CN115160392A

CN115160392A - Multifunctional cyclic dinucleotide and application thereof

Info

Publication number: CN115160392A
Application number: CN202111201201.4A
Authority: CN
Inventors: 张彦涛; 屈粒; 楼良; 郑普吉; 吕飞
Original assignee: Taili Biotechnology Shanghai Co ltd
Current assignee: Taili Biotechnology Shanghai Co ltd
Priority date: 2020-10-20
Filing date: 2021-10-15
Publication date: 2022-10-11
Also published as: CN117015383A

Abstract

The present invention relates to multifunctional cyclic dinucleotide compounds of formula (X) and derivatives thereof, which are useful as apoptosis inducers or prodrugs of cytotoxic agents for inducing apoptosis or for combating viruses, and which modulate the immune pathway to generate a therapeutically beneficial immune response. The disclosure further relates to pharmaceutical compositions and pharmaceutical combinations comprising the cyclic dinucleotide compounds of the present invention, methods for synthesizing the sameMethods and medical uses thereof.

Description

Multifunctional cyclic dinucleotide and application thereof

Technical Field

The present invention relates to multifunctional cyclic dinucleotide compounds and derivatives thereof, which function includes their use as pro-drugs of apoptosis-inducing or cytotoxic agents for inducing apoptosis of tumor cells or for combating viruses, and as immunomodulators, for modulating immune pathways leading to therapeutically beneficial immune responses, in particular for activating STING-mediated immune pathways. The present disclosure further relates to pharmaceutical compositions and pharmaceutical combinations comprising the cyclic-dinucleotide compounds of the present invention, methods for synthesizing the same, and medical uses thereof.

Background

Cancer is a malignant disease characterized biologically by abnormal cell differentiation and proliferation, uncontrolled growth, infiltrative and metastatic properties, has become one of the important causes of death in humans, and the incidence is still continuously increasing worldwide; at the same time, viral infections have also led to millions of human deaths worldwide.

Antimetabolic nucleoside analogs are one of the major therapeutic approaches in the treatment strategies against cancer/tumors and viral infections. Specifically, antimetabolic nucleoside drugs cannot directly act themselves, must be converted into triphosphorylated forms in vivo by various cellular kinases, become active substrates of polymerase as pseudo metabolites, are embedded into DNA or RNA through a nucleic acid biosynthesis pathway, inhibit modification and extension of DNA or RNA, or inhibit reverse transcriptase related to DNA or RNA synthesis, thereby inducing apoptosis of tumor cells or preventing replication of viruses, exhibit cytotoxicity, and can be used for treatment of cancer/tumor or viral infection; meanwhile, cell fragments generated by the cytotoxicity of the nucleoside drugs can cause the immune response of host cells, thereby further inhibiting the growth and reproduction of tumor cells or viruses.

The anticancer nucleosides currently used clinically include the following:

however, nucleoside drugs have a plurality of active groups such as hydroxyl and amino groups on the molecular structure, which causes low membrane permeation efficiency, poor stability and poor pharmacokinetic properties, and often need special or frequent administration, thus bringing inconvenience to patients. Therefore, the search for new nucleoside drug improvement schemes has been an active drug research and development field.

Endogenous Cyclic Dinucleotide (CDN) cGAMP is an important component of the innate immune system-the cGAS-STING (cyclic GMP-AMP synthase-interferon gene stimulator) signal pathway. Specifically, cGAS interacts with DNA from tumor cells, dying cells, viruses, bacteria, or mitochondria, catalyzing the synthesis of Cyclic Dinucleotide (CDN) cGAMP from ATP and GTP. The endogenous cGAMP produced further binds to STING on the Endoplasmic Reticulum (ER), which is activated, undergoes a conformational change, translocates to Golgi, induces activation of the key transcription factors IRF-3 and NF-. Kappa.B, which enter the nucleus, induces expression of type I interferons and proinflammatory cytokines such as IL-6, TNF-. Alpha.and IFN-. Gamma (Jiang et al, cGAS-STING, an antigenic pathway in cancer immunology, journal of Hematology & Oncology,2020, 13 81 Xiangling Cui et al, STING modulatories: diagnostic design in drug discovery, european Journal of Medicinal Chemistry 182 (2019) 111591.

