Multifunctional cyclic dinucleotides and their uses

Abstract

The present invention relates to multifunctional cyclic dinucleotide compounds of formula (X) and derivatives thereof, which can be used as prodrugs of apoptosis-inducing or cytotoxic agents to induce cell apoptosis or antiviral activity, and can also modulate immune pathways to generate therapeutically beneficial immune responses. The present disclosure further relates to pharmaceutical compositions and pharmaceutical combinations comprising the cyclic dinucleotide compounds of the present invention, methods for synthesizing them, and their medical uses. [Formula 1] TIFF2023546278000301.tif44142

Description

[Technical Field]

[0001] The present invention relates to multifunctional cyclic dinucleotide compounds and derivatives thereof that function as prodrugs of apoptosis inducers or cytotoxic agents for inducing apoptosis in tumor cells or for eliminating viruses, and as immunomodulators for modulating immune pathways that produce therapeutically beneficial immune responses, particularly for activating the STING-mediated immune pathway. The disclosure further relates to pharmaceutical compositions and pharmaceutical combinations comprising the cyclic dinucleotide compounds of the present invention, methods for synthesizing them, and their medical uses. [Background technology]

[0002] Cancer, a malignant disease biologically characterized by abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness, and metastasis, is a major cause of death in humans, and its incidence continues to increase worldwide. Meanwhile, viral infections also cause millions of deaths globally.

[0003] Antimetabolite nucleoside analogs represent one of the major therapeutic approaches in the treatment strategies for cancer / tumors and viral infections. In particular, antimetabolite nucleoside drugs do not act directly themselves, but need to be converted to their triphosphorylated form in vivo by various cell kinases, functioning as pseudometabolites that become active substrates for polymerases. They are then incorporated into DNA or RNA via the nucleic acid biosynthesis pathway, inhibiting DNA or RNA modification and elongation, or inhibiting reverse transcriptases involved in DNA or RNA synthesis. This induces apoptosis in tumor cells or prevents viral replication, thus exhibiting cytotoxicity and making them usable for the treatment of cancer / tumors or viral infections. On the other hand, the cell fragments resulting from the cytotoxicity of nucleoside drugs can trigger an immune response in host cells, further inhibiting the growth and proliferation of tumor cells or viruses.

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

[0005] [ka] TIFF0007875196000002.tif92158

[0006] However, nucleoside drugs have multiple active groups, such as hydroxyl and amino groups, on their molecular structure, which leads to low membrane permeability and insufficient stability and pharmacokinetic properties. This often necessitates special or frequent administration, causing considerable inconvenience to patients. Therefore, exploring new and improved nucleoside drug solutions has been an active area of ​​drug development.

[0007] cGAMP, an endogenous cyclic dinucleotide (CDN), is a key link in the cGAS-STING (cyclic GMP-AMP synthase-interferon gene-stimulating factor) signaling pathway, a key component of the innate immune system. Specifically, cGAS interacts with DNA from tumor cells, dead cells, viruses, bacteria, or mitochondria to catalyze the synthesis of the cyclic dinucleotide (CDN) cGAMP from ATP and GTP. The produced endogenous cGAMP further binds to STING on the endoplasmic reticulum (ER). STING bound to cGAMP is activated, undergoes a conformational change, and moves to the Golgi apparatus. Subsequently, it induces the activation of key transcription factors IRF-3 and NF-κB. These activated transcription factors enter the nucleus and induce the expression of type I interferons such as IL-6, TNF-α, and IFN-γ, as well as inflammatory cytokines (Jiang et al, cGAS-STING, an antigenic pathway in cancer immunology, Journal of Hematology & Oncology, 2020, 13:81; Xiangling Cui et al, STING modulatories: Predictive significance in drug discovery, European Journal of Chemical Chemistry 182 (2019) 111591).

[0008] [ka]

[0009] It is well known in this field that type I interferons not only exhibit antiviral activity, but also directly inhibit human tumor cell proliferation, significantly enhance the anti-tumor immune response by inducing the activation of adaptive and innate immune cells, and inhibit tumor invasion by regulating the expression of enzymes related to tissue remodeling, and for this reason are also useful as anticancer agents.

[0010] As described above, considering that endogenous cyclic dinucleotide (CDN) cGAMP is a key mediator of the innate immune system in response to viruses and tumors, ultimately contributing to the production of interferons or inflammatory cytokines, thereby achieving therapeutic benefits, a series of CDN STING agonists have been synthesized and their activity validated in the laboratory, 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 common understanding in this field, the STING pathway can be activated by exogenous DNA (tumors or viruses, etc.). Without mediation by protein neoantigens, downstream pro-inflammatory factors driven by interferons and other factors in the STING pathway lack targeting properties, leading to tolerance for insufficient autoimmune responses and a narrow therapeutic window. First-generation mono-STING agonists combined with PD-1 antibodies still focused on systemic immune activation, failing to address adaptive immune selectivity and not inducing responses originating from the tumor microenvironment.

[0012] On the one hand, the compounds of the present invention act as known highly active STING agonists by activating signaling pathways and releasing interferons and other cytokines to activate the immune system; secondly, their cytotoxic functions are subsequently activated to selectively kill tumors, releasing large amounts of tumor neoantigens and tumor DNA, establishing adaptive immune recognition and training the immune system's targeting ability; thirdly, the tumor neoantigens and tumor DNA continue to activate the STING pathway and other immune systems to kill tumor cells; and finally, the released tumor neoantigens are recognized by dendritic cells (DC cells) and interact with T cells to form immunological memory, thereby achieving long-term suppression of distant tumor and cancer cell migration.

[0013] In particular, in the search for novel cytotoxic nucleoside drugs with improved properties, the present invention inventively introduces cytotoxic nucleoside drugs as basic units into the molecular structure of CDNs, that is, introduces hidden cytotoxic pharmacophores at the molecular level of CDNs. The resulting novel CDN drug molecules first activate STING to induce the production of type I interferon, thereby achieving antiviral or antitumor immunotherapy effects. Even more groundbreaking, the products formed by the in vivo degradation of the molecule, namely cytotoxic nucleoside drugs, can specifically interfere with nucleic acid metabolism, prevent cell division and proliferation, induce tumor cell death or prevent viral replication, release tumor DNA to continuously activate STING, release tumor neoantigens to establish adaptive immune recognition function, and train the targeting ability of the immune system. This overcomes the drawbacks of single nucleoside drugs, such as low membrane permeability, insufficient stability and poor pharmacokinetic properties, and the need for frequent or specialized drug administration. At the same time, it also overcomes the issue of the source of exogenous DNA (e.g., tumor DNA) required for the continuous activation of the STING pathway. More importantly, the CDN structural skeleton of the molecule disclosed herein can activate STING, thereby inducing type I interferon production and achieving antiviral or antitumor immunotherapy effects. On the other hand, cytotoxic pharmacophores hidden within novel CDN molecules can be released in a timely manner to generate apoptotic fragments, providing antigens against tumors or viruses for the immune system, and in coordination with immunoleukocyte subsets, they can induce an antibody-antigen response, thereby providing the ability for "immunological memory" or persistent immunity against the antigen.

[0014] Accordingly, the present invention provides a novel class of CDN compounds that can simultaneously achieve antimetabolism therapy and immunosuppression of viral infections or tumors through drug combination at the submolecular level, and can provide enhanced and more synergistic effects compared to single cytotoxic agents or single CDN STING agonists.

[0015] It should be noted that the above discussion of the background of the invention is provided solely to aid in understanding the present invention and should not be interpreted as an endorsement of the prior art description or as constituting prior art with respect to the present invention. [Prior art documents] [Patent Documents]

[0016] [Patent Document 1] WO2014 / 189805 [Patent Document 2] WO2017 / 027645 [Patent Document 3] WO2018 / 060323 [Non-patent literature]

[0017] [Non-Patent Document 1] Jiang et al, cGAS-STING, an antigenic pathway in cancer immunology, Journal of Hematology & Oncology, 2020, 13:81 [Non-Patent Document 2] Xiangling Cui et al, STING modulators: Predictive significance in drug discovery, European Journal of chemical Chemistry 182 (2019) 111591 [Overview of the project] [Means for solving the problem]

[0018] Summary of the Invention The present invention aims to provide a group of novel cyclic dinucleotide-based antiviral or antitumor compounds.

[0019] In one embodiment, the present invention provides a group of cyclic dinucleotide compounds of the following formulas, stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, or solvates thereof,

[0020] [ka] In the formula, X1 and X2 are independently selected from -OH and -SH, respectively; at least one of the two nucleosides of the cyclic dinucleotide has a cytotoxic or antiviral effect. Compounds of such a structure can, on the one hand, generate a single cytotoxic or antiviral nucleoside compound after degradation in vivo and upon triphosphorylation activation by nucleoside kinases in cells, which can prevent cell division and proliferation by specifically interfering with nucleic acid metabolism, thereby inhibiting tumor cell proliferation or viral replication. On the other hand, the molecules of the present invention maintain the immunoactivating function of the cyclic dinucleotide, i.e., activate targeted STING, ultimately inducing type I interferon production through the STING signaling cascade, thereby generating tumor immune activity to inhibit tumor growth and metastasis or exert antiviral activity. Furthermore, apoptotic tissue fragments provide the immune system with antigens not naturally expressed in the host, eliciting an antigen-antibody response, thereby providing the ability of "memory" or persistent immunity against the antigen.

[0021] In particular, in this embodiment, the present invention relates to formula (Y):

[0022] [ka] (In the formula, X 1 , X 2 B1, B2, R 1 , R 1 ', R 2 , R 2 (' is as defined herein.) The present invention provides cyclic dinucleotide compounds, their stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts, prodrugs, or solvates.

[0023] More particularly, the present invention provides, in this embodiment, cyclic dinucleotide compounds of formulas (I), (II), (III), and (IV) and each of their subformulas; more particularly, the present invention provides, in this embodiment, cyclic dinucleotide compounds of each of the subformulas (I), (II), (III), and (IV); and each of their specific embodiments described below.

[0024] In another embodiment, the present invention provides methods 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.

[0025] In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of the present invention as described herein, its stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, prodrugs or solvates, and one or more pharmaceutically acceptable excipients.

[0026] In another embodiment, the present invention provides compounds or pharmaceutical compositions described herein for use as activators for the treatment or prevention of diseases related to or mediated by immune responses, particularly for the treatment or prevention of diseases related to or mediated by STING, and more particularly for use as activators for the treatment or prevention of inflammation, allergic diseases or autoimmune diseases, infectious diseases or cancer, especially for antiviral or antitumor applications, or for use as vaccine adjuvants.

[0027] In another embodiment, the present invention provides compounds or pharmaceutical compositions described herein for use as cytotoxic agents, particularly as anti-cancer agents, for the treatment or prevention of hyperproliferative diseases.

[0028] In another embodiment, the present invention provides compounds or pharmaceutical compositions described herein for use as cytotoxic agents for the treatment or prevention of viral infections.

[0029] In another embodiment, the present invention provides the use of compounds or pharmaceutical compositions described herein for the treatment or prevention of diseases related to or mediated by immune responses, for example, as STING agonists for the treatment or prevention of diseases related to or mediated by STING, and more particularly for the treatment or prevention of inflammation, allergic diseases or autoimmune diseases, infectious diseases or cancer, especially tumors or viral infections; or for use as vaccine adjuvants.

[0030] In another embodiment, the present invention provides the use of compounds or pharmaceutical compositions described herein as cytotoxic agents in the treatment or prevention of hyperproliferative diseases, particularly tumors; or as cytotoxic agents in the treatment or prevention of viral infections.

[0031] In another embodiment, the present invention provides a method for the treatment or prevention of diseases related to or mediated by an immune response in a subject, particularly diseases related to or mediated by STING, and more particularly inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, particularly tumors or viral infections, comprising administering a compound of the present invention or a pharmaceutical composition described herein to a human or animal.

[0032] In another embodiment, the present invention provides a method for the treatment or prevention of hyperproliferative diseases, particularly tumors, in a subject, comprising administering a compound or pharmaceutical composition described herein to a human or animal.

[0033] In another embodiment, the present invention provides a method for treating or preventing a viral infection in a subject, comprising administering a compound of the present invention or a pharmaceutical composition described herein to a human or animal.

[0034] In another embodiment, the present invention provides the use of compounds or pharmaceutical compositions described herein for use in the manufacture of pharmaceuticals for the treatment or prevention of diseases related to or mediated by immune responses, particularly STING-related or STING-mediated diseases, and more particularly inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, especially tumors or viral infections, or for use as vaccine adjuvants.

[0035] In another embodiment, the present invention provides the use of compounds or pharmaceutical compositions described herein for the manufacture of pharmaceuticals for the treatment or prevention of hyperproliferative diseases, particularly tumors.

[0036] In another embodiment, the present invention provides the use of compounds or pharmaceutical compositions described herein for the manufacture of pharmaceuticals for the treatment or prevention of viral infections.

[0037] In another embodiment, the present invention provides compounds or pharmaceutical compositions described herein for use as multifunctional agents having both immunotherapeutic and cytotoxic therapeutic activity, for example, for use in exerting antitumor and antiviral replication functions by activating the immune system by activating the STING signaling pathway; for use in inducing tumor cell death or preventing viral replication by releasing a cytotoxic agent; for use in killing tumor cells by subsequently releasing tumor DNA and continuously activating STING; and for use in providing the ability of "immunological memory" or persistent immunity against tumors by releasing tumor neoantigens and inducing an antibody-antigen response. In this embodiment, the present invention also provides the use of compounds or pharmaceutical compositions described herein for, for example, the treatment or prevention of viral infections or tumors, for example; methods for treating or preventing diseases related to or mediated by an immune response in a subject, particularly diseases related to or mediated by STING, and more particularly inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, tumors or viral infections, in particular, by administering compounds or pharmaceutical combinations described herein to humans or animals; and the use of compounds or pharmaceutical compositions described herein in the manufacture of a pharmaceutical for achieving the above-mentioned multiple functions, for example, the pharmaceutical being used, in particular, to treat or prevent viral infections or tumors.

[0038] In another embodiment, the present invention provides pharmaceutical combinations comprising the compounds of the present invention described herein, their stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts or solvates, and at least one other therapeutic agent.

[0039] In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of the present invention as described herein, its stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts or solvates, at least one other therapeutic agent, and one or more pharmaceutically acceptable excipients.

[0040] In another embodiment, the present invention provides pharmaceutical combinations described herein, comprising the compounds of the present invention and at least one other therapeutic agent, for use in the treatment or prevention of hyperproliferative diseases, viral infections, or diseases related to or mediated by STING, and more particularly in inflammatory, allergic or autoimmune diseases, infectious diseases, or cancer, especially tumors or viral infections.

[0041] In another embodiment, the present invention provides the use of pharmaceutical combinations described herein, 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, and more particularly for inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, tumors, or viral infections.

[0042] In another embodiment, the present invention provides a method for the treatment or prevention of hyperproliferative diseases, viral infections, or diseases related to or mediated by STING, and more particularly, inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, tumors or viral infections, comprising administering to a human or animal a pharmaceutical combination described herein comprising a compound of the present invention and at least one other therapeutic agent. [Brief explanation of the drawing]

[0043] [Figure 1] Figure 1 shows the interferon-stimulating activity of representative compounds of the present invention in THP-1 cells. [Figure 2A]Figure 2 shows the tumor growth inhibitory activity of representative compounds of the present invention in a mouse transplanted CT26 colorectal cancer model. 2A; Change in tumor volume in treated tumors. [Figure 2B] Changes in tumor volume in untreated tumors. [Figure 2C] Changes in the weight of mice. [Figure 2D] Changes in tumor volume in immunized and unimmunized mice. [Figure 3A] Figure 3 shows the immunological memory of representative compounds of the present invention in immunized and unimmunized mice. 3A: Immunological memory in immunocompetent mice. [Figure 3B] Immunotherapy in immunodeficient mice. [Figure 4A] Figure 4 shows the hepatocyte metabolic properties of a representative compound of the present invention. 4A: Metabolic stability in hepatocytes. [Figure 4B] Identification of metabolites in hepatocytes. [Modes for carrying out the invention]

[0044] Description of the Invention definition Unless otherwise stated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which the present invention pertains.

[0045] Unless otherwise stated, the nomenclature used in this application is based on IUPAC systematic names. IUPAC chemical names were generated using OpenEye Lexichem version 1.2.0, PerkinElmer E-notebook for Chemistry, or Insight for Excel 2017 R2.

[0046] Unless otherwise stated, any open valence observed on carbon, oxygen, sulfur, or nitrogen atoms in the structures herein indicates the presence of a hydrogen atom.

[0047] The term “immune system” has the ordinary meaning as understood by those skilled in the art and refers to the molecules, substances (e.g., body fluids), anatomical structures (e.g., cells, tissues or organs), and physiological processes as a whole, or to any one or more components related to preventing infection in vivo, protecting the body during infection or disease, and / or assisting the body's recovery after infection or disease.

[0048] 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 defense response against foreign or mutated autologous components. For the purposes of this invention, “disease related to or mediated by an immune response” means, in particular, a condition in humans or animals in which the function of the immune system is weakened, inactivated, or impaired, or the function of one or more immune components is weakened, inactivated, or impaired, or a disease caused thereby, in particular a disease that can be mitigated by inducing an immune response via the STING pathway.

[0049] The term "STING" is an abbreviation for "interferon gene stimulator." STING is a transmembrane protein receptor in humans, and activation of STING by cyclic dinucleotides (CDNs) leads to activation of the IRF3 pathway and the NF-κB pathway, thus resulting in the induction of type I interferons and inflammatory cytokines, respectively. The term "STING agonist" refers to any substance that activates STING in vitro or in vivo to induce a physiological response.

[0050] The term "diseases related to or mediated by STING" refers to diseases that can be mitigated by inducing an immune response via the STING pathway, i.e., diseases in which activation of STING reduces the incidence and lessens or eliminates disease symptoms, including, but not limited to, inflammation, allergic diseases or autoimmune diseases, infectious diseases or cancer. For the purposes of the present invention, "diseases related to or mediated by STING" are preferably selected from tumors or cancers.

[0051] The terms “hyperproliferative disorder,” “tumor,” or “cancer” refer to a physiological condition in which uncontrolled or disordered cell proliferation or cell death occurs, and include solid tumors and hematogenous tumors, whether malignant or benign, and include, but are not limited to, brain cancer, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, blood cancer, lung cancer, and bone cancer. Examples of the above cancer types include neuroblastoma, intestinal cancer (e.g., rectal cancer, colorectal cancer, familial adenomatous polyposis cancer, and hereditary nonlymphatic colorectal cancer), esophageal cancer, lip cancer, laryngeal cancer, nasopharyngeal cancer, oral cancer, salivary gland cancer, peritoneal cancer, soft tissue sarcoma, urothelial carcinoma, sweat adenoma, stomach cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, and corpus cancer. Carcinoma, endometrial cancer, pancreatic cancer, prostate cancer, testicular cancer, breast cancer (including HER2-negative breast cancer), urological cancers, melanoma, brain tumors (e.g., glioblastoma, astrocytoma, meningioma, medullary tumor, and peripheral neuroectodermal tumors), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia Examples include chronic myeloid leukemia (CLL), lymphocytic cancers, acute myeloid leukemia (AML), myeloid leukemia (CML), adult T-cell lymphoma, diffuse lymphoma (DLBCL), liver cancer, multiple myeloma, seminomas, osteosarcoma, chondrosarcoma, anal canal cancer, adrenocortical carcinoma, chordoma, fallopian tube cancer, gastrointestinal stromal tumors, myeloproliferative disorders, mesothelioma, biliary tract cancer, Ewing's sarcoma, and other rare tumor types.

[0052] The term "therapeutic agent" refers to one or more substances administered to humans or animals to obtain a specific therapeutic effect, such as substances that prevent, cure or alleviate the effects of a disease or improve a state of health. The therapeutic agents of the present invention include not only the CDN compound provided, but also therapeutic agents that can be used in combination with the CDN compound provided, such as, but not limited to, chemotherapeutic agents, immunotherapeutic agents (especially immunosuppressant anticancer agents), vaccines, adjuvants, and radiotherapy.

[0053] The term "chemotherapeutic agent" refers to one or more chemical substances administered to humans or animals to kill tumors, slow or stop tumor growth, and / or slow or stop cancer cell division, and / or prevent or delay metastasis (retarting).

[0054] The term "immune agent" refers to any endogenous or exogenous substance that can interact with any one or more components of the immune system, such as 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.

[0055] The term "immunotherapy" refers to any medical procedure in which one or more components of the human or animal immune system are intentionally modified to directly or indirectly obtain specific therapeutic benefits, such as systemic and / or local effects, and prophylactic and / or therapeutic effects. Immunotherapy can involve the administration of one or more immunizing agents, either systemically, topically, or in combination, to human and animal subjects via any route, such as orally, intravenously, transdermally, by injection, or by inhalation.

[0056] The term "vaccine" refers to a biological preparation administered to a human or animal to induce or enhance a specific immune response in the human or animal, and / or to protect against one or more antigens.

[0057] The term "adjuvant" refers to a secondary treatment substance administered in any order with a primary treatment substance to achieve specific complementary, synergistic, or other beneficial effects that cannot be achieved by the primary treatment substance alone. Adjuvants may be used with vaccines, chemotherapeutic agents, or other treatment substances to enhance the effectiveness of the primary treatment substance, reduce the toxic side effects of the primary treatment substance, or provide specific protection to the recipient of the primary treatment substance, for example, to improve the function of the immune system, etc.

[0058] As used herein, the terms “cytotoxic agent” or “apoptosis-inducing agent” or similar expressions refer to active agents useful in treating abnormal and uncontrolled cell development and proliferation. For the purposes of the present invention, “cytotoxic agent” particularly refers to nucleoside antimetabolitic cytotoxic agents or antiviral agents, including, but not limited to, cytarabine, azacitidine, phloxuridine, deoxyuridine, enocitabine, doxifluridine, pentostatin, fludarabine, cladribine, gemcitabine, capecitabine, clopharabine, nerarabine, trifluorothymidine, 8-chloroadenosine, trisirivine, folodesine, 5-fluorodeoxycytidine, ribavirin, or akadesine.

[0059] As used herein, the term “multifunctional activator” refers to compounds of the present invention that, based on their unique structural design, can exert multiple functions in a target, i.e., can exhibit both immunotherapeutic and cytotoxic therapeutic activity, for example, but not limited to, activating the immune system by activating the STING signaling pathway to exert antitumor and antiviral replication functions, inducing tumor cell death or preventing viral replication by releasing cytotoxic agents, continuously activating STING by releasing tumor DNA to kill tumor cells, and inducing an antibody-antigen response by releasing tumor neoantigens to provide “immunological memory” or persistent immunity against tumors.

[0060] The terms “treatment” or “treating” of a disease encompass inhibiting the disease state, that is, halting the progression of the disease or its clinical symptoms, or improving the disease state, that is, causing a temporary or permanent regression of the disease state or its clinical symptoms.

[0061] The terms "prevention" or "preventing" of a disease mean preventing the onset of clinical symptoms of a disease in an individual who may be exposed to or is susceptible to that disease but has not yet experienced or shown symptoms of that disease.

[0062] The term "therapeutic dose" means the amount of the 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. The therapeutic dose will vary depending on the compound, the condition being treated, the severity of the condition being treated, the age and relative health status of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors.

[0063] As used herein, the terms “subject,” “individual,” or “patient” refer to vertebrates. In certain embodiments, vertebrates are mammals. Examples of mammals include, but are not limited to, livestock (e.g., cows), sporting animals, pets (e.g., guinea pigs, cats, dogs, rabbits, and horses), primates, mice, and rats. In a preferred embodiment, the mammal is a human.

[0064] The terms “pharmaceutical composition” and “pharmaceutical preparation” (or “preparation”) are used interchangeably and refer to a mixture or solution containing a therapeutically effective amount of one or more pharmacovigilant active ingredients and one or more pharmaceutically acceptable excipients, administered to a mammal (e.g., human) that requires it.

[0065] The term "pharmaceutical combination" means that the compounds of the present invention can be combined with other activators to achieve the objectives of the present invention. The other activators may be one or more further compounds of the present invention, or second or further (e.g., third) compounds that are compatible with the compounds of the present invention, i.e., that do not adversely affect each other or have complementary activity. Such activators are preferably present in combination in amounts effective to achieve the intended purpose. The other activators may be co-administered with the compounds of the present invention as a single pharmaceutical composition, or they may be administered separately from the compounds of the present invention in separate units, and if administered separately, they may be administered simultaneously or sequentially.

[0066] The term "pharmaceutically acceptable" generally refers to the characteristics of a substance that is safe, non-toxic, not biologically or non-biologically undesirable, and is acceptable for medicinal use in animals and humans, and is useful in the preparation of pharmaceutical compositions.

[0067] The terms “pharmaceutically acceptable excipient,” “pharmaceutically acceptable carrier,” and “therapeutically inactive excipient” are interchangeable and refer to any pharmaceutically acceptable component in a pharmaceutical composition that is therapeutically inactive and non-toxic to the target to which it is administered, such as a disintegrant, binder, filler, solvent, buffer, isotonic agent, stabilizer, antioxidant, surfactant, carrier, diluent, or lubricant for the formulation of a pharmaceutical product.

[0068] As used herein, the term “pharmaceutically acceptable salt” refers to a salt of the compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and may be inorganic acid addition salts or organic acid addition salts and base addition salts, for example, but are not limited to, the following: (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, caproic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, butanediic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptanoic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc.; or (2) Salts formed in the presence of acidic protons in the parent compound are either substituted with metal ions such as alkali metal ions, alkaline earth metal ions, or aluminum ions, or coordinated with organic bases such as ethanolamine, diethanolamine, triethanolamine, or N-methylglucamine. General principles and techniques for preparing pharmaceutically acceptable salts are known to those skilled in the art, and are described, for example, in Berge et al., Pharm ScL, 66, 1-19. (1977).

[0069] As used herein, the term “pharmaceutically acceptable prodrug” refers to a compound having a cleavable group that becomes a pharmaceutically active compound of the present invention in vivo by solvolysis or under physiological conditions, and in particular, prodrugs include those that can be oxidized, reduced, ammoniated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce an active compound (including derivatives) of the present invention. Various forms of prodrugs are well known in the art, and preferred prodrug portions are described, for example, in “Prodrugs and Targeted Delivery”, J. Rautico, Ed., John Wiley & Sons, 2011.

