours. The reaction mixture was concentrated and poured into water (100 mL), and extracted twice with ethyl acetate (50 mL each time). The combined organic phases were washed twice with saturated brine (50 mL each time) and dried over anhydrous sodium sulfate. The crude product obtained from the concentration was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate (3:7) to give a white solid (2.50 g).

[0477] 1 H NMR (400MHz, DMSO-d6) δ8.19 (s, 1H), 7.83 (s, 1H), 7.50-7.10 (m, 18H), 6.95-6.7 8(m, 8H), 6.28-6.18(m, 1H), 4.36-4.28(m, 1H), 4.00-3.88(m, 1H), 3.80-3.65(m, 12H), 3.50-3.41(m, 1H), 3.40-3.30(m, 1H), 2.33-2.25(m, 1H), 1.88-1.76(m, 1H), 0.71(s, 9H), -0.14(s, 6H).

[0478] Preparation of intermediate 13 N6-bis(4-methoxyphenyl)benzyl-2-chloro-3'-bis(4-methoxyphenyl)benzyl-2'-deoxyadenosine

[0479]

[0480] Intermediate 13 is obtained from intermediate 12 by following the route of intermediate 11.

[0481] 1 H NMR (400MHz, DMSO-d6) δ8.32 (s, 1H), 7.87 (s, 1H), 7.50-7.10 (m, 18H), 6.95-6.77 (m, 8H), 6.24 (t, J=6.8Hz, 1H), 4.83(brs, 1H), 4.39-4.28(m, 1H), 3.85-3.65(m, 13H), 3.22-3.12(m, 1H), 2.35-2.21(m, 1H), 1.88-1.70(m, 1H).

[0482] Preparation of intermediate 14 3'-5'-di-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0483]

[0484] To a DMF solution (50 mL) of 2'-deoxy-2',2'-difluorocytidine hydrochloride (3.00 g, 10.0 mmol), imidazole (3.41 g, 50.0 mmol) and tert-butyldimethylchlorosilane (4.53 g, 30.0 mmol) were added sequentially. The resulting mixture was stirred at 20–25 °C for 2 hours, followed by stirring at 60 °C for 16 hours. The reaction mixture was poured into a mixture of ethyl acetate (100 mL) and water (100 mL). The organic phase was separated, washed sequentially with water (100 mL) and saturated brine (100 mL), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with dichloromethane:methanol (9:2) to give a white solid (2.70 g).

[0485] 1 H NMR (400MHz, DMSO-d6) δ7.53 (d, J=7.6Hz, 1H), 7.40 (brs, 2H), 6.17 (t, J=8.0H z, 1H), 5.78 (d, J=7.6Hz, 1H), 4.40-4.22 (m, 1H), 3.98-3.83 (m, 2H), 3.78-3.70 (m, 1H), 1.00-0.75 (m, 18H), 0.15-0.00 (m, 12H).

[0486] Preparation of intermediate 15 3'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0487]

[0488] Intermediate 14 (1.00 g, 2.04 mmol) was dissolved in a mixture of tetrahydrofuran solution (10 mL), trifluoroacetic acid (5 mL), and water (5 mL) at 0 °C, and stirred at 0–5 °C for 2 hours. The pH of the reaction mixture was adjusted to approximately 9 with a saturated sodium bicarbonate aqueous solution, and then poured into a mixture of ethyl acetate (50 mL) and water (50 mL). The organic phase was separated, washed with saturated brine (50 mL), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with ethyl acetate:methanol (17:3) to give a white solid (620 mg).

[0489] 1H NMR (400MHz, DMSO-d6) δ7.67 (d, J=7.6Hz, 1H), 7.50-7.35 (m, 2H), 6.14 (t, J=8.0Hz, 1H), 5.79 (d, J=7.6Hz, 1H), 5.26 (d, J=7.6Hz, 1H), 4.40-4.22 (m, 1H), 3.86-3.72(m, 2H), 3.68-3.50(m, 1H), 0.88(s, 9H), 0.11(s, 3H), 0.10(s, 3H). MS-ESI [M+H] + : 378.2.

[0490] Preparation of intermediate 16 N4-(2-propyl-valeryl)-3'-5'-di-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0491]

[0492] At 25°C, carbonyl diimidazole (363 mg, 2.24 mmol) was added to a 5 mL acetonitrile solution of 2-propylpentanoic acid (322 mg, 2.24 mmol), and the resulting mixture was stirred at 25°C for 2 hours. The resulting mixture was then added dropwise to a 10 mL pyridine solution of intermediate 14 (1.0 g, 2.03 mmol) at 0°C. The resulting mixture was stirred at 45°C for 16 hours. The resulting reaction solution was evaporated to dryness, diluted with 20 mL of water, and extracted twice with 30 mL of ethyl acetate each time. The combined organic phases were washed twice with 50 mL of water each time and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography with petroleum ether:ethyl acetate (1:1) to give a white solid (430 mg).

[0493] 1 H NMR (400MHz, CDCl3) δ8.09 (d, J=7.6Hz, 1H), 7.49 (d, J=7.6Hz, 1H), 6.33 (d, J=10.0Hz, 1H), 4.39-4.29 (m, 1H), 4.02 (d, J=12.0Hz, 1H), 3.96 (d, J=7.6Hz, 1H), 3.81 (d, J=11.2Hz, 1H), 2.50-2.30 (m, 1H), 1.49-1.44 (m, 2H), 1.33 (q, J=7.6 Hz, 6H), 0.99-0.80 (m, 24H), 0.13 (s, 9H), 0.10 (s, 3H). MS-ESI[M+H] + :619.4.

[0494] Preparation of intermediate 17N4-(2-propyl-valeryl)-3'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0495]

[0496] Intermediate 17 is obtained from intermediate 16 by following the route of intermediate 15.

[0497] 1 H NMR (400MHz, CDCl3) δ8.38 (s, 1H), 8.00 (d, J=7.6Hz, 1H), 7.50 (d, J=7.6 Hz, 1H), 6.25 (t, J=7.6Hz, 1H), 4.49-4.40 (m, 1H), 4.08 (d, J=12.4Hz, 1H), 4.02- 3.90 (m, 1H), 3.88-3.75 (m, 1H), 2.51 (s, 1H), 2.40-2.30 (m, 1H), 1.75-1.55 (m, 2H) , 1.52-1.41 (m, 2H), 1.38-1.28 (m, 4H), 0.95-0.85 (m, 15H), 0.14 (d, J=2.8Hz, 6H). MS-ESI[M+H] + :504.2.

[0498] Intermediate 18 5'-O-tert-butyldimethylsilyl-2'-tert-butyldimethylsilyl-adenosine, and

[0499] Intermediate 19 5'-O-tert-butyldimethylsilyl-3'-tert-butyldimethylsilyl-adenosine

[0500] Triethylenediamine (7.0 g, 62.5 mmol) and silver nitrate (5.08 g, 28.8 mmol) were added sequentially to a tetrahydrofuran solution (40 mL) of adenosine (3.35 g, 12.5 mmol) at room temperature (10 °C). The mixture was stirred at room temperature (10 °C) for 30 min, followed by the addition of tert-butyldimethylchlorosilane (4.71 g, 30 mmol), and the resulting mixture was stirred at 10 °C for 16 h. The reaction mixture was filtered through diatomaceous earth, the filtrate was quenched with water (50 mL), and then extracted twice with ethyl acetate (50 mL each time). The combined organic phases were washed twice with saturated brine (100 mL each time) and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate (1:1) to give intermediate 18 (1.0 g, white solid). Elution with ethyl acetate gave intermediate 19 (4.4 g, white solid).

[0501] Intermediate 18:

[0502]

[0503] 1 H NMR (400MHz, CDCl3) δ8.35 (s, 1H), 8.24 (s, 1H), 6.11 (d, J=4.8Hz, 1H), 5.72 (brs, 2H), 4.64 (t, J=4.8Hz, 1H), 4.31-4.2 6 (m, 1H), 4.24-4.20 (m, 1H), 4.02 (dd, J=11.6, 2.8Hz, 1H), 3.87 (dd, J=11.6, 2.8Hz, 1H), 2.76 (d, J=3.6Hz, 1H), 0.96 (s, 9H), 0.84(s, 9H), 0.18-0.11(m, 6H), -0.05(s, 3H), -0.12(s, 3H).

[0504] Intermediate 19:

[0505]

[0506] 1 H NMR (400MHz, CDCl3) δ8.35 (s, 1H), 8.10 (s, 1H), 6.03 (d, J=3.6Hz, 1H), 5.75 (b rs, 2H), 4.56 (d, J=1.6Hz, 1H), 4.12 (d, J=2.8Hz, 1H), 3.92 (dd, J=11.6, 3.6Hz, 1H), 3.77 (dd, J=11.2, 2.8Hz, 1H), 3.31 (brs, 1H), 0.95 (s, 9H), 0.90 (s, 9H), 0.17 (s, 6H), 0.06 (s, 3H), -0.01 (s, 3H).

[0507] Preparation of intermediate 20 N4-benzoyl-2'-5'-di-O-tert-butyldimethylsilyl-3'-benzoyl-adenosine

[0508]

[0509] Benzoyl chloride (354.4 mg, 2.52 mmol) was added dropwise to a solution of intermediate 18 (500 mg, 1.01 mmol) and N-methylimidazole (248 mg, 3.03 mmol) in dichloromethane (10 mL) at room temperature (25 °C). The resulting mixture was reacted at room temperature for 16 hours. The reaction solution was quenched with water (20 mL), the organic phase was separated, and the aqueous phase was extracted twice with dichloromethane (20 mL each time). The combined organic phases were washed once with saturated brine (20 mL) and then dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate (1:1) to give a white solid (400 mg).

[0510] 1 H NMR (400MHz, CDCl3) δ9.36 (s, 1H), 8.85 (s, 1H), 8.48 (s, 1H), 8.15-8.10 (m, 2H), 8.08-8.05 (m, 2H), 7.70-7.55 (m, 2H), 7 .55-7.47 (m, 4H), 6.33 (d, J=6.0Hz, 1H), 5.63-5.50 (m, 1H), 4.89 (d, J=1.2Hz, 1H), 4.48 (d, J=2.4Hz, 1H), 4.05 (d, J=2.4 Hz, 1H), 4.00 (d, J=2.4Hz, 1H), 0.99 (s, 9H), 0.65 (s, 9H), 0.19 (d, J=4.4Hz, 6H), -0.10 (s, 3H), -0.26 (s, 3H). MS-ESI[M+H] + :704.3.

[0511] Preparation of intermediate 21 N4-benzoyl-2'-O-tert-butyldimethylsilyl-3'-benzoyl-adenosine

[0512]

[0513] Intermediate 21 is obtained from intermediate 20 by following the route of intermediate 15.

[0514] 1H NMR (500MHz, CDCl3) δ8.86 (s, 1H), 8.17-8.09 (m, 3H), 8.08-8.03 (m, 2H), 7.66 -7.60 (m, 2H), 7.55 (t, J=7.6Hz, 2H), 7.50 (t, J=7.6Hz, 2H), 5.97 (d, J=7.6Hz, 1H), 5.75 (d, J=5.2Hz, 1H), 5.27 (dd, J=7.6, 5.2Hz, 1H), 4.51 (s, 1H), 4.08-4.03 (m, 1H), 3.93 (d, J=12.8Hz, 1H), 0.62 (s, 9H), -0.10 (s, 3H), -0.43 (s, 3H). MS-ESI [M+H] + :591.2.

[0515] Preparation of intermediate 22 N4-benzoyl-5'-O-tert-butyldimethylsilyl-3'-tert-butyldimethylsilyl-2'-benzoyl-adenosine

[0516]

[0517] Intermediate 22 is obtained by following the route of intermediate 20, from intermediate 19 and benzoyl chloride.

[0518] 1 H NMR (400MHz, CDCl3) δ9.02 (brs, 1H), 8.83 (s, 1H), 8.41 (s, 1H), 8.08-7.99 (m, 4 H), 7.64-7.49 (m, 4H), 7.47-7.39 (m, 2H), 6.54 (d, J=6.0Hz, 1H), 5.81 (t, J=5.6 Hz, 1H), 4.84 (dd, J=4.8, 3.6Hz, 1H), 4.26 (dd, J=6.0, 3.2Hz, 1H), 4.02 (dd, J= 11.6, 3.2Hz, 1H), 3.85 (dd, J=11.6, 2.8Hz, 1H), 0.95 (s, 9H), 0.84 (s, 9H), 0.18-0.11 (m, 6H), 0.03--0.05 (m, 6H).

[0519] Preparation of intermediate 23 N4-benzoyl-3'-tert-butyldimethylsilyl-2'-benzoyl-adenosine

[0520]

[0521] Intermediate 23 is obtained from intermediate 22 by following the route of intermediate 15.

[0522] 1 H NMR (400MHz, CDCl3) δ9.08 (brs, 1H), 8.84 (s, 1H), 8.18 (s, 1H), 8.08-7.98 (m, 4 H), 7.67-7.41 (m, 6H), 6.32 (d, J=7.6Hz, 1H), 6.01 (dd, J=7.6, 5.2Hz, 1H), 4.94 (d, J=5.2Hz, 1H), 4.35 (d, J=0.8Hz, 1H), 4.07 (dd, J=13.2, 1.6Hz, 1H), 3.84 (d, J=12.4Hz, 1H), 0.89 (s, 9H), 0.11 (s, 3H), 0.00 (s, 3H).

[0523] Preparation of intermediate 24 N2-Isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-guanosine

[0524]

[0525] Intermediate 24 was obtained via the route of intermediate 20, from N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-2'-O-tert-butyldimethylsilyl-guanosine. MS-ESI[M+H] + :874.3.

[0526] Preparation of intermediate 25 N2-Isobutyryl-3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-guanosine

[0527]

[0528] Intermediate 24 (5.50 g, 6.30 mmol) was dissolved in a mixture of glacial acetic acid (60 mL) and water (10 mL), and stirred at 25 °C for 2 hours. The reaction mixture was evaporated to dryness and then co-distilled twice with ethyl acetate (25 mL each time). The crude product was purified by silica gel column chromatography, eluting with ethyl acetate to give a white solid (2.2 g).

[0529] 1H NMR (400MHz, CDCl3) δ12.15 (brs, 1H), 8.35 (s, 1H), 8.15-8.02 (m, 2H), 7.74 (s, 1H), 7.68-7.56 (m, 1H), 7.55-7.42 (m, 2H), 5.80 (d, J=7.6Hz, 1H), 5.66 (dd, J= 5.6, 1.2Hz, 1H), 5.52 (d, J=9.2Hz, 1H), 4.98 (dd, J=7.2, 5.6Hz, 1H), 4.45 (d, J= 1.2Hz, 1H), 4.06-3.82(m, 2H), 2.78-2.62(m, 1H), 1.35-1.25(m, 6H), 0.66(s, 9H), - 0.09(s, 3H), -0.33(s, 3H). MS-ESI[M+H] + :572.3.

[0530] Preparation of intermediate 26 N2-Isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-tert-butyldimethylsilyl-2'-benzoyl-guanosine

[0531]

[0532] Intermediate 26 was obtained via the route of intermediate 20, from N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-tert-butyldimethylsilyl-guanosine. MS-ESI[M+H] + :874.3.

[0533] Preparation of intermediate 27 N2-Isobutyryl-3'-tert-butyldimethylsilyl-2'-benzoyl-guanosine

[0534]

[0535] Intermediate 27 is obtained from intermediate 26 by following the route of intermediate 25.

[0536] 1H NMR (400MHz, CDCl3) δ12.06 (brs, 1H), 8.24 (brs, 1H), 8.01-7.96 (m, 2H), 7.83 (s , 1H), 7.62-7.55 (m, 1H), 7.43 (d, J=8.0Hz, 2H), 6.13 (d, J=7.2Hz, 1H), 5.81 (dd, J=7.2, 5.6Hz, 1H), 4.80 (dd, J=4.8, 1.6Hz, 1H), 4.30-4.25 (m, 1H), 4.02 (dd, J= 12.8, 2.0Hz, 1H), 3.78 (d, J=12.4Hz, 1H), 2.71-2.60 (m, 1H), 1.30-1.28 (m, 6H), 0.83 (s, 9H), 0.07 (s, 3H), 0.00 (s, 3H).

[0537] Preparation of intermediate 28 2'-5'-di-O-tert-butyldimethylsilyl-guanosine

[0538]

[0539] Intermediate 28 is obtained from guanine nucleosides using the same route as intermediate 18. MS-ESI [M+H] + :513.3.

[0540] Preparation of intermediate 29 2'-5'-di-O-tert-butyldimethylsilyl-3'-benzoyl-guanosine

[0541]

[0542] Intermediate 29 is obtained from intermediate 28 by following the route of intermediate 20.

[0543] 1 H NMR (400MHz, CDCl3) δ12.04 (brs, 1H), 8.14-8.09 (m, 2H), 7.94 (s, 1H), 7.61 (t, J=7.6Hz, 1H), 7.49 (t, J=7.6Hz, 2H), 6.09 (s, 2H), 6.00 (d, J=6.0Hz, 1H), 5.53 (s, 1H), 4.76 (t, J = 5.6Hz, 1H), 4.41 (d, J = 2.8Hz, 1H), 4.01 (d, J = 11.6Hz, 1H), 3.93 (dd, J=11.6, 2.4Hz, 1H), 0.97 (s, 9H), 0.70 (s, 9H), 0.16 (d, J=3.2Hz, 6H), - 0.09 (s, 3H), -0.16 (s, 3H). MS-ESI[MH]- :615.3.

[0544] Preparation of intermediate 30 2'-O-tert-butyldimethylsilyl-3'-benzoyl-guanosine

[0545]

[0546] Intermediate 30 is obtained from intermediate 29 by following the route of intermediate 15.