It is well known in the art that type I interferons not only exhibit antiviral activity, but also directly inhibit human tumor cell proliferation, significantly enhance antitumor immune response by inducing the activation of adaptive and innate immune cells, and inhibit tumor invasion by modulating the expression of enzymes associated with tissue remodeling, and thus are useful as anticancer agents.

Given that endogenous Cyclic Dinucleotide (CDN) cGAMP is a key mediator of the above-mentioned innate immune system in response to viruses and tumors, ultimately contributing to the production of interferons or proinflammatory cytokines and thereby achieving therapeutic benefit, a range of CDN STING agonists have been synthesized and validated for laboratory activity, examples of which are described in WO2014/189805, WO2017/027645, and WO2018/060323, for example. However, existing CDN-based therapies still lack sufficient clinical therapeutic efficacy, and thus there is still a need for improved CDN-based STING agonists to provide safer and more potent antiviral or antitumor effects.

According to the consensus in the art, the STING pathway can be activated by foreign DNA (tumor or virus, etc.). If the mediation of the protein-free new antigen is adopted, downstream proinflammatory factors driven by interferon and the like in the STING pathway lack targeting, so that the autoimmunity response has poor tolerance and a narrow treatment window. The first generation of single STING agonists combined with PD-1 antibodies still focused on activation of systemic immunity, and did not address the selectivity of adaptive immunity and provoke a response from the tumor microenvironment.

The compounds of the invention act, on the one hand, as known high-activity STING agonists, activate signaling pathways, release interferons and other inflammatory factors, activate the immune system; secondly, the cytotoxic function of the tumor cell is started, tumors are killed selectively, a large amount of tumor neoantigens and tumor DNA are released, so that the recognition function of adaptive immunity is established, and the immune system is trained with a definite aim; thirdly, the tumor neoantigen and the tumor DNA continue to activate the STING pathway and other immune systems to kill tumor cells; finally, the released tumor neoantigen is recognized by DC cells and interacts with T cells to form immunological memory, thus achieving long-term control of remote tumor and cancer cell migration.

Specifically, in the process of searching for a new cytotoxic nucleoside drug with improved properties, the nucleoside drug with cytotoxic effect is creatively introduced into the molecular structure of the CDN as a construction unit, namely, a hidden cytotoxic pharmacophore is introduced at the molecular level of the CDN. The formed novel CDN drug molecule can firstly activate STING to induce I-type interferon to generate, and further realize antiviral or antitumor immunotherapy effect. More innovatively, the product formed by the decomposition of the molecule in vivo, namely the nucleoside cytotoxic drug, can specifically interfere the metabolism of nucleic acid, prevent the division and the propagation of cells, cause the death of tumor cells or prevent the replication of viruses, release tumor DNA to continuously activate STING, release tumor neoantigen to establish the recognition function of adaptive immunity, train the goal of the immune system, overcome the defects of low transmembrane efficiency, poor stability, poor pharmacokinetic property and frequent need of special or frequent administration of single nucleoside drugs, and simultaneously overcome the problem of the source of exogenous DNA (such as tumor DNA) required by the continuous activation of STING pathway. More importantly, the molecules disclosed herein have a CDN structural backbone that activates STING, thereby inducing type I interferon production, and thus achieving antiviral or antitumor immunotherapeutic effects. On the other hand, the hidden cytotoxic pharmacophore in the novel CDN molecule can timely release to generate apoptosis fragments, provide antigens aiming at tumors or viruses for an immune system, and generate antibody-antigen response under the coordination of immune leukocyte subgroups, thereby providing the capability of 'immunological memory' or lasting immunity aiming at the encountered antigens.

Therefore, the invention provides a novel CDN compound which can simultaneously realize the anti-metabolic treatment and immunosuppression of virus infection or tumor by the combination of medicines at a sub-molecular level, and can provide enhanced and even synergistic effects compared with single cytotoxic medicine or single CDN STING agonist.