[0070] The prodrugs of CDN compounds described herein can generally increase the activity, bioavailability, or stability of the compounds. Generally, alkylation, acylation, or other lipophilic modifications of the phosphate moiety, or the use of other analogues of the nucleotide, will help to enhance the stability of the nucleotide.

[0071] As used herein, the term “solvate” refers to a solubilization form containing a stoichiometric or non-stoichiometric solvent, such as a solvate with water, e.g., a hydrate, or a solvate with an organic solvent such as methanol, ethanol, or acetonitrile, i.e., a solvate as methanolate, ethanolate, or acetonitrile solvate, respectively; or a solvate of 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.

[0072] 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 an atomic mass or mass number different from that usually 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, etc., and these are intended to be included within the scope of the present invention regardless of whether they are radioactive. In some embodiments, the incorporated isotope is 2H (deuterium); in other embodiments, the incorporated isotope is 3H (tritium).

[0073] As used herein, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In a compound having one or more (e.g., 1, 2, 3, or 4) asymmetric centers, a racemic mixture, a single enantiomer, a mixture of diastereomers, and a single diastereoisomer may exist. Similarly, the compounds of the present invention may exist as a mixture of two or more different structural forms in rapid equilibrium (generally called tautomers). It should be understood that the scope of this application includes all such isomers or mixtures thereof in any ratio (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0074] The compounds of the present invention may have one or more asymmetric centers and, therefore, can be prepared as (R)- or (S)-stereoisomers, individually or as mixtures thereof. In the structural formulas or structural fragments of the compounds herein,

Chemical formula

[0075] [ka] The symbol indicates the stereoconfiguration of a chiral center, i.e., a chiral center. Correspondingly, in the naming of the compounds or intermediates provided by the present invention, R or S represents the stereoconfiguration of a chiral center. Those skilled in the art will understand that the phosphorothioate bonds in the compounds of the present invention are essentially chiral and may exist in either an R configuration or an S configuration, and therefore, the two phosphorothioate bonds can be in the forms R,R, S,S, S,R and R,S. The present invention encompasses the compounds of the present invention and their embodiments as substantially pure forms or mixtures, and the compounds containing two phosphorothioate bonds are preferably substantially pure forms of R,R, S,S, S,R and R,S stereoisomers, and particularly preferably substantially pure R,R stereoisomers (i.e., both phosphorus atoms have the R configuration). The assignment of their absolute configurations can be done according to the methods described in the literature (Zhao et al. Nucleosides, Nucleotides and Nucleic Acids 2009, 289, 352-378; Knouse et al. Science 2018, 361, 1234). It should be noted that incorrect configuration assignments resulting from errors in the literature's methods will not affect the actual configuration of the compounds of the present invention.

[0076] As used herein with respect to CDNs, the term “substantially pure” means that a particular stereochemistry is at least 75% pure of other possible stereochemistrys at the chiral centers shown in the above diagram. In preferred embodiments, substantially pure CDNs are 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 preparations of the present invention are “stereochemically pure” in the sense that all CDNs in the preparation have a particular stereochemistry at those chiral centers, and it is not intended to mean that all CDNs in the preparation having a particular stereochemistry at those chiral centers are otherwise identical. For example, a substantially pure CDN R,R cGAMP phosphorothioate preparation may include a combination of R,R c-di-GMP phosphorothioate and R,R c-di-AMP phosphorothioate and may still be a substantially pure cyclic pruridinibacterial preparation.

[0077] As used herein, the term “protecting group” usually refers to a group that selectively blocks a reaction site in a polyfunctional compound, in the sense of a protecting group in synthetic chemistry, allowing a chemical reaction to proceed selectively on another unprotected reaction site. Protecting groups can be removed at an appropriate time. Examples of protecting groups include amino protecting groups, such as, but are 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, etc.; as well as hydroxyl protecting groups, for example, but not limited to ester-forming groups and ether-forming groups, particularly tetrahydropyranyloxy, Bz (benzoyl), iExamples include -BuCO(isobutyryl), DMTr(bis(4-methoxyphenyl)benzyl), acetoxy, carbamoyloxy, benzyl ethers and silyl ethers, such as TBS(tert-butyldimethylsilyl) and TBDPS(tert-butyldiphenylsilyl). Further examples of these groups can be found in TW Greene and PGM Wuts, “Greene's protective groups in organic synthesis”, 5th edition. John Wiley & Sons., Inc., Hoboken, New Jersey, 2014.

[0078] The term "deprotection" refers to the process of removing a protecting group after a selective reaction has been completed. Examples of deprotecting agents include acids, bases, or hydrogen, particularly potassium carbonate or sodium carbonate, lithium hydroxide in alcohols, zinc in methanol, acetic acid, trifluoroacetic acid, palladium catalysts, or boron tribromide.

[0079] As used herein, the term "alkyl" refers to a linear or branched aliphatic hydrocarbon group having a specified number of carbon atoms. In particular, alkyl groups 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. Preferred C 1-14 Examples of 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, and others. Certain alkyl groups have 1 to 7 carbon atoms, for example, 1 to 6 carbon atoms or 1 to 4 carbon atoms.

[0080] As used herein, the terms "halo" or "halogen" refer to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). Specific halos are fluoro or chloro.

[0081] For clarity, the term "guanine" as used herein refers to

[0082] [ka] The term is,

[0083] [ka] It can also be expressed as follows. The phosphorothioate group used in the formula of the compound of the present invention is

[0084] [ka] It can be shown as follows.

[0085] The compound of the present invention Unless otherwise indicated, terms such as “compounds described herein,” “compounds of the invention,” and “compounds of the present invention,” as used throughout this application, encompass the compound of formula (X), the compound of formula (Y), and the compounds of formulas (I), (II), (III), and (IV) of specific embodiments, their respective subformulas, and their specific or preferred embodiments, their stereoisomers, tautomers, racemates, stable isotope variants, pharmaceutically acceptable salts or solvates, and pharmaceutically acceptable prodrugs, as defined in the above definitions section. The compounds of the present invention may be isolated as a mixture of isomers, or prepared by racemate resolution, for example by chromatography or fractional crystallization, or isolated as individual isomers synthesized from optically active starting materials. Similarly, references to “intermediates” herein are intended to encompass their free forms and the derivative forms described above, where the context allows, whether or not they are claimed.

[0086] Preferably, the compound of the present invention is in free form or a pharmaceutically acceptable salt or solvate thereof; most preferably, it is in free form or a pharmaceutically acceptable salt thereof.

[0087] Certain compounds of the present invention may exist in polymorphic or amorphous forms, which are also within the scope of the invention. If in solid crystalline form, the compounds of the present invention may also exist in cocrystal form with another chemical entity, and this specification encompasses all such cocrystals.

[0088] In particular, in one embodiment, the present invention relates to a cyclic dinucleotide compound of the following formula:

[0089] [ka] (wherein X1 and X2 are independently selected from -OH and -SH, respectively), providing stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, prodrugs or solvates thereof; at least one of the two nucleosides of the above cyclic dinucleotide has a cytotoxic or antiviral effect, preferably an antimetabolite anticancer drug thereof, i.e., nucleoside anticancer drugs or derivatives thereof, include, but are not limited to, enocitabine, fludarabine, cytarabine, nerarabine, doxifluridine, capecitabine, azacitidine, pentostatin, cladribine, gemcitabine and clofarabine, more preferably cladribine, gemcitabine and clofarabine or derivatives thereof. The nucleoside anticancer drug derivatives referred to herein retain anticancer cytotoxicity and refer to compounds derived from the substitution of a hydrogen atom or group of atoms in the compound structure with another atom or group of atoms.

[0090] In a particular embodiment, the present invention relates to the following formula (Y):

[0091] [ka] (In the formula, X 1 and X 2 These are independently selected from -OH and -SH; B1 is an adenine substituted with X.

[0092] [ka] (Here X is Cl, F, or -NHC) 1-6 (Selected from alkyl) or R a Cytosine is sometimes substituted.

[0093] [ka] (R here) a is H or -C(O)-C 1-14 (Selected from alkyl groups); R1 and R1' are each independently selected from H, F, or -OH; B2 is an adenine that may be substituted with X.

[0094] [ka] (Here X is selected from H, F, or Cl); R a Cytosine is sometimes substituted.

[0095] [ka] (R here) a is H or -C(O)-C 1-14 Selected from alkyl groups; or guanine

[0096] [ka] (Here OH is C 1-6 Selected from (sometimes substituted with alkyl);

[0097] [ka] In this case, the phosphate bond is attached to the 2' or 3' position of the pentose, and the position that is not cyclized with the phosphate is R 2 And it is shown that it is substituted with R2'; R2 and R2' are each independently selected from H, -OH, or F; However, if one of B1 or B2 is R a (If it is a cytosine that is substituted in some cases, the carbon atoms adjacent to it on the pentose ring to which it is bonded are substituted with two fluorine atoms.) The present invention provides cyclic dinucleotide compounds, or their stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, prodrugs, or solvates.

[0098] In a particular embodiment, the compound of formula (Y) is

[0099] [ka] It may be a compound of formula TIFF0007875196000020.tif52142 (wherein B1, B2, R1, R1', R2, R2' have the meanings defined above for compounds of formula (Y).

[0100] In certain embodiments, the two phosphorothioate bonds (if present) in the compound of the present invention are in an R,R,S,S,S,R or R,S configuration, or a mixture thereof. In preferred embodiments, the two phosphorothioate bonds (if present) in the compound of the present invention are in a substantially pure form in an R,R,S,S,S,R or R,S configuration, and particularly preferably in a substantially pure R,R configuration.

[0101] In certain embodiments, the single phosphorothioate bond present in the compound of the present invention may be in an R configuration or an S configuration, and is preferably in an R configuration.

[0102] In one embodiment of the compounds of formula (I), (II), (III), or (IV), B1 is an adenine substituted with X.

[0103] [ka] (wherein X is selected from Cl or F, preferably Cl) or R a Cytosine is sometimes substituted.

[0104] [ka] (In the formula, R a is H or -C(O)-C 1-14 (Selected from alkyl groups).

[0105] In one embodiment of the compounds of formula (I), (II), (III), or (IV), B1 is an adenine substituted with X.

[0106] [ka] (In the formula, X is -NHC) 1-6 Alkyl (for example, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, preferably -NHCH3)

[0107] In one embodiment of the compound of formula (I), (II), (III), or (IV), B1 is R a Cytosine is sometimes substituted.

[0108] [ka] (In the formula, R a is H or -C(O)-C 1-14 (Selected from alkyl groups).

[0109] In one embodiment of the compounds of formula (I), (II), (III), or (IV), R1 and R1' are both H, one is H and the other is F, or both are F.

[0110] In one embodiment of the compounds of formula (I), (II), (III), or (IV), B2 is an adenine optionally substituted with X.

[0111] [ka] (wherein X is selected from H, F, or Cl, and is preferably H or Cl).

[0112] In one embodiment of the compound of formula (I), (II), (III), or (IV), B2 is R a Cytosine is sometimes substituted.

[0113] [Chemical formula] (wherein, R a is selected from H or -C(O)-C 1-14 alkyl).

[0114] In one embodiment of the compound of formula (I), (II), (III) or (IV), B2 is guanine

[0115] [Chemical formula] is as follows.

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

[0117] In one embodiment of the compound of formula (I), (II), (III) or (IV), when R a is -C(O)C 1-14 alkyl, it may 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 formula (I), (II), (III) or (IV) of the present invention also include any combination of the above embodiments and their preferred or exemplary embodiments.

[0119] In a further specific embodiment, the present invention relates to a formula (Y) having the following general formula

[0120] [Chemistry] TIFF0007875196000029.tif168148(where B1, B2, R1, R1', R2, and R2' have the meanings defined above for the compounds of formula (I), (II), (III), or (IV) and specific embodiments thereof) provides a cyclic dinucleotide compound.

[0121] In one embodiment of a compound of formula (II-a) or formula (II-b), particularly a compound of formula (II-a), B1 is

[0122] [Chemistry] and B2 is

[0123] [Chemistry] In a particular such embodiment, both R1 and R1' are H; in another particular such embodiment, one of R1 and R1' is H and the other is F. Further, in each of such particular embodiments, both R2 and R2' are H, or one of them is H and the other is F, or one of them is H and the other is OH.

[0124] In one embodiment of a compound of formula (II-a) or formula (II-b), particularly a compound of formula (II-a), both B1 and B2 are cytosine optionally substituted with a R

[0125] [Chemistry] and in a particular such embodiment, R1, R1', R2, and R2' are all F 。

[0126] In one embodiment of the compound of formula (II-a) or formula (II-b), particularly the compound of formula (II-b), B1 is

[0127]

Chemical formula

[0128]

Chemical formula

[0129] In one embodiment of the compound of formula (II-a) or formula (II-b), particularly the compound of formula (II-b), B1 is cytosine optionally substituted by R a

[0130]

Chemical formula

[0131]

Chemical formula

[0132] In one embodiment of the compound of formula (II-a) or formula (II-b), R a is H, or -C(O)C 1-10 alkyl, for example -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 It is alkyl(2).

[0133] In one embodiment of the compound of formula (II-a) or formula (II-b), B1 is

[0134] [ka] Therefore, both R1 and R1' are H.

[0135] In one embodiment of the compound of formula (II-a) or formula (II-b), B1 is

[0136] [ka] And one of R1 and R1' is H and the other is F; in a particular such embodiment, F and B1 are on the same side of ribose.

[0137] In one embodiment of a compound of formula (II-a) or formula (II-b), particularly a compound of formula (II-b), B1 is

[0138] [ka] Preferably

[0139] [ka] Therefore, R1 and R1' are both 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.

[0140] In one embodiment of the compound of formula (II-a) or formula (II-b), B1 is R a Cytosine is sometimes substituted.

[0141] [ka] (In the formula, R a (is selected from H), and both R1 and R1' are F.

[0142] In one embodiment of the compound of formula (II-a) or formula (II-b), B is R a Cytosine is sometimes substituted.

[0143] [ka] (In the formula, R a is -C(O)C 1-10 Alkyl, preferably -C(O)CH(C 1-4 It is alkyl(2), and both R1 and R1' are F.

[0144] In an exemplary embodiment, R a These are -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.

[0145] In one embodiment of the compound of formula (II-a) or formula (II-b), B2 is guanine.

[0146] [ka] or adenine

[0147] [ka] In this configuration, one of R2 and R2' is H, and the other is selected from -OH or F.

[0148] In one embodiment of the compound of formula (II-a), B2 is guanine.

[0149] [ka] In this configuration, one of R2 and R2' is H, and the other is selected from -OH or F.

[0150] In one embodiment of the compound of formula (II-a), B2 is adenine.

[0151] [ka] In this configuration, one of R2 and R2' is H, and the other is selected from -OH or F.

[0152] In one embodiment of the compound of formula (II-a), B2 is an adenine substituted with halogen X.

[0153] [ka] (wherein X is Cl); one of R2 and R2' is H and the other is F, preferably F and B2 are on the same side as ribose.

[0154] In one embodiment of the compound of formula (II-a), B2 is an adenine substituted with halogen X.

[0155] [ka] (wherein X is Cl) and R2 and R2' are both H.

[0156] In one embodiment of the compound of formula (II-a), B2 is R aCytosine is sometimes substituted.

[0157] [ka] (In the formula, R a (is selected from H), and both R1 and R1' are F.

[0158] In one embodiment of the compound of formula (II-a), B2 is R a Cytosine is replaced by

[0159] [ka] (In the formula, R a is -C(O)C 1-10 Alkyl, preferably -C(O)CH(C 1-4 It is alkyl(2), and R1 and R1' are all F.

[0160] In an exemplary embodiment, R a These are -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.

[0161] In one embodiment of the compound of formula (II-b), B2 is guanine.

[0162] [ka] or adenine

[0163] [ka] Therefore, one of R2 and R2' is H, and the other is -OH.

[0164] In the compounds of formula (II-a) or formula (II-b) described above and in various specific embodiments thereof, the two phosphorothioate bonds are R,R,S,S,S,R or R,S configurations or a mixture thereof; preferably, a substantially pure form of R,R,S,S,S,R or R,S configuration, and particularly preferably, a substantially pure R,R configuration.

[0165] The specific embodiments and preferred or exemplary embodiments thereof shown above for compounds of formula (II-a) or formula (II-b) are also applicable to compounds of formula (Ia) or formula (Ib), compounds of formula (III-a) or formula (III-b), and compounds of formula (IV-a) or formula (IV-b), respectively. That is, the present invention also encompasses compounds of formula (Ia) or formula (Ib), compounds of formula (III-a) or formula (III-b), and compounds of formula (IV-a) or formula (IV-b), where each specific substituent utilizes the specific definitions shown above for compounds of formula (II-a) or formula (II-b) or its specific embodiments.

[0166] For example, in one embodiment of a compound of formula (Ia) or formula (Ib), formula (III-a) or formula (III-b), or formula (IV-a) or formula (IV-b), particularly a compound of formula (Ib), formula (III-b) or formula (IV-b), B1 is

[0167] [ka] Therefore, B2 is

[0168] [ka] Preferably [ka] In certain such embodiments, one of R1 and R1' is H and the other is F. Furthermore, in each of these embodiments, one of R2 and R2' is H and the other is OH.

[0169] The compounds of formula (Ia) / (Ib), (II-a) / (II-b), (III-a) / (III-b), or (IV-a) / (IV-b) of the present invention also encompass any combination of the above embodiments and their preferred or exemplary embodiments.

[0170] In further specific embodiments, the present invention relates to cyclic dinucleotide compounds of the following lower-order formulas:

[0171] [ka] Provided TIFF0007875196000057.tif100136, where B1, B2, R1, R1', R2, R2' have the meanings defined above with respect to compounds of formula (I), (II), (III), or (IV) and their particular embodiments; more particularly, with respect to compounds of formula (Ia) / (Ib), (II-a) / (II-b), (III-a) / (III-b), or (IV-a) / (IV-b) and their particular embodiments.

[0172] In one embodiment of a compound of formula (II-a') or formula (II-b'), particularly a compound of formula (II-a'), B1 is

[0173] [ka] Therefore, B2 is

[0174] [ka] In a particular such embodiment, R1 and R1' are both H; in another particular such embodiment, R1 is F and R1' is H, i.e., F and B1 are on the same side of ribose. Furthermore, in each of the particular embodiments, R2 and R2' are both 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.

[0175] A compound of formula (II-a') or formula (II-b'), in particular an embodiment of the compound of formula (II-a'), where both B1 and B2 are R a Cytosine is sometimes substituted.

[0176] [ka] And in certain such embodiments, R1, R1', R2, and R2' are all F.

[0177] In one embodiment of a compound of formula (II-a') or formula (II-b'), particularly a compound of formula (II-b'), B1 is

[0178] [ka] Therefore, B2 is

[0179] [ka] In a particular such embodiment, R1 and R1' are both H; in another particular such embodiment, R1 is F and R1' is H, i.e., F and B1 are on the same side of ribose. Furthermore, in each of these embodiments, R2 and R2' are both 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.

[0180] In one embodiment of the compound of formula (II-a') or formula (II-b'), particularly the compound of formula (II-b'), B1 is R a cytosine optionally substituted with

[0181]

Chemical formula

[0182]

Chemical formula

[0183] In one embodiment of the compound of formula (II-a') or formula (II-b'), R a is H, or -C(O)C 1-10 alkyl, 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.

[0184] In one embodiment of the compound of formula (II-a') or formula (II-b'), B1 is

[0185]

Chemical formula

[0186] In one embodiment of the compound of formula (II-a') or formula (II-b'), B1 is

[0187] [ka] Therefore, R1 is F and R1' is H, meaning that F and B1 are on the same side of ribose, and in this way B1, together with the ribose to which it is bound, forms the nucleoside anti-cancer drug clopharabine.

[0188] In one embodiment of a compound of formula (II-a') or formula (II-b'), particularly a compound of formula (II-b'), B1 is

[0189] [ka] Preferably

[0190] [ka] Therefore, R1 and R1' are both H, or R1 is F and R1' is H, preferably R1 is F and R1'' is H.

[0191] In one embodiment of the compound of formula (II-a') or formula (II-b'), B1 is R a Cytosine is sometimes substituted.

[0192] [ka] (In the formula, R a (is selected from H), and R1 and R1' are both F, and in this way this B1, together with the ribose to which it is bound, forms the nucleoside anti-cancer drug gemcitabine.

[0193] In one embodiment of the compound of formula (II-a') or formula (II-b'), B1 is Ra cytosine optionally substituted in this case

[0194]

Chem.

[0195] In one embodiment of the compound of formula (II-a'), B2 is guanine

[0196]

Chem.

[0197] In one embodiment of the compound of formula (II-a'), B2 is adenine

[0198]

Chem.

[0199] <00##In one embodiment of the compound of formula (II-a'), B2 is adenine substituted with halogen X

[0200] [ka] (wherein X is Cl), R2 is H, R2' is F, and F and B2 are on the same side of ribose, so that B2, together with the ribose to which it is bound, forms the nucleoside anti-cancer drug clopharabine.

[0201] In one embodiment of the compound of formula (II-a'), B2 is an adenine substituted with halogen X.

[0202] [ka] (wherein X is Cl), and R2 and R2' are both H, and in this way B2, together with the ribose to which it is bound, forms the nucleoside anti-cancer drug cladribine.

[0203] In one embodiment of the compound of formula (II-a'), B2 is R a Cytosine is sometimes substituted.

[0204] [ka] (In the formula, R a (is selected from H), and R1 and R1' are both F, and in this way B2, together with the ribose to which it is bound, forms the nucleoside anti-cancer drug gemcitabine.

[0205] In one embodiment of the compound of formula (II-a'), B2 is R a Cytosine is replaced by

[0206] [ka] (In the formula, R a is -C(O)C 1-10Alkyl, preferably -C(O)CH(C 1-4 B2 is alkyl)2) and both R1 and R1' are F, and in this way B2 together with the ribose to which it is bound forms an alkanoylated derivative of the nucleoside anti-cancer drug gemcitabine. In an exemplary embodiment, R a These are -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 This is -C(O)CH(CH2CH2CH3)2.

[0207] In one embodiment of the compound of formula (II-b'), B2 is guanine.

[0208] [ka] or adenine

[0209] [ka] Therefore, R2' is H and R2 is -OH.

[0210] The specific embodiments and preferred or exemplary embodiments thereof shown above for compounds of formula (II-a') or formula (II-b') are also applicable to compounds of formula (I-a') or formula (I-b'), compounds of formula (III-a') or formula (III-b'), and compounds of formula (IV-a') or formula (IV-b'), respectively. That is, the present invention also encompasses compounds of formula (I-a') or formula (I-b'), compounds of formula (III-a') or formula (III-b'), and compounds of formula (IV-a') or formula (IV-b'), where each specific substituent utilizes the specific definitions shown above for each compound of formula (II-a') or formula (II-b') or its specific embodiment.

[0211] For example, in one embodiment of a compound of formula (I-a') or formula (I-b'), formula (III-a') or formula (III-b'), or formula (IV-a') or formula (IV-b'), particularly formula (I-b'), formula (III-b') or formula (IV-b'), B1 is

[0212] [ka] Therefore, B2 is

[0213] [ka] TIFF0007875196000081.tif24144, preferably

[0214] [ka] In certain such embodiments, R1 is F and R1' is H. Furthermore, in each of these embodiments, one of R2 is OH and R2' is H.

[0215] It should be noted that the compounds of the present invention encompass each of the specific embodiments described above, encompass embodiments resulting from any combination or subcombination of the specific embodiments described above, and also encompass embodiments resulting from any combination of any of the preferred or exemplary embodiments described above.

[0216] Preferred specific embodiments of the compounds of the present invention include the following compounds, their stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, or solvates.

[0217] [ka] TIFF0007875196000084.tif216170

[0218] Pharmacological activity and advantageous effects of the compound of the present invention Through research, it has been found that the cyclic dinucleotide compounds provided by the present invention have the following pharmacological activities and beneficial effects:

[0219] - This demonstrates that THP-1 cells can be effectively stimulated to secrete IFN-β, and that the above-mentioned cyclic dinucleotide compound has high affinity for the STING receptor, effectively activates STING, and can induce the production of type I interferon; - Effectively inhibits in vitro proliferation of the mouse colorectal cancer cell line CT26, exhibits toxic effects on tumor cells, and prevents tumor cell division and proliferation; - In a bilateral transplant tumor model using immunocompetent CT26 syngeneic mice, the compound exhibited superior antitumor activity compared to that in an immunodeficient mouse model, demonstrating that it possesses both tumor immunoactivation and cytotoxicity through STING activation, resulting in additive and even synergistic antitumor effects. - In a CT26 syngeneic mouse transplant tumor model, it exhibits immunological memory function that can effectively prevent tumor recurrence; - In hepatocyte metabolism research, favorable pharmacokinetic properties, such as relatively long t 1 / 2 It exhibits low clearance, allowing for relatively long dosing intervals and relatively good patient compliance; - Allows for local administration to lesions with appropriate efficacy and reduced doses; the high polarity of the molecules limits their spread outside the lesion, resulting in limited toxicity and excellent safety.

[0220] Pharmaceutical composition In another embodiment, 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 using the compound of the present invention to prepare the composition.

[0221] The composition or dosage form is formulated, administered, and given in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of that condition, the site of drug delivery, the method of administration, the timing of administration, and other factors known to the physician.

[0222] 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, 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 enhancers, processing aids, colorants, sweeteners, fragrances, flavorings, diluents, and other known additives to provide a refined appearance of the drug (i.e., the compounds of the present invention or its pharmaceutical compositions) or to contribute to the preparation of the formulation (i.e., the drug).

[0223] The compounds of the present invention can be administered, if necessary, for local treatment and intra-lesional administration by any preferred method, such as orally, topically (including buccal and sublingual), rectally, vaginally, percutaneously, parenterally, subcutaneously, intraperitoneally, intrapulmonaryly, intradermally, intrathecally, and epidurally, as well as intranasally. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.

[0224] 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 (directly into the tumor mass) or peritumoral (around the tumor mass) administration of the compounds of the present invention can directly activate locally infiltrating dendritic cells, directly promote apoptosis of tumor cells, or sensitize tumor cells to cytotoxic agents.

[0225] The compounds of the present invention can be administered in any convenient dosage form, such as tablets, powders, capsules, sterile injection preparations, solutions, dispersants, suspensions, syrups, sprays, suppositories, gels, emulsions, and patches. Such compositions may contain conventional components in 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 activators. They may also contain other therapeutically beneficial substances. Various formulations can be prepared according to conventional methods in the field of pharmaceutical formulation; see, for example, Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000), Lippincott, Williams & Wilkins, Philadelphia, or various national pharmacopoeias.