[0547] 1 H NMR (400MHz, DMSO-d6) δ10.71 (brs, 1H), 8.05 (d, J=7.2Hz, 3H), 7.75-7.68 (m, 1H), 7.59 (t, J=7.6Hz, 2H), 6.51 (s, 2H), 5.84 (d, J=7.6Hz, 1H), 5.58 (dd, J= 5.6, 1.6Hz, 1H), 5.42 (t, J=5.6Hz, 1H), 4.96 (dd, J=7.6, 5.2Hz, 1H), 4.39-4.25 (m, 1H), 4.20-4.00(m, 1H), 0.57(s, 9H), -0.12(s, 3H), -0.29(s, 3H). MS-ESI[M-H] - :501.3.

[0548] Preparation of intermediate 31 3'-5'-di-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroadenosine

[0549]

[0550] Imidazole (1.81 g, 26.6 mmol) and 4-dimethylaminopyridine (50 mg, 0.41 mmol) were added to a DMF solution (10 mL) of 2'-deoxy-2'-fluoroadenosine (1.10 g, 4.09 mmol), followed by tert-butyldimethylchlorosilane (2.16 g, 14.3 mmol). The mixture was stirred at 15–20 °C for 16 hours. The reaction mixture was poured into water (100 mL) and extracted twice with ethyl acetate (100 mL each time). The combined organic phases were washed twice with saturated brine (100 mL) and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate (3:2) to give a white solid (1.90 g).

[0551] 1H NMR (400MHz, CDCl3) δ8.35 (s, 1H), 8.12 (s, 1H), 6.25 (dd, J=16.0, 2.4Hz, 1H), 5.66(brs, 2H), 5.48-5.20(m, 1H), 4.79-4.62(m, 1H), 4.18-3.95(m, 2H), 3.85- 3.72(m, 1H), 1.00-0.80(m, 18H), -0.05-0.18(m, 12H).MS-ESI[M+H] + :498.4.

[0552] Preparation of intermediate 32 3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroadenosine

[0553]

[0554] Intermediate 32 is obtained from intermediate 31 by following the route of intermediate 15.

[0555] 1 H NMR (400MHz, DMSO-d6) δ8.37 (s, 1H), 8.14 (s, 1H), 7.38 (brs, 2H), 6.22 (dd, J=16.4, 3.6Hz, 1H), 5.70-5.40 (m, 1H), 5.29 (t, J=5.6Hz, 1H), 4.80-4.62 (m, 1H), 4.02-3.90 (m, 1H), 3.79-3.68 (m, 1H), 3.63-3.48 (m, 1H), 0.90 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H).

[0556] Preparation of intermediate 33 :(2R,3aS,6R,7aS)-2-((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-3-((tert-butyldimethylsilyl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)-3a-methyl-6-(propyl-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxothiophosphazenecyclopentane 2-sulfide

[0557]

[0558] Intermediate 33 is obtained by following the route of intermediate 2, from intermediate 32 and (-)-PSI reagent.

[0559] 1H NMR (400MHz, CDCl3) δ8.36 (s, 1H), 8.05 (s, 1H), 6.30-6.18 (m, 1H), 5.58 (brs, 2H), 5.50-5.22 (m, 1H), 4.80-4.62 (m, 3H), 4.5 0-4.25(m, 4H), 2.59-2.48(m, 1H), 2.33-2.20(m, 1H), 2.15-2.02(m, 1H), 1.99-1.76(m, 3H), 1.75-1.55(m, 7H), 0.93(s, 9H), 0.19 (s, 3H), 0.15 (s, 3H).

[0560] Preparation of intermediate 34 3'-5'-di-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-guanosine

[0561]

[0562] Intermediate 34 is obtained from 2'-deoxy-2'-fluoroguanosine via the route of intermediate 31.

[0563] 1H NMR (400MHz, DMSO-d6) δ10.69 (brs, 1H), 7.84 (s, 1H), 6.52 (brs, 2H), 6.00 (d, J=16.8Hz, 1H), 5.36 (d, J=52.0Hz, 1H), 4.62-4.45 (m, 1H), 3.98-3.86 (m, 2H), 3.74 (d, J=11.6Hz, 1H), 0.93-0.85 (m, 18H), 0.20-0.00 (m, 12H). MS-ESI [M+H] + :515.3.

[0564] Preparation of intermediate 35 3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-guanosine

[0565]

[0566] Intermediate 35 is obtained from intermediate 34 by following the route of intermediate 15.

[0567] 1H NMR (400MHz, DMSO-d6) δ10.71 (brs, 1H), 7.97 (s, 1H), 6.56 (s, 2H), 5.36 (dd, J=52.5, 5.0Hz, 1H), 5.20 (s, 1H), 4.52 (dd, J=12.6, 6.4Hz, 1H), 3.96-3.89 (m, 1H), 3.70 (d, J=12.0Hz, 1H), 0.89 (s, 9H), 0.12 (s, 6H). MS-ESI[M+H] + :401.2.

[0568] Preparation of intermediate 36 2-Amino-9-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-fluoro-5-((((2R,3aS,6R,7aS)-3a-methyl-6-(propyl-1-en-2-yl)-2-thiohexahydrobenzo[d][1,3,2]oxothiophosphazenecyclopentan-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purine-6-one

[0569]

[0570] Intermediate 36 was obtained via the route of intermediate 2, from intermediate 35 and (-)-PSI reagent in DMF. MS-ESI[M+H] + :646.4.

[0571] Preparation of intermediate 37 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N6-bis(4-methoxyphenyl)benzyl-2-chloro-3'-bis(4-methoxyphenyl)benzyl-2'-deoxy-2'-fluoro-beta-adenosine]

[0572]

[0573] To a 3 mL acetonitrile solution of intermediate 2 (280 mg, 0.422 mmol) and intermediate 11 (383 mg, 0.422 mmol), 1,8-diazabicycloundec-7-ene (192 mg, 1.27 mmol) was added, and the resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was diluted with ethyl acetate (25 mL). The organic phase was washed successively with water (25 mL) and saturated brine (25 mL) and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with ethyl acetate:methanol (7:3) to give a white solid (260 mg).

[0574] 1 H NMR (400MHz, DMSO-d6) δ8.26 (s, 1H), 8.08 (s, 1H), 7.91 (brs, 3H), 7.45-7.10 (m, 18H), 6.95-6.76 (m, 8H), 6.32-6. 15(m, 2H), 5.40-5.18(m, 2H), 5.00-4.80(m, 2H), 4.40-4.02(m, 3H), 3.85-3.62(m, 15H), 0.84(s, 9H), 0.01(s, 6H).

[0575] Preparation of intermediate 38 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine]-(3',5')-[N6-bis(4-methoxyphenyl)benzyl-2-chloro-3'-bis(4-methoxyphenyl)benzyl-2'-deoxyadenosine]

[0576]

[0577] Intermediate 38 is obtained by following the route of intermediate 37, from intermediate 4 and intermediate 13 in a mixed solution of tetrahydrofuran and acetonitrile.

[0578] 1 H NMR (400MHz, DMSO-d6) δ8.68 (s, 1H), 8.22 (s, 1H), 7.82 (brs, 2H), 7.75 (s, 1H), 7.42(d, J=8.0Hz, 2H), 7.39-7.10(m, 16H), 6.95-6.78(m, 8H), 6.32-6.10(m, 2H), 4.90-4.80(m, 1H), 4.43-4.36(m, 1H), 4.15-4.08(m, 1H), 3.85-3.40(m, 17H), 2.50-2.20 (m, 4H), 0.81 (s, 9H), 0.00 (s, 3H), -0.02 (s, 3H).

[0579] Preparation of intermediate 39 [3'-O-thiophosphate diester-5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[3'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine]

[0580]

[0581] Intermediate 39 is obtained by following the route of intermediate 37, from intermediate 6 and intermediate 15 in a mixed solution of tetrahydrofuran and acetonitrile.

[0582] 1 H NMR (400MHz, DMSO-d6) δ9.58 (brs, 1H), 7.79-7.60 (m, 2H), 7.48-7.25 (m, 4H), 6.28-6.05 (m, 2H), 5.76 (dd, J=7.6, 1.6Hz, 2H), 5.00-4.85 (m, 1H), 4.38-4.25 (m, 1H), 4.15-3.80 (m, 6H), 0.95-0.80 (m, 18H), 0.18-0.00 (m, 12H).

[0583] Preparation of intermediate 40 [3'-O-thiophosphate diester-N4-(2-propylpentanoyl)-5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[N4-(2-propylpentanoyl)-3'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine]

[0584]

[0585] Intermediate 40 is obtained by following the route of intermediate 37, and is derived from intermediate 17 and intermediate 9.

[0586] 1 H NMR (500MHz, DMSO-d6) δ11.07 (brs, 1H), 11.02 (brs, 1H), 8.32 (d, J=7.6Hz, 1H ), 8.24 (d, J=7.6Hz, 1H), 7.36-7.30 (m, 2H), 6.28-6.15 (m, 2H), 4.97 (t, J=10.4 Hz, 1H), 4.40-4.30 (m, 1H), 4.17 (s, 1H), 4.08 (q, J=9.1, 8.5Hz, 2H), 4.04*3.99 (m, 2H), 3.93 (t, J=7.8Hz, 1H), 2.62 (d, J=3.2Hz, 2H), 1.60-1.45 (m, 4H), 1.42-1.27 (m, 4H), 1.25-1.20 (m, 9H), 1.00-0.75 (m, 30H), 0.11 (d, J=3.0Hz, 6H), 0.05 (s, 3H), 0.02 (s, 3H).

[0587] Preparation of intermediate 41 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N6-benzoyl-2'-O-tert-butyldimethylsilyl-3'-O-benzoyl-adenosine]

[0588]

[0589] Intermediate 41 is obtained by following the route of intermediate 37, and is derived from intermediate 21 and intermediate 2.

[0590] 1 H NMR (400MHz, DMSO-d6) δ11.19 (brs, 1H), 9.10 (brs, 1H), 8.76 (s, 1H), 8.12 (d, J=2.4Hz, 1H), 8.05 (dd, J=7.6, 2.0Hz, 4H), 7.89 (s, 2H), 7.75-7.61 (m, 2H), 7.60- 7.45 (m, 4H), 6.33 (dd, J=18.0, 4.0Hz, 1H), 6.23 (d, J=7.2Hz, 1H), 5.77 (d, J=5.2 Hz, 1H), 5.30 (dd, J=7.6, 5.2Hz, 1H), 5.04 (s, 1H), 4.53 (s, 1H), 4.21 (t, J=10.0Hz, 1H), 4.18-4.08(m, 3H), 4.00-3.92(m, 1H), 3.92-3.85(m, 1H), 0.85(s, 9H), 0.49(s, 9H), 0.05(s, 6H), -0.12(s, 3H), -0.42(s, 3H). MS-ESI[(M+2H) / 2] + : 543.7.

[0591] Preparation of intermediate 42 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine]-(3',5')-[N6-benzoyl-2'-O-tert-butyldimethylsilyl-3'-O-benzoyl-adenosine]

[0592]

[0593] Intermediate 42 is obtained by following the route of intermediate 37, and is derived from intermediates 21 and 4. MS-ESI [(M+H)] + : 1067.2.

[0594] Preparation of intermediate 43 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N6-benzoyl-3'-O-tert-butyldimethylsilyl-2'-O-benzoyl-adenosine]

[0595]

[0596] Intermediate 43 is obtained by following the route of intermediate 37, from intermediate 23 and intermediate 2 in N,N'-dimethylformamide solution.

[0597] 1 H NMR (400MHz, DMSO-d6) δ11.23 (brs, 1H), 8.96 (s, 1H), 8.76 (s, 1H), 8.11 (d, J=2.4Hz, 1H), 8.02 (d, J=7.6Hz, 2H), 7.88 (d, J=7.6Hz, 4H), 7.64 (dd, J=6.8, 6.0Hz, 2H), 7.65-7.45 (m, 4H), 6.51 (d, J=5.6Hz, 1H), 6.32 (dd, J=18.8, 3.6Hz, 1H ), 5.92 (t, J = 5.6Hz, 1H), 5.55-5.30 (m, 1H), 5.11-4.98 (m, 1H), 4.83 (t, J = 4.0Hz, 1H), 4.29(s, 1H), 4.28-4.15(m, 1H), 4.15-4.05(m, 1H), 4.00-3.89(m, 2H), 3.87-.80 (m, 1H), 0.85 (s, 9H), 0.76 (s, 9H), 0.08 (s, 3H), 0.04 (s, 6H), -0.09 (s, 3H). MS-ESI [M+H] + : 1085.3.

[0598] Preparation of intermediate 44 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-guanosine]

[0599]

[0600] Intermediate 44 is obtained by following the route of intermediate 37, from intermediate 2 and intermediate 25 in DMF solution.

[0601] 1H NMR (400MHz, DMSO-d6) δ12.59 (brs, 1H), 12.11 (brs, 1H), 8.37 (s, 1H), 8.14 (d, J=2.8Hz, 1H), 8.08-7.85 (m, 4H), 7.80-7.50 (m, 3H), 6.35 (dd, J=18.0, 4.0Hz, 1H), 5.92 (d, J=8.0Hz, 1H), 5.68 (d, J=5.2Hz, 1H), 5.55-5.38 (m, 2H), 5.15-4.98 (m, 1H), 4.52-4.40 (m, 1H), 4.20-3.95 (m, 5H), 2.90-2.80 (m, 1H), 1.25-1.05 (m, 6H), 0.87 (s, 9H), 0.53 (s, 9H), 0.07 (s, 6H), -0.13 (s, 3H), -0.38 (s, 3H). MS-ESI[(M- 2H) / 2] - :532.2.

[0602] Preparation of intermediate 45 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-guanosine]

[0603]

[0604] Intermediate 45 is obtained by following the route of intermediate 37, from intermediate 4 and intermediate 25 in DMF solution.

[0605] 1H NMR (400MHz, DMSO-d6) δ12.76 (brs, 1H), 12.12 (brs, 1H), 8.37 (s, 1H), 8.27 (s, 1H), 8.03 (d, J = 7.6Hz, 2H), 7.81 (brs, 2H), 7.70-7.50 (m, 3H), 6.29 (t, J = 6.8Hz, 1H), 5.92 (d, J=7.6Hz, 1H), 5.76-5.65 (m, 1H), 5.55-5.45 (m, 1H), 5.10-4.96 (m, 1H), 4.47(s, 1H), 4.30-4.20(m, 1H), 4.10-3.75(m, 3H), 2.98-2.83(m, 1H), 2.81-2.55(m, 2H), 1.20-1.05(m, 6H), 0.84(s, 9H), 0.54(s, 9H), 0.03(s, 6H), -0.11(s, 3H), -0.36(s, 3H). MS-ESI[(M+2H) / 2] + : 525.3.

[0606] Preparation of intermediate 46 [3'-O-thiophosphate diester-5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-guanosine]

[0607]

[0608] Intermediate 46 is obtained by following the route of intermediate 37, from intermediate 6 and intermediate 25 in DMF solution.

[0609] 1H NMR (400MHz, DMSO-d6) δ12.49(brs, 1H), 12.09(brs, 1H), 8.39(brs, 1H), 8.05(d , J=7.2Hz, 2H), 7.78-7.50 (m, 4H), 7.40 (brs, 2H), 6.19 (t, J=8.4Hz, 1H), 5.91 (d, J=8.0Hz, 1H), 5.78 (d, J=7.6Hz, 1H), 5.66 (d, J=4.8Hz, 1H), 5.49-5.35 (m, 1H), 5.10-4.90 (m, 1H), 4.45 (s, 1H), 4.20-3.80 (m, 5H), 2.95-2.77 (m, 1H), 1.12 (d, J=6.8 Hz, 6H), 0.89 (s, 9H), 0.54 (s, 9H), 0.15-0.05 (m, 6H), -0.10 (s, 3H), -0.37 (s, 3H). MS-ESI[(M+2H) / 2] + :514.3.

[0610] Preparation of intermediate 47 [3'-O-thiophosphate diester-N4-(2-propylpentanoyl)-5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[2'-O-tert-butyldimethylsilyl-3'-O-benzoyl-guanosine]

[0611]

[0612] Intermediate 47 is obtained by following the route of intermediate 37, from intermediate 30 and intermediate 9 in DMF solution.

[0613] 1H NMR (400MHz, DMSO-d6) δ11.10 (brs, 1H), 10.60 (brs, 1H), 8.22 (d, J=7.6Hz, 1H ), 8.16 (s, 1H), 8.02 (d, J = 7.6Hz, 2H), 7.68 (t, J = 7.6Hz, 1H), 7.56 (t, J = 7.6Hz, 2H), 7.35 (d, J=7.6Hz, 1H), 6.51 (brs, 2H), 6.23 (t, J=7.6Hz, 1H), 5.82 (d, J=7.6 Hz, 1H), 5.67 (d, J=5.2Hz, 1H), 5.09 (dd, J=7.6, 5.2Hz, 1H), 5.02-4.95 (m, 1H), 4.41(d, J=3.6Hz, 1H), 4.18-3.92(m, 4H), 2.70-2.55(m, 1H), 1.65-1.50(m, 2H), 1.45-1.30(m, 2H), 1.28-1.15(m, 4H), 0.96-0.88(m, 12H), 0.87-0.80(m, 9H), 0.53(s, 9H), -0.11(s, 3H), -0.31(s, 3H). MS-ESI[(M-2H) / 2] - : 540.3.

[0614] Preparation of intermediate 48 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N2-isobutyryl-3'-tert-butyldimethylsilyl-2'-benzoyl-guanosine]

[0615]

[0616] Intermediate 48 is obtained by following the route of intermediate 37, from intermediate 27 and intermediate 2 in DMF solution.