It should be noted that the above discussion of the background of the invention is provided merely to aid the reader in understanding the present invention and is not an admission that it is prior art to describe or constitute the present invention.

Summary of The Invention

The invention aims to provide a group of novel antiviral or antitumor compounds based on a cyclic dinucleotide structure.

In one aspect, the present invention provides a group of cyclic dinucleotide compounds having the general formula, stereoisomers, tautomers, stable isotopic variations, pharmaceutically acceptable salts or solvates thereof,

for example

Wherein X ¹ And X ² Each independently selected from-OH and-SH; and at least one of the two nucleosides in the two nucleotides in the loop has a cytotoxic or antiviral effect. On one hand, the compound with the structure can generate a single nucleoside cytotoxicity or antiviral compound after being decomposed in vivo, and after the compound is activated by intracellular nucleoside kinase triphosphorylation, the division and the reproduction of cells are prevented by specifically interfering the metabolism of nucleic acid, thereby inhibiting the proliferation of tumor cells or the replication of viruses. On the other hand, the molecule of the invention retains the immune activation function of cyclic dinucleotide, namely activates target STING and finally induces I-type interferon to generate through STING signal transduction cascade, thereby generating tumor immune activity, inhibiting the growth and metastasis of tumor, or exerting antiviral activity; furthermore, apoptotic tissue fragments provide the immune system with antigens that are not naturally expressed in the host, producing an antigen-antibody response, thereby providing the ability to "memory" or sustain immunity against the encountered antigen.

Specifically, the present invention provides in this aspect a cyclic dinucleotide compound of formula (Y),

wherein X ¹ 、X ² 、B ₁ 、B ₂ 、R ¹ 、R ¹ ’、R ² 、R ² ' as defined herein; stereoisomers, tautomers, stable isotopic variations, pharmaceutically acceptable salts, prodrugs or solvates thereof.

More specifically, the present invention provides in this aspect cyclic dinucleotide compounds of formulae (I), (II), (III) and (IV) and their respective sub-formulae; more specifically, the present invention provides in this aspect cyclic dinucleotide compounds of each of the subformulae of formulae (I), (II), (III) and (IV); as well as their respective specific embodiments, as described herein below.

In another aspect, the present invention provides a process for the preparation of a compound of the invention as described herein, and also provides a compound of the invention as described herein obtainable by a process as described herein.

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

In another aspect, the present invention provides a compound of the invention as described herein or a pharmaceutical composition as described herein for use as an active agent for the treatment or prevention of a disease associated with or mediated by an immune response, in particular for the treatment or prevention of a disease associated with or mediated by STING, more particularly for the treatment or prevention of an inflammatory, allergic or autoimmune disease, infectious disease or cancer, especially for use as an antiviral or antitumor agent, or as a vaccine adjuvant.

In another aspect, the present invention provides a compound of the invention as described herein or a pharmaceutical composition as described herein for use as a cytotoxic agent, especially an anti-neoplastic agent, for the treatment or prevention of a hyperproliferative disease.

In another aspect, the present invention provides a compound of the invention as described herein or a pharmaceutical composition as described herein for use as a cytotoxic agent for the treatment or prevention of a viral infection.

In another aspect, the present invention provides the use of a compound of the invention as described herein or a pharmaceutical composition as described herein for the treatment or prevention of a disease associated with or mediated by an immune response, for example as a STING agonist, in particular for the treatment or prevention of a disease associated with or mediated by STING, more particularly for the treatment or prevention of inflammation, allergic or autoimmune disease, infectious disease or cancer, especially tumour or viral infection; or as a vaccine adjuvant.

In another aspect, the present invention provides the use of a compound of the invention as described herein or a pharmaceutical composition as described herein as a cytotoxic agent in the treatment or prevention of a hyperproliferative disease, especially a tumor; or as a cytotoxic agent in the treatment or prevention of a viral infection.