[0226] In preferred embodiments, pharmaceutical compositions comprising one or more compounds of the present invention are provided, prepared as parenteral formulations, particularly in the form of sterile injectable formulations such as sterile injectable solutions or suspensions in non-toxic and parenterally acceptable diluents or solvents, or as lyophilized powders. Acceptable vehicles or solvents may include, for example, water, 1,3-butanediol, Ringer's solution, or isotonic sodium chloride solution; furthermore, sterile fixed oils have conventionally been used as solvents or suspension media, and for this purpose, any mild fixed oil may be used, such as synthetic monoglycerides or diglycerides, fatty acids, etc.

[0227] The dosage of the compound of the present invention can vary widely and, naturally, can be adjusted according to the individual needs in each specific case. Typically, the effective dose for systemic administration of the compound of the present invention is about 0.1 μg / kg / day to about 50 mg / kg / day, e.g., 0.5 μg / kg / day to about 10 mg / kg / day, or 1 μg / kg / day to about 1 mg / kg / day. Each dosage unit may conveniently contain 0.001 μg to 10 mg, e.g., 0.01 μg to 1 mg, e.g., 50 μg to 500 μg. The effective dose can be administered in one or more doses, i.e., one, two or more doses, and can be administered multiple times at equal or different intervals, for example, once or more times per day, once or multiple times per week, or every few days / weeks, according to a dosage regimen.

[0228] Usage and Method In view of the fact that the compounds of the present invention can activate STING and induce the expression of type I interferons and inflammatory cytokines such as IL-6, TNF-α, and IFN-γ, while also possessing cytotoxic activity, another aspect of the present invention provides therapeutic uses and methods of the compounds of the present invention.

[0229] In one embodiment, 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, particularly STING-related or STING-mediated diseases, such as inflammation, allergic diseases or autoimmune diseases, infectious diseases or cancer, or as vaccine adjuvants.

[0230] In a preferred embodiment, the compounds or compositions of the present invention are used as cytotoxic agents for the treatment or prevention of hyperproliferative 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.

[0231] In preferred embodiments, the compounds or compositions of the present invention are used as cytotoxic agents for the treatment or prevention of viral infections.

[0232] In another embodiment, the present invention therefore provides a method for inducing, stimulating, or assisting an immune response in an organism, comprising administering a compound or pharmaceutical composition of the present invention to the organism. In one embodiment, the compounds of the present invention are administered to an organism as immunotherapy to modulate the human or animal immune system and achieve a specific therapeutic effect by inducing in vivo production of various types of cytokines that are therapeutically useful in humans or animals, such as type I interferons and inflammatory cytokines, such as IL-6, TNF-α, and IFN-γ.

[0233] In another embodiment, the present invention therefore provides a method for the treatment or prevention of diseases related to or mediated by an immune response, particularly diseases related to or mediated by STING, such as inflammation, allergic diseases or autoimmune diseases, infectious diseases or cancer, comprising administering a therapeutically effective amount of the compound or pharmaceutical composition of the present invention to a subject in need thereof.

[0234] 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 the 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 recurrent tumors or preventing tumor recurrence, comprising administering a therapeutically effective amount of the compound or pharmaceutical composition of the present invention to a subject in need.

[0235] In a preferred embodiment, the present invention provides a method for treating or preventing a viral infection, comprising administering a therapeutically effective amount of the compound or pharmaceutical composition of the present invention to a subject in need thereof.

[0236] In another embodiment, the present invention therefore provides the use of compounds or pharmaceutical compositions of the present invention for the manufacture of pharmaceuticals for the treatment or prevention of diseases related to or mediated by immune responses, particularly diseases related to or mediated by STING, such as inflammation, allergic diseases or autoimmune diseases, infectious diseases or cancer.

[0237] In another embodiment, the present invention also provides the use of the compounds or pharmaceutical compositions of the present invention in the manufacture of vaccine adjuvants.

[0238] In a preferred embodiment, the present invention provides the use of the compounds or pharmaceutical compositions of the present invention in the manufacture of a pharmacopoeia for the treatment or prevention of hyperproliferative 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 manufacture of a pharmacopoeia for the treatment of recurrent tumors or for the prevention of tumor recurrence.

[0239] In a preferred embodiment, the present invention provides the use of the compound or pharmaceutical composition of the present invention in the manufacture of a pharmaceutical for treating or preventing a viral infection.

[0240] Regarding the above uses and methods, inflammation may be acute or chronic and may involve any organ or tissue of the body, such as musculoskeletal inflammation, vasculitis, neuritis, gastroenteritis, ophthalmitis, inflammation of the reproductive system or other inflammations, and naturally, autoimmune diseases as well as allergic diseases, such as contact dermatitis, urticaria and respiratory allergies of an inflammatory nature.

[0241] Regarding the use and methods described above, autoimmune diseases refer to diseases in which the body's immune response to its own antigens causes damage to its own tissues. Examples, but not limited to, include 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, Crohn's disease, morphea, scleroderma, and others.

[0242] With regard to the above use and method, hyperproliferative disorders refer to physiological conditions in subjects characterized by uncontrolled or disordered cell proliferation or cell death, particularly tumors or cancers such as solid tumors and hematogenous tumors, for example, but not limited to brain cancer, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colorectal cancer, hematological cancer, lung cancer, and bone cancer. Examples of the above cancer types include neuroblastoma, intestinal cancer (e.g., rectal cancer, colorectal cancer, familial adenomatous polyposis cancer, and hereditary nonlymphatic colorectal cancer), esophageal cancer, lip cancer, laryngeal cancer, nasopharyngeal cancer, oral cancer, salivary gland cancer, peritoneal cancer, soft tissue sarcoma, urothelial carcinoma, sweat adenoma, stomach cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, and corpus cancer. Carcinoma, endometrial cancer, pancreatic cancer, prostate cancer, testicular cancer, breast cancer (including HER2-negative breast cancer), urological cancers, melanoma, brain tumors (e.g., glioblastoma, astrocytoma, meningioma, medullary tumor, and peripheral neuroectodermal tumors), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CLL), Other examples include lymphocytic cancers, acute myeloid leukemia (AML), myeloid leukemia (chronic myeloid leukemia (CML)), adult T-cell lymphoma, diffuse lymphoma (DLBCL), liver cancer, multiple myeloma, seminomas, osteosarcoma, chondrosarcoma, anal canal cancer, adrenocortical carcinoma, chordoma, fallopian tube cancer, gastrointestinal stromal tumors, myeloproliferative disorders, mesothelioma, biliary tract cancer, Ewing's sarcoma, and other rare tumor types, as well as recurrent forms of the above tumors.

[0243] In preferred embodiments, the hyperproliferative diseases for which the above uses and methods are applicable 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 lymphoma, leukemia, or bladder cancer.

[0244] Regarding the above-described uses and methods, viral infection refers to the process by which a virus enters the body through various channels and replicates in susceptible host cells. Examples of viruses involved include, but are not limited to, double-stranded DNA viruses and single-stranded DNA viruses, positive-sense single-stranded RNA viruses, negative-sense single-stranded 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, swine fever virus, hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola virus, enterovirus and human immunodeficiency virus. The therapeutic uses and methods provided by the present invention can be used for the above-described viral infections and diseases caused thereby.

[0245] With regard to the use of the compounds or compositions of the present invention as vaccine adjuvants and their use in the manufacture of vaccine adjuvants, it means that the compounds or compositions of the present invention may be used as adjuvants in a vaccine-based therapeutic or preventive strategy, i.e., the compounds or compositions of the present invention may be used together with one or more vaccines selected to stimulate an immune response to one or more predetermined antigens, wherein the vaccines include inactivated or attenuated bacteria or viruses, for example, inactivated tumor cells expressing and secreting one or more GM-CSF, CCL-20, CCL3, IL-12p70, FLT-3 ligand, and cytokines.

[0246] Combinations of medications In view of the preferred pharmacological activity of the compounds of the present invention, in addition to their use alone in the therapeutic uses or methods described above, they may also be used in combination with at least one other therapeutic agent or therapy to provide further therapeutic effects.

[0247] Accordingly, in another embodiment, the present invention also provides pharmaceutical combinations comprising, or both comprising, a cyclic dinucleotide compound, its stereoisomer, tautomer, stable isotope variant, pharmaceutically acceptable salt, prodrug or solvate or pharmaceutical composition thereof, and at least one other therapeutic agent.

[0248] In another embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutical combination described herein and one or more pharmaceutically acceptable excipients.

[0249] In another embodiment, the present invention provides the use of pharmaceutical combinations or pharmaceutical compositions contained herein for the treatment or prevention of diseases such as hyperproliferative diseases, viral infections, or diseases related to or mediated by STING, and more particularly, inflammatory diseases, allergic diseases or autoimmune diseases, infectious diseases or cancer, or in the manufacture of pharmaceuticals for the treatment or prevention of diseases. In a preferred embodiment, the diseases to which the above use relates are tumors or viral infections.

[0250] In another embodiment, the present invention also provides a therapeutic method in which the compound of the present invention is administered together with one or more other therapeutic agents.

[0251] The use of the pharmaceutical combination and pharmaceutical composition containing the present invention for inflammation, autoimmune diseases, hyperproliferative diseases, and viral infections is as described above in relation to the use and methods of the present invention.

[0252] The compounds of the present invention can also be administered in conjunction with surgical procedures, radiotherapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), and immunosuppressant drugs.

[0253] Other therapeutic agents used in combination with the present invention may be administered simultaneously with, separately from, or sequentially to, the compound of the present invention, via the same or different routes of administration. The further therapeutic agents may be co-administered with the compound of the present invention as a single pharmaceutical composition, or may be administered separately from the compound of the present invention as separate units, for example, as a combination product, preferably in the form of a kit. If administered separately, they may be administered simultaneously or sequentially, and in the case of sequential administration, the administration times may be close together or spaced apart. These may be prepared and / or formulated by the same manufacturer or by different manufacturers. Furthermore, the compound of the present invention and other therapeutic agents may be added to the combination therapy (i) before the combination product is sent to the physician (for example, in the case of a kit containing the compound of the present invention and other agents); (ii) immediately before administration by the physician himself (or under the guidance of the physician); or (iii) by the patient himself, for example, during sequential administration of the compound of the present invention and other therapeutic agents.

[0254] Accordingly, in another embodiment, the present invention also provides a kit comprising two or more distinct pharmaceutical compositions, at least one of which comprises a cyclic dinucleotide compound of the present invention, its stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, prodrugs, or solvates, and the remaining coexisting pharmaceutical compositions comprising at least one other therapeutic agent, and a device containing each of the compositions, such as a container, a dispenser bottle, or a separate foil pack, such as a blister pack for tablets or capsules. The kit 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 dosing intervals.

[0255] The other therapeutic agents described above may be one or more further compounds of the present invention, or second or further (e.g., third) therapeutic agents that are compatible with (i.e., do not adversely affect) the compounds of the present invention, or have complementary or different activities.

[0256] Other therapeutic agents that can be used in combination with the compounds of the present invention in certain embodiments 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.

[0257] In certain embodiments, adjuvants used in combination with the compounds of the present invention are useful due to their properties of stimulating or utilizing the immune system to respond to cancer antigens present on tumor cells, and include, but are not limited to, lipids that induce innate immunity, liposomes, inactivated bacteria, and compounds that mediate the activation of innate immunity.

[0258] In certain embodiments, the immune checkpoint inhibitor used in combination with the compound of the present invention is selected from, for example, a CTLA-4 pathway antagonist, a PD-1 pathway antagonist, a Tim-3 pathway antagonist, a Vista pathway antagonist, a BTLA pathway antagonist, a LAG-3 pathway antagonist, or a TIGIT pathway antagonist.

[0259] Examples of T cell receptor agonists used in combination with the compounds of the present invention in certain embodiments include, but are not limited to, CD28 agonists, OX40 agonists, GITR agonists, CD137 agonists, CD27 agonists, or HVEM agonists.

[0260] Examples of TLR agonists used in combination with the compounds of the present invention in certain embodiments 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, reximod, loxoribine, and the like.

[0261] In certain embodiments, chemotherapeutic agents used in combination with the compounds of the present invention include, but are not limited to, alkylating agent-based anticancer agents, platinum-based anticancer agents, antimetabolites, antimicrotubule agents, antimitotic agents, topoisomerase inhibitors, and antitumor antibiotics.

[0262] Examples of therapeutic antibodies used in combination with the compounds of the present invention in specific embodiments include, but are not limited to, muromonab-CD3, infliximab (Remicade®), adalimumab (Humira®), omalizumab (Xolair®), daclizumab (Zenapax®), rituximab (trade name = Rituxan®), and ibritumomab (trade name = Zevalin®). Examples include tositumomab (Bexxar®), cetuximab (Erbitux®), trastuzumab (Herceptin®), Adcetris®, alemtuzumab (Campath-1H®), Lym-1 (Oncolym®), ipilimumab (Yervoy®), vitaxin, bevacizumab (Avastin®), and absiximab (ReoPro®). 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.

[0263] In other embodiments, the compounds of the present invention may also be combined 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.

[0264] Examples of the various 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, and the corresponding content is incorporated herein.

[0265] The compounds, pharmaceutical compositions, methods, uses, pharmaceutical combinations, and kits of the present invention described above are preferably cyclic dinucleotide compounds of the present invention in the various preferred embodiments described above, their stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, prodrugs or solvates, or pharmaceutical compositions thereof. More preferably, the compounds defined in a particular embodiment, i.e., the compounds of Examples 1 to 26, and most preferably, the compounds that exhibit excellent activity in the active examples.

[0266] With regard to the compounds, pharmaceutical compositions, methods, uses, pharmaceutical combinations, and kits of the present invention described above, it is preferable to use the free form or pharmaceutically acceptable salt or prodrug of the cyclic dinucleotide compound as defined herein, and more preferably, it is preferable to use the substantially pure free form or pharmaceutically acceptable salt or prodrug of the cyclic dinucleotide compound as defined herein.

[0267] Regarding the therapeutic use and method of the present invention described above, it is preferable that the target of use or treatment is a mammal, preferably a human.

[0268] Where a dosage of a drug or a pharmaceutically acceptable salt thereof is described herein, unless otherwise indicated, it is understood that such dosage is based on the weight of the free base excluding any hydrate or solvate thereof.

[0269] Method for preparing the compound of the present invention General synthesis methods

[0270] The compounds of the present invention, their stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, or solvates can be prepared by various methods well known in the art of organic synthesis, such as the methods shown below, the methods shown in the examples, or similar methods understood by those skilled in the art.

[0271] A general synthesis scheme for the synthesis of the compounds of the present invention is shown below. For each reaction step, suitable reaction conditions are known to those skilled in the art or can be routinely determined. In particular, the steps for the synthesis of the compounds of the present invention can be carried out under reaction conditions known in themselves, for example, under the reaction conditions specifically mentioned, in the absence or usually in the presence of a solvent or diluent (e.g., a solvent or diluent that is inert to the reagent used and can dissolve the reagent used), in the absence or in the presence of a catalyst, condensing agent or neutralizing agent (e.g., an ion exchanger, e.g., an H+ type cation exchanger), at low temperature, room temperature or high temperature (e.g., about -100°C to about 190°C, e.g., about -78°C to about 150°C, e.g., about 0°C to about 125°C, room temperature, -20 to 40°C or reflux temperature, etc.), and / or at atmospheric pressure or in a sealed container, under appropriate pressure, and / or under an inert atmosphere such as oxygen or nitrogen, depending on the properties of the reactants.

[0272] The starting materials and reagents used in the preparation of these compounds are generally commercially available or can be prepared by the following methods, similar methods, or methods known in the art. The starting materials and intermediates in the synthesis reaction scheme can be isolated and purified by conventional techniques, such as, but not limited to, filtration, distillation, crystallization, and chromatography, as needed. These substances can be characterized using conventional methods, such as physical constants and spectral data.

[0273] Unless otherwise stated in the description of the method, suitable solvents for any particular reaction include, for example: the solvents specifically listed, or, for example, water; esters, e.g., lower alkyl esters of lower fatty acids, e.g., ethyl acetate; ethers, e.g., aliphatic ethers, e.g., diethyl ether, or cyclic ethers, e.g., tetrahydrofuran or dioxane; liquid aromatic hydrocarbons, e.g., benzene or toluene; alcohols, e.g., methanol, ethanol, or 1- or 2-propanol, e.g., acetonitrile; halogenated hydrocarbons, e.g., dichloromethane or chloroform; amides, e.g., N,N-dimethylformamide or N,N-dimethylacetamide; bases, e.g., heterocyclic nitrogen bases, e.g., pyridine; carboxylic acid anhydrides, e.g., lower fatty acid anhydrides, e.g., acetic anhydride; cyclic, linear or branched hydrocarbons, e.g., cyclohexane, hexane or isopentane; or mixtures of these solvents, e.g., aqueous solutions. Such solvent mixtures can also be used for post-treatment, for example, by chromatography or partitioning.

[0274] Those skilled in the art will recognize the presence of stereocenters in the compound of formula I. 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 a racemate or a mixture of diastereomers; see, for example, EL Eliel, SH Wilen and LN Mander, “Stereochemistry of Organic Compounds” (Wiley-Interscience, 1994).

[0275] The following Scheme 1 shows a general synthetic route that can be used to prepare the compounds of Formula II as defined herein and various embodiments thereof. Unless otherwise stated, the variable elements of the general formula in the following scheme have the same meanings as they do in the compounds defined herein or in each of their embodiments.

[0276] [ka] In the formula, P1 and P2 are suitable hydroxyl protecting groups, and P3 and P4 are suitable hydroxyl protecting groups or amino protecting groups, for example, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis(4-methyloxyphenyl)benzyl), Bz (benzoyl), i- Examples include BuCO(isobutyryl). The deprotection used in the synthesis scheme of the compounds of the present invention is carried out under acidic conditions (e.g., acetic acid / water, trifluoroacetic acid / water, etc., not limited to these), under basic conditions (e.g., aqueous ammonia, ammonia / methanol solution, etc., not limited to these), or in the presence of a fluoride anion-containing compound (e.g., tetrabutylammonium fluoride, triethylamine hydrofluoric acid, etc., not limited to these).

[0277] Scheme 2 shows the synthesis routes for the compounds of formulas A and C in Scheme 1, as well as the synthesis of intermediates used further in those routes.

[0278] [ka]

[0279] Scheme 3 shows the synthetic route when the compound of formula H is the compound of formula H-1 (gemcitabine prodrug LY2334737).

[0280] [ka]

[0281] Schemes 4-7 show the synthesis route of the compound of formula B in Scheme 1, and the synthesis of intermediates used further in that route.

[0282] [ka] TIFF0007875196000089.tif47157

[0283] Scheme 8 shows the synthesis route of the compound of formula D.

[0284] [ka]

[0285] In particular, the present invention relates to a method for preparing the above-mentioned compounds of the present invention, Compound of formula A

[0286] [ka] (In the formula, B1, R1, and R1' have the meanings defined above for the compound of formula (II) of the present invention or various specific embodiments thereof; P1 is a preferred hydroxyl protecting group, for example, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), Bz (benzoyl), i BuCO (isobutyryl) And the compound of formula B

[0287] [ka] (In the formula, B2, R2, and R2' have the meanings defined above for the compound of formula (II) of the present invention or various embodiments thereof; P2 is a preferred hydroxyl protecting group, and P3 and P4 are preferred hydroxyl protecting groups or amino protecting groups, respectively, for example, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bi(4-methoxyphenyl)benzyl), Bz (benzoyl), i- BuCO (isobutyryl) The two are reacted in the presence of a base such as DBU; Alternatively, the compound of formula C.

[0288] [ka] (In the formula, B1, R1, R1', and P1 are as defined above for the compound of formula A.) And the compound of formula D

[0289] [ka] (In the formula, B2, R2, R2', P2, P3, and P4 are as defined above for the compound of formula B.) These are reacted in the presence of a base such as DBU to form the compound of formula E.

[0290] [ka] This is obtained and selectively deprotected under conditions such as trifluoroacetic acid / water, tetrabutylammonium fluoride, or triethylamine hydrofluoride to obtain the compound of formula F.

[0291] [ka] (In the formula, each unit has the meaning defined above.) Obtained, a) When R2'=-O(H) and protecting group P3 is benzoyl, the compound of formula F is cyclized with a (-)-PSI reagent in the presence of a base such as DBU to form the cyclic dinucleotide compound of formula G.

[0292] [ka] After obtaining the compound, the benzoyl is deprotected in aqueous ammonia or ammonia-methanol solution to obtain the cyclic dinucleotide compound of formula II.

[0293] [ka] (In the formula, R2' = -O(H), and each other group has the meaning defined above for the compound of formula II or various specific embodiments thereof.) To obtain, Alternatively, b) If R2'=-F or -H, the compound of formula F is cyclized with a (-)-PSI reagent in the presence of a base such as DBU to form the cyclic dinucleotide compound of formula II.

[0294] [ka] (wherein R2' = -F or -H, and each other group has the meaning defined above for the compound of formula II or various specific embodiments thereof.) The present invention provides a method that includes obtaining the above.

[0295] Compounds of formulas A and C can be prepared as follows: Primary alcohol of compound H of formula

[0296] [ka] The compound of formula C is selectively protected in the presence of a base such as imidazole.

[0297] [ka] This is obtained and reacted with a (+)-PSI reagent in the presence of a base such as DBU to form the compound of formula A.

[0298] [ka] (In the formula, B1, R1, R1', and P1 are as defined above for the compound of formula A.) To obtain.

[0299] If the compound of formula H is the compound of formula H-1 (gemcitabine prodrug LY2334737),

[0300] [ka] It is a compound of formula H-1-1.

[0301] [ka] It is prepared using 2-propylpentanoic acid.

[0302] The compound of formula B can be prepared in the form of formula B-1 as follows: Compound of formula H

[0303] [ka] The two hydroxyl groups are protected in the presence of a base such as imidazole, resulting in the compound of formula B-1-1.

[0304] [ka] To obtain the compound of formula B-1-1, the protecting group on the primary alcohol is selectively removed under conditions such as trifluoroacetic acid / water, and the compound of formula B-1 is obtained.

[0305] [ka] (In the formula, B2, R2, R2', and P2 are as defined above for the compound of formula B.) To obtain.

[0306] The compound of formula B can also be prepared in the form of formula B-2, as follows: In the presence of a base such as DBU / pyridine, the compound of formula C

[0307] [ka] Protects the secondary alcohol of the compound of formula B-2-1

[0308] [ka] To obtain the compound of formula B-2-1, the protecting group on the primary alcohol is selectively removed under conditions such as acetic acid / water, and the compound of formula B-2 is obtained.

[0309] [ka] (In the formula, B2, R2, R2', P1, and P2 are as defined above for compounds of formula A or formula B.) To obtain.

[0310] The compound of formula B can also be prepared in the form of formula B-3 as follows: Compound of formula B-3-1

[0311] [ka] Selectively protect one of the primary and secondary alcohols in the presence of a base such as imidazole to form the compound of formula B-3-2.

[0312] [ka] To obtain the compound of formula B-3-2, the unprotected secondary alcohol and the amino group on the base are reacted with a protecting group such as benzoyl chloride in the presence of a base such as N-methylimidazole to obtain the compound of formula B-3-3.

[0313] [ka] To obtain the compound of formula B-3-3, the protecting group on the primary alcohol is selectively removed under conditions such as trifluoroacetic acid / water, and the compound of formula B-3 is obtained.

[0314] [ka] (In the formula, B2, R 2、 R2', P1, P2, and P3 are defined above for compounds of formula A or formula B. To obtain.

[0315] The compound of formula B can also be prepared in the form of formula B-4 as follows: Compound of formula B-4-1

[0316] [ka] The unprotected secondary alcohol is reacted with a protecting group such as benzoyl chloride in the presence of a base such as N-methylimidazole to form the compound of formula B-4-2.

[0317] [ka] To obtain the compound of formula B-4-2, the protecting group on the primary alcohol is selectively removed under conditions such as acetic acid / water, and the compound of formula B-4 is obtained.

[0318] [ka] (In the formula, B2, R 2、 R2', P1, P2, and P4 are defined above for compounds of formula A or formula B. To obtain.

[0319] The compound of formula D can be prepared as follows: Compound of formula B

[0320] [ka] This is reacted with a (-)-PSI reagent in the presence of a base such as DBU to form the compound of formula D.

[0321] [ka] (In the formula, B2, R2, R2', P2, P3, and P4 are as defined above for the compound of formula B.) To obtain.

[0322] The following scheme 9 shows a general synthetic route that can be used to prepare the compounds of formulas (Ib), (II-b), (III-b), and (IV-b) as defined herein, and various embodiments thereof. Unless otherwise stated, the variable elements of the general formulas in the following scheme have the same meanings as they do in the compounds defined herein or in their respective embodiments.

[0323] [ka] In each formula, X is independently a hydroxyl or a mercapto; R1, R1', B1 and B2 are as defined above for each of formulas (Ib), (II-b), (III-b), (IV-b) and their sub-general formulas and specific embodiments; P1 and P2 are preferred hydroxyl protecting groups, and P3 and P4 are preferred hydroxyl protecting groups or amino protecting groups, for example, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), Bz (benzoyl), i- Examples include BuCO(isobutyryl). P5 is a suitable hydroxyl protecting group or mercapto protecting group on the phosphate, and examples include, but are not limited to, cyanoethyl.

[0324] The deprotection used in the above-described synthesis scheme of the compounds of the present invention is carried out under acidic conditions (e.g., acetic acid / water, trifluoroacetic acid / water), under basic conditions (e.g., aqueous ammonia, ammonia / methanol solution, methylamine / ethanol solution, lithium hydroxide), or in the presence of a fluorine anion-containing compound (e.g., tetrabutylammonium fluoride, triethylamine hydrofluoride, ammonium fluoride). The oxidation used in the synthesis scheme of the compounds of the present invention is carried out under conditions such as the presence of iodine (e.g., iodine), and the sulfidation is carried out under conditions such as the presence of 3H-1,2-benzodisulfonol-3-one (e.g., 3H-1,2-benzodisulfonol-3-one).

[0325] Scheme 10 shows the synthesis route of the compound of formula J in Scheme 9, and the synthesis of intermediates used further in that route.