[0617] 1H NMR (400MHz, DMSO-d6) δ12.53 (brs, 1H), 12.13 (brs, 1H), 8.36 (s, 1H), 8.14 (d, J=2.8Hz, 1H), 7.98-7.80 (m, 4H), 7.64 (t, J=7.6Hz, 1H), 7.48 (t, J=8.0Hz, 2H), 6.36 (dd, J=18.0, 4.0Hz, 1H), 6.22 (d, J=7.2Hz, 1H), 5.88 (dd, J=7.2, 5.2Hz, 1H), 5.60--5.43(m, 1H), 5.19-5.05(m, 1H), 4.72(d, J=4.8Hz, 1H), 4.29-4.22(m, 1H), 4.22-4.09(m, 2H), 4.08-3.91(m, 3H), 2.96-2.86(m, 1H), 1.18-1.09(m, 6H), 0.87 (s, 9H), 0.72 (s, 9H), 0.07 (s, 6H), 0.00 (s, 3H), -0.02 (s, 3H).

[0618] Preparation of intermediate 49 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-adenosine]-(3',5')-[N2-isobutyryl-3'-tert-butyldimethylsilyl-2'-benzoyl-guanosine]

[0619]

[0620] Intermediate 49 is obtained by following the route of intermediate 37, from intermediate 27 and intermediate 4 in a DMF solution.

[0621] 1H NMR (400MHz, DMSO-d6) δ12.55 (brs, 1H), 12.14 (brs, 1H), 8.37 (s, 1H), 8.28 (s, 1H), 7.94-7.76 (m, 4H), 7.65 (t, J=7.6Hz, 1H), 7.49 (t, J=8.0Hz, 2H), 6.33-6.21 (m, 2H), 5.95-5.85 (m, 1H), 5.10-5.02 (m, 1H), 4.75 (d, J=4.8Hz, 1H), 4.31-4.23 (m, 2H), 4.22-4.15(m, 1H), 4.14-4.09(m, 1H), 3.91-3.78(m, 2H), 2.93-2.86(m, 1H), 2.7 6-2.71 (m, 1H), 2.68-2.60 (m, 1H), 1.20-1.10 (m, 6H), 0.84 (s, 9H), 0.74 (s, 9H), 0.07- 0.00 (m, 9H), -0.10 (s, 3H).

[0622] Preparation of intermediate 50 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroadenosine]

[0623]

[0624] Intermediate 50 is obtained by following the route of intermediate 37, from intermediate 32 and intermediate 2 in DMF solution.

[0625] 1 H NMR (400MHz, DMSO-d6) δ8.49 (s, 1H), 8.18-8.08 (m, 2H), 7.92 (brs, 2H), 7.34 (brs, 2H), 6.32-6.20 (m, 2H), 5.50 (dt, J=52.0 , 4.4Hz, 1H), 5.30 (d, J=52.0Hz, 1H), 5.05-4.94 (m, 1H), 4.78-4.65 (m, 1H), 4.14-4.06 (m, 3H), 3.95-3.70 (m, 3H), 0.90-0.75 (m, 18H), 0.10 (d, J=8.8Hz, 6H), 0.04 (s, 6H).

[0626] Preparation of intermediate 51[3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine]-(3',5')-[3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroadenosine]

[0627]

[0628] Intermediate 51 is obtained by following the route of intermediate 37, and is derived from intermediate 3 and intermediate 33.

[0629] 1 H NMR (400MHz, DMSO-d6) δ8.51 (s, 1H), 8.25 (s, 1H), 8.13 (s, 1H), 7.82 (brs, 2H), 7.31(brs, 2H), 6.30-6.15(m, 2H), 5.69(dt, J=52.4, 4.0Hz, 1H), 5.00-4.86(m, 1H), 4.80-4.65(m, 1H), 4.19-4.00(m, 3H), 3.99-3.85(m, 1H), 3.78-3.62(m, 2H), 2.75- 2.62 (m, 1H), 2.60-2.50 (m, 1H), 0.88 (s, 9H), 0.81 (s, 9H), 0.14 (s, 3H), 0.11 (s, 3H), 0.00 (s, 3H), -0.01 (s, 3H).

[0630] Preparation of intermediate 52 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroguanosine]

[0631]

[0632] Intermediate 52 is obtained by following the route of intermediate 37, from intermediate 1 and intermediate 36 in DMF solution.

[0633] 1H NMR (400MHz, DMSO-d6) δ10.64 (brs, 1H), 8.12 (s, 1H), 8.05 (s, 1H), 7.93-7.82 (m, 3H), 6.54 (brs, 2H), 6.36-6.25 (m, 1H), 6. 00(dd, J=14.8, 4.8Hz, 1H), 5.58-5.28(m, 2H), 5.15-4.98(m, 1H), 4.60-4.50(m, 1H), 4.15-4.00(m, 3H), 3.98-3.88(m, 3H), 0.86 (s, 18H), 0.10-0.04 (m, 12H).

[0634] Preparation of intermediate 53 [3'-O-thiophosphate diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine]-(3',5')-[3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroguanosine]

[0635]

[0636] Intermediate 53 is obtained by following the route of intermediate 37, from intermediates 3 and 36 in DMF solution.

[0637] 1 H NMR (400MHz, DMSO-d6) δ10.65 (brs, 1H), 8.25 (s, 1H), 8.05 (s, 1H), 7.80 (brs, 2H), 6.55 (brs, 2H), 6.25 (t, J=7.2Hz, 1H), 6.03-5.95 (m, 1H), 5.47 (d, J=52.0Hz, 1H), 4.97 (s, 1H), 4.65-4.50 (m, 1H), 4.19 (s, 1H), 4.15-4.00 (m, 3H), 3.99-3.85 (m, 1H), 3.82 (d, J = 11.2Hz, 1H), 3.76-3.69 (m, 1H), 2.75-2.60 (m, 1H), 0.88 (s, 9H), 0.80 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H), 0.02 (s, 6H). MS-ESI[(MH)] - :875.4.

[0638] Preparation of intermediate 54 [3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0639]

[0640] Intermediate 37 (260 mg, 0.188 mmol) was dissolved in a mixture of tetrahydrofuran (2 mL), trifluoroacetic acid (2 mL), and water (1 mL), and stirred at 25 °C for 2 hours. The reaction mixture was evaporated to dryness and diluted with methanol (5 mL). The pH of the mixture was adjusted to approximately 8-9 with a saturated sodium bicarbonate aqueous solution. The solution was evaporated to dryness again and diluted with methanol (5 mL), then filtered. The filtrate was concentrated, and the crude product was purified by silica gel column chromatography, eluting with ethyl acetate:methanol (7:3) to give a white solid (120 mg).

[0641] 1 H NMR (400MHz, DMSO-d6) δ8.30 (s, 1H), 8.23 ​​(s, 1H), 7.88 (brs, 4H), 6.40-6.18 ( m, 2H), 6.06 (d, J=5.2Hz, 1H), 5.45-5.12 (m, 2H), 5.04 (t, J=6.0Hz, 1H), 5.00- 4.85 (m, 1H), 4.50-4.40 (m, 1H), 4.18-3.90 (m, 4H), 3.75-3.58 (m, 2H). MS-ESI[M-H] - : 682.8.

[0642] Preparation of intermediate 55 [3'-O-thiophosphate diester-2-chloro-2'-deoxyadenosine]-(3',5')-[2-chloro-2'-deoxyadenosine]

[0643]

[0644] Tetrabutylammonium fluoride (34 mg, 0.132 mmol) was added to a tetrahydrofuran solution (5 mL) of intermediate 38 (150 mg, 0.110 mmol), and the resulting mixture was stirred at 25 °C for 3 hours. The reaction mixture was evaporated to dryness and then dissolved in a mixture of glacial acetic acid (2 mL) and water (0.5 mL), and stirred at 25 °C for 0.5 hours. The reaction mixture was evaporated to dryness, and the crude product was purified by silica gel column chromatography, eluting with ethyl acetate:methanol (3:1) to give a white solid (45 mg). MS-ESI [MH] - : 647.0.

[0645] Preparation of intermediate 56 [3'-O-thiophosphate diester-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[2'-deoxy-2',2'-difluorocytidine]

[0646]

[0647] Tetrabutylammonium fluoride (329 mg, 1.26 mmol) was added to a tetrahydrofuran solution (5 mL) of intermediate 39 (350 mg, 0.421 mmol), and the resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was evaporated to dryness, and the crude product was purified by silica gel column chromatography, eluting with ethyl acetate:methanol (3:1) to give a white solid (130 mg).

[0648] 1 H NMR (400MHz, DMSO-d6) δ7.77 (d, J=7.2Hz, 1H), 7.65 (d, J=7.6Hz, 1H), 7.45-7.25 (m, 4H), 6.30 (d, J=6.4Hz, 1H), 6.20-6.05 (m, 2H), 5.89-5.70 (m, 2H), 5.13 (t, J=6.0Hz, 1H), 4.90-4.75 (m, 1H), 4.20-4.00 (m, 2H), 4.00-3.60 (m, 5H).

[0649] Preparation of intermediate 57 [3'-O-thiophosphate diester-N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]

[0650]

[0651] Intermediate 57 is obtained from intermediate 40 using the route of intermediate 56. MS-ESI[M+H] + : 858.2, [(M+2H) / 2] + :429.1.

[0652] Preparation of intermediate 58 [3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N6-benzoyl-3'-O-benzoyl-adenosine]

[0653]

[0654] Intermediate 58 is obtained from intermediate 41 using the route of intermediate 56. MS-ESI [M+H] + :857.0.

[0655] Preparation of intermediate 59 [3'-O-thiophosphate diester-2-chloro-2'-deoxyadenosine]-(3',5')-[N6-benzoyl-3'-O-benzoyl-adenosine]

[0656]

[0657] Intermediate 59 is obtained from intermediate 42 using the route of intermediate 56. MS-ESI [M+H] + :839.0.

[0658] Preparation of intermediate 60 [3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N6-benzoyl-2'-O-benzoyl-adenosine]

[0659]

[0660] Triethylamine trihydrofluoride (223 mg, 1.38 mmol) was added to a tetrahydrofuran solution (5 mL) of intermediate 43 (250 mg, 0.23 mmol), and the mixture was stirred at 30 °C for 16 hours. The reaction mixture was neutralized to pH 7-8 with triethylamine. The resulting mixture was evaporated to dryness, and the crude product was purified by silica gel column chromatography with ethyl acetate:methanol (8:2) to give a white solid (160 mg). MS-ESI [M+H] + :857.1.

[0661] Preparation of intermediate 61 [3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-guanosine]

[0662]

[0663] Intermediate 61 is obtained from intermediate 44 by following the route of intermediate 60.

[0664] 1 H NMR (400MHz, DMSO-d6) δ12.56 (brs, 1H), 12.11 (brs, 1H), 8.35-8.20 (m, 2H) , 8.10-8.00(m, 2H), 7.91(brs, 2H), 7.75-7.45(m, 3H), 6.38-6.18(m, 1H), 6 .00-5.75(m, 2H), 5.70-5.21(m, 2H), 5.16-4.90(m, 2H), 4.50-4.36(m, 1H), 4.28-4.00(m, 2H), 3.90-3.58(m, 2H), 3.00-2.82(m, 1H), 1.36-1.05(m, 6H). MS-ESI[MH] - :837.0.

[0665] Preparation of intermediate 62 [3'-O-thiophosphate diester-2-chloro-2'-deoxyadenosine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-guanosine]

[0666]

[0667] Intermediate 62 is obtained from intermediate 45 by following the route of intermediate 60.

[0668] 1 H NMR (400MHz, DMSO-d6) δ12.72 (brs, 1H), 12.13 (brs, 1H), 8.34 (s, 1H), 8.32 (s, 1H), 8.03 (d, J=7.6Hz, 1H), 7.98-7.81 (m, 3H), 7.71-7.62 (m, 1H), 7.60-7.43 (m, 1H), 6.38-6.20(m, 1H), 5.99-5.70(m, 2H), 5.70-5.57(m, 1H), 5.40-5.28(m, 1H), 5.18- 4.93 (m, 2H), 4.42 (s, 1H), 4.32-4.00 (m, 3H), 3.75-3.52 (m, 2H), 2.93-2.81 (m, 1H), 2.80-2.62 (m, 1H), 1.20-1.05 (m, 6H). MS-ESI[M+H] + :821.2.

[0669] Preparation of intermediate 63 [3'-O-thiophosphate diester-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-guanosine]

[0670]

[0671] Intermediate 63 is obtained from intermediate 46 by following the route of intermediate 60.

[0672] 1H NMR (400MHz, DMSO-d6) δ12.42 (brs, 1H), 12.12 (brs, 1H), 8.35 (s, 1H), 8.08 (d, J=7.6Hz, 2H), 7.80-7.53 (m, 4H), 7.48-7.34 (m, 2H), 6.24-6.12 (m, 1H), 5.97- 5.87 (m, 1H), 5.86-5.76 (m, 1H), 5.57 (d, J = 5.2Hz, 1H), 5.30-5.18 (m, 1H), 5.17- 5.05 (m, 1H), 4.95-4.75 (m, 1H), 4.41 (s, 1H), 4.25-3.70 (m, 6H), 2.90-2.77 (m, 1H), 1.20-1.05 (m, 6H).

[0673] Preparation of intermediate 64 [3'-O-thiophosphate diester-N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[3'-O-benzoyl-guanosine]

[0674]

[0675] Intermediate 64 is obtained from intermediate 47 using the route of intermediate 56. MS-ESI [(M+2H) / 2] + :428.0.

[0676] Preparation of intermediate 65 [3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N2-isobutyryl-2'-benzoyl-guanosine]

[0677]

[0678] Intermediate 65 is obtained from intermediate 48 using the route of intermediate 56. MS-ESI[MH] - :837.0.

[0679] Preparation of intermediate 66 [3'-O-thiophosphate diester-2-chloro-2'-deoxy-adenosine]-(3',5')-[N2-isobutyryl-2'-benzoyl-guanosine]

[0680]

[0681] Intermediate 66 is derived from intermediate 49 using the same route as intermediate 56. MS+ESI [M+H] + :821.1.

[0682] Preparation of intermediate 67 [3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[2'-deoxy-2'-fluoroadenosine]

[0683]

[0684] Intermediate 67 is obtained from intermediate 50 by following the route of intermediate 60.

[0685] 1 H NMR (400MHz, DMSO-d6) δ8.47 (s, 1H), 8.24 (s, 1H), 8.15 (s, 1H), 7.91 (brs, 2H), 7.35(brs, 2H), 6.32-6.20(m, 2H), 5.88(brs, 1H), 5.55-5.30(m, 2H), 5.18-4.87 (m, 2H), 4.53 (d, J=16.4Hz, 1H), 4.18-4.08 (m, 2H), 4.10-4.02 (m, 1H), 4.01-3.92 (m, 1H), 3.75-3.59 (m, 2H). MS-ESI[M+H] + :651.0.

[0686] Preparation of intermediate 68 [3'-O-thiophosphate diester-2-chloro-2'-deoxyadenosine]-(3',5')-[2'-deoxy-2'-fluoroadenosine]

[0687]

[0688] Intermediate 68 is obtained from intermediate 51 by following the route of intermediate 56.

[0689] 1 H NMR (400MHz, DMSO-d6) δ8.50 (s, 1H), 8.34 (s, 1H), 8.15 (s, 1H), 7.84 (brs, 2H), 7.31 (brs, 2H), 6.30-6.18 (m, 2H), 5.90 (d, J=5.6Hz, 1H), 5.45 (dt, J=52.0, 3.6 Hz, 1H), 5.12-4.90 (m, 2H), 4.60-4.45 (m, 1H), 4.18-4.00 (m, 3H), 3.99-3.85 (m, 1H), 3.65-3.50 (m, 2H), 2.70-2.55 (m, 1H), 2.50-2.40 (m, 1H).

[0690] Preparation of intermediate 69[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[2'-deoxy-2'-fluoroguanosine]

[0691]

[0692] Intermediate 69 is obtained from intermediate 52 by following the route of intermediate 60.

[0693] 1 H NMR (400MHz, DMSO-d6) δ10.64 (s, 1H), 8.24 (s, 1H), 8.00 (s, 1H), 7.88 (brs, 2H ), 6.54(brs, 2H), 6.28(dd, J=18.0, 3.6Hz, 1H), 6.02(dd, J=16.0, 3.6Hz, 1H), 5.80 (d, J=5.4Hz, 1H), 5.44 (d, J=4.4Hz, 1H), 5.30 (d, J=7.6Hz, 1H), 5.04 (d, J =6.0Hz, 1H), 4.95 (t, J = 14.8Hz, 1H), 4.42 (dt, J = 10.4, 5.6Hz, 1H), 4.11-3.99 (m, 3H), 3.98-3.89 (m, 1H), 3.77-3.60 (m, 2H). MS-ESI[M+H] + :667.2.

[0694] Preparation of intermediate 70 [3'-O-thiophosphate diester-2-chloro-2'-deoxyadenosine]-(3',5')-[2'-deoxy-2'-fluoroguanosine]

[0695]

[0696] Intermediate 70 is obtained from intermediate 53 by following the route of intermediate 60.

[0697] 1H NMR (400MHz, DMSO-d6) δ10.67 (brs, 1H), 8.37 (s, 1H), 8.04 (s, 1H), 7.85 (brs, 2 H), 6.58 (brs, 2H), 6.26 (t, J=7.2Hz, 1H), 6.03 (d, J=15.6Hz, 1H), 5.85 (d, J=5.6 Hz, 1H), 5.38 (d, J = 52.8Hz, 1H), 5.10 (s, 1H), 4.97 (s, 1H), 4.46 (d, J = 14.8Hz, 1H), 4.20-4.00(m, 4H), 4.00-3.90(m, 1H), 3.70-3.50(m, 2H), 2.78-2.60(m, 1H). MS-ESI[MH] - :647.2.

[0698] Preparation of intermediate 71 (2',3')-cyclo-[2'-O-thiophosphate diester-N6-benzoyl-3'-benzoyl-adenosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0699]

[0700] To a DMF solution (10 mL) of intermediate 58 (95 mg, 0.11 mmol) and (-)-PSI reagent (150 mg, 0.33 mmol), 1,8-diazabicycloundec-7-ene (253 mg, 1.66 mmol) was added, and the resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was evaporated to dryness and reprecipitated with ethyl acetate (20 mL). The resulting solid was slurried with ethyl acetate (20 mL), filtered, and dried to obtain a crude product (200 mg), which was used directly for the next reaction. MS-ESI[M+2+H] + : 937.0.