In another aspect, the present invention provides a method of treating or preventing a disease associated with or mediated by an immune response, in particular a disease associated with or mediated by STING, more particularly an inflammatory, allergic or autoimmune disease, infectious disease or cancer, especially a tumour or a viral infection in a subject, the method comprising administering a compound of the invention as described herein or a pharmaceutical composition as described herein to a human being or animal.

In another aspect, the present invention provides a method of treating or preventing a hyperproliferative disease, particularly a tumor, in a subject, the method comprising administering to a human or animal a compound of the invention as described herein or a pharmaceutical composition as described herein.

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

In another aspect, the present invention provides the use of a compound of the invention as described herein or a pharmaceutical composition as described herein in the manufacture of a medicament for the treatment or prevention of a disease associated with or mediated by an immune response, in particular a disease associated with or mediated by STING, more particularly an inflammatory, allergic or autoimmune disease, an infectious disease or cancer, especially a tumour or viral infection, or for use as a vaccine adjuvant.

In another aspect, the present invention provides the use of a compound of the invention as described herein or a pharmaceutical composition as described herein for the manufacture of a medicament for the treatment or prevention of a hyperproliferative disease, particularly a tumor.

In another aspect, the present invention provides the use of a compound of the invention as described herein or a pharmaceutical composition as described herein in the manufacture of a medicament for the treatment or prevention of a viral infection.

In another aspect, the present invention provides the compounds of the invention described herein or the pharmaceutical compositions described herein for use as multifunctional active agents, having both immunotherapeutic and cytotoxic therapeutic activity, including the ability to activate the immune system by agonizing the STING signaling pathway to exert anti-tumor and anti-viral replication functions, to cause tumor cell death or prevent viral replication by releasing cytotoxic agents, to subsequently continuously activate STING by releasing tumor DNA to kill tumor cells, and to generate antibody-antigen responses to provide "immunological memory" or sustained immunity to tumors by releasing tumor neoantigens. In this respect, the present invention also provides the use of a compound of the invention as described herein or a pharmaceutical composition as described herein for performing the various functions described above, such as in particular for the treatment or prevention of a viral infection or a tumour; a method of treating or preventing a disease associated with or mediated by an immune response, in particular a disease associated with or mediated by STING, more particularly an inflammatory, allergic or autoimmune disease, an infectious disease or cancer, especially a tumor or viral infection in a subject by various functions as described herein, the method comprising administering a compound of the invention as described herein or a pharmaceutical composition as described herein to a human being or animal; and the use of a compound of the invention as described herein or a pharmaceutical composition as described herein in the manufacture of a medicament for performing a plurality of the functions described above, for example in particular for the treatment or prevention of a viral infection or a tumour.

In another aspect, the present invention provides a pharmaceutical combination comprising a compound of the invention, stereoisomers, tautomers, stable isotopic variations, pharmaceutically acceptable salts or solvates thereof, and at least one other therapeutic agent as described herein,

in another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, stereoisomers, tautomers, stable isotopic variations, pharmaceutically acceptable salts or solvates thereof, at least one additional therapeutic agent, and one or more pharmaceutically acceptable excipients, as described herein.

In another aspect, the present invention provides a pharmaceutical combination as described herein comprising a compound of the invention and at least one other therapeutic agent for the treatment or prevention of a hyperproliferative disease, a viral infection or a disease associated with or mediated by STING, more particularly an inflammatory, allergic or autoimmune disease, an infectious disease or a cancer, especially a tumor or a viral infection.

In another aspect, the present invention provides the use of a pharmaceutical combination as described herein comprising a compound of the invention and at least one other therapeutic agent for the treatment or prevention of a hyperproliferative disease, a viral infection or a disease associated with or mediated by STING, more specifically an inflammatory, allergic or autoimmune disease, an infectious disease or cancer, especially a tumor or a viral infection.

In another aspect, the present invention provides a method of treating or preventing a hyperproliferative disease, a viral infection or a disease associated with or mediated by STING, more particularly an inflammatory, allergic or autoimmune disease, an infectious disease or a cancer, especially a tumor or a viral infection in a subject, the method comprising administering to a human or animal a pharmaceutical combination comprising a compound of the invention and at least one further therapeutic agent as described herein.