[0326] [ka]

[0327] Scheme 11 shows the synthesis route of the compound of formula K, and the synthesis of intermediates used therein.

[0328] [ka]

[0329] In particular, the present invention relates to a method for preparing the above-mentioned compounds of the present invention: Compound of formula J

[0330] [ka] (In the formulas, 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 specific embodiments thereof; P3 is a suitable hydroxyl protecting group, for example, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), Bz (benzoyl), i- (Examples include BuCO (isobutyryl)) and the compound of formula K

[0331] [ka] (wherein B2 has the meaning defined above for compounds of formula (Ib), (II-b), (III-b), (IV-b) of the present invention or specific embodiments thereof; P1, P2 are preferred hydroxyl protecting groups, and P4 is a preferred hydroxyl protecting group or amino protecting group, for example, but not limited to TBS (tert-butyldimethylsilyl), DMTr (bis(4-methoxyphenyl)benzyl), Bz (benzoyl), i- (Examples include BuCO (isobutyryl)) You can react them in the presence of a base (for example, DBU), Alternatively, a compound of formula J and a compound of formula L

[0332] [ka] (In the formula, B2, P1, P2, and P4 are as defined above for the compound of formula K.) The two are reacted in the presence of tetrazole to oxidize or sulfurize (wherein the oxidation conditions are not limited, but examples include the use of iodine, tert-butyl hydroperoxide, etc., and the sulfurization conditions are not limited, but examples include the use of N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formamidine (DDTT), 3H-1,2-benzodisulfonal (disulfonol)-3-one, etc.; the introduction and removal of protecting groups are carried out according to standard methods well known to those skilled in the art), Compound of formula M

[0333] [ka] (In the formula, X is OH or SH, and P5 is a suitable hydroxyl protecting group or mercapto protecting group on the phosphoric acid / phosphate, such as, but not limited to, cyanoethyl.) To obtain the compound of formula M, selectively deprotect it under conditions such as lithium hydroxide to obtain the compound of formula N.

[0334] [ka] (In the formula, each unit has the meaning defined above.) Obtained, a) Reacting the compound of formula N with a (+)-PSI reagent or a (+)-PSI reagent in the presence of a base such as DBU to obtain the compound of formula O

[0335] [ka] The compound of formula P is obtained, and then the P1 protection (e.g., DMTr) is removed in acetic acid / aqueous solution to obtain the compound of formula P.

[0336] [ka] To obtain the compound of formula P, in the presence of a base such as DBU, the compound of formula Q is cyclized to form a cyclic dinucleotide compound of formula Q.

[0337] [ka] Obtain a cyclic dinucleotide compound of formula (Ib), (II-b), (III-b), or (IV-b) where R2 is OH and R2' is H, by removing the P4 protection (e.g., TBS) under conditions such as ammonium fluoride.

[0338] [ka] (In the formula, each unit has the meaning defined above.) Obtained, b) Alternatively, react the compound of formula N with diphenyl phosphite in the presence of a base such as DBU to obtain the compound of formula R.

[0339] [ka] The compound of formula S is obtained by removing the P1 protection (e.g., DMTr) in acetic acid / aqueous solution.

[0340] [ka] The compound of formula S is obtained, and the compound is cyclized in the presence of an activating reagent such as pivaloyl chloride, and then oxidized or sulfurized to obtain the cyclic dinucleotide compound of formula Q.

[0341] [ka] The following is obtained: the P4 protection (e.g., TBS) is removed under conditions such as ammonium fluoride to obtain a cyclic dinucleotide compound of formula (Ib), (II-b), (III-b), or (IV-b) where R2 is OH and R2' is H.

[0342] [ka] (In the formula, each unit has the meaning defined above.) To obtain The present invention provides the above method, which includes the following:

[0343] The compound of formula J can be prepared as follows: Compound of formula C

[0344] [ka] The secondary alcohol is protected in the presence of a base such as N-methylimidazole to form the compound of formula J-1.

[0345] [ka] To obtain the compound of formula J-1, selective deprotection is performed to obtain the compound of formula J.

[0346] [ka] (In the formula, each group is as defined above.) To obtain.

[0347] The compound of formula K can be prepared as follows: Compound of formula K-1

[0348] [ka] This is reacted with a (+)-PSI reagent in the presence of a base such as DBU to form the compound of formula K.

[0349] [ka] (In the formula, each group is as defined above.) To obtain.

[0350] Unless otherwise specified, the experimental materials and reagents used in the above synthesis methods and schemes can be commercially available, prepared according to prior art methods, or prepared according to methods similar to those disclosed herein. Unless otherwise specified, the synthesis conditions used in the above synthesis methods and schemes can be routinely determined by those skilled in the art.

[0351] The present invention also relates to a preparation method in which the remaining process steps are carried out using a compound that can be obtained as an intermediate in any step of the various preparation methods and schemes described herein as a starting material, or the starting material is formed in situ under reaction conditions, or used in the form of a derivative, such as a protected form or a salt form, or the compound that can be obtained by the method according to the present invention is generated under process conditions and further processed in situ. [Examples]

[0352] The present invention is further illustrated below with reference to examples. It should be noted that the following examples are not intended to limit the scope of protection of the present invention.

[0353] Unless there is an obvious error in the structural formula, if the chemical name of any compound in the present invention does not match a given structural formula, the structural formula shall take precedence.

[0354] In the following examples, experimental methods for which no specific conditions are indicated generally follow conventional conditions for this type of reaction or conditions suggested by the manufacturer. Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercially available sources, prepared according to prior art methods, or prepared according to methods similar to those disclosed in this application.

[0355] Unless otherwise indicated, percentages and parts are based on weight; ratios of liquids are based on volume; and unless otherwise indicated, all temperatures are given in degrees Celsius.

[0356] In the following examples, 1 H NMR spectrum 31P NMR spectra were typically recorded using Bruker 400 MHz and 500 MHz nuclear magnetic resonance spectrometers, with chemical shifts expressed in δ (ppm); mass spectra were recorded using Agilent 1290 liquid chromatography + 6120B mass spectrometer LCMS liquid chromatography-mass spectrometry. Silica gel column purification was performed using Biotage SelektSEL-2SV or ISO-1SV; preparative liquid chromatography purification was performed using Gilson 281 (column: Waters Xbridge 19 x 250 mm, 5 μm or WELCH C18, 21.2 x 250 mm, 10 μm; mobile phase A: water (10 mM NH4HCO3 or 0.05% formic acid), B: acetonitrile (or containing 0.05% formic acid); flow rate: 20-30 mL / min; detection wavelength: 214 nm / 254 nm) or as otherwise specified.

[0357] The following abbreviations are used in the synthesis examples below, and each abbreviation not listed has a meaning that will be generally understood by those skilled in the art.

[0358] List of abbreviations TIFF0007875196000141.tif191150 Synthesis Example Preparation of Intermediate 1 :2-Chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine

[0359] [ka]

[0360] To a 15 mL DMF solution 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 successively added, and the resulting mixture was stirred at 20-25°C for 16 hours. The reaction mixture was poured into 150 mL of water and extracted twice with ethyl acetate (100 mL each). The organic phases were combined, washed twice with saturated brine (100 mL each), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column elution with petroleum ether:ethyl acetate (2:3) to obtain a white solid (2.53 g). 1 H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.89 (brs, 2H), 6.33 (dd, J = 12.0, 4.8 Hz, 1H), 6.00 (d, J = 4.8 Hz, 1H), 5.28 (dt, J = 52, 4.8 Hz, 1H), 4.50-4.30 (m, 1H), 3.98-3.73 (m, 3H), 0.89 (s, 9H), 0.07 (s, 6H).

[0361] Preparation of Intermediate 2 :(2S,3aR,6S,7aR)-2-((2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purine-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]oxathiophosphoran 2-sulfide

[0362] [ka]

[0363] To a 10 mL solution (1.00 g, 2.40 mmol) of intermediate 1 (1.00 g, 2.40 mmol) and (+)-PSI reagent (1.39 g, 3.12 mmol) in THF at 0°C, 1,8-diazabicycloundeca-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 successively washed 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 elution with petroleum ether:ethyl acetate (3:7) to obtain a white solid (1.50 g). 1 H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 6.52-6.40 (m, 1H), 6.23 (brs, 2H), 5.55-5.40 (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 (162 MHz, CDCl3) δ 101.9.

[0364] Preparation of Intermediate 3 :2-Chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine

[0365] [ka]

[0366] Intermediate 3 was obtained from 2'-deoxyadenosine via the pathway of intermediate 1. 1H NMR (400 MHz, DMSO-d6) δ 8.29 (s, 1H), 7.82 (brs, 2H), 6.26 (t, J = 6.4 Hz, 1H), 5.37 (d, J = 4.0 Hz, 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).

[0367] Preparation of intermediate 4 :(2S,3aR,6S,7aR)-2-((2R,3S,5R)-5-(6-amino-2-chloro-9H-purine-9-yl)-2-((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-3-yl)oxy)-3a-methyl-6-(propyl-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiophosphoran 2-sulfide

[0368] [ka]

[0369] Intermediate 4 was obtained from intermediate 3 and the (+)-PSI reagent via the pathway of intermediate 2. 1 H NMR (400 MHz, CDCl3) δ 8.65 (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 (162 MHz, CDCl3) δ 100.6.

[0370] Preparation of intermediate 5 :5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0371] [ka]

[0372] Intermediate 5 was obtained from 2'-deoxy-2',2'-difluorocytidine via the pathway of intermediate 1. 1 H NMR (400 MHz, DMSO-d6) δ 7.63 (d, J = 7.6 Hz, 1H), 7.37 (brs, 2H), 6.29 (d, J = 6.8 Hz, 1H), 6.14 (t, J = 7.6 Hz, 1H), 5.75 (d, J = 7.6 Hz, 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.

[0373] 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]oxythiophosphoran-2-yl)oxy)tetrahydrofuran-2-yl)pyrimidine-2(1H)-one

[0374] [ka]

[0375] Intermediate 6 was obtained from intermediate 5 and the (+)-PSI reagent via the intermediate pathway. 1H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J = 8.0 Hz, 1H), 6.34 (t, J = 8.0 Hz, 1H), 6.21 (d, J = 8.0 Hz, 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 (162 MHz, CDCl3) δ 103.0. MS-ESI [M+H] + : 624.2.

[0376] Preparation of intermediate 7 :N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine

[0377] [ka]

[0378] Trimethylchlorosilane (3.61 g, 33.4 mmol) was added dropwise to a solution of 2'-deoxy-2',2'-difluorocytidine hydrochloride (2.00 g, 6.67 mmol) in pyridine (20 mL) at 0°C, and the resulting mixture was stirred at 0-5°C for 2 hours. Meanwhile, carbonyldiimidazole (1.19 g, 7.33 mmol) was gradually added to a solution of 2-propylvaleric acid (1.06 g, 7.33 mmol) in acetonitrile (20 mL), and the resulting mixture was stirred at 25°C for 2 hours. Next, the resulting acetonitrile mixture was added dropwise to the pyridine mixture at 0°C, and the resulting mixture was stirred at 45°C for 16 hours. Ethanol (20 mL) was added to the above mixture and stirred at 45°C for 0.5 hours. Then, water (20 mL) was added to the above mixture and stirred at 45°C for 1 hour. The resulting reaction solution was dried by rotary evaporation 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). The organic phases were combined, washed twice with water (50 mL each), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified using a silica gel column eluted with petroleum ether:ethyl acetate (2:3) to obtain a white solid (1.20 g). 1 H NMR (400 MHz, DMSO-d6) δ 11.06 (brs, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 6.32 (d, J = 8.0 Hz, 1H), 6.17 (t, J = 8.0 Hz, 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 = 7.6 Hz, 6H). MS-ESI [M+H] + : 390.2.

[0379] Preparation of intermediate 8:N4-(2-propylpentanoyl)-5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0380] [ka]

[0381] Intermediate 8 was obtained from intermediate 7 via the pathway of intermediate 1. 1 H NMR (400 MHz, CDCl3) δ 9.07 (brs, 1H), 8.27 (brs, 1H), 7.51 (d, J = 8.0 Hz, 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, 2.4 Hz, 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).

[0382] 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]oxythiophosphoran-2-yl)oxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidine-4-yl)-2-propylpentanamide

[0383] [ka]

[0384] Intermediate 9 was obtained from intermediate 8 and the (+)-PSI reagent via the pathway of intermediate 2. 1H NMR (400 MHz, CDCl3) δ 9.63 (brs, 1H), 8.29-8.15 (m, 1H), 7.60-7.48 (m, 1H), 6.48-6.39 (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 = 4.0 Hz, 6H).

[0385] 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

[0386] [ka]

[0387] To a solution of intermediate 1 (1.53 g, 3.67 mmol) in pyridine (10 mL), bis(4-methoxyphenyl)benzyl chloride (1.49 g, 4.40 mmol) was added, and the resulting mixture was stirred at 25°C for 2 hours. Then, bis(4-methoxyphenyl)benzyl chloride (497 mg, 1.47 mmol) and 1,8-diazabicycloundeca-7-ene (669 mg, 4.40 mmol) were added successively, and the resulting mixture was stirred at 20-25°C for 16 hours. Subsequently, bis(4-methoxyphenyl)benzyl chloride (1.49 g, 4.40 mmol) and 1,8-diazabicycloundeca-7-ene (669 mg, 4.40 mmol) were added successively, and the resulting 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) and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column elution with petroleum ether:ethyl acetate (5:1) to obtain a white solid (2.60 g). 1 H NMR (400 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.95 (s, 1H), 7.50-7.10 (m, 18H), 6.90-6.78 (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).

[0388] Preparation of intermediate 11 : N6-bis(4-methoxyphenyl)benzyl-2-chloro-3'-bis(4-methoxyphenyl)benzyl-2'-deoxy-2'-fluoro-beta-adenosine

[0389] [ka]

[0390] To a solution of intermediate 10 (2.60 g, 2.55 mmol) in tetrahydrofuran (15 mL), tetrabutylammonium fluoride (1.33 g, 5.10 mmol) was added, 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 by concentration was purified by eluting with petroleum ether:ethyl acetate (2:3) using a silica gel column to obtain a white solid (1.20 g). 1 H NMR (400 MHz, DMSO-d6) δ 8.17 (d, J = 2.4 Hz, 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 = 6.0 Hz, 1H), 4.30-4.10 (m, 3H), 3.76-3.68 (m, 12H), 3.49-3.30 (m, 2H).

[0391] Preparation of intermediate 12 : N6-bis(4-methoxyphenyl)benzyl-2-chloro-5'-O-tert-butyldimethylsilyl-3'-bis(4-methoxyphenyl)base)benzyl-2'-deoxyadenosine

[0392] [ka]

[0393] To a solution of intermediate 3 (1.80 g, 4.51 mmol) in pyridine (10 mL), bis(4-methoxyphenyl)benzyl chloride (3.81 g, 11.3 mmol) and 1,8-diazabicycloundeca-7-ene (743 mg, 11.3 mmol) were added, and 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). The organic phases were combined, washed twice with saturated brine (50 mL each), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column elution with petroleum ether:ethyl acetate (3:7) to obtain a white solid (2.50 g). 1 H NMR (400 MHz, DMSO-d6) δ 8.19 (s, 1H), 7.83 (s, 1H), 7.50-7.10 (m, 18H), 6.95-6.78 (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).

[0394] Preparation of intermediate 13 : N6-bis(4-methoxyphenyl)benzyl-2-chloro-3'-bis(4-methoxyphenyl)benzyl-2'-deoxyadenosine

[0395] [ka]

[0396] Intermediate 13 was obtained from intermediate 12 via the pathway of intermediate 11. 1H NMR (400 MHz, DMSO-d6) δ 8.32 (s, 1H), 7.87 (s, 1H), 7.50-7.10 (m, 18H), 6.95-6.77 (m, 8H), 6.24 (t, J = 6.8 Hz, 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).

[0397] Preparation of intermediate 14 :3'-5'-di-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0398] [ka]

[0399] To a solution of 2'-deoxy-2',2'-difluorocytidine hydrochloride (3.00 g, 10.0 mmol) in DMF (50 mL), imidazole (3.41 g, 50.0 mmol) and tert-butyldimethylsilyl chloride (4.53 g, 30.0 mmol) were successively added. The resulting mixture was stirred at 20-25°C for 2 hours, and then 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 successively 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 elution with dichloromethane:methanol (9:2) to obtain a white solid (2.70 g). 1H NMR (400 MHz, DMSO-d6) δ 7.53 (d, J = 7.6 Hz, 1H), 7.40 (brs, 2H), 6.17 (t, J = 8.0 Hz, 1H), 5.78 (d, J = 7.6 Hz, 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).

[0400] Preparation of intermediate 15 :3'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0401] [ka]

[0402] At 0°C, intermediate 14 (1.00 g, 2.04 mmol) was dissolved in a mixture of tetrahydrofuran (10 mL), trifluoroacetic acid (5 mL), and water (5 mL), and the mixture was stirred at 0-5°C for 2 hours. The pH of the reaction mixture was adjusted to approximately 9 with 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 elution with ethyl acetate:methanol (17:3) to obtain a white solid (620 mg). 1 H NMR (400 MHz, DMSO-d6) δ 7.67 (d, J = 7.6 Hz, 1H), 7.50-7.35 (m, 2H), 6.14 (t, J = 8.0 Hz, 1H), 5.79 (d, J = 7.6 Hz, 1H), 5.26 (d, J = 7.6 MS-ESI [M+H]+ :378.2.

[0403] Preparation of intermediate 16 : N4-(2-propyl-pentanoyl)-3'-5'-di-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0404] [ka]

[0405] At 25°C, carbonyl diimidazole (363 mg, 2.24 mmol) was added to a solution of 2-propylvaleric acid (322 mg, 2.24 mmol) in acetonitrile (5 mL), and the resulting mixture was stirred at 25°C for 2 hours. The resulting mixture was added dropwise to a solution of intermediate 14 (1.0 g, 2.03 mmol) in pyridine (10 mL) at 0°C. The resulting mixture was stirred at 45°C for 16 hours, and the resulting reaction solution was dried by rotary evaporation, diluted with water (20 mL), and extracted twice with ethyl acetate (30 mL each). The organic phases were combined, washed twice with water (50 mL each), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column with petroleum ether:ethyl acetate (1:1) to obtain a white solid (430 mg). 1 H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 7.6 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 6.33 (d, J = 10.0 Hz, 1H), 4.39-4.29 (m, 1H), 4.02 (d, J = 12.0 Hz, 1H), 3.96 (d, J = 7.6 Hz, 1H), 3.81 (d, J = 11.2 Hz, 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.

[0406] Preparation of intermediate 17 : N4-(2-propyl-pentanoyl)-3'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine

[0407] [ka]

[0408] Intermediate 17 was obtained from intermediate 16 via the pathway of intermediate 15. 1 H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 6.25 (t, J = 7.6 Hz, 1H), 4.49-4.40 (m, 1H), 4.08 (d, J = 12.4 Hz, 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 = 2.8 Hz, 6H). MS-ESI [M+H] + :504.2.

[0409] Intermediate 18 : 5'-O-tert-butyldimethylsilyl-2'-tert-butyldimethylsilyl-adenosine, and Intermediate 19 : 5'-O-tert-butyldimethylsilyl-3'-tert-butyldimethylsilyl-adenosine At room temperature (10°C), triethylenediamine (7.0 g, 62.5 mmol) and silver nitrate (5.08 g, 28.8 mmol) were successively added to a solution of adenosine (3.35 g, 12.5 mmol) in tetrahydrofuran (40 mL). This mixture was stirred at room temperature (10°C) for 30 minutes, then tert-butyldimethylsilyl chloride (4.71 g, 30 mmol) was added, and the resulting mixture was stirred at 10°C for 16 hours. The reaction mixture was filtered through Celite, the filtrate was quenched with water (50 mL), and then extracted twice with ethyl acetate (50 mL each). The organic phases were combined, washed twice with saturated brine (100 mL each), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column elution with petroleum ether:ethyl acetate (1:1) to obtain intermediate 18 (1.0 g, white solid). Elution with ethyl acetate yielded intermediate 19 (4.4 g, white solid).

[0410] Intermediate 18:

[0411] [ka] 1 H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 8.24 (s, 1H), 6.11 (d, J =4.8 Hz, 1H), 5.72 (brs, 2H), 4.64 (t, J = 4.8 Hz, 1H), 4.31-4.26 (m, 1H), 4.24-4.20 (m, 1H), 4.02 (dd, J = 11.6, 2.8 Hz, 1H), 3.87 (dd, J = 11.6, 2.8 Hz, 1H), 2.76 (d, J = 3.6 Hz, 1H), 0.96 (s, 9H), 0.84 (s, 9H), 0.18-0.11 (m, 6H), -0.05 (s, 3H), -0.12 (s, 3H).

[0412] Intermediate 19:

[0413] [ka] 1 H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 8.10 (s, 1H), 6.03 (d, J = 3.6 Hz, 1H), 5.75 (brs, 2H), 4.56 (d, J = 1.6 Hz, 1H), 4.12 (d, J = 2.8 Hz, 1H), 3.92 (dd, J = 11.6, 3.6 Hz, 1H), 3.77 (dd, J = 11.2, 2.8 Hz, 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).

[0414] Preparation of Intermediate 20 : N4-benzoyl-2'-5'-di-O-tert-butyldimethylsilyl-3'-benzoyl-adenosine

[0415] [ka]

[0416] At room temperature (25°C), 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). 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). The combined organic phase was 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 elution with petroleum ether:ethyl acetate (1:1) to obtain a white solid (400 mg). 1H NMR (400 MHz, CDCl3) δ 9.36 (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 = 6.0 Hz, 1H), 5.63-5.50 (m, 1H), 4.89 (d, J = 1.2 Hz, 1H), 4.48 (d, J = 2.4 Hz, 1H), 4.05 (d, J = 2.4 Hz, 1H), 4.00 (d, J = 2.4 Hz, 1H), 0.99 (s, 9H), 0.65 (s, 9H), 0.19 (d, J = 4.4 Hz, 6H), -0.10 (s, 3H), -0.26 (s, 3H). MS-ESI [M+H] + :704.3.

[0417] Preparation of intermediate 21 : N4-benzoyl-2'-O-tert-butyldimethylsilyl-3'-benzoyl-adenosine

[0418] [ka]

[0419] Intermediate 21 was obtained from intermediate 20 via the pathway of intermediate 15. 1H NMR (500 MHz, CDCl3) δ 8.86 (s, 1H), 8.17-8.09 (m, 3H), 8.08-8.03 (m, 2H), 7.66 -7.60 (m, 2H), 7.55 (t, J = 7.6 Hz, 2H), 7.50 (t, J = 7.6 Hz, 2H), 5.97 (d, J = 7.6 Hz, 1H), 5.75 (d, J = 5.2 Hz, 1H), 5.27 (dd, J = 7.6, 5.2 Hz, 1H), 4.51 (s, 1H), 4.08-4.03 (m, 1H), 3.93 (d, J = 12.8 Hz, 1H), 0.62 (s, 9H), -0.10 (s, 3H), -0.43 (s, 3H). MS-ESI [M+H] + :591.2.

[0420] Preparation of intermediate 22 : N4-benzoyl-5'-O-tert-butyldimethylsilyl-3'-tert-butyldimethylsilyl-2'-benzoyl-adenosine

[0421] [ka]

[0422] Intermediate 22 was obtained from intermediate 19 and benzoyl chloride via the route of intermediate 20. 1H NMR (400 MHz, CDCl3) δ 9.02 (brs, 1H), 8.83 (s, 1H), 8.41 (s, 1H), 8.08-7.99 (m, 4H), 7.64-7.49 (m, 4H), 7.47-7.39 (m, 2H), 6.54 (d, J = 6.0 Hz, 1H), 5.81 (t, J = 5.6 Hz, 1H), 4.84 (dd, J = 4.8, 3.6 Hz, 1H), 4.26 (dd, J = 6.0, 3.2 Hz, 1H), 4.02 (dd, J = 11.6, 3.2 Hz, 1H), 3.85 (dd, J = 11.6, 2.8 Hz, 1H), 0.95 (s, 9H), 0.84 (s, 9H), 0.18-0.11 (m, 6H), 0.03--0.05 (m, 6H).

[0423] Preparation of intermediate 23 : N4-benzoyl-3'-tert-butyldimethylsilyl-2'-benzoyl-adenosine

[0424] [ka]

[0425] Intermediate 23 was obtained from intermediate 22 via the pathway of intermediate 15. 1 H NMR (400 MHz, CDCl3) δ 9.08 (brs, 1H), 8.84 (s, 1H), 8.18 (s, 1H), 8.08-7.98 (m, 4H), 7.67-7.41 (m, 6H), 6.32 (d, J = 7.6 Hz, 1H), 6.01 (dd, J = 7.6, 5.2 Hz, 1H), 4.94 (d, J = 5.2 Hz, 1H), 4.35 (d, J = 0.8 Hz, 1H), 4.07 (dd, J = 13.2, 1.6 Hz, 1H), 3.84 (d, J = 12.4 Hz, 1H), 0.89 (s, 9H), 0.11 (s, 3H), 0.00 (s, 3H).

[0426] Preparation of intermediate 24 : N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-guanosine

[0427] [ka]

[0428] Intermediate 24 was obtained from N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-2'-O-tert-butyldimethylsilyl-guanosine via the route of intermediate 20. MS-ESI [M+H] + :874.3.

[0429] Preparation of intermediate 25 : N2-isobutyryl-3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-guanosine

[0430] [ka]

[0431] 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 by rotation and dried, and then evaporated twice with ethyl acetate (25 mL each). The crude product was purified by silica gel column elution with ethyl acetate to obtain a white solid (2.2 g). 1H NMR (400 MHz, CDCl3) δ 12.15 (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 = 7.6 Hz, 1H), 5.66 (dd, J = 5.6, 1.2 Hz, 1H), 5.52 (d, J = 9.2 Hz, 1H), 4.98 (dd, J = 7.2, 5.6 Hz, 1H), 4.45 (d, J = 1.2 Hz, 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.

[0432] Preparation of intermediate 26 : N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-tert-butyldimethylsilyl-2'-benzoylguanosine

[0433] [ka]

[0434] Intermediate 26 was obtained from N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-tert-butyldimethylsilyl-guanosine via the route of intermediate 20. MS-ESI [M+H] + :874.3.