[0701] Preparation of intermediate 72 (2',3')-cyclo-[2'-O-thiophosphate diester-N6-benzoyl-3'-O-benzoyl-adenosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxyadenosine]

[0702]

[0703] Add 1,8-diazabicycloundec-7-ene (570 mg, 3.75 mmol) to a DMF solution (10 mL) of intermediate 59 (210 mg, 0.25 mmol) and (-)-PSI reagent (335 mg, 0.75 mmol). Stir the resulting mixture at 25 °C for 1 hour. After rotary evaporation to dryness, reprecipitate with ethyl acetate (20 mL). The resulting solid was slurryed with ethyl acetate (20 mL), filtered, and dried to obtain the crude product. The crude product was purified by preparative liquid chromatography (10 mM ammonium bicarbonate as an additive), and lyophilized to obtain a white solid (15.8 mg), which was directly used in the next reaction. MS-ESI [M+H] + : 917.0.

[0704] Preparation of intermediate 73 (3',3')-cyclo-bis-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-[3'-O-thiophosphate diester-N6-benzoyl-2'-O-benzoyl-adenosine]

[0705]

[0706] Intermediate 73 was obtained via the route of intermediate 71, from intermediate 60 and (-)-PSI reagent. MS-ESI [MH] - : 932.8.

[0707] Intermediate 74 :(2',3')-cyclo-[2'-O-thiophosphate diester-3'-O-benzoyl-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-isomer 1 and Intermediate 75 Preparation of (2',3')-cyclo-[2'-O-thiophosphate diester-3'-O-benzoyl-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-isomer 2

[0708]

[0709] Intermediates 74 and 75 were obtained from intermediate 61 and (-)-PSI reagent via the same route as intermediate 72.

[0710] Intermediate 74: MS-ESI[MH] - : 844.8, retention time: 4.96min;

[0711] Intermediate 75: MS-ESI[MH] -844.9, retention time: 5.20 min. Analytical LCMS: Agilent 1100+G1946D LCMS, 4.6 x 150 mm Waters XBridge C18 3.5 μm analytical column, mobile phase A was 10 mM NH4HCO3 aqueous solution, and B was acetonitrile, flow rate 1.0 mL / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0–0.1 min, 5% B; 0.1–8 min, 5–95% B; 8–15 min, 95% B.

[0712] Intermediate 76 :(2',3')-cyclo-[2'-O-thiophosphate diester-N2-isobutyryl-3'-O-benzoyl-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-adenosine]-isomer 1, and

[0713] Intermediate 77 Preparation of (2',3')-cyclo-[2'-O-thiophosphate diester-N2-isobutyryl-3'-O-benzoyl-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-adenosine]-isomer 2

[0714]

[0715] Intermediates 76 and 77 were obtained from intermediate 62 and (-)-PSI reagent via the same route as intermediate 72.

[0716] Intermediate 76: MS-ESI[MH] - 897.2, retention time: 1.69 min;

[0717] Intermediate 77: MS-ESI[MH] - 897.2, retention time: 1.74 min. Analytical LCMS: Agilent 1100+G1946D LCMS, 4.6 x 50 mm Waters XBridge C18 3.5 μm analytical column, mobile phase A was 10 mM NH4HCO3 aqueous solution, B was acetonitrile, flow rate 1.8 min / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0-0.1 min, 5% B; 0.1-2.5 min, 5-95% B; 2.5-5 min, 95% B.

[0718] Intermediate 78 Preparation of: (2',3')-cyclo-[2'-O-thiophosphate diester-N2-isobutyryl-3'-O-benzoyl-guanosine]-[3'-O-thiophosphate diester-2'-deoxy-2',2'-difluorocytidine]

[0719]

[0720] Intermediate 78 was obtained via the same route as intermediate 72, from intermediate 63 and (-)-PSI reagent. Intermediate 78: MS-ESI[(M-2H) / 2] - :437.0.

[0721] Intermediate 79 Preparation of: (2',3')-cyclo-[2'-O-thiophosphate diester-3'-O-benzoyl-guanosine]-[3'-O-thiophosphate diester-N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]

[0722]

[0723] Intermediate 79 was obtained via the route of intermediate 72, from intermediate 64 and (-)-PSI reagent. MS-ESI [MH] - : 931.0.

[0724] Intermediate 80 :(3',3')-cyclo-[3'-O-thiophosphate diester-N2-isobutyryl-2'-benzoyl-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-isomer 1, and

[0725] Intermediate 81 :(3',3')-cyclo-[3'-O-thiophosphate diester-N2-isobutyryl-2'-benzoyl-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-isomer 2

[0726]

[0727] Intermediates 80 and 81 are obtained via the route used for intermediate 72, derived from intermediate 65 and (-)-PSI reagent. Intermediate 80: MS-ESI[MH] - : 915.0, Retention time: 1.50 min;

[0728] Intermediate 81: MS-ESI[MH] -915.0, retention time: 1.53 min. Analytical LCMS: Agilent 1100+G1946D LCMS, 4.6 x 50 mm Waters XBridge C18 3.5 μm analytical column, mobile phase A was 10 mM NH4HCO3 aqueous solution, B was acetonitrile, flow rate 1.8 ml / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0-0.2 min, 5% B; 0.2-1.5 min, 5-95% B; 1.5-3 min, 95% B.

[0729] Preparation of intermediate 82 (3',3')-cyclo-[3'-O-thiophosphate diester-N2-isobutyryl-2'-benzoyl-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-adenosine]

[0730]

[0731] Intermediate 82 was obtained via the same route as intermediate 72, from intermediate 66 and (-)-PSI reagent. MS-ESI [MH] - :896.8.

[0732] Preparation of intermediate 83 2-Chloro-5'-O-tert-butyldimethylsilyl-3'-O-benzoyl-2'-deoxy-2'-fluoro-beta-adenosine

[0733]

[0734] Intermediate 83 is obtained from intermediate 1 by following the route of intermediate 20.

[0735] 1 H NMR (400MHz, DMSO-d6) δ8.21 (d, J=2.4Hz, 1H), 8.08 (d, J=7.2Hz, 2H), 7.95 (brs, 2H), 7.72 (t, J=7.6Hz, 1H), 7.58 (t, J=7.6Hz, 2H), 6.50 (dd, J=16.6, 4.0 Hz, 1H), 5.80-5.77 (m, 1H), 5.75-5.66 (m, 1H), 4.32 (q, J=4.8Hz, 1H), 3.97 (d, J= 4.4Hz, 2H), 0.86 (s, 9H), 0.06 (s, 6H).

[0736] Preparation of intermediate 84 2-Chloro-3'-O-benzoyl-2'-deoxy-2'-fluoro-beta-adenosine

[0737]

[0738] Intermediate 84 is obtained from intermediate 83 by following the route of intermediate 60.

[0739] 1 H NMR (400MHz, DMSO-d6) δ8.40 (d, J=2.4Hz, 1H), 8.12 (d, J=7.6Hz, 2H), 7.99 (brs, 2H), 7.74 (t, J=7.2Hz, 1H), 7.60 (t, J=7.6Hz, 2H), 6.55 (dd, J=18.2, 3.6 Hz, 1H), 5.81-5.78 (m, 1H), 5.77-5.66 (m, 1H), 4.35 (q, J=4.4Hz, 1H), 3.89-3.80 (m, 2H).

[0740] Preparation of intermediate 85 :N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-thiohexahydrobenzo[d][1,3,2]oxothiophosphazenecyclopentan-2-yl)oxy)tetrahydrofuran-2-yl)-6-oxy-6,9-dihydro-1H-purine-2-yl)isobutyramide

[0741]

[0742] Intermediate 85 was obtained via the route of intermediate 2, from N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-tert-butyldimethylsilyl-guanosine and (+)-PSI reagent.

[0743] 1H NMR (400MHz, DMSO-d6) δ12.11 (s, 1H), 11.62 (s, 1H), 8.14 (s, 1H), 7.38 (d, J =8.0Hz, 2H), 7.31-7.22 (m, 7H), 6.86 (d, J = 8.4Hz, 4H), 6.00 (d, J = 6.8Hz, 1H), 5.67-5.59 (m, 1H), 4.89 (s, 1H), 4.75 (s, 1H), 4.42 (d, J=4.0Hz, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.06-4.02 (m, 1H), 3.73 (s, 6H), 3.41-3.35 (m, 2H), 3.24 (dd, J=12.0Hz, 1H), 2.82-2.73(m, 1H), 2.07(d, J=12.0Hz, 1H), 1.93-1.83(m, 2H), 1.76-1.65(mf, 6H), 1.54 (s, 3H), 1.12 (d, J=6.8Hz, 6H), 0.83 (s, 9H), 0.08 (s, 3H), 0.04 (s, 3H).

[0744] Preparation of intermediate 86 [2'-O-Rp-thiophosphate diester-N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-tert-butyldimethylsilyl-guanosine]-(2',5')-[2-chloro-3'-O-benzoyl-2'-deoxy-2'-fluoro-beta-adenosine]

[0745]

[0746] Intermediate 86 is obtained by following the route of intermediate 37, from intermediates 84 and 85 in DMF solution.

[0747] MS-ESI[M+H] + :1255.0.

[0748] Preparation of intermediate 87 [2'-O-Rp-thiophosphate diester-N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-tert-butyldimethylsilyl-guanosine]-(2',5')-[2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0749]

[0750] Intermediate 86 (1.0 g, 0.8 mmol) was added to a tetrahydrofuran / water / methanol mixture (8:4:1) in 10 mL. The mixture was cooled to 0 °C, and lithium hydroxide monohydrate (54 mg, 1.6 mmol) was added. The resulting mixture was stirred at 0 °C for 20 min. The reaction mixture was poured into water (50 mL) and extracted twice with ethyl acetate (50 mL each time). The organic phases were combined, washed twice with saturated brine (100 mL each time), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluting with ethyl acetate:methanol (10:1) to give a white solid (733 mg).

[0751] 1 H NMR (400MHz, DMSO-d6) δ12.02 (s, 1H), 11.67 (s, 1H), 8.21 (t, J=5.2Hz, 1H), 8.1 4(s, 1H), 7.84(s, 2H), 7.37(d, J=7.6Hz, 2H), 7.29-7.14(m, 7H), 6.87-6.77(m, 4H), 6.23 (dd, J=13.9, 4.5Hz, 1H), 6.08-5.98 (m, 2H), 5.49-5.38 (m, 1H), 5.23-5.04 (m, 1H), 4.66-4.59 (m, 1H), 4.35-4.24 (m, 1H), 3.99-3.95 (m, 1H), 3.77-3.71 (m, 2H), 3.71-3.63 (m, 7H), 3.22-3.00 (m, 2H), 2.79-2.68 (m, 1H), 1.12-1.07 (m, 6H), 0.82 (s, 9H), 0.13 (d, J=13.4Hz, 6H).

[0752] Preparation of intermediate 88 O-((2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-fluoro-3-((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2-yl)-2-thiohexahydrobenzo[d][1,3,2]oxothiophosphazene-2-yl)methyl)-O-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutylamino-6-oxy-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)-(R)-thiophosphate diester

[0753]

[0754] Intermediate 88 is prepared via the route of intermediate 2, from intermediate 87 and (-)-PSI reagent in tetrahydrofuran, yielding a crude product that is directly used in the next reaction. MS-ESI[M+H-DMTr] + :1095.0.

[0755] Preparation of intermediate 89 O-((2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-fluoro-3-((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2-yl)-2-thiohexahydrobenzo[d][1,3,2]oxothiophosphazenecyclopentane-2-yl)methyl)-O-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutylamino-6-oxy-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)-(R)-thiophosphate diester

[0756]

[0757] Intermediate 89 is obtained from intermediate 88 by following the route of intermediate 25.

[0758] MS-ESI[M+H] + :1095.0.

[0759] Preparation of intermediate 90 :(2',3')-cyclo-(Rp,Sp)-[2'-O-thiophosphate diester-N2-isobutyryl-3'-O-tert-butyldimethylsilyl-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0760]

[0761] Intermediate 89 (90 mg, 0.082 mmol) was dissolved in anhydrous DMF (7 mL), and 4A molecular sieve (90 mg) was added. The mixture was stirred and dried for 0.5 hours at room temperature (28 °C) under nitrogen protection. Then, DBU (75.0 mg, 0.492 mmol) was added using a syringe, and the mixture was stirred for 0.5 hours at room temperature (28 °C). After filtration with diatomaceous earth, the filtrate was evaporated to dryness to obtain crude intermediate 90 (100 mg, crude product), which was directly used in the next reaction.

[0762] MS-ESI[M+H] + : 926.8.

[0763] Preparation of intermediate 91:(2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutylamino-6-oxy-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylbenzoate

[0764]

[0765] Intermediate 84 (500 mg, 1.23 mmol) and (2R, 3R, 4R, 5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutylamino-6-oxy-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphonamide (1.78 g, 1.84 mmol) were dissolved in a mixed solution of acetonitrile / tetrahydrofuran (8 mL / 8 mL), and 4A molecular sieve (500 mg) was added. The mixture was stirred for 0.5 hours at room temperature (28 °C) under nitrogen protection. Then, a tetrazolium acetonitrile solution (8.17 mL, 0.45 M) was added using a syringe; after stirring at room temperature (28 °C) for 2 hours, 70% tert-butanol peroxide (520.9 mg, 4.05 mmol, 0.544 mL) was added, and after stirring at room temperature (28 °C) for 20 minutes, the solution was quenched with 50% sodium thiosulfate pentahydrate aqueous solution (5 mL); the solution was filtered with diatomaceous earth, the filtrate was diluted with ethyl acetate (30 mL), and washed with water (20 mL, twice). The organic phase was dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate (10:1-1:10), and the resulting mixture (1.9 g) was used directly in the next step.

[0766] MS-ESI[M+H] + : 1296.0.

[0767] Preparation of intermediate 92 [2'-O-phosphodiester-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-tert-butyldimethylsilyl-guanosine]-(2',5')-[2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0768]

[0769] 33% methylamine ethanol solution (8 mL) was added to intermediate 91 (1 g, 0.775 mmol), and the mixture was stirred at room temperature for 3 hours. The reaction solution was evaporated to dryness and purified by C18 reversed-phase column elution with water (containing 0.1% ammonium bicarbonate):acetonitrile (3:2). The solution was lyophilized to give a pink solid (550 mg).

[0770] 1 H NMR (400MHz, DMSO-d6) δ10.65 (s, 1H), 8.18 (d, J=2.0Hz, 1H), 7.93-7.77 (m, 2H), 7.38-7.15 (m, 10H), 7.13-6.92 (m, 2H), 6.90-6.77 (m, 4H), 6.49-6.20 (m, 4H), 5.92 (d, J=5.6Hz, 1H), 5.26-5.09 (m, 2H), 4.61-4.24 (m, 3H), 3.98-3.82 (m, 3H), 3.79-3.67 (m, 7H), 0.79 (s, 9H), 0.14-0.02 (m, 6H). MS-ESI[M+H] + : 1064.8.

[0771] Preparation of intermediate 93 [2'-O-phosphodiester-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-tert-butyldimethylsilyl-guanosine]-(2',5')-[2-chloro-3'-O-phosphite monoester-2'-deoxy-2'-fluoro-beta-adenosine]

[0772]

[0773] Diphenyl phosphite (137 mg, 0.47 mmol) was added to intermediate 92 (250 mg, 0.235 mmol), DBU (143 mg, 0.94 mmol), and pyridine (8 mL) in a 4A molecular sieve at 0 °C under nitrogen protection. The resulting mixture was stirred at room temperature for 2 hours. The reaction solution was filtered and evaporated to dryness, and used directly for the next reaction.

[0774] MS-ESI[M+H] + :1129.8.

[0775] Preparation of intermediate 94 [2'-O-phosphodiester-3'-O-tert-butyldimethylsilyl-guanosine]-(2',5')-[2-chloro-3'-O-phosphite monoester-2'-deoxy-2'-fluoro-beta-adenosine]

[0776]

[0777] Intermediate 94 is obtained from intermediate 93 by following the route of intermediate 25.

[0778] 1 H NMR (400MHz, DMSO-d6) δ8.26 (s, 1H), 7.99 (s, 1H), 7.88 (s, 2H), 6.55 (s, 2H), 6. 27(dd, J=17.2, 4.0Hz, 1H), 6.00-5.70(m, 3H), 5.44-5.21(m, 1H), 5.09-4.98(m, 1H), 4.87-4.71 (m, 1H), 4.47 (d, J=4.0Hz, 1H), 4.07-3.97 (m, 1H), 3.91-3.73 (m, 3H), 3.56-3.49 (m, 5H), 0.89 (s, 9H), 0.14 (s, 6H). 31 P NMR (162MHz, DMSO-d6) δ1.72, -1.58. MS-ESI[M+H] + : 826.8.

[0779] Preparation of intermediate 95 (2',3')-cyclo-[2'-O-phosphodiester-3'-O-tert-butyldimethylsilyl-guanosine]-[3'-O-phosphodiester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0780]

[0781] At 0 °C, 48 mg of pentanoyl chloride (0.40 mmol) was added to a pyridine solution (15 mL) containing 55 mg (0.067 mmol) of intermediate 94, and the reaction mixture was stirred at room temperature for 3 hours. Then, a mixture of 0.5 mL of acetonitrile (26 mg, 0.10 mmol) and 0.1 mL of water was added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with a 10% sodium thiosulfate aqueous solution (5 mL), evaporated to dryness, purified by a C18 reversed-phase column, eluted with water (containing 0.1% ammonium bicarbonate):acetonitrile (4:1), and lyophilized to give a white solid (20 mg). MS-ESI [M+H] + :825.0.