Drawings

FIG. 1 shows interferon stimulating activity of representative compounds of the invention in THP-1 cells.

FIG. 2 shows the tumor growth inhibitory activity of representative compounds of the invention in a mouse transplanted CT26 colon cancer model. 2A: tumor volume change of the treated tumor; 2B: tumor volume change of untreated tumors; 2C: body weight change in mice; 2D: tumor volume changes in immunized/non-immunized mice.

FIG. 3 shows the immunological memory of representative compounds of the invention in immunized/non-immunized mice. 3A: immunological memory of immune healthy mice; 3B: immunological memory in non-immunized mice.

FIG. 4 shows the hepatocyte metabolic properties of representative compounds of the invention. 4A: stability of hepatocyte metabolism; 4B: and (4) identifying the metabolite of the liver cell.

FIG. 5 is a drawing: schematic diagram of cGAS-STING signal path.

Disclosure of Invention

Definition of

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

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

Any open valency appearing on a carbon, oxygen, sulfur or nitrogen atom in the structures herein indicates the presence of a hydrogen atom, unless otherwise indicated.

The term "immune system" has the ordinary meaning as understood by those skilled in the art and refers to the whole or any one or more components of molecules, substances (e.g., bodily fluids), anatomical structures (e.g., cells, tissues or organs) and physiological processes involved in preventing infections in vivo, protecting the body during infections or diseases, and/or helping the body to regain health after infections or diseases.

The term "disease associated with or mediated by an immune response" means a disease associated with or mediated by the body's immune system's defense response to the isohexide or variant autologous components. For the purposes of the present invention, "diseases which are associated with or mediated by an immune response" refers in particular to the state of a human or animal or a disease state resulting from a state in which the function of the immune system is impaired, inactivated or otherwise impaired, or in which the function of one or more immune components is impaired, inactivated or otherwise impaired, and in particular to diseases in which the induction of an immune response by the STING pathway can be alleviated.

The term "STING" is an abbreviation for stimulator of interferon genes. STING is a receptor for transmembrane proteins in humans, and activation of STING by a Cyclic Dinucleotide (CDN) leads to activation of the IRF3 and NF- κ B pathways, and thus to induction of type I interferon and proinflammatory cytokines, respectively. The term "STING agonist" refers to any substance that activates STING in vitro or in vivo to elicit a physiological response.

The term "disease associated with or mediated by STING" means a disease in which induction of an immune response by the STING pathway can be alleviated, i.e., a disease in which activation of STING will reduce the incidence of disease, reduce or eliminate disease symptoms, including but not limited to inflammation, allergic or autoimmune diseases, infectious diseases, or cancer, and the like. For the purposes of the present invention, a "disease associated with or mediated by STING" is preferably selected from a tumor or cancer.

The term "hyperproliferative disease," "tumor," or "cancer" refers to a physiological condition characterized by uncontrolled or deregulated cell growth or death in a subject, including solid and blood-borne tumors, whether malignant or benign, including, but not limited to, brain, skin, bladder, ovarian, breast, stomach, pancreatic, prostate, colon, blood, lung, and bone cancers. Examples of the above cancer types include neuroblastoma, intestinal cancers such as rectal cancer, colon cancer, familial adenomatous polyposis carcinoma and hereditary non-lymphoid colorectal cancer, esophageal cancer, lip cancer, larynx cancer, nasopharynx cancer, oral cancer, salivary gland cancer, peritoneal cancer, soft tissue sarcoma, urothelial cancer, sweat gland cancer, stomach cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, testicular cancer, breast cancer (including HER2 negative breast cancer), urinary cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, neuroblastoma and peripheral neuroectodermal tumor, hodgkin's lymphoma, non-hodgkin's lymphoma, burkitt's lymphoma, acute Lymphocytic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), chronic myelogenous leukemia (CLL) and lymphocytic carcinomas, acute Myelogenous Leukemia (AML), myelogenous leukemia (chronic myelogenous leukemia (CML), adult T-cell lymphoma, diffuse lymphoma (DLBCL), hepatoma, multiple myeloma, seminoma, osteosarcoma, chondrosarcoma, anal canal cancer, adrenocortical cancer, chordoma, fallopian tube cancer, gastrointestinal stromal tumor, myeloproliferative disorders, mesothelioma, biliary tract cancer, ewing's sarcoma, and other rare tumor types.