[0435] Preparation of intermediate 27 : N2-isobutyryl-3'-tert-butyldimethylsilyl-2'-benzoyl guanosine

[0436] [ka]

[0437] Intermediate 27 was obtained from intermediate 26 via the pathway of intermediate 25. 1 H NMR (400 MHz, CDCl3) δ 12.06 (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 = 8.0 Hz, 2H), 6.13 (d, J = 7.2 Hz, 1H), 5.81 (dd, J = 7.2, 5.6 Hz, 1H), 4.80 (dd, J = 4.8, 1.6 Hz, 1H), 4.30-4.25 (m, 1H), 4.02 (dd, J = 12.8, 2.0 Hz, 1H), 3.78 (d, J = 12.4 Hz, 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).

[0438] Preparation of intermediate 28 : 2'-5'-di-O-tert-butyldimethylsilyl-guanosine

[0439] [ka]

[0440] Intermediate 28 was obtained from guanosine via the pathway of intermediate 18. MS-ESI [M+H] + :513.3.

[0441] Preparation of intermediate 29 : 2'-5'-di-O-tert-butyldimethylsilyl-3'-benzoyl-guanosine

[0442] [ka]

[0443] Intermediate 29 was obtained from intermediate 28 via the pathway of intermediate 20. 1 H NMR (400 MHz, CDCl3) δ 12.04 (brs, 1H), 8.14-8.09 (m, 2H), 7.94 (s, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.49 (t, J = 7.6 Hz, 2H), 6.09 (s, 2H), 6.00 (d, J = 6.0 Hz, 1H), 5.53 (s, 1H), 4.76 (t, J = 5.6 Hz, 1H), 4.41 (d, J = 2.8 Hz, 1H), 4.01 (d, J = 11.6 Hz, 1H), 3.93 (dd, J = 11.6, 2.4 Hz, 1H), 0.97 (s, 9H), 0.70 (s, 9H), 0.16 (d, J = 3.2 Hz, 6H), -0.09 (s, 3H), -0.16 (s, 3H). - :615.3.

[0444] Preparation of intermediate 30 : 2'-O-tert-butyldimethylsilyl-3'-benzoyl-guanosine

[0445] [ka]

[0446] Intermediate 30 was obtained from intermediate 29 via the pathway of intermediate 15. 1H NMR (400 MHz, DMSO-d6) δ 10.71 (brs, 1H), 8.05 (d, J = 7.2 Hz, 3H), 7.75-7.68 (m, 1H), 7.59 (t, J = 7.6 Hz, 2H), 6.51 (s, 2H), 5.84 (d, J = 7.6 Hz, 1H), 5.58 (dd, J = 5.6, 1.6 Hz, 1H), 5.42 (t, J = 5.6 Hz, 1H), 4.96 (dd, J = 7.6, 5.2 Hz, 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 [MH] - :501.3.

[0447] Preparation of intermediate 31 : 3'-5'-di-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroadenosine

[0448] [ka]

[0449] To a solution of 2'-deoxy-2'-fluoroadenosine (1.10 g, 4.09 mmol) in DMF (10 mL), imidazole (1.81 g, 26.6 mmol) and 4-dimethylaminopyridine (50 mg, 0.41 mmol) were added, followed by the addition of tert-butyldimethylsilyl chloride (2.16 g, 14.3 mmol). The resulting 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). The organic phases were combined, 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 elution with petroleum ether:ethyl acetate (3:2) to obtain a white solid (1.90 g). 1H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 8.12 (s, 1H), 6.25 (dd, J = 16.0, 2.4 Hz, 1H), 5.66 (brs, 2H), 5.48-5.20 (m, 1H), 4.79-4.62 (m, MS-ESI [M+H] + :498.4.

[0450] Preparation of intermediate 32 : 3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroadenosine

[0451] [ka]

[0452] Intermediate 31 was obtained from intermediate 31 via the pathway of intermediate 15. 1 H NMR (400 MHz, DMSO-d6) δ 8.37 (s, 1H), 8.14 (s, 1H), 7.38 (brs, 2H), 6.22 (dd, J = 16.4, 3.6 Hz, 1H), 5.70-5.40 (m, 1H), 5.29 (t, J = 5.6 Hz, 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).

[0453] Preparation of intermediate 33: (2R,3aS,6R,7aS)-2-((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3-((tertbutyldimethylsilyl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)-3a-methyl-6-(propyl-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiophosphoran 2-sulfide

[0454] [ka]

[0455] Intermediate 33 was obtained from intermediate 32 and the (-)-PSI reagent via the pathway of intermediate 2. 1 H NMR (400 MHz, CDCl3) δ 8.36 (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), 4.50-4.25 (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).

[0456] Preparation of intermediate 34: 3'-5'-di-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroguanosine

[0457] [ka]

[0458] Intermediate 34 was obtained from 2'-deoxy-2'-fluoroguanosine via the pathway of intermediate 31. 1H NMR (400 MHz, DMSO-d6) δ 10.69 (brs, 1H), 7.84 (s, 1H), 6.52 (brs, 2H), 6.00 (d, J = 16.8 Hz, 1H), 5.36 (d, J = 52.0 Hz, 1H), 4.62-4.45 (m, 1H), 3.98-3.86 (m, 2H), 3.74 (d, J = 11.6 Hz, 1H), 0.93 -0.85 (m, 18H), 0.20-0.00 (m, 12H). MS-ESI [M+H] + :515.3.

[0459] Preparation of intermediate 35 : 3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroguanosine

[0460] [ka]

[0461] Intermediate 35 was obtained from intermediate 34 via the pathway of intermediate 15. 1 H NMR (400 MHz, DMSO-d6) δ 10.71 (brs, 1H), 7.97 (s, 1H), 6.56 (s, 2H), 5.36 (dd, J = 52.5, 5.0 Hz, 1H), 5.20 (s, 1H), 4.52 (dd, J = 12.6, MS-ESI [M+H] + :401.2.

[0462] 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]oxathiophosphoran-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one

[0463] [ka]

[0464] Intermediate 36 was obtained from intermediate 35 in DMF and a (-)-PSI reagent via the pathway of intermediate 2. MS-ESI [M+H] + :646.4.

[0465] Preparation of intermediate 37 : [3'-O-phosphorothioate-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]

[0466] [ka]

[0467] To a solution of intermediate 2 (280 mg, 0.422 mmol) and intermediate 11 (383 mg, 0.422 mmol) in acetonitrile (3 mL), 1,8-diazabicycloundeca-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 successively washed 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 elution with ethyl acetate:methanol (7:3) to obtain a white solid (260 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.26 (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).

[0468] Preparation of intermediate 38 : [3'-O-phosphorothioate-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]

[0469] [ka]

[0470] Intermediate 38 was obtained from intermediates 4 and 13 in a mixed solution of tetrahydrofuran and acetonitrile, via the route of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H), 8.22 (s, 1H), 7.82 (brs, 2H), 7.75 (s, 1H), 7.42 (d, J = 8.0 Hz, 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).

[0471] Preparation of intermediate 39 : [3'-O-phosphorothioate-diester-5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[3'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine]

[0472] [ka]

[0473] Intermediate 39 was obtained from intermediates 6 and 15 in a mixed solution of tetrahydrofuran and acetonitrile, via the route of intermediate 37. 1 H NMR (400 MHz, DMSO-d6) δ 9.58 (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, 1.6 Hz, 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).

[0474] Preparation of intermediate 40: [3'-O-phosphorothioate-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]

[0475] [ka]

[0476] Intermediate 40 was obtained from intermediates 17 and 9 via the pathway of intermediate 37. 1 H NMR (500 MHz, DMSO-d6) δ 11.07 (brs, 1H), 11.02 (brs, 1H), 8.32 (d, J = 7.6 Hz, 1H), 8.24 (d, J = 7.6 Hz, 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, 8.5 Hz, 2H), 4.04*3.99 (m, 2H), 3.93 (t, J = 7.8 Hz, 1H), 2.62 (d, J = 3.2 Hz, 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 = 3.0 Hz, 6H), 0.05 (s, 3H), 0.02 (s, 3H).

[0477] Preparation of intermediate 41 : [3'-O-phosphorothioate-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]

[0478] [ka]

[0479] Intermediate 41 was obtained from intermediates 21 and 2 via the pathway of intermediate 37. 1 H NMR (400 MHz, DMSO-d6) δ 11.19 (brs, 1H), 9.10 (brs, 1H), 8.76 (s, 1H), 8.12 (d, J = 2.4 Hz, 1H), 8.05 (dd, J = 7.6, 2.0 Hz, 4H), 7.89 (s, 2H), 7.75-7.61 (m, 2H), 7.60-7.45 (m, 4H), 6.33 (dd, J = 18.0, 4.0 Hz, 1H), 6.23 (d, J = 7.2 Hz, 1H), 5.77 (d, J = 5.2 Hz, 1H), 5.30 (dd, J = 7.6, 5.2 Hz, 1H), 5.04 (s, 1H), 4.53 (s, 1H), 4.21 (t, J = 10.0 Hz, 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.

[0480] Preparation of intermediate 42 : [3'-O-phosphorothioate-diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine]-(3',5')-[N6-benzoyl-2'-O-tert-butyldimethylsilyl-3'-O-benzoyl-adenosine]

[0481] [ka]

[0482] Intermediate 42 was obtained from intermediates 21 and 4 via the pathway of intermediate 37. MS-ESI [(M+H)] + : 1067.2.

[0483] Preparation of intermediate 43 : [3'-O-phosphorothioate-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]

[0484] [ka]

[0485] Intermediate 43 was obtained from intermediates 23 and 2 in N,N'-dimethylformamide via the route of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 11.23 (brs, 1H), 8.96 (s, 1H), 8.76 (s, 1H), 8.11 (d, J = 2.4 Hz, 1H), 8.02 (d, J = 7.6 Hz, 2H), 7.88 (d, J = 7.6 Hz, 4H), 7.64 (dd, J = 6.8, 6.0 Hz, 2H), 7.65-7.45 (m, 4H), 6.51 (d, J = 5.6 Hz, 1H), 6.32 (dd, J = 18.8, 3.6 Hz, 1H), 5.92 (t, J = 5.6 Hz, 1H), 5.55-5.30 (m, 1H), 5.11-4.98 (m, 1H), 4.83 (t, J = 4.0 Hz, 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.

[0486] Preparation of intermediate 44 : [3'-O-phosphorothioate-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]

[0487] [ka]

[0488] Intermediate 44 was obtained from intermediates 2 and 25 in DMF via the pathway of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 12.59 (brs, 1H), 12.11 (brs, 1H), 8.37 (s, 1H), 8.14 (d, J = 2.8 Hz, 1H), 8.08-7.85 (m, 4H), 7.80-7.50 (m, 3H), 6.35 (dd, J = 18.0, 4.0 Hz, 1H), 5.92 (d, J = 8.0 Hz, 1H), 5.68 (d, J = 5.2 Hz, 1H), 5.55-5.38 (m, 2H), 5.15-4.98 (m, 1H), 4.52-4.40 (m, 1H), 4.20-3.95 MS-ESI [(M-2H) / 2] - : 532.2.

[0489] Preparation of intermediate 45 : [3'-O-phosphorothioate-diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-guanosine]

[0490] [ka]

[0491] Intermediate 45 was obtained from intermediates 4 and 25 in DMF via the pathway of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 12.76 (brs, 1H), 12.12 (brs, 1H), 8.37 (s, 1H), 8.27 (s, 1H), 8.03 (d, J = 7.6 Hz, 2H), 7.81 (brs, 2H), 7.70-7.50 (m, 3H), 6.29 (t, J = 6.8 Hz, 1H), 5.92 (d, J = 7.6 Hz, 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.

[0492] Preparation of intermediate 46 : [3'-O-phosphorothioate-diester-5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-guanosine]

[0493] [ka]

[0494] Intermediate 46 was obtained from intermediates 6 and 25 in DMF via the pathway of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 12.49 (brs, 1H), 12.09 (brs, 1H), 8.39 (brs, 1H), 8.05 (d, J = 7.2 Hz, 2H), 7.78-7.50 (m, 4H), 7.40 (brs, 2H), 6.19 (t, J = 8.4 Hz, 1H), 5.91 (d, J = 8.0 Hz, 1H), 5.78 (d, J = 7.6 Hz, 1H), 5.66 (d, J = 4.8 Hz, 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.

[0495] Preparation of intermediate 47 : [3'-O-phosphorothioate-diester-N4-(2-propylpentanoyl)-5'-O-tert-butyldimethylsilyl-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[2'-O-tert-butyldimethylsilyl-3'-O-benzoyl-guanosine]

[0496] [ka]

[0497] Intermediate 47 was obtained from intermediates 30 and 9 in DMF, via the pathway of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 11.10 (brs, 1H), 10.60 (brs, 1H), 8.22 (d, J = 7.6 Hz, 1H), 8.16 (s, 1H), 8.02 (d, J = 7.6 Hz, 2H), 7.68 (t, J = 7.6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.35 (d, J = 7.6 Hz, 1H), 6.51 (brs, 2H), 6.23 (t, J = 7.6 Hz, 1H), 5.82 (d, J = 7.6 Hz, 1H), 5.67 (d, J = 5.2 Hz, 1H), 5.09 (dd, J = 7.6, 5.2 Hz, 1H),5.02-4.95 (m, 1H), 4.41 (d, J = 3.6 Hz, 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.

[0498] Preparation of intermediate 48 : [3'-O-phosphorothioate-diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N2-isobutyryl-3'-tert-butyldimethylsilyl-2'-benzoyl-guanosine]

[0499] [ka]

[0500] Intermediate 48 was obtained from intermediates 27 and 2 in DMF, via the pathway of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 12.53 (brs, 1H), 12.13 (brs, 1H), 8.36 (s, 1H), 8.14 (d, J = 2.8 Hz, 1H), 7.98-7.80 (m, 4H), 7.64 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 8.0 Hz, 2H), 6.36 (dd, J = 18.0, 4.0 Hz, 1H), 6.22 (d, J = 7.2 Hz, 1H), 5.88 (dd, J = 7.2, 5.2 Hz, 1H), 5.60--5.43 (m, 1H), 5.19-5.05 (m, 1H), 4.72 (d, J = 4.8 Hz, 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).

[0501] Preparation of intermediate 49 : [3'-O-phosphorothioate-diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-adenosine]-(3',5')-[N2-isobutyryl-3'-tert-butyldimethylsilyl-2'-benzoyl-guanosine]

[0502] [ka]

[0503] Intermediate 49 was obtained from intermediates 27 and 4 in DMF, via the pathway of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 12.55 (brs, 1H), 12.14 (brs, 1H), 8.37 (s, 1H), 8.28 (s, 1H), 7.94-7.76 (m, 4H), 7.65 (t, J = 7.6 Hz, 1H), 7.49 (t, J = 8.0 Hz, 2H), 6.33-6.21 (m, 2H), 5.95-5.85 (m, 1H), 5.10-5.02 (m,1H), 4.75 (d, J = 4.8 Hz, 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), 2.76-2.71 (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).

[0504] Preparation of intermediate 50 : [3'-O-phosphorothioate-diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroadenosine]

[0505] [ka]

[0506] Intermediate 50 was obtained from intermediates 32 and 2 in DMF, via the pathway of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 8.49 (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, 4.4 Hz, 1H), 5.30 (d, J = 52.0 Hz, 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 = 8.8 Hz, 6H), 0.04 (s, 6H).

[0507] Preparation of intermediate 51 :[3'-O-phosphorothioate-diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine]-(3',5')-[3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroadenosine]

[0508] [ka]

[0509] Intermediate 51 was obtained from intermediates 3 and 33 via the pathway of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (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 = 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).

[0510] Preparation of intermediate 52 : [3'-O-phosphorothioate-diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroguanosine]

[0511] [ka]

[0512] Intermediate 52 was obtained from intermediates 1 and 36 in DMF via the pathway of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 10.64 (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, 4.8 Hz, 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).

[0513] Preparation of intermediate 53 : [3'-O-phosphorothioate-diester-2-chloro-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine]-(3',5')-[3'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoroguanosine]

[0514] [ka]

[0515] Intermediate 53 was obtained from intermediates 3 and 36 in DMF via the pathway of intermediate 37. 1H NMR (400 MHz, DMSO-d6) δ 10.65 (brs, 1H), 8.25 (s, 1H), 8.05 (s, 1H), 7.80 (brs, 2H), 6.55 (brs, 2H), 6.25 (t, J = 7.2 Hz, 1H), 6.03-5.95 (m, 1H), 5.47 (d, J = 52.0 Hz, 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 = 11.2 Hz, 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.

[0516] Preparation of intermediate 54 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0517] [ka]

[0518] 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 the mixture was stirred at 25°C for 2 hours. The reaction mixture was evaporated by rotary evaporation and dried, diluted with methanol (5 mL), and the pH of the mixture was adjusted to approximately 8-9 with saturated sodium bicarbonate aqueous solution. After evaporating by rotary evaporation and drying, it was diluted again with methanol (5 mL) and filtered. The filtrate was concentrated, and the resulting crude product was purified by silica gel column elution with ethyl acetate:methanol (7:3) to obtain a white solid (120 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.30 (s, 1H), 8.23 ​​(s, 1H), 7.88 (brs, 4H), 6.40-6.18 (m, 2H), 6.06 (d, J = 5.2 Hz, 1H), 5.45-5.12 (m, 2H), 5.04 (t, J = 6.0 Hz, 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 [MH] - :682.8.

[0519] Preparation of intermediate 55 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxyadenosine]-(3',5')-[2-chloro-2'-deoxyadenosine]

[0520] [ka]

[0521] To a solution of intermediate 38 (150 mg, 0.110 mmol) in tetrahydrofuran (5 mL), tetrabutylammonium fluoride (34 mg, 0.132 mmol) was added, and the resulting mixture was stirred at 25°C for 3 hours. The reaction mixture was rotated and dried, then dissolved in a mixed solution of glacial acetic acid (2 mL) and water (0.5 mL), and stirred at 25°C for 0.5 hours. The reaction mixture was rotated and dried, and the resulting crude product was purified by silica gel column elution with ethyl acetate:methanol (3:1) to obtain a white solid (45 mg). MS-ESI [MH]-: 647.0.

[0522] Preparation of intermediate 56 : [3'-O-phosphorothioate-diester-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[2'-deoxy-2',2'-difluorocytidine]

[0523] [ka]

[0524] To a solution of intermediate 39 (350 mg, 0.421 mmol) in tetrahydrofuran (5 mL), tetrabutylammonium fluoride (329 mg, 1.26 mmol) was added, and the resulting mixture was stirred at 25°C for 2 hours. The reaction mixture was rotated and evaporated to dry, and the resulting crude product was purified by silica gel column elution with ethyl acetate:methanol (3:1) to obtain a white solid (130 mg). 1 H NMR (400 MHz, DMSO-d6) δ 7.77 (d, J = 7.2 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.45-7.25 (m, 4H), 6.30 (d, J = 6.4 Hz, 1H), 6.20-6.05 (m, 2H), 5.89-5.70 (m, 2H), 5.13 (t, J = 6.0 Hz, 1H), 4.90-4.75 (m, 1H), 4.20-4.00 (m, 2H), 4.00-3.60 (m, 5H).

[0525] Preparation of intermediate 57 : [3'-O-phosphorothioate-diester-N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]

[0526] [ka]

[0527] Intermediate 57 was obtained from intermediate 40 via the pathway of intermediate 56. MS-ESI [M+H] + :858.2, [(M+2H) / 2] + : 429.1.

[0528] Preparation of intermediate 58: [3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N6-benzoyl-3'-O-benzoyl-adenosine]

[0529] [ka]

[0530] Intermediate 58 was obtained from intermediate 41 via the pathway of intermediate 56. MS-ESI [M+H] + :857.0.

[0531] Preparation of intermediate 59 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxyadenosine]-(3',5')-[N6-benzoyl-3'-O-benzoyl-adenosine]

[0532] [ka]

[0533] Intermediate 59 was obtained from intermediate 42 via the pathway of intermediate 56. MS-ESI [M+H] + :839.0.

[0534] Preparation of intermediate 60 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N6-benzoyl-2'-O-benzoyl-adenosine]

[0535] [ka]

[0536] To a solution of intermediate 43 (250 mg, 0.23 mmol) in tetrahydrofuran (5 mL), triethylamine hydrofluoric acid (223 mg, 1.38 mmol) was added and the mixture was stirred at 30°C for 16 hours. The reaction mixture was neutralized with triethylamine to pH 7-8. The resulting mixture was dried by rotary evaporation, and the crude product was purified by silica gel column elution with ethyl acetate:methanol (8:2) to obtain a white solid (160 mg). MS-ESI [M+H] + :857.1.

[0537] Preparation of intermediate 61 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-guanosine]

[0538] [ka]

[0539] Intermediate 61 was obtained from intermediate 44 via the pathway of intermediate 60. 1 H NMR (400 MHz, DMSO-d6) δ 12.56 (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.

[0540] Preparation of intermediate 62: [3'-O-phosphorothioate-diester-2-chloro-2'-deoxyadenosine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-guanosine]

[0541] [ka]

[0542] Intermediate 62 was obtained from intermediate 45 via the pathway of intermediate 60. 1 H NMR (400 MHz, DMSO-d6) δ 12.72 (brs, 1H), 12.13 (brs, 1H), 8.34 (s, 1H), 8.32 (s, 1H), 8.03 (d, J = 7.6 Hz, 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.

[0543] Preparation of intermediate 63 : [3'-O-phosphorothioate-diester-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[N2-isobutyryl-3'-O-benzoyl-guanosine]

[0544] [ka]

[0545] Intermediate 63 was obtained from intermediate 46 via the pathway of intermediate 60. 1 H NMR (400 MHz, DMSO-d6) δ 12.42 (brs, 1H), 12.12 (brs, 1H), 8.35 (s, 1H), 8.08 (d, J = 7.6 Hz, 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 = 5.2 Hz, 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).

[0546] Preparation of intermediate 64 : [3'-O-phosphorothioate-diester-N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]-(3',5')-[3'-O-benzoyl-guanosine]

[0547] [ka]

[0548] Intermediate 64 was obtained from intermediate 47 via the pathway of intermediate 56. MS-ESI [(M+2H) / 2] + :428.0.

[0549] Preparation of intermediate 65 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[N2-isobutyryl-2'-benzoyl-guanosine]

[0550] [ka]

[0551] Intermediate 65 was obtained from intermediate 48 via the pathway of intermediate 56. MS-ESI[MH]-:837.0.

[0552] Preparation of intermediate 66 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-adenosine]-(3',5')-[N2-isobutyryl-2'-benzoyl-guanosine]

[0553] [ka]

[0554] Intermediate 66 was obtained from intermediate 49 via the pathway of intermediate 56. MS+ESI [M+H] + :821.1.

[0555] Preparation of intermediate 67 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[2'-deoxy-2'-fluoroadenosine]

[0556] [ka]

[0557] Intermediate 67 was obtained from intermediate 50 via the pathway of intermediate 60. 1 H NMR (400 MHz, DMSO-d6) δ 8.47 (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 = 16.4 Hz, 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.

[0558] Preparation of intermediate 68 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxyadenosine]-(3',5')-[2'-deoxy-2'-fluoroadenosine]

[0559] [ka]

[0560] Intermediate 68 was obtained from intermediate 51 via the pathway of intermediate 56. 1 H NMR (400 MHz, DMSO-d6) δ 8.50 (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 = 5.6 Hz, 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).

[0561] Preparation of intermediate 69 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-(3',5')-[2'-deoxy-2'-fluoroguanosine]

[0562] [ka]

[0563] Intermediate 69 was obtained from intermediate 52 via the pathway of intermediate 60. 1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.24 (s, 1H), 8.00 (s, 1H), 7.88 (brs, 2H), 6.54 (brs, 2H), 6.28 (dd, J = 18.0, 3.6 Hz, 1H), 6.02 (dd, J = 16.0, 3.6 Hz, 1H), 5.80 (d, J = 5.4 Hz, 1H), 5.44 (d, J = 4.4 Hz, 1H), 5.30 (d, J = 7.6 Hz, 1H), 5.04 (d, J = 6.0 Hz, 1H), 4.95 (t, J = 14.8 Hz, 1H), 4.42 (dt, J = 10.4, 5.6 Hz, 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.

[0564] Preparation of intermediate 70 : [3'-O-phosphorothioate-diester-2-chloro-2'-deoxyadenosine]-(3',5')-[2'-deoxy-2'-fluoroguanosine]

[0565] [ka]

[0566] Intermediate 69 was obtained from intermediate 53 via the pathway of intermediate 60. 1H NMR (400 MHz, DMSO-d6) δ 10.67 (brs, 1H), 8.37 (s, 1H), 8.04 (s, 1H), 7.85 (brs, 2H), 6.58 (brs, 2H), 6.26 (t, J = 7.2 Hz, 1H), 6.03 (d, J = 15.6 Hz, 1H), 5.85 (d, J = 5.6 Hz, 1H), 5.38 (d, J = 52.8 Hz, 1H), 5.10 (s, 1H), 4.97 (s, 1H), 4.46 (d, J = 14.8 Hz, 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.

[0567] Preparation of intermediate 71 : (2',3')-cyclo-[2'-O-phosphorothioate-diester-N6-benzoyl-3'-benzoyl-adenosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0568] [ka]

[0569] To a solution of intermediate 58 (95 mg, 0.11 mmol) and (-)-PSI reagent (150 mg, 0.33 mmol) in DMF (10 mL), 1,8-diazabicycloundeca-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 by rotation and dried, and reprecipitation was performed with ethyl acetate (20 mL). The resulting solid was slurryed with ethyl acetate (20 mL), filtered, and dried to obtain the crude product (200 mg), which was used directly in the next reaction: MS-ESI [M+2+H] + : 937.0.

[0570] Preparation of intermediate 72: (2',3')-cyclo-[2'-O-phosphorothioate-diester-N6-benzoyl-3'-O-benzoyl-adenosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxyadenosine]

[0571] [ka]

[0572] To a solution of intermediate 59 (210 mg, 0.25 mmol) and (-)-PSI reagent (335 mg, 0.75 mmol) in DMF (10 mL), 1,8-diazabicycloundeca-7-ene (570 mg, 3.75 mmol) was added, and the resulting mixture was stirred at 25°C for 1 hour. The reaction mixture was evaporated by rotation and dried, and reprecipitation was performed 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 (with 10 mM ammonium bicarbonate as an additive), and after lyophilization, a white solid (15.8 mg) was obtained, which was used directly in the following reaction: MS-ESI [M+H] + : 917.0.