[0782] Preparation of intermediate 96 (2',3')-cyclo-[2'-O-Rp-thiophosphate diester-N2-isobutyryl-3'-O-tert-butyldimethylsilyl-guanosine]-[3'-O-phosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0783]

[0784] Intermediate 96 is obtained via the route of intermediate 95, from [2'-O-phosphodiester-N2-isobutyryl-3'-O-tert-butyldimethylsilyl-guanosine]-(2',5')-[N6-benzoyl-2-chloro-3'-O-phosphite monoester-2'-deoxy-2'-fluoro-beta-adenosine].

[0785] 1 H NMR (400MHz, DMSO-d6) δ12.92(s, 1H), 12.12(s, 1H), 11.54(s, 1H), 8.55(d, J=2.0Hz, 1H), 8.23 ​​(s, 1H), 8.06 (d, J=7.2Hz, 2H), 7.71-7.61 (m, 1H), 7.59-7.51 (m, 3H), 6.47 (dd, J=21.6, 2.4Hz, 1H), 5.97-5.74 (m, 2H), 5.60-5.40 (m, 1H), 5.12 -5.00 (m, 1H), 4.41 (d, J=3.2Hz, 1H), 4.27-4.15 (m, 1H), 4.13-3.94 (m, 5H), 2.97- 2.84 (m, 1H), 1.65-1.54 (m, 1H), 1.09 (d, J=7.2Hz, 3H), 0.98 (d, J=6.8Hz, 3H), 0.93 (s, 9H), 0.20 (d, J=8.6Hz, 6H).

[0786] Preparation of intermediate 97 :(2',3')-cyclo-[2'-O-phosphodiester-3'-O-tert-butyldimethylsilyl-guanosine]-[3'-O-Rp-thiophosphatediester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0787]

[0788] Intermediate 97 was synthesized using the same route as intermediate 95, consisting of intermediate 92 reacting with a (+)-PSI reagent. Following deprotection and cyclization, the mixture was purified by preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Waters XBridge C18 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and mobile phase B being acetonitrile, flow rate 25 mL / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0–2 min, 20% B; 2–14 min, 20–40% B; 14–14.2 min, 40–95% B; 14.2–18 min, 95% B. The retention time of this compound was 12.5 min).

[0789] 1 H NMR (400MHz, DMSO-d6) δ10.59 (s, 1H), 8.18 (d, J=2.4Hz, 1H), 8.11 (s, 1H), 7. 45-6.98(m, 5H), 6.33-6.23(m, 1H), 5.82(d, J=8.6Hz, 1H), 5.42(s, 1H), 5.32- 5.23 (m, 2H), 4.33 (d, J=4.0Hz, 1H), 4.19-3.70 (m, 9H), 0.91 (s, 9H), 0.15 (s, 6H). 19 F NMR (376MHz, DMSO) δ-196.51 (s). 31 P NMR (162MHz, DMSO) δ53.76(s), -0.49(s). MS-ESI[M+H] + :841.0.

[0790] Preparation of intermediate 98 :(2',3')-cyclo-(Rp,Rp)-[2'-O-thiophosphate diester-N2-isobutyryl-3'-O-tert-butyldimethylsilyl-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0791]

[0792] Intermediate 98 is synthesized using the same route as intermediate 90, by reacting intermediate 87 with a (+)-PSI reagent, followed by deprotection and cyclization reactions.

[0793] 1H NMR (400MHz, DMSO-d6) δ12.41 (brs, 1H), 11.90 (s, 1H), 11.31 (s, 1H), 8.35 (d, J=2.0Hz, 1H), 8.03 (s, 1H), 7.85 (d, J=7.2Hz, 2H), 7.45 (t, J=7.2Hz, 1H), 7.35 (t, J=7.6Hz, 2H), 6.88 (t, J=48Hz, 6H), 6.25 (dd, J=20.0, 4.0Hz, 1H), 5.65 (d, J =8.4Hz, 1H), 5.59-5.46 (m, 1H), 5.32 (d, J = 50.0Hz, 1H), 5.02 (t, J = 11.2Hz, 1H), 4.15(d, J=4.0Hz, 1H), 4.07-4.01(m, 1H), 3.97-3.64(m, 5H), 2.74-2.62(m, 1H), 0.86 (d, J=6.4Hz, 3H), 0.81-0.66 (m, 12H), -0.01 (d, J=9.6Hz, 6H). MS+ESI--[M+H]: 1030.8.

[0794] Example 1 :(3',3')-cyclo-(Rp,Rp)-bis-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]1,5-ammonium-0.5-1,8-diazabicycloundec-7-ene-salt

[0795]

[0796] Add 1,8-diazabicycloundec-7-ene (342 mg, 2.25 mmol) to an 8 mL DMF solution of intermediate 54 (120 mg, 0.150 mmol) and (-)-PSI reagent (201 mg, 0.450 mmol), and stir the resulting mixture at 25 °C for 1 hour. After evaporating the reaction mixture to dryness, reprecipitate it with ethyl acetate (20 mL). The solid was collected, and the crude product was purified by preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A: 10mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate: 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min, 0-3% B; 3-14min, 3-30% B; 14-14.3min, 30-95% B; 14.3-20min, 95% B. The retention time of the compound was 13.5min). After lyophilization, a white solid (20mg) was obtained.

[0797] 1H NMR (400MHz, DMSO-d6) δ8.37 (s, 1H), 8.22 (s, 1H), 7.90 (brs, 4H), 7.12 (brs, 6H), 6.33-6.20 (m, 2H), 5.56-5.30 (m, 2H), 5.20 -5.00(m, 2H), 4.22-3.85(m, 6H), 3.55-3.40(m, 2H), 3.35-3.15(m, 1H), 2.70-2.55(m, 1H), 1.96-1.85(m, 1H), 1.73-1.52(m, 3H). 31 P NMR (162MHz, DMSO-d6) δ 53.47. MS-ESI[MH] - : 760.9.

[0798] Example 2 (3',3')-cyclo-(Rp,Rp)-bis-[3'-O-thiophosphate diester-2-chloro-2'-deoxyadenosine]

[0799]

[0800] Example 2 was prepared using the route of Example 1, involving the reaction of intermediate 55 with (-)-PSI reagent, followed by purification by preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 0.05% formic acid aqueous solution, and mobile phase B being 0.05% formic acid in acetonitrile solution, flow rate 25 ml / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0-3 min, 0-5% B; 3-14 min, 5-50% B; 14-14.3 min, 50-95% B; 14.3-20 min, 95% B. The retention time of the compound was 12 min).

[0801] 1 H NMR (400MHz, DMSO-d6) δ8.53 (s, 1H), 8.38 (s, 1H), 7.84 (brs, 4H), 6.33-6.18 (m, 2H), 5.20-4.75(m, 2H), 4.30-3.75(m, 6H), 3.00-2.70(m, 2H), 2.70-2.50(m, 2H). 31 P NMR (162MHz, DMSO-d6) δ 53.68, 52.69. MS-ESI[MH] - : 724.9.

[0802] Example 3(3',3')-cyclo-(Rp,Rp)-bis-[3'-O-thiophosphate diester-2'-deoxy-2',2'-difluorocytidine]

[0803]

[0804] Example 3 was prepared using the route of Example 1, involving the reaction of intermediate 56 with (-)-PSI reagent, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 0.05% formic acid aqueous solution, and mobile phase B being 0.05% formic acid in acetonitrile solution, flow rate 25 ml / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0-3 min, 0-5% B; 3-14 min, 5-30% B; 14-14.3 min, 30-95% B; 14.3-20 min, 95% B. The retention time of the compound was 18 min).

[0805] 1 H NMR (400MHz, DMSO-d6) δ9.20-8.80 (m, 2H), 8.31 (d, J=6.4Hz, 1H), 8.28- 8.00 (m, 2H), 7.90 (d, J=6.0Hz, 1H), 6.20-6.05 (m, 3H), 6.00 (d, J=6.4Hz, 1H), 4.95-4.68 (m, 2H), 4.30-4.05 (m, 4H), 3.90-3.70 (m, 2H). 31 P NMR (162MHz, DMSO-d6) δ 53.27, 52.67. MS-ESI[M+H] + :683.0.

[0806] Example 4 (3',3')-cyclo-(Rp,Rp)-bis-[3'-O-thiophosphate diester-N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]diammonium salt

[0807]

[0808] Example 4 was prepared using the route of Example 1, involving the reaction of intermediate 57 with (-)-PSI reagent, followed by purification by preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min, 0-35% B; 3-14min, 35-70% B; 14-14.3min, 70-95% B; 14.3-20min, 95% B. The retention time of the compound was 14min).

[0809] 1H NMR (400MHz, DMSO-d6) δ11.05 (s, 1H), 11.00 (s, 1H), 8.39 (d, J=7.6Hz, 1H), 8.16 (d, J=7.6Hz, 1H), 7.40 (d, J=7.6Hz, 1H), 7.34 (d, J=7.6Hz, 1H), 7.12 (t, J=52.0Hz, 8H), 6.30-6.18(m, 2H), 5.02-4.75(m, 2H), 4.35-4.10(m, 4H), 3.83(t, J = 11.2Hz, 2H), 2.70-2.52 (m, 2H), 1.62-1.45 (m, 4H), 1.40-1.17 (m, 12H), 0.85 (t, J = 7.2Hz, 12H). 31P NMR (162MHz, DMSO) δ 54.54, 53.29. MS-ESI[M+H] + : 935.1.

[0810] Example 5 :(2',3')-cyclo-(Rp,Rp)-[2'-O-thiophosphate diester-adenosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0811]

[0812] 7M ammonia-methanol solution (2 mL) was added to intermediate 71 (200 mg, 0.214 mmol), and the resulting mixture was stirred at 25 °C for 6 hours. The reaction mixture was evaporated to dryness to obtain the crude product. The crude product was purified by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A was 0.05% formic acid aqueous solution, and mobile phase B was 0.05% formic acid in acetonitrile solution, flow rate 25 mL / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0–3 min, 0–5% B; 3–14 min, 5–30% B; 14–14.3 min, 30–95% B; 14.3–20 min, 95% B. The retention time of the compound was 14 min). After lyophilization, a white solid (4.0 mg) was obtained.

[0813] 1 H NMR (500MHz, DMSO-d6) δ8.63 (s, 1H), 8.23 ​​(s, 1H), 8.17 (s, 1H), 7.92 (brs, 2H), 7.29(brs, 2H), 6.33-6.28(m, 1H), 6.12(d, J=8.5Hz, 1H), 5.40-5.15(m, 2H), 5.05-4.95 (m, 1H), 4.55 (t, J=3.5Hz, 1H), 4.36-4.24 (m, 2H), 4.19 (s, 1H), 4.10-3.95 (m, 2H), 3.90-3.78 (m, 1H), 3.72 (d, J=12.0Hz, 1H). 31 P NMR (162MHz, DMSO-d6) δ 53.08, 48.81. MS-ESI[MH] - : 724.8.

[0814] Example 6 (2',3')-cyclo-(Rp,Rp)-[2'-O-thiophosphate diester-adenosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxyadenosine]diammonium salt

[0815]

[0816] Example 6 was prepared using the route of Example 5, by reacting intermediate 72 with 28% ammonia, followed by purification using preparative liquid chromatography (10 mM ammonium bicarbonate as an additive). Analytical LCMS: Agilent 1100+G1946D LCMS, 4.6 x 50 mm Waters XBridge C18 3.5 μm column, mobile phase A was 10 mM ammonium bicarbonate aqueous solution, and mobile phase B was acetonitrile, flow rate 1.8 mL / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0–1.5 min, 5–95% B; 1.5–3 min, 95% B. The retention time of this compound was 0.35 min.

[0817] 1 H NMR (400MHz, DMSO-d6) δ8.61 (s, 1H), 8.40 (s, 1H), 8.17 (s, 1H), 7.86 (brs, 2H), 7.41-7.06(m, 10H), 6.30-6.18(m, 1H), 6.09(d, J=8.4Hz, 1H), 5.42-5.30(m, 2H), 5.30-5.20 (m, 1H), 4.27 (d, J=4.4Hz, 1H), 4.20-4.05 (m, 2H), 4.04-3.90 (m, 2H), 3.72-3.62 (m, 2H), 2.85-2.55 (m, 2H). 31P NMR (162MHz, DMSO-d6) δ 56.62, 53.65. MS-ESI[MH] - : 707.0.

[0818] Example 7 :(3',3')-cyclo-(Rp,Rp)-[3'-O-thiophosphate diester-adenosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0819]

[0820] Example 7 was prepared using the route of Example 5, by reacting intermediate 73 with 28% ammonia, followed by purification using preparative liquid chromatography (10 mM ammonium bicarbonate as an additive). Analytical LCMS: Agilent 1100+G1946D LCMS, 4.6 x 150 mm Waters XBridge C18 3.5 μm column, mobile phase A was 10 mM ammonium bicarbonate aqueous solution, and mobile phase B was acetonitrile, flow rate 1 ml / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0–8 min, 5–95% B; 8–15 min, 95% B. The retention time of this compound was 4.1 min.

[0821] 1 H NMR (400MHz, DMSO-d6) δ8.62 (s, 1H), 8.23 ​​(s, 1H), 8.19 (s, 1H), 7.95 (brs, 2H), 7.65 (brs, 2H), 7.14 (t, J=52.0Hz, 8H), 6.30-6.22 (m, 1H), 6.12 (d, J=8.4Hz, 1H), 5.50-5.32(m, 2H), 5.30-5.15(m, 1H), 5.08-4.92(m, 1H), 4.30-4.00(m, 5H), 3.95- 3.86(m, 1H), 3.68(d, J=12.0Hz, 1H). 31 P NMR (162MHz, DMSO-d6) δ 57.14, 54.13. MS-ESI[(M-2H) / 2] - :362.0.

[0822] Example 8 :(2',3')-cyclo-(Sp,Rp)-[2'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt, and

[0823] Example 9 (2',3')-cyclo-(Sp,Sp)-[2'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0824] Examples 8 and 9 were obtained by following the route of Example 5, from the reaction of intermediate 74 with 28% ammonia, followed by purification by preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min, 0-2% B; 3-14min, 2-37% B; 14-14.3min, 37-95% B; 14.3-20min, 95% B. The retention time of compound 8 was 13min, and the retention time of compound 9 was 15min).

[0825] Example 8:

[0826]

[0827] 1H NMR (400MHz, DMSO-d6) δ10.62 (brs, 1H), 8.19 (s, 1H), 8.11 (s, 1H), 7.94 (brs, 2H ), 7.10 (t, J=50.8Hz, 8H), 6.63 (brs, 2H), 6.25 (dd, J=24.0, 2.8Hz, 1H), 5.85 (d, J=8.8Hz, 1H), 5.50-5.15 (m, 4H), 4.30-4.13 (m, 2H), 4.12-3.92 (m, 4H), 3.72 (d, J= 12.0Hz, 1H). 31 P NMR (162MHz, DMSO-d6) δ 56.84, 54.05. MS-ESI[MH] - :741.0.

[0828] Example 9:

[0829]

[0830] 1 H NMR (400MHz, DMSO-d6) δ10.67 (brs, 1H), 8.23 ​​(s, 1H), 8.08 (s, 1H), 7.93 (brs, 2H ), 7.10 (t, J=51.2Hz, 8H), 6.67 (brs, 2H), 6.30 (dd, J=18.8, 3.6Hz, 1H), 5.77 (d, J=8.0Hz, 1H), 5.58-5.38 (m, 1H), 5.02-4.92 (m, 1H), 4.86-4.78 (m, 1H), 4.69-4.60 (m, 1H), 4.33-3.75 (m, 7H). 31 P NMR (162MHz, DMSO-d6) δ 59.03, 54.74. MS-ESI [MH] - : 740.9.

[0831] Example 10 (2',3')-cyclo-(Rp,Rp)-[2'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0832]

[0833] Example 10 was obtained by reacting intermediate 75 with 28% ammonia via the route of Example 5, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A: 10mM ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile, flow rate: 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min, 0-3% B; 3-14min, 3-38% B; 14-14.3min, 38-95% B; 14.3-20min, 95% B. The retention time of the compound was 13min). Example 10 was also obtained by preparative liquid chromatography following the route of Example 25, after deprotection of intermediate 98. The two different synthetic routes yielded identical products.

[0834] 1 H NMR (400MHz, DMSO-d6) δ10.63 (brs, 1H), 8.22 (s, 1H), 8.18 (s, 1H), 7.93 (brs, 2H ), 7.12 (t, J=51.2Hz, 8H), 6.69 (brs, 2H), 6.28 (dd, J=24.0, 2.4Hz, 1H), 5.88 (d, J=8.4Hz, 1H), 5.42-4.80 (m, 4H), 4.45 (d, J=4.0Hz, 1H), 4.39-4.10 (m, 3H), 4.08-3.94 (m, 1H), 3.93-3.70 (m, 2H). 31 P NMR (162MHz, DMSO-d6) δ 54.07, 50.13. MS-ESI[MH] - :741.0.

[0835] Example 11 (2',3')-cyclo-(Sp,Rp)-[2'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-adenosine]diammonium salt

[0836]

[0837] Example 11 was prepared using the route of Example 5, by reacting intermediate 76 with a 7M ammonia-methanol solution, followed by purification using preparative liquid chromatography (10mM ammonium bicarbonate as an additive). LC-MS analysis was performed using an Agilent 1100+G1946D LCMS, a 4.6 x 150 mm Waters XBridge C18 3.5 μm column, with mobile phase A being 10mM ammonium bicarbonate aqueous solution and mobile phase B being acetonitrile, at a flow rate of 1 mL / min. UV absorption was monitored at 214 and 254 nm, with gradient elution: 0–8 min, 5–95% B; 8–15 min, 95% B. The retention time of this compound was 3.3 min.

[0838] 1 H NMR (400MHz, DMSO-d6) δ10.63 (brs, 1H), 8.39 (s, 1H), 8.21 (s, 1H), 7.83 (brs, 2 H), 7.10 (t, J=50.8Hz, 8H), 6.67 (brs, 2H), 6.30-6.18 (m, 1H), 5.87 (d, J=8.4Hz, 1H), 5.35-5.20 (m, 2H), 4.28 (d, J=4.0Hz, 1H), 4.21-3.92 (m, 4H), 3.80-3.60 (m, 2H), 2.92-2.70 (m, 1H), 2.69-2.55 (m, 1H). 31 P NMR (162MHz, DMSO-d6) δ 56.96, 54.34. MS-ESI[MH] - : 722.9.

[0839] Example 12 (2',3')-cyclo-(Rp,Rp)-[2'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-adenosine]diammonium salt

[0840]

[0841] Example 12 was prepared using the route of Example 5, by reacting intermediate 77 with a 7M ammonia-methanol solution, followed by purification using preparative liquid chromatography (10mM ammonium bicarbonate as an additive). Analytical LCMS: Agilent 1100+G1946D LCMS, 4.6 x 150 mm Waters XBridge C18 3.5 μm column, mobile phase A was 10mM ammonium bicarbonate aqueous solution, and mobile phase B was acetonitrile, flow rate 1 mL / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0–8 min, 5–95% B; 8–15 min, 95% B. The retention time of this compound was 3.3 min.

[0842] 1 H NMR (400MHz, DMSO-d6) δ10.61 (brs, 1H), 8.39 (s, 1H), 8.20 (s, 1H), 7.82 (brs, 2 H), 7.11 (t, J=51.2Hz, 8H), 6.70 (brs, 2H), 6.30-6.18 (m, 1H), 5.87 (d, J=8.4Hz, 1H), 5.25-5.13(m, 2H), 4.96(s, 1H), 4.50(d, J=4.4Hz, 1H), 4.31-3.71(m, 6H), 3.03-2.82(m, 1H), 2.65-2.50(m, 1H). 31 P NMR (162MHz, DMSO-d6) δ54.26, 49.51. MS-ESI[MH] - : 722.8.

[0843] Example 13 (2',3')-cyclo-(Rp,Rp)-[2'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2'-deoxy-2',2'-difluorocytidine]diammonium salt

[0844]

[0845] Example 13 was prepared using the route of Example 5, by reacting intermediate 78 with 28% ammonia, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min, 0-3% B; 3-14min, 3-25% B; 14-14.3min, 25-95% B; 14.3-20min, 95% B. The retention time of the compound was 2min).

[0846] 1 H NMR (400MHz, DMSO-d6) δ8.16 (s, 1H), 7.60-6.70 (m, 11H), 6.58 (brs, 2H), 6.35-6.20(m, 1H), 5.90-5.70(m, 2H), 5.22-5.10(m, 1H), 5.00-4.80(m, 2H), 4.52(s, 1H), 4.32-4.15(m, 2H), 4.11(s, 1H), 4.00-3.82(m, 2H), 3.78-3.65(m, 1H). 31P NMR (162MHz, DMSO-d6) δ 53.24, 48.05. MS-ESI[(M-2H) / 2] - 350.0.

[0847] Example 14 (2',3')-cyclo-(Rp,Rp)-[2'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]diammonium salt

[0848]

[0849] Examples 14 and 5 were obtained by reacting intermediate 79 with a 7M ammonia-methanol solution, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min, 0-10% B; 3-14min, 10-55% B; 14-14.3min, 55-95% B; 14.3-20min, 95% B. The retention time of the compound was 11min).

[0850] 1 H NMR (400MHz, DMSO-d6) δ11.07 (brs, 1H), 10.62 (s, 1H), 8.68-7.97 (m, 2H), 7.40-6 .97(m, 9H), 6.59(brs, 2H), 6.34-6.20(m, 1H), 5.90-5.70(m, 1H), 5.47-5.09(m, 2 H), 5.05-4.74 (m, 1H), 4.29 (d, J = 4.4Hz, 1H), 4.20 (d, J = 9.6Hz, 1H), 4.13-3.99 (m , 2H), 3.95-3.80(m, 1H), 3.85-3.65(m, 1H), 2.70-2.55(m, 1H), 1.60-1.46(m, 2H), 1.45-1.10 (m, 6H), 0.86 (t, J=7.2Hz, 6H). 31 P NMR (162MHz, DMSO-d6) δ 56.16, 53.39. MS-ESI[MH] - :827.0.

[0851] Example 15:(3',3')-cyclo-(Sp,Rp)-[3'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0852]

[0853] Example 15 was prepared using the route of Example 5, by reacting intermediate 80 with a 7M ammonia-methanol solution, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min, 0-3% B; 3-14min, 3-33% B; 14-14.3min, 33-95% B; 14.3-20min, 95% B. The retention time of the compound was 11min). 1 H NMR (400MHz, DMSO-d6) δ8.20 (d, J=2.8Hz, 1H), 8.04 (s, 1H), 8.03-6.87 (m, 10H), 6.60 (brs, 2H), 6.22 (dd, J=24.0, 2.0Hz, 1H), 5.85 (d, J=8.8Hz, 1H), 5.51-5.15 (m, 3H), 4.35-3.89 (m, 6H), 3.72 (d, J=12.4Hz, 1H). 31 P NMR (162MHz, DMSO-d6) δ 56.65, 54.03. MS-ESI[MH] - :741.0.

[0854] Example 16 :(3',3')-cyclo-(Rp,Rp)-[3'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0855]

[0856] Example 16 was prepared using the route of Example 5, by reacting intermediate 81 with a 7M ammonia-methanol solution, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min, 0-3% B; 3-14min, 3-33% B; 14-14.3min, 33-95% B; 14.3-20min, 95% B. The retention time of the compound was 13min). 1 H NMR (400MHz, DMSO-d6) δ10.63 (brs, 1H), 8.22 (s, 1H), 8.09 (s, 1H), 7.94 (brs, 2H), 7.38-6.94 (m, 8H), 6.65 (brs, 2H), 6.28 (dd, J=24.0, 2.4Hz, 1H), 5.87 (d, J=8.8 Hz, 1H), 5.42-5.11 (m, 3H), 5.00 (s, 1H), 4.56-4.42 (m, 1H), 4.39-4.21 (m, 2H), 4.13 (s, 1H), 4.05-3.94 (m, 1H), 3.89-3.71 (m, 2H). 31 P NMR (162MHz, DMSO-d6) δ 53.97, 49.08. MS-ESI[MH] - : 740.9.

[0857] Example 17 :(3',3')-cyclo-(Rp,Rp)-[3'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-adenosine]diammonium salt

[0858]

[0859] Example 17 followed the route of Example 5, reacting intermediate 82 with a 7M ammonia-methanol solution, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min, 0-3% B; 3-14min, 3-33% B; 14-14.3min, 33-95% B; 14.3-20min, 95% B. The retention time of this compound was 11min).

[0860] 1H NMR (400MHz, DMSO-d6) δ10.59 (brs, 1H), 8.41 (s, 1H), 8.11 (s, 1H), 7.85 (brs, 2H ), 7.15 (brs, 8H), 6.66 (brs, 2H), 6.25 (dd, J = 12.8, 6.0Hz, 1H), 5.85 (d, J = 8.8Hz, 1H), 5.35-5.11(m, 2H), 4.91(s, 1H), 4.59-4.50(m, 1H), 4.31-3.71(m, 6H), 3.03-2.87 (m, 1H). 31 P NMR (162MHz, DMSO-d6) δ53.46, 47.84. MS-ESI[MH] - : 740.9.

[0861] Example 18 :(3',3')-cyclo-(Rp,Rp)-[3'-O-thiophosphate diester-2'-deoxy-2'-fluoroadenosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-betaadenosine]-0.5-ammonium-1.5-1,8-diazabicycloundec-7-ene-salt

[0862]

[0863] Example 18 was prepared by following the route of Example 1, reacting intermediate 67 with (-)-PSI reagent, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min, 0-5% B; 3-14min, 5-33% B; 14-14.3min, 33-95% B; 14.3-20min, 95% B. The retention time of the compound was 11min).

[0864] 1H NMR (400MHz, DMSO-d6) δ9.62 (brs, 2H), 8.39 (s, 1H), 8.26 (d, J=2.0Hz, 1H), 8.18 (s , 1H), 7.36 (brs, 2H), 7.15 (t, J=52.0Hz, 2H), 6.31-6.20 (m, 2H), 5.46-5.25 (m, 2H), 5.15-5.00 (m, 1H), 4.95-4.83 (m, 1H), 4.42-4.25 (m, 1H), 4.10-3.98 (m, 2H), 3.80-3 .60(m, 2H), 3.60-3.40(m, 6H), 3.30-3.20(m, 3H), 2.70-2.55(m, 3H), 1.96-1.85(m, 3H), 1.73-1.50(m, 9H). 31 P NMR (162MHz, DMSO-d6) δ 53.37, 52.10. MS-ESI [M+H] + : 729.0.

[0865] Example 19 :(3',3')-cyclo-(Rp,Rp)-[3'-O-thiophosphate diester-2'-deoxy-2'-fluoroadenosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxyadenosine]0.5-1,8-diazabicycloundec-7-ene-salt

[0866]

[0867] Example 19 was prepared by following the route of Example 1, involving the reaction of intermediate 68 with (-)-PSI reagent, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Welch 10μm preparative column, mobile phase A being 0.05% formic acid aqueous solution, and mobile phase B being 0.05% formic acid in acetonitrile solution, flow rate 25 ml / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0-3 min, 0-10% B; 3-14 min, 10-50% B; 14-14.3 min, 50-95% B; 14.3-20 min, 95% B. The retention time of the compound was 10 min).

[0868] 1H NMR (400MHz, DMSO-d6) δ9.56 (s, 1H), 8.60 (s, 1H), 8.50-8.35 (m, 1H), 8.28 (s, 1H), 7.85 (brs, 2H), 6.40-6.20 (m, 2H), 5.70-5.40 (m, 1H), 5.18-4.80 (m, 2H), 4.60- 3.95(m, 6H), 3.65-3.45(m, 2H), 3.30-3.20(m, 1H), 2.85-2.71(m, 1H), 2.70-2.55(m, 2H), 1.96-1.85(m, 1H), 1.73-1.50(m, 3H). 31 P NMR (162MHz, DMSO-d6) δ 54.78, 54.00. MS-ESI[M+H] + :710.8.

[0869] Example 20 :(3',3')-cyclo-(Rp,Rp)-[3'-O-thiophosphate diester-2'-deoxy-2'-fluoroguanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine] diammonium salt

[0870]

[0871] Example 20 was prepared by following the route of Example 1, involving the reaction of intermediate 69 with (-)-PSI reagent, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Waters XBridge C18 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-2min, 5% B; 2-9min, 5-15% B; 9-19min, 15-25% B; 19-19.5min, 25-95% B; 19.5-22.5min, 95% B. The retention time of the compound was 9min).

[0872] 1H NMR (400MHz, DMSO-d6) δ10.69 (brs, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.92 (brs, 2H), 7.15 (t, J=52.0Hz, 6H), 6.64 (brs, 2H), 6.26 (dd, J=17.2, 3.6Hz, 1H), 6.08 (d, J=16.8Hz, 1H), 5.43-5.35 (m, 1H), 5.31-5.22 (m, 1H), 5.18-5.02 (m, 1H), 4.99-4.85 (m, 1H), 4.42-4.30 (m, 1H), 4.26 (d, J=8.8Hz, 1H), 4.15-4.00 (m, 2H), 3.90-3.70 (m, 2H). 31 P NMR (162MHz, DMSO-d6) δ 54.20, 53.14. MS-ESI[MH] - : 743.2.

[0873] Example 21 :(3',3')-cyclo-(Rp,Rp)-[3'-O-thiophosphate diester-2'-deoxy-2'-fluoroguanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxyadenosine]diammonium salt

[0874]

[0875] Example 21 was prepared by following the route of Example 1, reacting intermediate 70 with (-)-PSI reagent, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Waters XBridge C18 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-2min, 5% B; 2-10min, 5-20% B; 10-10.6min, 20-95% B; 10.6-12.6min, 95% B. The retention time of the compound was 7.3min).

[0876] 1H NMR (400MHz, DMSO-d6) δ10.67 (brs, 1H), 8.49 (s, 1H), 7.92 (s, 1H), 7.82 (brs, 2H ), 7.14 (t, J=52.0Hz, 6H), 6.62 (s, 2H), 6.25 (dd, J=6.4, 4.8Hz, 1H), 6.05 (d, J=1 6.8Hz, 1H), 5.44 (dd, J=52.0, 4.0Hz, 1H), 5.05-4.85 (m, 2H), 4.33 (d, J=12.4Hz, 1H), 4.23(d, J=9.2Hz, 1H), 4.10-3.92(m, 2H), 3.98-3.50(m, 2H), 2.78-2.54(m, 2H). 31 P NMR (162MHz, DMSO-d6) δ 52.56, 52.51. MS-ESI[MH] - : 725.2.

[0877] Example 22 (2',3')-cyclo-[2'-O-phosphodiester-guanosine]-[3'-O-phosphodiester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0878]

[0879] Example 22 was prepared using the route of Example 9, by reacting intermediate 95 with ammonium fluoride in methanol solution, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Waters XBridge C18 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-2min, 5% B; 2-15.6min, 5-20% B; 15.6-15.8min, 20-95% B; 15.8-18min, 95% B. The retention time of the compound was 6.6min).

[0880] 1H NMR (400MHz, DMSO-d6) δ10.77 (s, 1H), 8.20 (d, J=2.8Hz, 1H), 7.99 (s, 1H), 7.93 (brs, 2H), 6.71 (brs, 2H), 6.33 (dd, J=24.0, 2.4Hz, 1H), 5.90 (d, J=8.0Hz, 1H), 5.40 (d, J=49.6Hz, 1H), 5.20-5.00 (m, 2H), 4.42-4.30 (m, 2H), 4.20-3.80 (m, 6H). 31 P NMR (162MHz, DMSO-d6) δ1.61, -0.07. MS-ESI[M+H] + : 710.9.

[0881] Example 23 (2',3')-cyclo-[2'-O-Rp-thiophosphate diester-guanosine]-[3'-O-phosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine] diammonium salt

[0882]

[0883] Intermediate 96 (75 mg, 0.074 mmol) was added to a 33% methylamine ethanol solution (3 mL) and stirred at room temperature for 3 hours. The reaction mixture was evaporated to dryness and then ammonium fluoride (82 mg, 2.2 mmol) and methanol (3 mL) were added, and the mixture was stirred at 60 °C for 16 hours. The mixture was purified by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Waters XBridge C18 10 μm preparative column, mobile phase A: 10 mM ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile, flow rate: 25 mL / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0–2 min, 5% B; 2–18.9 min, 5–15% B; 18.9–19.4 min, 15–95% B; 19.4–22.4 min, 95% B. The retention time of the compound was 8.5 min). The purified solid was lyophilized to give 30.3 mg of a white solid.

[0884] 1H NMR (400MHz, DMSO-d6) δ10.60 (s, 1H), 8.21 (d, J=2.8Hz, 1H), 8.01 (s, 1H), 7.91 (s, 2H), 7.35-6.94 (m, 6H), 6.63 (s, 2H), 6.29 (dd, J=24.4, 1.6Hz, 1H), 5.86 (d, J =8.0Hz, 1H), 5.42-5.17 (m, 2H), 5.09-4.90 (m, 2H), 4.48 (d, J = 4.0Hz, 1H), 4.36 (dd, J=10.8, 5.2Hz, 1H), 4.28-4.17 (m, 1H), 4.09 (s, 1H), 3.98-3.73 (m, 3H). 31 P NMR (162MHz, DMSO-d6) δ48.92, -2.42. MS-ESI[M+H] + : 726.8.

[0885] Example 24 (2',3')-cyclo-[2'-O-phosphodiester-guanosine]-[3'-O-Rp-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0886]

[0887] Example 24 was prepared using the route of Example 9, by reacting intermediate 97 with ammonium fluoride in methanol solution, followed by purification using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Waters XBridge C18 10μm preparative column, mobile phase A being 10mM ammonium bicarbonate aqueous solution, and B being acetonitrile, flow rate 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-2min, 20% B; 2-14min, 20-40% B; 14-14.2min, 40-95% B; 14.2-18min, 95% B. The retention time of the compound was 2min).

[0888] 1H NMR (400MHz, DMSO-d6) δ10.58 (s, 1H), 8.19 (d, J=2.8Hz, 1H), 8.04-7.82 (m, 2H), 7.37-7.00 (m, 4H), 6.73-6.53 (m, 1H), 6.27 (dd, J=24.0, 2.4Hz, 1H), 5.83 (d, J=8.3Hz, 1H), 5.44-5.19 (m, 2H), 5.18-5.06 (m, 1H), 5.04-4.95 (m, 1H), 4.36 -4.27(m, 2H), 4.12(s, 1H), 4.09-3.96(m, 2H), 3.79-3.65(m, 2H). 31 P NMR (162 MHz, DMSO-d6) δ53.89 (s), -1.20 (s). 19F NMR (376MHz, DMSO-d6) δ-195.45 (s). MS-ESI[M+H] + :726.

[0889] Example 25 (2',3')-cyclo-(Rp,Sp)-[2'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0890]

[0891] Intermediate 90 (100 mg, crude product) was dissolved in 7 M ammonia-methanol solution (3 mL), stirred at room temperature (28 °C) for 4 hours, and then evaporated to dryness. The residue was dissolved in methanol (1 mL), and ammonium fluoride (60.5 mg, 1.63 mmol) was added. The mixture was heated (60 °C) and stirred for 20 hours. After the reaction was complete as monitored by LCMS and HPLC, the reaction solution was purified by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Waters XBridge C18 10 μm preparative column, mobile phase A was 10 mM ammonium bicarbonate aqueous solution, and B was acetonitrile, flow rate 25 mL / min, dual-wavelength UV absorption monitoring at 214 and 254 nm, gradient elution: 0–2 min, 5% B; 2–18.9 min, 5–15% B; 18.9–19.4 min, 15–95% B; 19.4–22.4 min, 95% B. The retention time of the compound was 10 min). After lyophilization, a white solid (21 mg) was obtained.