[0573] Preparation of intermediate 73 : (3',3')-cyclo-bis-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-[3'-O-phosphorothioate-diester-N6-benzoyl-2'-O-benzoyl-adenosine]

[0574] [ka]

[0575] Intermediate 73 was obtained from intermediate 60 and the (-)-PSI reagent via the pathway of intermediate 71. MS-ESI [MH]-: 932.8.

[0576] Intermediate 74: (2',3')-cyclo-[2'-O-phosphorothioate-diester-3'-O-benzoyl-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-isomer 1, and Intermediate 75 Preparation of (2',3')-cyclo-[2'-O-phosphorothioate-diester-3'-O-benzoyl-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-isomer 2

[0577] [ka]

[0578] Intermediates 74 and 75 were obtained from intermediate 61 and the (-)-PSI reagent via the pathway of intermediate 72.

[0579] Intermediate 74: MS-ESI [MH]-: 844.8, retention time: 4.96 min;

[0580] Intermediate 75: MS-ESI [MH] - : 844.9, Retention time: 5.20 min. Analysis LCMS: Agilent 1100+G1946D LCMS, 4.6 x 150 mm Waters XBridge C18 3.5 μm analytical column, mobile phase A: 10 mM NH4HCO3 aqueous solution, B: acetonitrile, flow rate 1.0 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0~0.1 min, 5% B; 0.1~8 min, 5~95% B; 8~15 min, 95% B.

[0581] Intermediate 76 : (2',3')-cyclo-[2'-O-phosphorothioate-diester-N2-isobutyryl-3'-O-benzoyl-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-adenosine]-isomer 1, and Intermediate 77Preparation of (2',3')-cyclo-[2'-O-phosphorothioate-diester-N2-isobutyryl-3'-O-benzoyl-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-adenosine]-isomer 2

[0582] [ka]

[0583] Intermediates 76 and 77 were obtained from intermediate 62 and a (-)-PSI reagent via the pathway of intermediate 72.

[0584] Intermediate 76:MS-ESI [MH]-: 897.2, retention time: 1.69 min;

[0585] Intermediate 77:MS-ESI [MH] - : 897.2, Retention time: 1.74 min. Analysis LCMS: Agilent 1100+G1946D LCMS, 4.6 x 50 mm Waters XBridge C18 3.5 μm analytical column, Mobile phase A: 10 mM NH4HCO3 aqueous solution, B: Acetonitrile, Flow rate 1.8 min / min, Dual wavelength UV absorption monitoring at 214 nm 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.

[0586] Preparation of intermediate 78 : (2',3')-Cyclo-[2'-O-phosphorothioate-diester-N2-isobutyryl-3'-O-benzoyl-guanosine]-[3'-O-phosphorothioate-diester-2'-deoxy-2',2'-difluorocytidine]

[0587] [ka]

[0588] Intermediate 78 was obtained from intermediate 63 and a (-)-PSI reagent via the pathway of intermediate 72. Intermediate 78: MS-ESI [(M-2H) / 2]-: 437.0.

[0589] Preparation of intermediate 79 : (2',3')-Cyclo-[2'-O-phosphorothioate-diester-3'-O-benzoyl-guanosine]-[3'-O-phosphorothioate-diester-N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]

[0590] [ka]

[0591] Intermediate 79 was obtained from intermediate 64 and the (-)-PSI reagent via the pathway of intermediate 72. MS-ESI [MH]-: 931.0.

[0592] Intermediate 80 : (3',3')-cyclo-[3'-O-phosphorothioate-diester-N2-isobutyryl-2'-benzoyl-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-isomer 1, and Intermediate 81: (3',3')-Cyclo-[3'-O-phosphorothioate-diester-N2-isobutyryl-2'-benzoyl-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-isomer 2

[0593] [ka]

[0594] Intermediates 80 and 81 were obtained from intermediate 65 and the (-)-PSI reagent via the pathway of intermediate 72.

[0595] Intermediate 80: MS-ESI [MH]-: 915.0, retention time: 1.50 min;

[0596] Intermediate 81: MS-ESI [MH] - :915.0, retention time:1.53 min. Analysis LCMS: Agilent 1100+G1946D LCMS, 4.6 x 50 mm Waters XBridge C18 3.5 μm analytical column, mobile phase A: 10 mM NH4HCO3 aqueous solution, B: acetonitrile, flow rate 1.8 mL / min, dual wavelength UV absorption monitoring at 214 nm 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.

[0597] Preparation of intermediate 82 : (3',3')-cyclo-[3'-O-phosphorothioate-diester-N2-isobutyryl-2'-benzoyl-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-adenosine]

[0598] [ka]

[0599] Intermediate 82 was obtained from intermediate 66 and a (-)-PSI reagent via the pathway of intermediate 72. MS-ESI [MH]-: 896.8.

[0600] Preparation of intermediate 83 : 2-Chloro-5'-O-tert-butyldimethylsilyl-3'-O-benzoyl-2'-deoxy-2'-fluoro-beta-adenosine

[0601] [ka]

[0602] Intermediate 83 was obtained from intermediate 1 via the pathway of intermediate 20. 1H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J = 2.4 Hz, 1H), 8.08 (d, J = 7.2 Hz, 2H), 7.95 (brs, 2H), 7.72 (t, J = 7.6 Hz, 1H), 7.58 (t, J = 7.6 Hz, 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 = 4.8 Hz, 1H), 3.97 (d, J = 4.4 Hz, 2H), 0.86 (s, 9H), 0.06 (s, 6H).

[0603] Preparation of intermediate 84 : 2-Chloro-3'-O-benzoyl-2'-deoxy-2'-fluoro-beta-adenosine

[0604] [ka]

[0605] Intermediate 84 was obtained from intermediate 83 via the pathway of intermediate 60. 1 H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 2.4 Hz, 1H), 8.12 (d, J = 7.6 Hz, 2H), 7.99 (brs, 2H), 7.74 (t, J = 7.2 Hz, 1H), 7.60 (t, J = 7.6 Hz, 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 = 4.4 Hz, 1H), 3.89-3.80 (m, 2H).

[0606] 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-(propa-1-en-2-yl)-2-thio-hexahydrobenzo[d][1,3,2]oxathiophosphoran-2-yl)oxy)tetrahydrofuran-2-yl)-6-oxy-6,9-dihydro-1H-purine-2-yl)isobutylamide

[0607] [ka]

[0608] Intermediate 85 was obtained from N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-tert-butyldimethylsilyl-guanosine and a (+)-PSI reagent via the pathway of intermediate 2. 1 H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 11.62 (s, 1H), 8.14 (s, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.31-7.22 (m, 7H), 6.86 (d, J = 8.4 Hz, 4H), 6.00 (d, J = 6.8 Hz, 1H), 5.67-5.59 (m, 1H), 4.89 (s, 1H), 4.75 (s, 1H), 4.42 (d, J = 4.0 Hz, 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 = 12.0 Hz, 1H), 2.82-2.73 (m, 1H), 2.07 (d, J = 12.0 Hz, 1H), 1.93-1.83 (m, 2H), 1.76-1.65 (mf, 6H), 1.54 (s, 3H), 1.12 (d, J = 6.8 Hz, 6H), 0.83 (s, 9H), 0.08 (s, 3H), 0.04 (s, 3H).

[0609] Preparation of intermediate 86 : [2'-O-Rp-phosphorothioate-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]

[0610] [ka]

[0611] Intermediate 86 was obtained from intermediates 84 and 85 in DMF via the pathway of intermediate 37. MS-ESI [M+H] + : 1255.0.

[0612] Preparation of intermediate 87 : [2'-O-Rp-phosphorothioate-diester-N2-isobutyryl-5'-O-bis(4-methoxyphenyl)benzyl-3'-O-tert-butyldimethylsilyl-guanosine]-(2',5')-[2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0613] [ka]

[0614] Intermediate 86 (1.0 g, 0.8 mmol) was added to a tetrahydrofuran / water / methanol mixture (10 mL), 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 minutes. The reaction mixture was poured into water (50 mL) and extracted twice with ethyl acetate (50 mL each). The organic phases were combined, washed twice with saturated brine (100 mL each), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column elution with ethyl acetate:methanol (10:1) to obtain a white solid (733 mg). 1H NMR (400 MHz, DMSO-d6) δ 12.02 (s, 1H), 11.67 (s, 1H), 8.21 (t,J= 5.2 Hz, 1H), 8.14 (s, 1H), 7.84 (s, 2H), 7.37 (d,J= 7.6 Hz, 2H), 7.29-7.14 (m, 7H), 6.87-6.77 (m, 4H), 6.23 (dd,J= 13.9, 4.5 Hz, 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= 13.4 Hz, 6H).

[0615] Preparation of intermediate 88 ; O-((2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purine-9-yl)-4-fluoro-3-((2R,3aS,6R,7aS)-3a-methyl-6-(propa-1-en-2-yl)-2-thiohexahydrobenzo[d][1,3,2]oxathiophosphoran-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-purine-9-yl)tetrahydrofuran-3-yl)-(R)-phosphorothioate-diester

[0616] [ka]

[0617] Intermediate 88 was prepared as a crude product from intermediate 87 in tetrahydrofuran and a (-)-PSI reagent via the pathway of intermediate 2, and was used directly in the following reaction: MS-ESI [M+H-DMTr] + : 1095.0.

[0618] Preparation of intermediate 89 : O-((2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purine-9-yl)-4-fluoro-3-((2R,3aS,6R,7aS)-3a-methyl-6-(propa-1-en-2-yl)-2-thiohexahydrobenzo[d][1,3,2]oxathiophosphoran-2-yl)methyl)-O-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutylamino-6-oxy-1,6-dihydro-9H-purine-9-yl)tetrahydrofuran-3-yl)-(R)-phosphorothioate-diester

[0619] [ka]

[0620] Intermediate 89 was obtained from intermediate 88 via the pathway of intermediate 25. MS-ESI [M+H] + :1095.0.

[0621] Preparation of intermediate 90 : (2',3')-Cyclo-(Rp,Sp)-[2'-O-phosphorothioate-diester-N2-isobutyryl-3'-O-tert-butyldimethylsilyl-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0622] [ka]

[0623] Intermediate 89 (90 mg, 0.082 mmol) was dissolved in anhydrous DMF (7 mL), 4A molecular sieve (90 mg) was added, the mixture was stirred, and it was dried under nitrogen at room temperature (28°C) for 0.5 hours. Then, DBU (75.0 mg, 0.492 mmol) was added by syringe, the mixture was stirred at room temperature (28°C) for 0.5 hours, and Celite was added for filtration. The filtrate was dried by rotary evaporation to obtain crude intermediate 90 (100 mg, crude product), which was used directly in the next reaction. MS-ESI [M+H] + :926.8.

[0624] Preparation of intermediate 91 : (2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purine-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-purine-9-yl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylbenzoate

[0625] [ka]

[0626] 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-purine-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramide (1.78 g, 1.84 mmol) were dissolved in a mixed solution of acetonitrile / tetrahydrofuran (8 mL / 8 mL), a 4A molecular sieve (500 mg) was added, and the mixture was stirred at room temperature (28°C) for 0.5 hours under nitrogen protection. Next, a solution of tetrazole in acetonitrile (8.17 mL, 0.45 M) was added by 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 the mixture was stirred at room temperature (28°C) for 20 minutes. The mixture was then quenched with 50% sodium thiosulfate pentahydrate aqueous solution (5 mL); Celite was added for filtration, the filtrate was diluted with ethyl acetate (30 mL), washed with water (20 mL, twice), and the organic phase was dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by silica gel column elution with petroleum ether:ethyl acetate (10:1~1:10) to obtain a mixture (1.9 g), which was used directly in the next step. MS-ESI [M+H] + :1296.0.

[0627] 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]

[0628] [ka]

[0629] Intermediate 91 (1 g, 0.775 mmol) was mixed with 33% methylamine in ethanol (8 mL) and stirred at room temperature for 3 hours. The reaction solution was dried by rotary evaporation, purified using a C18 reversed-phase column, eluted with water (containing 0.1% ammonium bicarbonate):acetonitrile (3:2), and a pink solid (550 mg) was obtained after lyophilization. 1 H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 8.18 (d, J = 2.0 Hz, 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 = 5.6 Hz, 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.

[0630] 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]

[0631] [ka]

[0632] Under nitrogen protection at 0°C, diphenyl phosphite (137 mg, 0.47 mmol) was added to a solution of intermediate 92 (250 mg, 0.235 mmol), DBU (143 mg, 0.94 mmol), and 4A molecular sieve in pyridine (8 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction solution was filtered, dried by rotary evaporation, and used directly in the next reaction: MS-ESI [M+H]+ : 1129.8.

[0633] 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]

[0634] [ka]

[0635] Intermediate 94 was obtained from intermediate 93 via the pathway of intermediate 25. 1 H NMR (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.99 (s, 1H), 7.88 (s, 2H), 6.55 (s, 2H), 6.27 (dd, J = 17.2, 4.0 Hz, 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 = 4.0 Hz, 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 (162 MHz, DMSO-d6) δ 1.72, -1.58. MS-ESI [M+H] + : 826.8.

[0636] 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]

[0637] [ka]

[0638] At 0°C, pivaloyl chloride (48 mg, 0.40 mmol) was added to a solution of intermediate 94 (55 mg, 0.067 mmol) in pyridine (15 mL), and the resulting reaction solution was stirred at room temperature for 3 hours. Next, a mixed solution of iodine (26 mg, 0.10 mmol) and water (0.1 mL) in acetonitrile (0.5 mL) was added, and the resulting reaction solution was stirred at room temperature for 16 hours. The reaction solution was quenched with 10% sodium thiosulfate aqueous solution (5 mL), then dried by rotary evaporation, purified on a C18 reversed-phase column eluted with water (containing 0.1% ammonium bicarbonate):acetonitrile (4:1), and freeze-dried to obtain a white solid (20 mg). MS-ESI [M+H] + : 825.0.

[0639] Preparation of intermediate 96 : (2',3')-Cyclo-[2'-O-Rp-phosphorothioate-diester-N2-isobutyryl-3'-O-tert-butyldimethylsilyl-guanosine]-[3'-O-phosphodiester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0640] [ka]

[0641] Intermediate 96 was obtained 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] via the pathway of intermediate 95. 1H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H), 12.12 (s, 1H), 11.54 (s, 1H), 8.55 (d, J = 2.0 Hz, 1H), 8.23 ​​(s, 1H), 8.06 (d, J = 7.2 Hz, 2H), 7.71-7.61 (m, 1H), 7.59-7.51 (m, 3H), 6.47 (dd, J = 21.6, 2.4 Hz, 1H), 5.97-5.74 (m, 2H), 5.60-5.40 (m, 1H), 5.12-5.00 (m, 1H), 4.41 (d, J = 3.2 Hz, 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 = 7.2 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.93 (s, 9H), 0.20 (d, J = 8.6 Hz, 6H).

[0642] Preparation of intermediate 97 : (2',3')-Cyclo-[2'-O-phosphodiester-3'-O-tert-butyldimethylsilyl-guanosine]-[3'-O-Rp-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0643] [ka]

[0644] Intermediate 97 was obtained via the pathway of intermediate 95 by reacting intermediate 92 with a (+)-PSI reagent, then deprotecting and cyclizing it, and purifying it 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, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm 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; retention time of compound: 12.5 min). 1 H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.18 (d,J = 2.4 Hz, 1H), 8.11 (s, 1H), 7.45 - 6.98 (m, 5H), 6.33 - 6.23 (m, 1H), 5.82 (d,J = 8.6 Hz, 1H), 5.42 (s, 1H), 5.32 - 5.23 (m, 2H), 4.33 (d,J = 4.0 Hz, 1H), 4.19 - 3.70 (m, 9H), 0.91 (s, 9H), 0.15 (s, 6H). 19 F NMR (376 MHz, DMSO) δ -196.51 (s). 31 P NMR (162 MHz, DMSO) δ 53.76 (s), -0.49 (s). MS-ESI [M+H] + : 841.0.

[0645] Preparation of intermediate 98 : (2',3')-Cyclo-(Rp,Rp)-[2'-O-phosphorothioate-diester-N2-isobutyryl-3'-O-tert-butyldimethylsilyl-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0646] [ka]

[0647] Intermediate 98 was obtained via the synthetic route of intermediate 90 by reacting intermediate 87 with a (+)-PSI reagent, followed by deprotection and cyclization. 1 H NMR (400 MHz, DMSO-d6) δ 12.41 (brs, 1H), 11.90 (s, 1H), 11.31 (s, 1H), 8.35 (d, J = 2.0 Hz, 1H), 8.03 (s, 1H), 7.85 (d, J = 7.2 Hz, 2H), 7.45 (t, J = 7.2 Hz, 1H), 7.35 (t, J = 7.6 Hz, 2H), 6.88 (t, J = 48 Hz, 6H), 6.25 (dd, J = 20.0, 4.0 Hz, 1H), 5.65 (d, J = 8.4 Hz, 1H), 5.59-5.46 (m, 1H), 5.32 (d, J = 50.0 Hz, 1H), 5.02 (t, J = 11.2 Hz, 1H), 4.15 (d, J = 4.0 Hz, 1H), 4.07-4.01 (m, 1H), 3.97-3.64 (m, 5H), 2.74-2.62 (m, 1H), 0.86 (d, J = 6.4 Hz, 3H), 0.81-0.66 (m, 12H), -0.01 (d, J = 9.6 Hz, 6H). MS+ESI--[M+H]: 1030.8.

[0648] Example 1 : (3',3')-cyclo-(Rp,Rp)-bis-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]1.5-ammonium-0.5-1,8-diazabicycloundeca-7-ene salt

[0649] [ka]

[0650] To a solution (8 mL) of intermediate 54 (120 mg, 0.150 mmol) in DMF and (-)-PSI reagent (201 mg, 0.450 mmol), 1,8-diazabicycloundeca-7-ene (342 mg, 2.25 mmol) was added, and the resulting mixture was stirred at 25°C for 1 hour. The reaction mixture was rotated and evaporated to dry, and reprecipitation was performed with ethyl acetate (20 mL). The solid was recovered, and the resulting crude product was purified by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 10 mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-3 min, 0-3% B; 3-14 min, 3-30% B; 14-14.3 min, 30-95% B; 14.3-20 min, 95% B; retention time of compound: 13.5 min), and lyophilized to obtain a white solid (20 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.37 (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 (162 MHz, DMSO-d6) δ 53.47. MS-ESI [MH] - : 760.9.

[0651] Example 2 : (3',3')-cyclo-(Rp,Rp)-bis-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxyadenosine]

[0652] [ka]

[0653] Example 2 was obtained via the same route as Example 1 by reacting intermediate 55 with a (-)-PSI reagent and then purifying it by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 0.05% formic acid aqueous solution, B: 0.05% formic acid in acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm 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; retention time of compound: 12 min). 1 H NMR (400 MHz, DMSO-d6) δ 8.53 (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 (162 MHz, DMSO-d6) δ 53.68, 52.69. MS-ESI [MH] - : 724.9.

[0654] Example 3 : (3',3')-cyclo-(Rp,Rp)-bis-[3'-O-phosphorothioate-diester-2'-deoxy-2',2'-difluorocytidine]

[0655] [ka]

[0656] Example 3 was obtained via the route of Example 1 by reacting intermediate 56 with a (-)-PSI reagent and then purifying it by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 0.05% formic acid aqueous solution, B: 0.05% formic acid in acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm 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; retention time of compound: 18 min). 1 H NMR (400 MHz, DMSO-d6) δ 9.20-8.80 (m, 2H), 8.31 (d, J = 6.4 Hz, 1H), 8.28-8.00 (m, 2H), 7.90 (d, J = 6.0 Hz, 1H), 6.20-6.05 (m, 3H), 6.00 (d, J = 6.4 Hz, 1H), 4.95-4.68 (m, 2H), 4.30-4.05 (m, 4H), 3.90-3.70 (m, 2H). 31 P NMR (162 MHz, DMSO-d6) δ 53.27, 52.67. MS-ESI [M+H] + : 683.0.

[0657] Example 4 : (3',3')-cyclo-(Rp,Rp)-bis-[3'-O-phosphorothioate-diester-N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]diammonium salt

[0658] [ka]

[0659] Example 4 was obtained via the route of Example 1 by reacting intermediate 57 with the (-)-PSI reagent and then purifying it by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 10 mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-3 min, 0-35% B; 3-14 min, 35-70% B; 14-14.3 min, 70-95% B; 14.3-20 min, 95% B; retention time of compound: 14 min). 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 11.00 (s, 1H), 8.39 (d, J = 7.6 Hz, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.12 (t, J = 52.0 Hz, 8H), 6.30-6.18 (m, 2H), 5.02-4.75 (m, 2H), 4.35-4.10 (m, 4H), 3.83 (t, J = 11.2 Hz, 2H), 2.70-2.52 (m, 2H), 1.62-1.45 (m, 4H), 1.40-1.17 (m, 12H), 0.85 (t, J = 7.2 Hz, 12H). 31P NMR (162 MHz, DMSO) δ 54.54, 53.29. MS-ESI [M+H] + : 935.1.

[0660] Example 5 : (2',3')-cyclo-(Rp,Rp)-[2'-O-phosphorothioate-diester-adenosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0661] [ka]

[0662] Intermediate 71 (200 mg, 0.214 mmol) was mixed with 7 M ammonia in methanol (2 mL), and the resulting mixture was stirred at 25°C for 6 hours. The reaction mixture was rotated and dried 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: 0.05% formic acid aqueous solution, B: 0.05% formic acid in acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm 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; retention time of compound: 14 min), and lyophilized to obtain a white solid (4.0 mg). 1 H NMR (500 MHz, DMSO-d6) δ 8.63 (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 = 8.5 Hz, 1H), 5.40-5.15 (m, 2H), 5.05-4.95 (m, 1H), 4.55 (t, J = 3.5 Hz, 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 (= 12.0 Hz, 1H). 31 P NMR (162 MHz, DMSO-d6) δ 53.08, 48.81. MS-ESI [MH] - : 724.8.

[0663] Example 6 :(2',3')-cyclo-(Rp,Rp)-[2'-O-phosphorothioate-diester-adenosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxyadenosine]diammonium salt

[0664] [ka]

[0665] Example 6 was obtained via the route of Example 5 by reacting intermediate 72 with 28% aqueous ammonia and then purifying it by preparative liquid chromatography (with 10 mM ammonium bicarbonate as an additive). Analysis LCMS: Agilent 1100+G1946D LCMS, 4.6 x 50 mm Waters XBridge C18 3.5 μm analytical column, mobile phase A: 10 mM aqueous ammonium bicarbonate, B: acetonitrile, flow rate 1.8 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-15 min, 5-95% B; 1.5-3 min, 95% B. Retention time of compound: 0.35 min. 1 H NMR (400 MHz, DMSO-d6) δ 8.61 (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 = 8.4 Hz, 1H), 5.42-5.30 (m, 2H), 5.30-5.20 (m, 1H), 4.27 (d, J = 4.4 Hz, 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 (162 MHz, DMSO-d6) δ 56.62, 53.65. MS-ESI [MH] - : 707.0.

[0666] Example 7 : (3',3')-cyclo-(Rp,Rp)-[3'-O-phosphorothioate-diester-adenosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0667] [ka]

[0668] Example 7 was obtained via the route of Example 5 by reacting intermediate 73 with 28% aqueous ammonia and then purifying it by preparative liquid chromatography (with 10 mM ammonium bicarbonate as an additive). Analysis LCMS: Agilent 1100+G1946D LCMS, 4.6 x 150 mm Waters XBridge C18 3.5 μm analytical column, mobile phase A: 10 mM aqueous ammonium bicarbonate, B: acetonitrile, flow rate 1 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-8 min, 5-95% B; 8-15 min, 95% B. Retention time of compound: 4.1 min. 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.23 ​​(s, 1H), 8.19 (s, 1H), 7.95 (brs, 2H), 7.65 (brs, 2H), 7.14 (t, J = 52.0 Hz, 8H), 6.30-6.22 (m, 1H), 6.12 (d, J = 8.4 Hz, 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 = 12.0 Hz, 1H). 31 P NMR (162 MHz, DMSO-d6) δ 57.14, 54.13. MS-ESI [(M-2H) / 2] - : 362.0.

[0669] Example 8 : (2',3')-cyclo-(Sp,Rp)-[2'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt, and Example 9: (2',3')-cyclo-(Sp,Sp)-[2'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0670] Examples 8 and 9 were obtained via the route of Example 5 by reacting intermediate 74 with 28% aqueous ammonia, followed by purification by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 10 mM aqueous ammonium bicarbonate, B: acetonitrile, flow rate 25 mL / min, dual-wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-3 min, 0-2% B; 3-14 min, 2-37% B; 14-14.3 min, 37-95% B; 14.3-20 min, 95% B; retention time for compound 8: 13 min, and retention time for compound 9: 15 min).

[0671] Example 8:

[0672] [ka] 1 H NMR (400 MHz, DMSO-d6) δ 10.62 (brs, 1H), 8.19 (s, 1H), 8.11 (s, 1H), 7.94 (brs, 2H), 7.10 (t, J = 50.8 Hz, 8H), 6.63 (brs, 2H), 6.25 (dd, J = 24.0, 2.8 Hz, 1H), 5.85 (d, J = 8.8 Hz, 1H), 5.50-5.15 (m, 4H), 4.30-4.13 (m, 2H), 4.12-3.92 (m, 4H), 3.72 (d, J = 12.0 Hz, 1H). 31 P NMR (162 MHz, DMSO-d6) δ 56.84, 54.05. MS-ESI [MH] - : 741.0.

[0673] Example 9:

[0674] [ka] 1 H NMR (400 MHz, DMSO-d6) δ 10.67 (brs, 1H), 8.23 ​​(s, 1H), 8.08 (s, 1H), 7.93 (brs, 2H), 7.10 (t, J = 51.2 Hz, 8H), 6.67 (brs, 2H), 6.30 (dd, J = 18.8, 3.6 Hz, 1H), 5.77 (d, J = 8.0 Hz, 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 (162 MHz, DMSO-d6) δ 59.03, 54.74. MS-ESI [MH] - : 740.9.