[0892] 1H NMR (400MHz, DMSO-d6) δ10.71 (s, 1H), 8.44 (s, 1H), 8.23 ​​(s, 1H), 7.92 (s, 2H), 7.11 (t, J=52Hz, 6H), 6.56 (s, 2H), 6.29 (d, J=28Hz, 1H), 5.95 (d, J=8.4Hz, 1H), 5.35 (d, J=50.4Hz, 1H), 5.20 (t, J=11.8Hz, 1H), 5.11-5.05 (m, 1H), 4.48 (d, J=3.6 Hz, 1H), 4.39-4.28 (m, 2H), 4.14 (s, 1H), 4.02-3.96 (m, 1H), 3.88-3.83 (m, 1H), 3.81- 3.76 (m, 1H). 31 P NMR (162MHz, DMSO-d6) δ 53.30, 50.07. 19 F NMR (376MHz, DMSO-d6) delta-196.87. MS-ESI[M+H] + : 742.8.

[0893] Example 26 :(2',3')-cyclo-(Rp,Rp)-[2'-O-thiophosphate diester-guanosine]-[3'-O-thiophosphate diester-2-methylamino-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0894]

[0895] Example 26 describes the deprotection reaction of intermediate 98 with an ethanolic solution of methylamine, following the route of Example 25. The byproduct was purified using preparative liquid chromatography (Gilson 281 preparative HPLC, 19x250mm Waters XBridge C18 10μm preparative column, mobile phase A: 10mM ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile, flow rate: 25ml / min, dual-wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-2min, 5% B; 2-15.6min, 5-15% B; 15.6-15.8min, 15-95% B; 15.8-18min, 95% B. The retention time of this compound was 14.1min). The purified product was lyophilized to obtain a white solid.

[0896] 1H NMR (400MHz, DMSO-d6) δ10.69 (brs, 1H), 8.27 (s, 1H), 8.02 (s, 1H), 7.34-6.97 ( m, 3H), 6.31 (brs, 1H), 6.28 (d, J = 2.4Hz, 1H), 5.90 (d, J = 0.8Hz, 1H), 5.44-5.15 (m, 3H), 4.44 (d, J=0.4Hz, 1H), 4.35-4.27 (m, 1H), 4.24-4.12 (m, 2H), 4.03-3.94 (m, 1H), 3.93-3.79 (m, 2H), 2.85 (s, 3H). 31 P NMR (162MHz, DMSO-d6) δ 54.02, 51.06. MS+ESI-.[M+H]: 738.0.

[0897] Active Examples

[0898] Example 1: Activation effect of the compound of the present invention on IFN-β secretion by THP-1 cells

[0899] THP-1 cells are a human monocytic leukemia cell line with the HAQ STING phenotype, namely R71H-G230A-R293Q. This experiment used the Human IFN-beta DuoSet ELISA kit (R&D, catalog number #DY814-05) and the DuoSet ELISA Ancillary Reagent Kit 2 (R&D, catalog number #DY008) to evaluate the activation effect of representative compounds of this invention on IFN-β secretion in THP-1 cells.

[0900] When thawing THP-1 cells (ATCC#TIB-202), the cell cryovials were rapidly shaken in a 37°C water bath to thaw within 1 minute. The thawed cell suspension was mixed with RPMI 1640 medium (Hyclone, #SH30027.01) containing 10% FBS (Lifetechnology, catalog number #10099-141), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 5 mL of complete medium (RPMI 1640 medium containing 10% FBS) and placed in a container with a bottom area of ​​25 cm². 2 The cells were placed in cell culture flasks and cultured in a cell culture incubator at 37°C, 5% CO2, and 95% humidity. Cell passage was performed when the cell confluence reached approximately 80%. For cell passage, all cells in the culture flask were transferred to a 15mL centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 5mL of fresh complete culture medium, and 1mL was placed in a container with a bottom area of ​​25cm².2 Add 4 mL of fresh complete culture medium to the cell culture flask and continue culturing. When the cell confluence reaches approximately 80% again, plate the cells. When plateding, follow the cell passage method, retaining 1 / 5 of the cell suspension for further culture, and transferring the remaining 4 / 5 of the cell suspension to a 15 mL centrifuge tube. Centrifuge, discard the old culture medium, wash the cells once with RPMI 1640 medium (without serum), centrifuge to remove the supernatant. Resuspend the cells in RPMI 1640 medium (without serum). Assess cell viability using the trypan blue rejection method, and plate the cells when the viability is above 95%. Prepare RPMI 1640 medium (without serum) with a density of 1.1 × 10⁶ cells / mL. 6 A cell suspension of 180 μL was added to each well of a 96-well cell culture plate (NUNC, catalog number #167008) to achieve a cell density of 2 × 10⁶ cells / mL. 5 One live cell per well.

[0901] First, the DMSO stock solution containing the 10 mM compound was serially diluted 3.16 times with DMSO (Sigma, catalog number #D2650) to the 5th concentration, and a 6th concentration of DMSO was set up as a control. Then, the DMSO solutions containing different concentrations of the compound were diluted 10 times with PBS to ensure that the DMSO content in each compound solution was 10%. Finally, 20 μL of the above solution was added to the corresponding cell culture plates to make the initial compound concentration 100 μM, with adjacent concentrations diluted 3.16 times, and the DMSO content in the cell culture plate 1%. The cell culture plates were then placed in a cell culture incubator and cultured for 24 hours.

[0902] The ELISA testing process should be performed in accordance with the R&D System, Cat#DY814-05 instruction manual.

[0903] ELISA Plate Coating: Dilute the capture antibody (mouse anti-human IFN-β capture antibody PART#844508) with PBS (R&D System Cat#DY006) to prepare a working concentration. Add 100 μL of the capture antibody working solution to each 96-well ELISA plate, seal the plate, and incubate overnight at room temperature. Discard the capture antibody working solution. Wash the cell plate three times with washing buffer (0.05% Tween-20 in PBS solution, pH 7.2-7.4, R&D System Cat#WA126), using 400 μL of washing buffer per well. Thoroughly remove the washing buffer after each wash. For the final wash, invert the plate on clean paper and tap it to completely remove the washing buffer. Add 300 μL of blocking buffer (1% BSA in PBS solution, pH 7.2-7.4, R&D System Cat#DY995) to each well and incubate at room temperature for 1–2 hours. Repeat the above washing steps to prepare each plate for sample addition.

[0904] Sample detection: Add 100 μL of sample or standard (recombinant human IFN-β standard, PART#844510) to each well, seal the plate, and incubate at room temperature for 2 hours. Repeat the washing steps of the plate coating method above. Then, add 100 μL of detection antibody (biotinylated mouse anti-human IFN-β detection antibody, PART#844509) to each well, seal the plate, and incubate at room temperature for 2 hours. Repeat the washing steps of the plate coating method above. Subsequently, add 100 μL of Streptavidin-HRP (PART#893975) working solution to each well, seal the plate, and incubate at room temperature for 20 min, protecting from light during this process. Repeat the washing steps of the plate coating method above. Next, add 100 μL of substrate solution (a 1:1 mixture of Colar Reagent A (H2O2) and Color Reagent B (tetramethylbenzidine), R&D System Cat#DY999) to each well, seal the plate, and incubate at room temperature for 20 min, protected from light. Finally, add 50 μL of stop solution (2N H2SO4, R&D System Cat#DY994) to each well and gently tap the cell plate to ensure thorough mixing.

[0905] Measure the OD450 of each well using a multi-sensor microplate reader (Molecular Devices, Spectramax M3). If wavelength calibration is available, set it to 540 nm or 570 nm. If wavelength calibration is unavailable, subtract OD540 or OD570 from OD450. This process should be completed within 30 minutes after adding the stop solution.

[0906] 2′3′-cGAMP (Invivogen, catalog number #tlrl-nacga23) was used as a positive control compound, and ADU-S100 (MCE, catalog number #HY12885A) was used as a control compound.

[0907]

[0908] Data were analyzed using GraphPad Prism 7.0 software, and a standard curve for ELISA IFN-β content was obtained by double logarithmic plotting. The OD values ​​obtained from each sample well were substituted into the standard curve equation to calculate the corresponding IFN-β concentration. A dose-response curve of IFN-β concentration versus compound concentration was obtained by fitting the data using a nonlinear S-curve regression, and the EC50 value was calculated.

[0909] Table 1 - Activation effect of compounds from representative examples on IFN-β secretion in THP-1 cells (EC50, μM)

[0910] Example THP-1 activity Example THP-1 activity 5 B 16 A 8 B 17 A 9 B 18 B 10 A 19 B 11 B 21 B 12 A 2′,3′-cGAMP B 13 B ADU-S100 A 15 B 26 A

[0911] Where: A: EC50 1~10μM; B: EC50 10.1~100μM

[0912] Experimental results showed that the compounds in the examples activated the secretion of IFN-β in THP-1 cells. (See Table 1 and...) Figure 1 It is evident that the compounds in Examples 10, 12, 16, 17 and 26 exhibit particularly significant STING agonist activity: their EC50 values ​​are stronger than 2′,3′-cGAMP and comparable to ADU-S100; among them, the compounds in Examples 10, 16 and 17 stimulated THP-1 cells to secrete IFN-β at higher concentrations than ADU-S100.

[0913] Example 2: Inhibitory activity of the compound of the present invention on the proliferation of CT26 cells

[0914] CT26 cells are a mouse colorectal cancer cell line. In this study, the CellTiter-Glo Luminescence Cell Viability Assay kit from Promega was used to evaluate the inhibitory activity of compounds on CT26 cell proliferation.

[0915] When thawing CT26 cells (ATCC#CRL-2638), the cell cryovials were rapidly shaken in a 37°C water bath to thaw within 1 minute. The thawed cell suspension was mixed with DMEM medium (GE Healthcare, catalog #SH30243.01) containing 10% FBS (GIBCO, catalog #10099-141), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 5 mL of complete medium (DMEM medium containing 10% FBS) and placed in a container with a bottom area of ​​25 cm². 2 The cells were placed in cell culture flasks and cultured in a cell culture incubator at 37°C, 5% CO2, and 95% humidity. Cell passage was performed when the cell confluence reached 70%–80%. For cell passage, all cells in the culture flask were transferred to 15 mL centrifuge tubes and centrifuged at 1000 rpm for 5 min, discarding the supernatant. The cell pellet was resuspended in 5 mL of fresh complete culture medium, and 1 mL was placed in a container with a bottom area of ​​25 cm². 2 Add 4 mL of fresh complete culture medium to the cell culture flask and continue culturing. When the cell confluence reaches 70%–80% again, plate the cells. When plateding, follow the cell passage method, retaining 1 / 5 of the cell suspension for further culture, and transferring the remaining 4 / 5 of the cell suspension to a 15 mL centrifuge tube. Centrifuge, discard the old culture medium, wash the cells once with DMEM medium (without serum), and centrifuge to remove the supernatant. Resuspend the cells in DMEM medium (without serum). Assess cell viability using the trypan blue rejection method, and plate the cells when the viability is above 95%. Prepare DMEM medium (without serum) with a density of 1.1 × 10⁶ cells / mL. 4 Add 90 μL of cell suspension (1000 live cells / mL) to each well of a 96-well clear, flat-bottomed, black-walled cell culture plate (Coming, catalog number #3603) to achieve a cell density of 1000 live cells / well. Set up a control group (culture medium control) containing only complete culture medium and no cells, and a control group (cell control) containing only cells and no compounds. Incubate the cell culture plates overnight in a cell culture incubator.

[0916] First, the 10 mM DMSO stock solution was serially diluted 3.16-fold with DMSO (Sigma, catalog number #D2650) to the 9th concentration, with a 10th concentration of DMSO as a control. Then, the DMSO solutions containing different concentrations of the compound were diluted 10-fold with PBS (Solarbio, catalog number #P1020) to ensure each concentration contained 10% DMSO. Finally, 10 μL of the above solutions were added to the corresponding cell culture plates, resulting in an initial compound concentration of 100 μM, with adjacent concentrations diluted 3.16-fold, and the cell culture plate containing 1% DMSO. The cell culture plates were then incubated for 120 hours.

[0917] After 120 hours, melt the CellTiter-Glo reagent (Promega, catalog number #G7572) and move the cell plate to room temperature for equilibration for 30 minutes. Add 100 μL of CellTiter-Glo to each well of the cell plate and shake on a track-mounted shaker for 5 minutes to fully lyse the cells. Place the cell plate at room temperature for 20 minutes to stabilize the cold light signal and scan the cold light value of each well at all wavelengths using a multi-functional microplate reader (Molecular Devices, Spectramax M3).

[0918] The following compounds and ADU-S100 (MCE, catalog number #HY12885A) were used as controls: Clofarabine (Wuhu Huaren Technology, catalog number #HR-00701002), Cladribine (CSNpharm, catalog number #CSN10004), Gemcitabine Hydrochloride (Shaoyuan, catalog number #SY014538), and gemcitabine prodrug LY2334737 (self-made, intermediate 7).

[0919] Calculate cell viability under different concentrations of the compound using the following formula:

[0920] Cell viability (%) = (Lum) 待测药 -Lum 培养液对照 ) / (Lum 细胞对照 -Lum 培养液对照 )×100%.

[0921] The data were analyzed using GraphPad Prism 7.0 software. Nonlinear S-curve regression was used to fit the data to obtain the dose-response curve, and the IC50 value was calculated from it.

[0922] Table 2. Inhibitory effect of the examples on CT26 cell proliferation (EC50, μM)

[0923]

[0924]

[0925] Where: A: IC50 < 1 μM; B: IC50 1–10 μM; NA: IC50 > 10 μM

[0926] Experimental results showed that the compounds in the representative examples were able to inhibit the in vitro growth of CT26 tumor cells; among them, the inhibitory abilities of compounds in Examples 3, 10, and 13 were particularly significant, while ADU-S100 showed no obvious tumor cell inhibitory ability. Combined with the results of the active example 1, these results indicate that the compounds in the examples possess multifunctional antitumor properties, namely the tumor immunomodulatory activity of a STING agonist and the cytotoxic effects of an antimetabolite anticancer drug.

[0927] Example 3: Antitumor activity of the compounds of the present invention in a bilateral xenograft tumor model of CT26 syngeneic mice.

[0928] Six- to eight-week-old BALB / c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were subcutaneously inoculated with 5 × 10⁵ cells per 10⁻⁶ ... 5 CT26 cells (provided by Taicang Zexin Biotechnology Co., Ltd., ATCC#CRL-2638) were seeded at a volume of 0.1 mL per side. When the tumor grew to an average volume of 100 mm²... 3 Mice were randomly assigned to groups based on tumor size and body weight and began drug administration. The day of the first administration was designated day 0. Two administrations were administered: on days 0 and 4, 50 μg / mouse (2.5 mg / kg) of the compound was injected intratumorally into the right tumor, with an equal volume of PBS (Hyclone, catalog number #SH30258.01) as a control. The left tumor received no treatment. Control group mice were sacrificed 13 days after administration, and treatment group mice were sacrificed 21 days later. During the experiment, tumor volume and body weight were measured three times per week on both the left and right sides. Tumor volume was calculated using the formula V = D × d × d / 2 (where D represents the long diameter of the tumor, and d represents the short diameter).

[0929] Data from day 13 post-administration showed that all the compounds tested in the embodiments of the present invention significantly inhibited bilateral tumor growth. The right-sided administration group showed more significant effects, with most tumors regressing. Figure 2-A In the ADU-S100 treatment group, the tumor growth inhibition rate was 99.1%, in Example 17 it was 99.5%, and in the remaining groups the tumors were 100% inhibited, with no palpable tumors. Simultaneously, the growth of the untreated left-sided tumor slowed (…). Figure 2-B On day 13 after administration, the growth inhibition rates of the left-sided tumor groups were as follows: ADU-S100, 64.2%; Example 10, 91.2%; Example 12, 75.3%; Example 16, 71.7%; Example 17, 67.4%. Additionally, in this experiment, a decrease in body weight was observed in some mice after administration, within 15%, and the weight recovered after discontinuation of administration (Figure 2-C). These results indicate that the compounds of the present invention exhibited tumor inhibition capabilities comparable to or superior to ADU-S100 in the CT26 syngeneic bilateral tumor model.

[0930] Example 4: Antitumor activity of the compounds of the present invention in the CT26 nude mouse xenograft model

[0931] 6-8 week old BALB / c nude mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were subcutaneously inoculated with 5×10⁵ cells / mL of the right posterior dorsal surface. 5 One CT26 cell (provided by Taicang Zexin Biotechnology Co., Ltd., ATCC#CRL-2638) was seeded at a volume of 0.1 mL. When the tumor grew to an average volume of 100 mm²... 3 Mice were randomly assigned to groups based on tumor size and body weight for drug administration. The day of the first administration was designated day 0. Two administrations were administered: on days 0 and 4, 50 μg / mouse of the compound (2.5 mg / kg) was injected intratumorally into the right side of the tumor, with an equal volume of PBS (Hyclone, catalog number #SH30258.01) as a control. Nine days after administration, mice in the control group were sacrificed. Mice in the ADU-S100 and Example 10 compound treatment groups were sacrificed on days 14 and 16, respectively. Tumor volume and body weight were measured three times weekly during the experiment. Tumor volume was calculated using the formula V = D × d × d / 2 (as above).

[0932] Data from day 9 post-administration showed that the compound of Example 10 of this invention significantly inhibited tumor growth in T-cell immunodeficient nude mice, with an inhibition rate of 94.4%. In contrast, ADU-S100 only showed partial tumor inhibition ability, with an inhibition rate of 61.4%, possibly due to its residual immune activity or other unknown reasons. Both Example 10 and ADU-S100 showed better tumor growth inhibition in immunocompetent mice than in nude mice. Figure 2-D This experiment verified that ADU-S100's main mechanism of action is to activate T cells to mediate immunity; and the compound in Example 10, due to its multifunctional mechanism of tumor suppression activity, demonstrated in this case to have a superior tumor suppression ability compared to the STING agonist ADU-S100.