[0675] Example 10 : (2',3')-cyclo-(Rp,Rp)-[2'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0676] [ka]

[0677] Example 10 was obtained via the route of Example 5 by reacting intermediate 75 with 28% aqueous ammonia and then purifying it by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 10 mM aqueous ammonium bicarbonate, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-3 min, 0-3% B; 3-14 min, 3-38% B; 14-14.3 min, 38-95% B; 14.3-20 min, 95% B; retention time of compound: 13 min). Example 10 was also obtained via the route of Example 25 by deprotecting intermediate 98 and purifying it by preparative liquid chromatography. The two different synthetic routes yielded exactly the same product. 1 H NMR (400 MHz, DMSO-d6) δ 10.63 (brs, 1H), 8.22 (s, 1H), 8.18 (s, 1H), 7.93 (brs, 2H), 7.12 (t, J = 51.2 Hz, 8H), 6.69 (brs, 2H), 6.28 (dd, J = 24.0, 2.4 Hz, 1H), 5.88 (d, J = 8.4 Hz, 1H), 5.42-4.80 (m, 4H), 4.45 (d, J = 4.0 Hz, 1H), 4.39-4.10 (m, 3H), 4.08-3.94 (m, 1H), 3.93-3.70 (m, 2H). 31 P NMR (162 MHz, DMSO-d6) δ 54.07, 50.13. MS-ESI [MH] - : 741.0.

[0678] Example 11 : (2',3')-cyclo-(Sp,Rp)-[2'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-adenosine]diammonium salt

[0679] [ka]

[0680] Example 11 was obtained via the route of Example 5 by reacting intermediate 76 with a 7M ammonia-methanol solution and then purifying it by preparative liquid chromatography (with 10 mM ammonium bicarbonate as an additive). Analysis LCMS: Agilent 1100+G1946D LCMS, 4.6 x 150 mm Waters XBridge C18 3.5 μm analytical column, mobile phase A: 10 mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate 1 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-8 min, 5-95% B; 8-15 min, 95% B; retention time of compound: 3.3 min. 1 H NMR (400 MHz, DMSO-d6) δ 10.63 (brs, 1H), 8.39 (s, 1H), 8.21 (s, 1H), 7.83 (brs, 2H), 7.10 (t, J = 50.8 Hz, 8H), 6.67 (brs, 2H), 6.30-6.18 (m, 1H), 5.87 (d, J = 8.4 Hz, 1H), 5.35-5.20 (m, 2H), 4.28 (d, J = 4.0 Hz, 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 (162 MHz, DMSO-d6) δ 56.96, 54.34. MS-ESI [MH] - : 722.9.

[0681] Example 12 : (2',3')-cyclo-(Rp,Rp)-[2'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-adenosine]diammonium salt

[0682] [ka]

[0683] Example 12 was obtained via the route of Example 5 by reacting intermediate 77 with a 7M ammonia-methanol solution, followed by purification by preparative liquid chromatography (with 10 mM ammonium bicarbonate as an additive). Analysis LCMS: Agilent 1100+G1946D LCMS, 4.6 x 150 mm Waters XBridge C18 3.5 μm analytical column, mobile phase A: 10 mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate 1 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-8 min, 5-95% B; 8-15 min, 95% B; retention time of compound: 3.3 min. 1 H NMR (400 MHz, DMSO-d6) δ 10.61 (brs, 1H), 8.39 (s, 1H), 8.20 (s, 1H), 7.82 (brs, 2H), 7.11 (t, J = 51.2 Hz, 8H), 6.70 (brs, 2H), 6.30-6.18 (m, 1H), 5.87 (d, J = 8.4 Hz, 1H), 5.25-5.13 (m, 2H), 4.96 (s, 1H), 4.50 (d, J = 4.4 Hz, 1H), 4.31-3.71 (m, 6H), 3.03-2.82 (m, 1H), 2.65-2.50 (m, 1H). 31 P NMR (162 MHz, DMSO-d6) δ 54.26, 49.51. MS-ESI [MH] - : 722.8.

[0684] Example 13 : (2',3')-cyclo-(Rp,Rp)-[2'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2'-deoxy-2',2'-difluorocytidine]diammonium salt

[0685] [ka]

[0686] Example 13 was obtained via the route of Example 5 by reacting intermediate 78 with 28% aqueous ammonia, followed by purification by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 10 mM aqueous ammonium bicarbonate, B: acetonitrile, flow rate 25 mL / min, dual-wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-3 min, 0-3% B; 3-14 min, 3-25% B; 14-14.3 min, 25-95% B; 14.3-20 min, 95% B; retention time of compound: 2 min). 1 H NMR (400 MHz, DMSO-d6) δ 8.16 (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). 31 P NMR (162 MHz, DMSO-d6) δ 53.24, 48.05. MS-ESI [(M-2H) / 2] - : 350.0.

[0687] Example 14 : (2',3')-Cyclo-(Rp,Rp)-[2'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-N4-(2-propylpentanoyl)-2'-deoxy-2',2'-difluorocytidine]diammonium salt

[0688] [ka]

[0689] Example 14 was obtained via the route of Example 5 by reacting intermediate 79 with a 7M ammonia-methanol solution, followed by purification by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 10 mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate 25 mL / min, dual-wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-3 min, 0-10% B; 3-14 min, 10-55% B; 14-14.3 min, 55-95% B; 14.3-20 min, 95% B; retention time of compound: 11 min). 1 H NMR (400 MHz, DMSO-d6) δ 11.07 (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, 2H), 5.05-4.74 (m, 1H), 4.29 (d, J = 4.4 Hz, 1H), 4.20 (d, J = 9.6 Hz, 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 = 7.2 Hz, 6H). 31 P NMR (162 MHz, DMSO-d6) δ 56.16, 53.39. MS-ESI [MH] - : 827.0.

[0690] Example 15 : (3',3')-cyclo-(Sp,Rp)-[3'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0691] [ka]

[0692] Example 15 was obtained via the route of Example 5 by reacting intermediate 80 with a 7M ammonia-methanol solution, followed by purification by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 10 mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate 25 mL / min, dual-wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-3 min, 0-3% B; 3-14 min, 3-33% B; 14-14.3 min, 33-95% B; 14.3-20 min, 95% B; retention time of compound: 11 min). 1 H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J = 2.8 Hz, 1H), 8.04 (s, 1H), 8.03-6.87 (m, 10H), 6.60 (brs, 2H), 6.22 (dd, J = 24.0, 2.0 Hz, 1H), 5.85 (d, J = 8.8 Hz, 1H), 5.51-5.15 (m, 3H), 4.35-3.89 (m, 6H), 3.72 (d, J = 12.4 Hz, 1H). 31 P NMR (162 MHz, DMSO-d6) δ 56.65, 54.03. MS-ESI [MH] - : 741.0.

[0693] Example 16 : (3',3')-cyclo-(Rp,Rp)-[3'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0694] [ka]

[0695] Example 16 was obtained via the route of Example 5 by reacting intermediate 81 with a 7M ammonia-methanol solution, and then purifying it by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 10 mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-3 min, 0-3% B; 3-14 min, 3-33% B; 14-14.3 min, 33-95% B; 14.3-20 min, 95% B; retention time of compound: 13 min). 1 H NMR (400 MHz, DMSO-d6) δ 10.63 (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, 2.4 Hz, 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 (162 MHz, DMSO-d6) δ 53.97, 49.08. MS-ESI [MH] - : 740.9.

[0696] Example 17 : (3',3')-cyclo-(Rp,Rp)-[3'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-adenosine]diammonium salt

[0697] [ka]

[0698] Example 17 was obtained via the route of Example 5 by reacting intermediate 82 with a 7M ammonia-methanol solution, and then purifying it by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 10 mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-3 min, 0-3% B; 3-14 min, 3-33% B; 14-14.3 min, 33-95% B; 14.3-20 min, 95% B; retention time of compound: 11 min). 1 H NMR (400 MHz, DMSO-d6) δ 10.59 (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, 6.0 Hz, 1H), 5.85 (d, J = 8.8 Hz, 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 (162 MHz, DMSO-d6) δ 53.46, 47.84. MS-ESI [MH] - : 740.9.

[0699] Example 18 : (3',3')-cyclo-(Rp,Rp)-[3'-O-phosphorothioate-diester-2'-deoxy-2'-fluoroadenosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]-0.5-ammonium-1.5-1,8-diazabicycloundeca-7-ene salt

[0700] [ka]

[0701] Example 18 was obtained via the route of Example 1 by reacting intermediate 67 with the (-)-PSI reagent and then purifying it by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 10 mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-3 min, 0-5% B; 3-14 min, 5-33% B; 14-14.3 min, 33-95% B; 14.3-20 min, 95% B; retention time of compound: 11 min). 1 H NMR (400 MHz, DMSO-d6) δ 9.62 (brs, 2H), 8.39 (s, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.18 (s, 1H), 7.36 (brs, 2H), 7.15 (t, J = 52.0 Hz, 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 (162 MHz, DMSO-d6) δ 53.37, 52.10. MS-ESI [M+H] + : 729.0.

[0702] Example 19 : (3',3')-cyclo-(Rp,Rp)-[3'-O-phosphorothioate-diester-2'-deoxy-2'-fluoroadenosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxyadenosine] 0.5-1,8-diazabicycloundeca-7-ene salt

[0703] [ka]

[0704] Example 19 was obtained via the route of Example 1 by reacting intermediate 68 with the (-)-PSI reagent and then purifying it by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Welch 10 μm preparative column, mobile phase A: 0.05% formic acid aqueous solution, B: 0.05% formic acid in acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm 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; retention time of compound: 10 min). 1 H NMR (400 MHz, DMSO-d6) δ 9.56 (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 (162 MHz, DMSO-d6) δ 54.78, 54.00. MS-ESI [M+H] + : 710.8.

[0705] Example 20 : (3',3')-cyclo-(Rp,Rp)-[3'-O-phosphorothioate-diester-2'-deoxy-2'-fluoroguanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0706] [ka]

[0707] Example 20 was obtained via the route of Example 1 by reacting intermediate 69 with the (-)-PSI reagent and then purifying it 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, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-2 min, 5% B; 2-9 min, 5-15% B; 9-19 min, 15-25% B; 19-19.5 min, 25-95% B; 19.5-22.5 min, 95% B; retention time of compound: 9 min). 1 H NMR (400 MHz, DMSO-d6) δ 10.69 (brs, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.92 (brs, 2H), 7.15 (t, J = 52.0 Hz, 6H), 6.64 (brs, 2H), 6.26 (dd, J = 17.2, 3.6 Hz, 1H), 6.08 (d, J = 16.8 Hz, 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 = 8.8 Hz, 1H), 4.15-4.00 (m, 2H), 3.90-3.70 (m, 2H). 31 P NMR (162 MHz, DMSO-d6) δ 54.20, 53.14. MS-ESI [MH] - : 743.2.

[0708] Example 21 : (3',3')-cyclo-(Rp,Rp)-[3'-O-phosphorothioate-diester-2'-deoxy-2'-fluoroguanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxyadenosine]diammonium salt

[0709] [ka]

[0710] Example 21 was obtained via the route of Example 1 by reacting intermediate 70 with the (-)-PSI reagent and then purifying it 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, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-2 min, 5% B; 2-10 min, 5-20% B; 10-10.6 min, 20-95% B; 10.6-12.6 min, 95% B; retention time of compound: 7.3 min). 1 H NMR (400 MHz, DMSO-d6) δ 10.67 (brs, 1H), 8.49 (s, 1H), 7.92 (s, 1H), 7.82 (brs, 2H), 7.14 (t, J = 52.0 Hz, 6H), 6.62 (s, 2H), 6.25 (dd, J = 6.4, 4.8 Hz, 1H), 6.05 (d, J = 16.8 Hz, 1H), 5.44 (dd, J = 52.0, 4.0 Hz, 1H), 5.05-4.85 (m, 2H), 4.33 (d, J = 12.4 Hz, 1H), 4.23 (d, J = 9.2 Hz, 1H), 4.10-3.92 (m, 2H), 3.98-3.50 (m, 2H), 2.78-2.54 (m, 2H). 31 P NMR (162 MHz, DMSO-d6) δ 52.56, 52.51. MS-ESI [MH] - : 725.2.

[0711] Example 22 : (2',3')-cyclo-[2'-O-phosphodiester-guanosine]-[3'-O-phosphodiester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0712] [ka]

[0713] Example 22 was obtained via the route of Example 9 by reacting intermediate 95 with ammonium fluoride in methanol solution, and then purifying it by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Waters XBridge C18 10 μm preparative column, mobile phase A: 10 mM aqueous ammonium bicarbonate, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-2 min, 5% B; 2-15.6 min, 5-20% B; 15.6-15.8 min, 20-95% B; 15.8-18 min, 95% B; retention time of compound: 6.6 min). 1 H NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.20 (d, J = 2.8 Hz, 1H), 7.99 (s, 1H), 7.93 (brs, 2H), 6.71 (brs, 2H), 6.33 (dd, J = 24.0, 2.4 Hz,1H), 5.90 (d, J = 8.0 Hz, 1H), 5.40 (d, J = 49.6 Hz, 1H), 5.20 - 5.00 (m, 2H), 4.42-4.30 (m, 2H), 4.20-3.80 (m, 6H). 31 P NMR (162 MHz, DMSO-d6) δ 1.61, -0.07MS-ESI [M+H] + : 710.9.

[0714] Example 23 : (2',3')-cyclo-[2'-O-Rp-phosphorothioate-diester-guanosine]-[3'-O-phosphodiester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0715] [ka]

[0716] Intermediate 96 (75 mg, 0.074 mmol) was mixed with 33% methylamine ethanol solution (3 mL) and stirred at room temperature for 3 hours. The reaction solution was dried by rotary evaporation, and 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 compound 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, B: acetonitrile, flow rate 25 mL / min, dual-wavelength UV absorption monitoring at 214 nm 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; retention time of compound: 8.5 min) and lyophilized to obtain a white solid (30.3 mg). 1 H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.21 (d, J = 2.8 Hz, 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, 1.6 Hz,1H), 5.86 (d, J = 8.0 Hz, 1H), 5.42-5.17 (m, 2H), 5.09-4.90 (m, 2H), 4.48 (d, J = 4.0 Hz, 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 (162 MHz, DMSO-d6) δ 48.92, -2.42. MS-ESI [M+H] + : 726.8.

[0717] Example 24 : (2',3')-cyclo-[2'-O-phosphodiester-guanosine]-[3'-O-Rp-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]

[0718] [ka]

[0719] Example 24 was obtained via the route of Example 9 by reacting intermediate 97 with ammonium fluoride in methanol solution, and then purifying it by preparative liquid chromatography (Gilson 281 preparative HPLC, 19 x 250 mm Waters XBridge C18 10 μm preparative column, mobile phase A: 10 mM aqueous ammonium bicarbonate, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm 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; retention time of compound: 2 min). 1 H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 8.19 (d, J = 2.8 Hz, 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, 2.4 Hz, 1H), 5.83 (d, J = 8.3 Hz, 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) δ 53.89 (s), -1.20 (s). 19 F NMR (376 MHz, DMSO-d6) δ -195.45 (s). MS-ESI [M+H] + : 726.

[0720] Example 25: (2',3')-cyclo-(Rp,Sp)-[2'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2-chloro-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0721] [ka]

[0722] 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 dried by rotary evaporation. The resulting residue was dissolved in methanol (1 mL), ammonium fluoride (60.5 mg, 1.63 mmol) was added, and the mixture was stirred under heating (60°C) for 20 hours. After monitoring the completion of the reaction by LC-MS 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: 10 mM ammonium bicarbonate aqueous solution, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm 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; retention time of compound: 10 min), and then lyophilized to obtain a white solid (21 mg). 1H NMR (400 MHz, DMSO-d6) δ 10.71 (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 = 8.4 Hz, 1H), 5.35 (d, J = 50.4 Hz, 1H), 5.20 (t, J = 11.8 Hz, 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 (162 MHz, DMSO-d6) δ 53.30, 50.07. 19 F NMR (376 MHz, DMSO-d6) δ -196.87MS-ESI [M+H] + :742.8.

[0723] Example 26 : (2',3')-cyclo-(Rp,Rp)-[2'-O-phosphorothioate-diester-guanosine]-[3'-O-phosphorothioate-diester-2-methylamino-2'-deoxy-2'-fluoro-beta-adenosine]diammonium salt

[0724] [ka]

[0725] Example 26 was obtained via the route of Example 25 as a byproduct of the deprotection reaction between intermediate 98 and a methylamine ethanol solution. It 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, B: acetonitrile, flow rate 25 mL / min, dual wavelength UV absorption monitoring at 214 nm and 254 nm, gradient elution: 0-2 min, 5% B: 2-15.6 min, 5-15% B: 15.6-15.8 min, 15-95% B: 15.8-18 min, 95% B; retention time of compound: 14.1 min), and lyophilized to obtain a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.69 (brs, 1H), 8.27 (s, 1H), 8.02 (s, 1H), 7.34-6.97 (m, 3H), 6.31 (brs, 1H), 6.28 (d, J = 2.4 Hz, 1H), 5.90 (d, J = 0.8 Hz, 1H), 5.44-5.15 (m, 3H), 4.44 (d, J = 0.4 Hz, 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 (162 MHz, DMSO-d6) δ 54.02, 51.06. MS+ESI--[M+H]: 738.0.

[0726] Examples of activation Example 1: Activation effect of the compound of the present invention on IFN-β secretion by THP-1 cells The human monocytic leukemia cell line THP-1 cells possess the HAQ-type STING phenotype, namely R71H-G230A-R293Q. This assay evaluated the activation of IFN-β secretion from THP-1 cells by representative compounds of the present invention, using the Human IFN-β DuoSet ELISA Kit (R&D, catalog no. DY814-05) and DuoSet ELISA Reagent Kit 2 (R&D, catalog no. DY008) from R&D.

[0727] For thawing THP-1 cells (ATCC No. TIB-202), the cell freezing tubes were rapidly shaken in a 37°C water bath, and the cells were thawed within 1 minute. The thawed cell suspension was taken and homogeneously mixed with RPMI 1640 medium (Hyclone, product No. SH30027.01) containing 10% FBS (Life Technology, catalog No. 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 25 cm² substrate area. 2 The cells were placed in a cell culture flask and incubated in a cell incubator at 37°C, 5% CO2, and 95% humidity. When cell confluence reached approximately 80%, the cells were subcultured. All cells in the culture flask were transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 5 mL of fresh complete medium was added to resuspend the cell pellet, and 1 mL of this medium was taken and placed in a container with a base area of ​​25 cm². 2The cells were placed in a cell culture flask and supplemented with 4 mL of fresh complete medium for further incubation. When cell confluence again reached approximately 80%, 1 / 5 of the cell suspension was retained for further incubation, and 4 / 5 of the cell suspension was left in a 15 mL centrifuge tube. Plating was then performed after this cell subculturing procedure. The cell suspension was centrifuged to discard the old culture medium, washed once with RPMI1640 culture medium (serum-free), and centrifuged to remove the supernatant. The cells were then resuspended in RPMI1640 culture medium (serum-free). Cell viability was then detected by the trypan blue exclusion test, and cells were used for plating only if cell viability was confirmed to be above 95%. 1.1 × 10 6 A cell suspension with a viable cell density of 2 × 10⁶ cells / mL was prepared using RPMI1640 culture medium (serum-free). 180 μL of this cell suspension was added to a 96-well cell culture plate (NUNC Company, catalog No. 167008), resulting in a cell density of 2 × 10⁶ cells in the plate. 5 The number of surviving cells per well was determined.

[0728] A 10 mM stock solution of the compound in DMSO was first serially diluted 3.16 times with DMSO (Sigma, catalog no. D2650) up to the fifth concentration, with the sixth concentration set as the DMSO control (compound-free). Next, DMSO solutions containing different concentrations of the compound were diluted 10-fold with PBS, resulting in a DMSO content of 10% in each compound solution at various concentrations. Finally, 20 μL of each of these solutions was added to the corresponding cell culture plate to provide an initial compound concentration of 100 μM, with a dilution ratio of 3.16 to adjacent concentrations, and the DMSO content in the cell culture plate was 1%. The cell plates were then placed in a cell incubator for a further 24 hours.

[0729] ELISA detection was performed referring to the instructions for use of catalog No. DY814-05 from R&D Systems.

[0730] ELISA plate coating: Using PBS (R&D Systems, catalog No. DY006), the capture antibody (mouse anti-human IFN-β capture antibody PART No. 844508) was diluted to the working concentration, and 100 μL of the capture antibody working solution was added to a 96-well ELISA plate. This was then sealed and incubated overnight at room temperature. The capture antibody working solution was discarded, and the cell plate was washed three times with washing buffer (0.05% Tween 20 in PBS, pH 7.2-7.4, R&D Systems, catalog No. WA126) at a rate of 400 μL per well. After each wash, the washing buffer was completely removed. For the final wash, the plate was inverted and tapped against a clean piece of paper to completely remove the washing buffer. 300 μL of blocking buffer (1% BSA in PBS, pH 7.2-7.4, R&D Systems, catalog No. DY995) was added to each well, and incubated at room temperature for 1-2 hours. The above washing steps were repeated to prepare each plate for sample addition.

[0731] Sample detection: Add 100 μL of sample or standard (recombinant human IFN-beta standard, PART NO. 844510) to each cell, seal, incubate at room temperature for 2 hours, and repeat the washing step of the plate coating procedure described above. Then, add 100 μL of detection antibody (biotinylated mouse anti-human IFN-β detection antibody, PART No. 844509) to each well, seal the plate, leave it to incubate at room temperature for 2 hours, and repeat the washing step of the plate coating procedure described above. Subsequently, add 100 μL of streptavidin-HRP (PART No. 893975) action solution to each well, seal the plate, leave it to incubate at room temperature for 20 minutes, protect this procedure from light, and repeat the washing step of the plate coating procedure described above. Next, 100 μL of substrate solution (a 1:1 mixture of colorimetric reagent A (H2O2) and colorimetric reagent B (tetramethylbenzidine), R&D Systems, catalog no. DY999) was added to each well, the plate was sealed, and left to stand and incubated at room temperature for 20 minutes, protecting this step from light. Finally, 50 μL of stop solution (2N H2SO4, R&D Systems, catalog no. DY994) was added to each well, and the cell plate was tapped to ensure complete mixing.

[0732] The OD450 of each well was measured using a multifunction microplate reader (Molecular Devices, Spectramax M3). Wavelength calibration was set to 540 nm or 570 nm if available, or OD540 or OD570 was subtracted from OD450 if unavailable. This measurement was completed within 30 minutes of adding the stop solution.

[0733] 2',3'-cGAMP (Invivogen, catalog No. tlrl-nacga23) was used as the positive control compound, and ADU-S100 (MCE, catalog No. HY12885A) was used as the reference compound:

[0734] [ka]

[0735] Data were analyzed using GraphPad Prism 7.0 software, and a standard curve for ELISA IFN-β content was created using log-log plots. The OD values ​​tested from each sample well were fitted to the standard curve equation, giving the corresponding IFN-β concentrations. Nonlinear sigmoid regression was fitted to the data to obtain dose-response curves of IFN-β concentration versus compound concentration, and EC50 values ​​were calculated.

[0736] [Table 1]

[0737] The experimental results indicate that the compounds in this example have an activating effect on IFN-beta secretion by THP-1 cells. As can be seen from Table 1 and Figure 1, the compounds in Examples 10, 12, 16, 17, and 26 exhibit particularly important STING agonist activity: their EC50s were higher than those of 2',3'-cGAMP and equivalent to those of ADU-S100; among these, the compounds in Examples 10, 16, and 17 stimulated THP-1 cells to secrete IFN-β, and their peak concentrations were higher than those achieved by ADU-S100.

[0738] Example 2: Inhibitory activity of the compound of the present invention on the proliferation of CT26 cells CT26 cells are a mouse colorectal cancer cell line. This assay evaluated the inhibitory activity of compounds on CT26 cell proliferation using the CellTiter-Glo Luminescence Cell Vitality Assay Kit from Promega.

[0739] For thawing CT26 cells (ATCC No. CRL-2638), the cell freezing tubes were rapidly shaken in a 37°C water bath, and the cells were thawed within 1 minute. The thawed cell suspension was taken and homogeneously mixed with DMEM medium (GE, catalog No. SH30243.01) containing 10% FBS (GIBCO, catalog No. 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 incubated on a 25 cm² substrate. 2 The cells were placed in a cell culture flask and incubated in a cell incubator at 37°C, 5% CO2, and 95% humidity. When the cell confluence reached 70%-80%, the cells were subcultured. All cells in the culture flask were transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 5 mL of fresh complete medium was added to resuspend the cell pellet, and 1 mL of this medium was taken and placed in a 25 cm² substrate. 2 The cells were placed in a cell culture flask and supplemented with 4 mL of fresh complete medium for further incubation. When the cell confluence again reached 70%–80%, 1 / 5 of the cell suspension was retained for further incubation, and 4 / 5 of the cell suspension was left in a 15 mL centrifuge tube. Plating was then performed after this cell subculturing procedure. The cell suspension was centrifuged to discard the old culture medium, washed once with DMEM medium (serum-free), and centrifuged again to remove the supernatant. The cells were then resuspended in DMEM medium (serum-free). Cell viability was then detected by the trypan blue exclusion test, and cells were used for plating only if cell viability was confirmed to be over 95%. 1.1 × 10 4 A cell suspension with a viable cell density of 1000 cells / mL was prepared using DMEM medium (serum-free). 90 μL of this cell suspension was added to a 96-well transparent flat-bottom black-wall cell culture plate (Corning, catalog No. 3603), resulting in a cell density of 1000 viable cells / well in the plate. A control group containing only complete medium (i.e., culture medium control) and a control group containing cells but no compounds (i.e., cell control) were established. The cell plates were placed in a cell incubator overnight.

[0740] A stock solution of the compound in 10 mM DMSO was first serially diluted 3.16 times with DMSO (Sigma, catalog no. D2650) up to the 9th concentration, with the 10th concentration set as a DMSO control (compound-free). Next, DMSO solutions containing different concentrations of the compound were diluted 10 times with PBS (Solarbio, catalog no. P1020), resulting in a DMSO content of 10% in each compound solution at various concentrations. Finally, 10 μL of each of these solutions was added to the corresponding cell culture plate to provide an initial compound concentration of 100 μM, with a dilution ratio of 3.16 to adjacent concentrations, and the DMSO content in the cell culture plate was 1%. The cell plates were then placed in a cell incubator for a further 120 hours.

[0741] After 120 hours, the CellTiter-Glo reagent (Promega, catalog No. G7572) was thawed, and the plate was equilibrated at room temperature for 30 minutes. 100 μL of CellTiter-Glo was added to each well of the plate, and the cells were completely lysed by shaking on an orbital shaker for 5 minutes. The plate was left at room temperature for 20 minutes to stabilize the luminescence signal, and the luminescence value of each well was scanned at full wavelength using a multi-function microplate reader (Molecular Devices, Spectramax M3).

[0742] The following compounds were used as controls against ADU-S100 (MCE, catalog No. HY 12885A): clofarabine (Wuhu Huaren Science, catalog No. HR-00701002), cladribine (CSNpharm, catalog No. CSN10004), gemcitabine hydrochloride (ShaoYuan, catalog No. SY014538), and gemcitabine prodrug LY2334737 (proprietary product, intermediate 7).

[0743] For each compound concentration, cell viability was calculated using the following formula: Cell viability (%) = (Lum 試験薬 - Lum 培養培地対照 ) / (Lum 細胞対照 - Lum 培養培地対照 ) ×100%

[0744] The data was analyzed using GraphPad Prism 7.0 software, and dose-response curves were derived by fitting nonlinear S-curve regression, and IC50 values ​​were calculated.

[0745] [Table 2]

[0746] The experimental results showed that representative example compounds could inhibit the in vitro proliferation of CT26 tumor cells; among these, compounds in Examples 3, 10, and 13 showed particularly remarkable inhibitory activity, while ADU-S100 did not show significant tumor cell inhibitory activity. Combined with the results of Activity Example 1, the above results indicate that these compounds possess multifunctional antitumor properties, namely the tumor immunological activity of a STING agonist and the cytotoxic effect of an antimetabolite anticancer drug.

[0747] Example 3: Antitumor activity of the compound of the present invention in a bilateral transplanted tumor model of CT26 syngeneic mice 6-8 week old BALB / c mice (purchased from Shanghai Ling Chang Biotech Co., Ltd.) were given 5 × 10 5 CT26 cells (supplied by Taicang Zexin Biotechnology Co., Ltd., ATCC No. CRL-2638) were subcutaneously inoculated into the left and right dorsal regions, respectively, with an inoculation volume of 0.1 mL / side. The tumors had an average volume of 100 mm². 3When the tumors proliferated, they were randomized according to tumor size and mouse body weight, and treatment was initiated. The first dose was administered on day 0. Two doses were administered on days 0 and 4: 50 μg of the compound / mouse (i.e., 2.5 mg / kg) was injected into the right-side tumor, and the same volume of PBS (Hyclone, catalog no. SH30258.01) was administered as a control. The left-side tumor was not treated. Control mice were sacrificed 13 days after administration, and treated mice were sacrificed 21 days after administration. During the experiment, the left and right-side tumor volumes and body weight were measured three times per week, and the tumor volume was calculated as V = D × d × d / 2 (where D represents the tumor length and d represents the tumor width).

[0748] Data from 13 days post-administration showed that all tested example compounds significantly inhibited bilateral tumor growth. The right-sided treatment group showed a more pronounced effect, with most tumors regressing (Figure 2-A). Among these, the ADU-S100 treatment group showed 99.1% inhibition of tumor growth, Example 17 showed 99.5% inhibition, and the remaining groups showed 100% inhibition of tumor growth in tumors without palpable tumors. Simultaneously, the untreated left-sided tumors showed slow growth (Figure 2-B). On 13 days post-administration, the inhibition rates of growth in the left-sided tumor group were: ADU-S100, 64.2%; Example 10, 91.2%; Example 12, 75.3%; Example 16, 71.7%; Example 17, 67.4%. Furthermore, in this experiment, it was observed that the post-administration body weight of individual mice decreased by less than 15% and recovered after discontinuation of administration (Figure 2-C). The above results indicate that the compound of the present invention showed tumor inhibitory activity equivalent to or better than ADU-S100 in a CT26 syngeneic bilateral tumor model.

[0749] Example 4: Antitumor activity of the compound of the present invention in a transplanted tumor model of CT26 nude mice

[0750] 6-8 week old BALB / c nude mice (purchased from Shanghai Ling Chang Biotech Co., Ltd.) were given 5 × 10 5Each CT26 cell (supplied by Taicang Zexin Biotechnology Co., Ltd., ATCC No. CRL-2638) was subcutaneously inoculated into the right dorsal side, with an inoculation volume of 0.1 mL. The tumor had an average volume of 100 mm². 3 When the tumors proliferated, they were randomized according to tumor size and mouse body weight and administered the treatment. The first dose was administered on day 0. Two doses were administered on days 0 and 4, with 50 μg of the compound / mouse (i.e., 2.5 mg / kg) injected into the right-side tumor and the same volume of PBS (Hyclone, catalog no. SH30258.01) injected as a control. Control mice were sacrificed 9 days after administration, ADU-S100 treated mice were sacrificed 14 days after administration, and Example 10 treated mice were sacrificed 16 days after administration. During the experiment, tumor volume and body weight were measured three times per week, and tumor volume was calculated as V = D × d × d / 2 (ibid.).

[0751] Data from day 9 after administration show that the compound in Example 10 tested was able to significantly inhibit tumor growth in T-cell immunodeficient nude mice by a rate of 94.4%. In contrast, ADU-S100 showed only partial tumor suppression with an inhibition rate of 61.4%, possibly due to its remaining immune activity or other unknown reasons. Whether it was Example 10 or ADU-S100, they showed superior tumor growth inhibition in immunocompetent mice compared to tumor growth inhibition in nude mice (Figure 2D). This experiment demonstrates that ADU-S100 acts primarily by activating T-cell-mediated immunity; on the other hand, the compound in Example 10 inhibits tumors through its multifunctional mechanism, demonstrating superior antitumor activity compared to the STING agonist ADU-S100 in this case.

[0752] Example 5: Immunological memory function of the compound of the present invention in transplanted tumor models of CT26 syngeneic mice or nude mice. 6-8 week old BALB / c immunized mice or nude mice (purchased from Shanghai Ling Chang Biotech Co., Ltd.) were given 5 × 105 Each CT26 cell (supplied by Taicang Zexin Biotechnology Co., Ltd., ATCC No. CRL-2638) was subcutaneously inoculated into the right dorsal side, with an inoculation volume of 0.1 mL. The tumor had an average volume of 100 mm². 3 When the tumors proliferated, they were randomized according to tumor size and mouse body weight and administered the drug. The first dose was administered on day 0. Two doses were administered on days 0 and 4, with 50 μg of the compound / mouse (i.e., 2.5 mg / kg) injected into the tumor, and the same volume of PBS (Hyclone, catalog no. SH30258.01) injected as a control. The nude mouse group or the BALB / c mouse group was sacrificed on day 9 or day 13 after administration, respectively. On day 21 after administration, the mice were given 0.1 mL of 5 × 10⁴ 5 Each CT26 cell was subcutaneously inoculated again into the left dorsal side. Blank BALB / c mice or nude mice that had not received any treatment were also inoculated as controls. During the experiment, tumor volume and body weight were measured three times per week, and tumor volume was calculated as V = D × d × d / 2 (same as above).

[0753] The results show that the compound in Example 10 significantly inhibited CT26 tumor growth in both immunocompetent and immunodeficient mice. The effect was even better in immunocompetent mice (Figure 3-A). On day 5 after administration, tumors completely disappeared in mice treated with Example 10. On day 21, CT26 cells were re-inoculated into each group treated with compound 10. After 7 days (day 30), the mean tumor volume of unimmunized blank BALB / c mice was 75 mm². 3 However, the average tumor volume of the mice treated in Example 10 was 29 mm². 3 The results were as follows: 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 mm², respectively. 3 and 101mm 3 This experiment demonstrated that, after treatment with the compound of Example 10, immunocompetent mice generated immunological memory and exhibited strong immunological rejection to reinoculated allogeneic cells, thereby effectively preventing tumor recurrence.

[0754] On the other hand, in immunodeficient nude mice, continuous tumor growth occurred in individual mice after discontinuation of administration (Figure 3-B). Finally, three mice with relatively small tumor volumes were selected and re-inoculated with CT26 cells on day 21. This result indicates that the re-inoculated CT26 tumor cells showed a proliferation rate similar to that in blank mice. The above results indicate that no immunological memory was induced in the nude mouse experiment and further demonstrate that the compound in Example 10 has cytotoxic tumor inhibitory ability even without the involvement of the immune system in this model.

[0755] Example 6: Hepatocyte metabolic stability assay for the compound of the present invention The compounds of the present invention were similarly tested for hepatocyte metabolic stability in five species (mouse, rat, dog, monkey, and human) according to standard methods for in vitro metabolic stability studies commonly used in the art, such as (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 High Throughput Microsomal Stability Screening Assay for Profiling Drug Discovery Candidates, J Biomol Screen. 2003, 8(4), 453).

[0756] The hepatocytes used in this experiment were: human hepatocytes (SHQY, lot No. HEP190006), canine hepatocytes (IVT, lot No. ZMB); monkey hepatocytes (Xenotech, lot No. 2010022), rat hepatocytes (SHQY, lot No. HEP134045), and mouse hepatocytes (BioIVT, catalog No. M005052, lot No. MEO).

[0757] The cryopreserved liver cell tubes were removed from the liquid nitrogen tank and immediately placed in a water bath at 37±1°C with shaking for 2 minutes ±15 seconds. Hepatocytes were thawed in 50 mL of hepatocyte thawing medium (composition: Williams E medium, 35 mL, Invitrogen, catalog No. A1217601; isotonic Percoll solution, 13.5 mL, GE, catalog No. 17-0891-01; Dulbecco phosphate buffer, 1.5 mL, Invitrogen, catalog No. 14200-075; Glutamax, 500 μL, Invitrogen, catalog No. 35050061; HEPES, 750 μL, Invitrogen, catalog No. 15630106; fetal bovine serum, 2.5 mL, Invitrogen, catalog No. 10091130; recombinant human insulin, 50 μL, Invitrogen, catalog No. 12585014; dexamethasone (10 mM) Transfer 5 μL of DMSO solution (formulated as DMSO solution, Sigma, catalog No. D1756) to the solution, mix gently, and centrifuge at 500 rpm for 3 minutes. After centrifugation, carefully aspirate the supernatant (without disturbing the cell pellet), add 10 × 10⁻¹ volume of pre-warmed KHB buffer (Krebs-Henseleit buffer, Sigma, catalog No. K3753-10X1L) and 5.6 g / L HEPES, resuspend the cell pellet, mix gently, and centrifuge at 500 rpm for 3 minutes. Aspirate the supernatant without touching the cell pellet, and determine the cell viability and number. Count the hepatocytes, and then adjust the cell suspension to an appropriate density in KHB buffer (viable cell density = 2 × 10⁻¹⁶ cells). 6 The solution was diluted to the specified concentration (cells / mL). The hepatocyte solution was kept on ice until use.

[0758] A 2x medicinal solution was prepared in pre-warmed KHB (1% dimethyl sulfoxide) and centrifuged at 5594g for 15 minutes (Thermo Multifuge x 3R). 200 μM spiking solution: 20 μL of compound stock solution (10 mM in DMSO) was added to 980 μL of dimethyl sulfoxide; 2x medicinal solution: 10 μL of 200 μM spiking solution was added to 990 μL of KHB (2 μM after dilution).

[0759] 50 μL of pre-warmed 2× 10⁻¹ medicinal solution was added to the wells designated at different time points. 6 Cells / mL were added to the wells designated for detection at 15, 30, 60, and 120 minutes, then the timer was started and the reaction plate was placed in a 37°C incubator.

[0760] Add IS (osalmid or imipramine) (Merck, catalog no. CN34854-4L) in 100 μL of acetonitrile to the well specified at 0 min, mix gently, and then add 50 μL of pre-warmed hepatocyte solution (2 × 10⁻¹⁶). 6 Cells / mL were added, and the plate was blocked. At 15, 30, 60, and 120 minutes, 100 μL of IS-containing acetonitrile was added to each well, followed by blocking. After quenching, the plate was shaken on a shaker (IKA, MTS 2 / 4) for 10 minutes (600 rpm). The plate was sonicated for 2 minutes, then centrifuged at 5594 g for 15 minutes (Thermo, Multifuge × 3R). For LC / MS analysis, 50 μL of supernatant per well was transferred to a 96-well sample plate containing 50 μL of ultrapure water (Millicore, ZMQS 50F01).

[0761] The concentration of the test compound at time T0 was taken as 100% (C0), and the concentrations at other incubation time points were converted to residual percentages. Then, the natural logarithm of these percentages was processed against the incubation time using linear regression to obtain the slope K. Subsequently, the hepatocyte clearance rate (Clint) and in vitro half-life (T1 / 2) were calculated according to the following formulas: T1 / 2 = 1n2 / K = 0.693 / K Cl int = (0.693 / T1 / 2) × (1 / hepatocyte density) × scaling factor In the formula, hepatocyte density is the final concentration of hepatocytes in the experimental incubation system: 1 × 10⁻⁶ 6 The value is / mL. Scaling factor = number of hepatocytes × liver weight (for each of the 5 types of hepatocytes, 11812.5 × 10⁻¹⁰). 6 / kg (mouse), 4680×10 6 / kg (rat), 6880 x 10 6 / kg (dog), 3900 x 10 6 / kg (monkey), 2544.3 × 10 6 / kg (human).

[0762] As can be seen from Figure 4A, the compounds of the present invention, such as the compound in Example 10, showed excellent metabolic stability in five types of hepatocytes, exhibiting a long metabolic half-life and a low clearance rate.

[0763] Example 7: Discovery and identification of metabolites of the compound of the present invention The main 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 for in vitro metabolic stability studies commonly used in the art, e.g., (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 High Throughput Microsomal Stability Screening Assay for Profiling Drug Discovery Candidates, J Biomol Screen. 2003, 8(4), 453).

[0764] The hepatocytes used in this experiment were: human hepatocytes (SHQY, catalog No. BQHPCH10, lot No. HEP190006-TA05), canine hepatocytes (BioIVT, catalog No. M00205, lot No. ZMB); monkey hepatocytes (XENOTECH, catalog No. PPCH2000, lot No. 2010022), rat hepatocytes (SHQY, catalog No. BQR1000·H15, lot No. HEP134049), and mouse hepatocytes (BioIVT, catalog No. M005052, lot No. MEO).

[0765] HI hepatocyte maintenance medium (BIOIVT, catalog No. Z99009; lot No. C02060C) was preheated to 37°C. The cryopreserved hepatocyte tubes were removed from the liquid nitrogen tank and immediately placed in a water bath shaking at 37±1°C for 2 minutes ±15 seconds. The hepatocytes were transferred to 50 mL of HI medium, gently mixed, and centrifuged at 500 rpm for 3 minutes. After centrifugation, the supernatant was carefully aspirated (without disturbing the cell pellet), 10 × volume of preheated HI medium was added, the cell pellet was resuspended, gently mixed, and centrifuged at 500 rpm for 3 minutes. The supernatant was aspirated and discarded without touching the cell pellet. The hepatocytes were counted, and the cell suspension was then mixed in HI medium to an appropriate density (viable cell density = 2 × 10⁶). 6 The solution was diluted to the specified concentration (cells / mL). The hepatocyte solution was kept on ice until use.

[0766] The 2× medicinal solution (20 μM) was prepared in pre-warmed HI medium by adding 8 μL of 10 mM compound stock solution to 3992 μL of HI medium (20 μM, 0.2% dimethyl sulfoxide after dilution).

[0767] The hepatocyte solution and the 2× medicinal solution are preheated, and 200 μL of the preheated 2× medicinal solution is added to T 240 and T 240-w / o The solution was added to the wells designated for use. For T0, 1200 μL of acetonitrile (Merck, catalog no. CN34854-4L) and 200 μL of hepatocyte solution (2 × 10⁻¹⁶) were added. 6 Add the cells (2 × 10) to the wells, then add 200 μL of pre-warmed 2× medicinal solution and block the plate. 6 Cells / mL) to T 240 Add 200 μL of pre-warmed HI medium to the well designated for use. 240-w / o The wells designated for the reaction were added, and the timer was started. The reaction plate was placed in a CO2 incubator at 37°C.

[0768] After 240 minutes, 1200 μL of acetonitrile was added to each designated well, and then the plate was blocked. After quenching, the plate was sonicated for 2 minutes, and then centrifuged at 1400 rpm for 5 minutes. 1200 μL of supernatant was evaporated under a stream of nitrogen until dry. The dried extract was then redissolved in 200 μL of acetonitrile:water (1:3 v / v), vortexed for 2 minutes, and centrifuged at 14000 rpm for 5 minutes. For analysis, 2 / 5 μL of supernatant was injected into LC-UV-MS.

[0769] As can be seen from Figure 4-B, the cytotoxic small molecule clofarabine, produced by the degradation of the compounds of the present invention, such as the compound of Example 10, can be detected in five types of hepatocytes, and is the main metabolite of the compound of Example 10. Combined with the results of Activity Example 6, it is highly likely that the CDN molecule with STING agonist activity and the small cell toxic molecule are both present in vivo for the duration of the experiment, thereby achieving a molecular-level combination and providing an enhanced, synergistic antitumor effect. This result also explains why the compound functioned so well in mouse tumor suppression experiments.

[0770] Example 8: Comparison of antitumor activity between the compound of the present invention and a simple combination of a CDN STING agonist and a cytotoxic agent in a bilateral transplanted tumor model of CT26 syngeneic mice. 6-8 week old BALB / c mice (purchased from Shanghai Ling Chang Biotech Co., Ltd.) were given 5 × 10 5 CT26 cells (supplied by Taicang Zexin Biotechnology Co., Ltd., ATCC No. CRL-2638) were subcutaneously inoculated into the left and right dorsal regions, respectively, with an inoculation volume of 0.1 mL / side. The tumors had an average volume of 100 mm². 3When the tumors proliferated, they were randomized according to tumor size and mouse body weight (compound group of the present invention; single CDN STING agonist group; single cytotoxic agent group; simple combination group of CDN STING agonist and cytotoxic agent), and treatment was initiated. The first dose was administered on day 0. Three doses were administered on days 0, 4, and 7, injected into the right-side tumor, and the same volume of PBS (Hyclone, catalog no. SH30258.01) was administered as a control. The left-side tumor was not treated. Control mice were sacrificed 13 days after administration, and treated mice were sacrificed 21 days after administration. During the experiment, the left and right-side tumor volumes and body weight were measured three times per week, and the tumor volume was calculated as V = D × d × d / 2 (ibid.).

[0771] The experimental results clearly show that the tumor growth inhibition rate of the compounds of the present invention is significantly higher than that of the single CDN STING group and the single cytotoxic agent group. This indicates that the activity of the compounds of the present invention is equivalent to or higher than the additive activity of the latter two groups, resulting in improved and even synergistic tumor inhibitory activity.

[0772] The general or preferred definitions of certain features in the various enumerated embodiments of the present invention may be combined with the general or preferred definitions of other certain features to give rise to further embodiments of the present invention. Unless the context clearly indicates otherwise, such combinations are presented as if they were specifically and individually described herein.

[0773] Several prior publications are referenced herein. These publications are not considered relevant to the patentability of the present invention, but are incorporated herein by reference in their entirety. Any reference herein to any prior publication (or information derived therefrom) is not, and should not be construed as, an acknowledgment, admission, or any form of suggestion that the corresponding prior publication (or information derived therefrom) forms part of the common general knowledge in the art to which this specification relates. The present invention includes, for example, the following embodiments. [Section 1] Formula (II): [ka] (In the formula: B1 is an adenine substituted with X. [ka] (Here X is Cl, F, or -NHC) 1-6 (Selected from alkyl) or R a Cytosine is sometimes substituted. [ka] (R here) a is H or -C(O)-C 1-14 (Selected from alkyl groups); R1 and R1' are each independently selected from H, F, or -OH; B2 is an adenine that may be substituted with X. [ka] (Here X is selected from H, F, or Cl); R a Cytosine is sometimes substituted. [ka] (R here) a is H or -C(O)-C 1-14 Selected from alkyl groups; or guanine [ka] (Here OH is C 1-6 Selected from (sometimes substituted with alkyl); [ka] In this case, the phosphate bond is attached to the 2' or 3' position of the pentose, and the position that is not cyclized with the phosphate is R2 And it is shown that it is substituted with R2'; R2 and R2' are each independently selected from H, -OH, or F; However, if one of B1 or B2 is R a (If it is a cytosine that is substituted in some cases, the carbon atoms adjacent to it on the pentose ring to which it is bonded are substituted with two fluorine atoms.) A cyclic dinucleotide compound thereof, or its stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, prodrugs, or solvates. [Section 2] below: [ka] However, this shows that the phosphate bond is attached to the 3' position of the pentose, and that equation (II) is the following equation: [ka] Compounds of formula (II) as described in item 1, stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, prodrugs, or solvates thereof. [Section 3] B1 is [ka] The compound described in item 2, wherein both R1 and R1' are H, or one of R1 and R1' is H and the other is F. [Section 4] B1 is an adenine substituted with X. [ka] (In the formula, X is -NHC) 1-6 The compound according to item 2, wherein the alkyl group is preferably selected from -NHCH3, and both R1 and R1' are H, or one of R1 and R1' is H and the other is F. [Section 5] B1 is R a Cytosine is sometimes substituted. [ka] (In the formula, R a is H or -C(O)-C 1-14 The compound described in item 2, wherein R1 and R1' are both F (selected from alkyl). [Section 6] B2 is guanine [ka] or adenine [ka] The compound according to any one of items 2 to 5, wherein one of R2 and R2' is H and the other is selected from -OH or F. [Section 7] B2 is an adenine substituted with X. [ka] A compound described in any one of items 2 to 5, wherein (wherein X is Cl) one of R2 and R2' is H and the other is F, or both R2 and R2' are H. [Section 8] B2, R a Cytosine is sometimes substituted. [ka] (In the formula, R a is H or -C(O)-C 1-14 A compound according to any one of items 2 to 5, wherein R2 and R2' are both F (selected from alkyl). [Section 9] below: [ka] However, this shows that the phosphate bond is attached to the 2' position of the pentose, and equation (II) becomes the following equation: [ka] Compounds of formula (II) as described in item 1, stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, prodrugs, or solvates thereof. [Section 10] B1 is [ka] The compound described in item 9, wherein both R1 and R1' are H, or one of R1 and R1' is H and the other is F. [Section 11] B1 is an adenine substituted with X. [ka] (In the formula, X is -NHC) 1-6 The compound according to item 9, wherein the alkyl group (preferably -NHCH3) is such that both R1 and R1' are H, or one of R1 and R1' is H and the other is F. [Section 12] B1 is R a Cytosine is sometimes substituted. [ka] (In the formula, R a is H or -C(O)-C 1-14 The compound described in item 9, wherein R1 and R1' are both F (selected from alkyl). [Section 13] B2 is guanine [ka] or adenine [ka] The compound described in any one of items 9 to 12, wherein one of R2 and R2' is H and the other is -OH. [Section 14] below: [ka] The compounds described in item 1, 2, or 9, or their pharmaceutically acceptable salts or solvates. [Section 15] B1 is [ka] The compound according to item 14, wherein both R1 and R1' are H, or R1 is F and R1' is H. [Section 16] B1 is an adenine substituted with X. [ka] (In the formula, X is -NHC) 1-6 The compound according to item 14, wherein the compound is alkyl (preferably -NHCH3), and both R1 and R1' are H, or R1 is F and R1' is H. [Section 17] B1 is R a Cytosine is sometimes substituted. [ka] (In the formula, R a is H or -C(O)-C 1-14 The compound described in item 14, wherein R1 and R1' are both F (selected from alkyl). [Section 18] In equation (II-a'), B2 is guanine. [ka] or adenine [ka] And R2' is H, and R 2 A compound according to any one of items 14 to 17, wherein is selected from -OH or F. [Section 19] In equation (II-a'), B2 is adenine substituted with X. [ka] A compound as described in any one of items 14 to 17, wherein (wherein X is Cl), R2 is H, R2' is F, or both R2 and R2' are H. [Section 20] In equation (II-b'), B2 is guanine. [ka] or adenine [ka] A compound as described in any one of items 14 to 17, wherein R2' is H and R2 is -OH. [Section 21] below: [ka] A cyclic dinucleotide compound selected from the group consisting of TIFF0007875196000299.tif168167, or a pharmaceutically acceptable salt or solvate thereof. [Section 22] A pharmaceutical composition comprising a compound described in any one of items 1 to 21 of the therapeutically effective amount, together with a pharmaceutically acceptable excipient. [Section 23] A pharmaceutical composition as described in item 22, in the form of local administration. [Section 24] A method for the treatment or prevention of diseases related to or transmitted by STING in mammals, particularly humans, particularly viral infections or tumors, comprising administering an effective amount of any one of the compounds described in any one of the items 1 to 21 or any one of the pharmaceutical compositions described in any one of the items 22 to 23. [Section 25] Use of a compound described in any one of items 1 to 21 or a pharmaceutical composition described in any one of items 22 to 23 as a STING agonist for the treatment or prevention of diseases related to or mediated by STING, particularly viral infections or tumors. [Section 26] Use of a compound described in any one of items 1 to 21 or a pharmaceutical composition described in any one of items 22 to 23 as a cytotoxic agent for the treatment or prevention of viral infections or tumors. [Section 27] Use of a compound described in any one of items 1 to 21 or a pharmaceutical composition described in any one of items 22 to 23 as a multifunctional activator for immunotherapy and cytotoxic therapy. [Section 28] The use described in paragraph 27, in which a multifunctional activator is used to activate the immune system by activating the STING signaling pathway to exert antitumor and antiviral replication functions, induce tumor cell death or prevent viral replication by releasing cytotoxic agents, kill tumor cells by sustained activation of STING by releasing tumor DNA, and provide the ability of “immunological memory” or persistent immunity against tumor by releasing tumor neoantigens to produce an antibody-antigen response. [Section 29] Use of a compound described in any one of items 1 to 21 or a pharmaceutical composition described in any one of items 22 to 23 in the manufacture of a pharmaceutical for the treatment or prevention of diseases related to or transmitted by STING, particularly viral infections or tumors. [Section 30] Use of a compound described in any one of items 1 to 21 or a pharmaceutical composition described in any one of items 22 to 23 in the manufacture of a cytotoxic agent for the treatment or prevention of viral infections or tumors. [Section 31] The method or use described in any one of paragraphs 24-26 and 28-30, wherein the tumor is selected from brain cancer, head and neck cancer, skin cancer, melanoma, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colorectal cancer, hematological cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bone cancer, colorectal cancer, liver cancer, renal cell carcinoma, pancreatic cancer, Hodgkin lymphoma, or leukemia.

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