[0933] Example 5: Immune memory function of the compounds of the present invention in CT26 syngeneic mouse or nude mouse xenograft models

[0934] 6-8 week old BALB / c-immunized healthy mice or nude mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were subcutaneously injected with 5×10⁻⁶ dredges on the right posterior back. 5 One CT26 cell (provided by Taicang Zexin Biotechnology Co., Ltd., ATCC#CRL-2638) was seeded at a volume of 0.1 mL. When the tumor grew to an average volume of 100 mm²... 3Mice were randomly grouped according to tumor size and body weight and administered the drug. The day of the first administration was designated day 0. Two administrations were given, on days 0 and 4, with an intratumoral injection of 50 μg of the compound per mouse (i.e., 2.5 mg / kg). An equivalent volume of PBS (Hyclone, catalog number #SH30258.01) was used as a control. Nude mice or control mice from the BALB / c experiment were sacrificed 9 or 13 days after administration. 21 days later, 5 × 10⁵ mg / kg of the compound was subcutaneously injected again into the left posterior dorsal side of the mice. 5 CT26 cells were inoculated at a volume of 0.1 mL. Simultaneously, untreated BALB / c mice or nude mice were inoculated as controls. During the experiment, tumor volume and body weight were measured three times per week on both left and right sides. Tumor volume was calculated using the formula V = D × d × d / 2 (same as above).

[0935] The results showed that the compound of Example 10 of this invention significantly inhibited CT26 tumor growth in both immunocompetent and immunodeficient mice. The efficacy was more pronounced in immunocompetent mice (Figure 3-A). On day 5 post-administration, all tumors in the Example 10 treatment group disappeared. On day 21, CT26 cells were re-inoculated in the Example 10 compound treatment groups. After 7 days (day 30), the average tumor volume in unimmunized blank BALB / c mice was 75 mm. 3 The average tumor volume of mice treated in Example 10 was 29 mm. 3 By the end of the experiment on day 33, the average tumor volume of mice in the control group and the immunization group of Example 10 was 648 mm. 3 and 101mm 3 This experiment demonstrates that treatment with the compound of Example 10 induces immune memory in immunocompetent mice, resulting in a strong immune rejection of re-inoculated cells of the same type, effectively preventing tumor recurrence.

[0936] On the other hand, in immunodeficient nude mice, some mice showed continued tumor growth after drug administration was stopped. Figure 3-B Three mice with smaller tumors were selected and re-inoculated with CT26 cells on day 21. The results showed that the re-inoculated CT26 tumor cells grew at a similar rate to the tumor cells from the control mice. This indicates that no immune memory was generated in this nude mouse experiment, and further verifies the tumor-suppressive ability of the compound in Example 10 with cytotoxic effects even without the involvement of the immune system in this model.

[0937] Example 6: Hepatocyte metabolic stability test of the compounds of the present invention

[0938] The hepatic metabolic stability assays of the compounds of the present invention in five species (mice, rats, dogs, monkeys, and humans) were performed similarly according to standard methods conventional in the field for in vitro metabolic stability studies, such as those described in (Kerns, Edward H. and Di Li (2008). Drug-like Properties: Concepts, Structure Design and Methods: From ADME to Toxicity optimization. San Diego: Academic Press; Di, Li et al., Optimization of a Higher Throughput Microsomal Stability Screening Assay for Profiling Drug Discovery Candidates, J Biomol. Screen. 2003, 8(4), 453.).

[0939] The hepatocytes used in the experiment were: human hepatocytes (SHQY, lot#HEP190006); dog hepatocytes (IVT, lot#ZMB); monkey hepatocytes (Xenotech, lot#2010022); rat hepatocytes (SHQY, lot#HEP134045); and mouse hepatocytes (BioIVT, Cat.#M005052, Lot.#MEO).

[0940] The cryopreserved hepatocytes were removed from the liquid nitrogen tank and immediately placed in a shaking water bath at 37±1℃ for 2 minutes ± 15 seconds. Hepatocytes were transferred to 50 mL of hepatocyte thawing medium (composition: Williams E medium, 35 mL, Invitrogen, catalog #A1217601; isotonic Percoll solution, 13.5 mL, General Electric, catalog #17-0891-01; DuPont phosphate buffer, 1.5 mL, Invitrogen, catalog #14200-075; Glutamax, 500 μL, Invitrogen, catalog #35050061; HEPES, 750 μL, Invitrogen, catalog #15630106; fetal bovine serum, 2.5 mL, Invitrogen, catalog #10091130; recombinant human insulin, 50 μL, Invitrogen, catalog #12585014; dexamethasone (prepared to 10 mM)). Add 5 μL of DMSO solution (Sigma, Catalog No. #D1756), mix gently, and centrifuge at 500 rpm for 3 min. After centrifugation, carefully aspirate the supernatant (without disturbing the cell pellet), add 10 × 10 volumes of preheated KHB buffer (Krebs-Henseleit buffer, Sigma, Cat#K3753-10X1L) and 5.6 g / L HEPES, resuspend the cell pellet, mix gently, and centrifuge at 500 rpm for 3 min. Discard the supernatant, without touching the cell pellet, and determine cell viability and number. Count the hepatocytes, and then dilute the cell suspension with KHB buffer to an appropriate density (viable cell density = 2 × 10⁻⁶). 6 (cells / mL). Keep the hepatocyte solution on ice until used.

[0941] 2× dosing solutions were prepared in preheated KHB (1% dimethyl sulfoxide) and centrifuged at 5594 g for 15 min (Thermo Multifuge×3R). The 200 μM spiking solution was prepared by adding 20 μL of the compound stock solution (10 mM, DMSO solution) to 980 μL of dimethyl sulfoxide. The 2× dosing solution was prepared by adding 10 μL of 200 μM spiking solution (diluted to 2 μM) to 990 μL of KHB.

[0942] Add 50 μL of preheated 2× drug dosing solution to the wells at different specified time points. Add 50 μL of preheated hepatocyte solution (2×10⁻⁶) to each well. 6 Add cells / mL to the designated wells for 15 min, 30 min, 60 min and 120 min detection, then start timing and place the reaction plate in a 37°C incubator.

[0943] Add 100 μL of acetonitrile (Merck, Cat.#CN34854-4L) containing IS (isosaminoglycan or imipramine) to a well designed for 0 min, mix gently, and then add 50 μL of preheated hepatocyte solution (2×10⁻⁶). 6 Cells / mL), and sealed the wells. At 15 min, 30 min, 60 min, and 120 min, 100 μL of acetonitrile containing IS was added to each well, followed by sealing. After quenching, the plate was shaken on a vibrator (IKA, MTS 2 / 4) for 10 min (600 rpm). The plate was sonicated for 2 min and then centrifuged at 5594 g for 15 min (Thermo Multifuge × 3R). 50 μL of supernatant from each well was transferred to a 96-well plate containing 50 μL of ultrapure water (Milipore, ZMQS50F01) for LC / MS analysis.

[0944] The concentration of the analyte at time T0 (C0) was taken as 100%, and the concentrations at other incubation time points were converted to percentage residues. A linear regression was performed on the natural logarithm of the percentage residues at each time point against the incubation time to obtain the slope K. Then, the hepatocyte clearance rate (CI) was calculated using the following formula. int ) and in vitro half-life (T1 / 2):

[0945] T1 / 2 = 1 / n² / K = 0.693 / K

[0946] Cl int = (0.693 / T1 / 2) × (1 / hepatocyte density) × proportionality factor

[0947] The hepatocyte density refers to the final concentration of hepatocytes in the incubation system used in this experiment: 1 × 10⁻⁶. 6 Hepatocytes / mL. Proportion factor = number of hepatocytes × liver weight (for hepatocytes of 5 species, the values ​​are: mouse 118 × 12.5 × 10⁻⁶). 6 rats 4680 × 10⁶ per kg 6 Individuals / kg, 6880×10 dogs 6 Individuals / kg, Monkeys 3900×10 6 Individuals / kg, 2544.3 × 10 6 (pieces / kg).

[0948] Depend on Figure 4-A It is evident that the compounds of the present invention, such as the compound of Example 10, exhibited good metabolic stability in hepatocytes of five species; they also exhibited a long metabolic half-life and a low clearance rate.

[0949] Example 7: Discovery and Identification of Metabolites of the Compounds of the Invention

[0950] The major metabolites of the compounds of the present invention in hepatocytes of five species (mouse, rat, dog, monkey, and human) were similarly identified according to standard methods conventional in the field for in vitro metabolic stability studies, such as those described in (Kerns, Edward H. and Di Li (2008). Drug-like Properties: Concepts, Structure Design and Methods: From ADME to Toxicity optimization. San Diego: Academic Press; Di, Li et al., Optimization of a Higher Throughput Microsomal Stability Screening Assay for Profiling Drug Discovery Candidates, J Biomol. Screen. 2003, 8(4), 453.).

[0951] The hepatocytes used in the experiment were: human hepatocytes (SHQY, Cat.#BQHPCH10, Lot.#HEP190006-TA05); dog hepatocytes (BioIVT, Cat.#M00205, Lot.#ZMB); monkey hepatocytes (XENOTECH, Cat.#PPCH2000, Lot.#2010022); rat hepatocytes (SHQY, Cat.#BQR1000·H15, Lot.#HEP134049); and mouse hepatocytes (BioIVT, Cat.#M005052, Lot.#MEO).

[0952] Preheat HI hepatocyte maintenance medium (BIOIVT, Cat.#Z99009; Lot.#C02060C) to 37°C. Remove the cryopreserved hepatocytes from the liquid nitrogen container and immediately place them in a shaking water bath at 37±1°C for 2 min±15 seconds. Transfer the hepatocytes to 50 ml of HI medium, mix gently, and centrifuge at 500 rpm for 3 min. After centrifugation, carefully aspirate the supernatant (without disturbing the cell pellet). Add 10× volumes of preheated HI medium, resuspend the cell pellet, mix gently, and centrifuge at 500 rpm for 3 min. Discard the supernatant, avoiding touching the cell pellet. Count the hepatocytes, and then dilute the cell suspension with HI medium to an appropriate density (viable cell density = 2×10⁻⁶). 6 (cells / mL), keep the hepatocyte solution on ice until used.

[0953] Prepare a 2× dosing solution in preheated HI medium. Prepare a 2× dosing solution (20 μM) as follows: Add 8 μL of 10 mM compound stock solution (20 μM, i.e., diluted 0.2% dimethyl sulfoxide) to 3992 μL of HI medium.

[0954] Preheat the hepatocyte solution and 2× drug dosing solution, and administer to the designated T. 240 and T 240-w / o Add 200 μL of preheated 2× dosing solution to each well. For T0, add 1200 μL of acetonitrile (Merck, Cat.#CN34854-4L) and 200 μL of hepatocyte solution (2×10⁴ oz) to each well. 6 Add 200 μL of preheated 2× drug solution (cells / mL) and then seal the wells. Add 200 μL of preheated hepatocyte solution (2×10⁶ cells / mL) to the wells. 6 Add (cells / mL) to the specified T 240 In the hole; to the hole designated for T 240-w / o Add 200 μL of preheated HI medium to each well and start timing; place the reaction plate in a CO2 incubator at 37°C.

[0955] At 240 min, 1200 μL of acetonitrile was added to each designated well, and the plate was then sealed. After quenching, the plate was sonicated for 2 min, followed by centrifugation at 1400 rpm for 5 min. 1200 μL of the supernatant was evaporated under a nitrogen stream until dry. The dried extract was then redissolved in 200 μL of acetonitrile:water (1:3 v / v), vortexed for 2 min, and centrifuged at 14000 rpm for 5 min. 2 / 5 μL of the supernatant was injected into LC-UV-MS for analysis.

[0956] Depend on Figure 4-B It was found that the compounds of the present invention, such as the compound of Example 10, could be detected in hepatocytes of five species as the cytotoxic small molecule clofarabine, which is the major metabolite of the compound of Example 10. Combined with the results of Activity Example 6, it is highly likely that CDN molecules with STING agonist activity and cytotoxic small molecules coexist within a certain period after in vivo, thereby achieving a molecular-level synergistic effect and providing an enhanced or even synergistic antitumor effect. This result also mechanistically explains its good efficacy in mouse tumor suppression experiments.

[0957] Example 8: Comparison of the antitumor activity of the compounds of the present invention in combination with CDN STING agonists and cytotoxic drugs in a CT26 syngeneic mouse bilateral xenograft model.

[0958] Six- to eight-week-old BALB / c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were subcutaneously inoculated with 5 × 10⁵ cells per 10⁻⁶ ... 5 CT26 cells (provided by Taicang Zexin Biotechnology Co., Ltd., ATCC#CRL-2638) were seeded at a volume of 0.1 mL per side. When the tumor grew to an average volume of 100 mm²... 3 Mice were randomly assigned to groups based on tumor size and body weight (the compound group of this invention; the CDN STING agonist monotherapy group; the cytotoxic drug monotherapy group; and the CDN STING agonist and cytotoxic drug combination group) and started drug administration. The day of the first administration was designated day 0. A total of three administrations were given, on days 0, 4, and 7, via intratumoral injection on the right side, with an equal volume of PBS (Hyclone, catalog number #SH30258.01) as a control. The left tumor was left untreated. Mice in the control group were sacrificed 13 days after administration, and mice in the treatment group were sacrificed 21 days later. During the experiment, tumor volume and body weight were measured three times a week on both the left and right sides. Tumor volume was calculated using the formula V = D × d × d / 2 (as above).

[0959] Experimental results show that the tumor growth inhibition rate of the compound group of the present invention is significantly higher than that of the CDN STING monotherapy group and the cytotoxic drug monotherapy group. Its activity is comparable to or even higher than the sum of the activities of the latter two, resulting in enhanced or even synergistic tumor inhibition activity.

[0960] The general or preferred definitions of the specified features in the various enumerated embodiments of the present invention can be combined with other general or preferred definitions of the specified features to obtain other embodiments of the present invention. Just as these combinations are specifically and individually listed herein, unless the context clearly indicates otherwise.

[0961] Several prior publications are referenced in this specification. These publications are not considered to be related to the patentability of the present invention, but their entire contents are incorporated herein by reference. References to any prior publications (or information derived therefrom) in this specification are not, and should not be construed as, an endorsement or acknowledgment of any form of teaching that the corresponding prior publication (or information derived therefrom) constitute common general knowledge in the technical field to which this specification pertains.

Claims

1. A cyclic dinucleotide compound of formula (II), wherein B1 is adenine substituted with X wherein X is selected from Cl or F; R1and R1' are each independently selected from H or F; B2 is selected from adenine optionally substituted with X wherein X is selected from H, F or CI; or guanine represents a phosphodiester linkage to the 2' or 3' position of the pentose, whichever is not involved in the ring with the phosphate; and R2and R2' are independently selected from the group consisting of H, OH, SH, OR5, SR5, NHR5, NR5R6, and halogen; R2and R2' are each independently selected from H, -OH or F; a stereoisomer or a pharmaceutically acceptable salt thereof.

2. The compound of formula (II) according to claim 1, a stereoisomer or a pharmaceutically acceptable salt thereof, wherein represents a phosphoric acid linkage to the 3' position of the pentose, formula (II) has 3. The compound according to claim 2, wherein B1 is R1 and R1' are both H, or one of R1 and R1' is H and the other is F.

4. The compound according to any one of claims 2-3, wherein B2 is guanine or adenine one of R2 and R2' is H and the other is selected from -OH or F.

5. The compound according to any one of claims 2-3, wherein B2 is adenine substituted with X wherein X is CI, one of R2 and R2 is H and the other is F, or both R2 and R2 are H.

6. The compound of formula (II) according to claim 1, a stereoisomer or a pharmaceutically acceptable salt thereof, wherein represents a phosphoric acid linkage to the 2' position of the pentose sugar, formula (II) has 7. The compound according to claim 6, wherein B1 is R1 and R1' are both H, or one of R1 and R1' is H and the other is F.

8. The compound according to any one of claims 6-7, wherein B2 is guanine or adenine one of R2 and R2' is H and the other is -OH.

9. The compound according to claim 1, which is or a pharmaceutically acceptable salt thereof.

10. The compound according to claim 9, wherein B1 is R1 and R1' are both H, or R1 is F and R1' is H.

11. The compound according to any one of claims 9-10, wherein in formula (II-a') B2 is guanine or adenine R2' is H, R2 is selected from -OH or F.

12. The compound according to any one of claims 9-10, wherein in formula (II-a') B2 is adenine substituted with X wherein X is CI, R2 is H, R2' is F, or R2 and R2' are both H.

13. The compound according to any one of claims 9-10, wherein in formula (II-b') B2 is guanine or adenine R2' is H, R2 is -OH.

14. A cyclic dinucleotide compound selected from or a stereoisomer or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 14 or a stereoisomer or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

16. The pharmaceutical composition according to claim 15, which is in a form for topical administration.

17. Use of a compound according to any one of claims 1 to 14 or a stereoisomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to any one of claims 15 to 16 for the manufacture of a STING agonist for the treatment or prevention of colorectal cancer.

18. Use of a compound according to any one of claims 1 to 14 or a stereoisomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to any one of claims 15 to 16 for the manufacture of a multifunctional active agent for use as a STING agonist and a cytotoxic agent, wherein the multifunctional active agent is for the treatment or prevention of colorectal cancer.

19. The use according to claim 18, wherein the multifunctional active agent is for activating the STING signaling pathway to activate the immune system to exert anti-tumor functions, causing tumor cell death by releasing the cytotoxic agent, in turn, continuously activating STING to kill tumor cells by releasing tumor DNA, and providing the ability for "immunological memory" or durable immunity to the tumor by releasing tumor neoantigens to generate antibody-antigen responses.

20. Use of a compound according to any one of claims 1 to 14 or a stereoisomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to any one of claims 15 to 16 for the manufacture of a cytotoxic agent for the treatment or prevention of colorectal cancer.

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