The term "therapeutic agent" refers to one or more substances that are administered to a human or animal to achieve a therapeutic effect, including substances that prevent, cure or mitigate the effects of a disease, improve a health condition. Therapeutic agents of the present invention include not only the CDN compounds provided per se, but also therapeutic agents that may be used in combination with the CDN compounds provided, including but not limited to chemotherapeutic agents, immunological agents (particularly immunotumoral agents), vaccines, adjuvants, and radiation therapy.

The term "chemotherapeutic agent" refers to one or more chemical substances that are administered to a human or animal to kill a tumor, or slow or prevent the growth of a tumor, and/or slow or prevent division of cancer cells, and/or prevent or slow metastasis.

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

The term "immunotherapy" refers to any medical treatment in which one or more components of the human or animal immune system are deliberately modulated in order to directly or indirectly obtain a therapeutic benefit, including systemic and/or local effects as well as prophylactic and/or therapeutic effects. Immunotherapy can be by any route, such as oral, intravenous, cutaneous, injection, inhalation, etc., alone or in any combination, administering one or more immunizing agents to human and animal subjects, whether systemically, topically, or a combination of both.

The term "vaccine" refers to a biological agent administered to a human or animal to elicit or enhance a specific immune system response and/or protection against one or more antigens in the human or animal.

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

The term "cytotoxic agent" or "apoptosis-inducing agent" or the like as used herein refers to an agent used to treat abnormal and uncontrolled developmental growth of cells. For the purposes of the present invention, "cytotoxic agent" refers in particular to nucleoside antimetabolic cytotoxic or antiviral agents, including but not limited to cytarabine, azacitidine, fluorouracil, deoxyuridine, enocitabine, deoxyfluorouracil, pentostatin, fludarabine, cladribine, gemcitabine, capecitabine, clofarabine, nelarabine, trifluorothymidine, 8-chloroadenosine, triciribine, forodesine, 5-fluorodeoxycytidine, ribavirin or acadesine.

The term "multifunctional agent" as used herein means a compound molecule of the present invention, based on its unique structural design, capable of multiple functions in a subject, with both immunotherapeutic and cytotoxic therapeutic activities, including but not limited to the ability to activate the immune system by agonizing the STING signaling pathway to exert anti-tumor and anti-viral replication functions, to cause tumor cell death or prevent viral replication by releasing cytotoxic agents, to continuously activate STING by releasing tumor DNA to kill tumor cells, to generate antibody-antigen responses by releasing tumor neoantigens to provide "immunological memory" or sustained immunity to tumors.

The term "treatment" of a disease includes inhibiting the disease state, i.e., arresting the development of the disease state or its clinical symptoms, or relieving the disease state, i.e., causing temporary or permanent regression of the disease state or its clinical symptoms.

The term "prevention" or "preventing" of a disease means that clinical symptoms of the disease state are not developed in a subject that may be exposed to or predisposed to the disease state but does not yet experience or display symptoms of the disease state.

The term "therapeutically effective amount" means an amount of a compound or molecule of the invention that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The therapeutically effective amount will vary depending upon the compound, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors.

The term "subject", "individual" or "patient" as used herein refers to a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (e.g., cattle), sport animals, pets (e.g., guinea pigs, cats, dogs, rabbits, and horses), primates, mice, and rats. In a preferred embodiment, the mammal is a human.

The terms "pharmaceutical composition" and "pharmaceutical formulation" (or "formulation") are used interchangeably and refer to a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient, together with pharmaceutically acceptable excipients, to be administered to a mammal (e.g., a human) in need thereof.

The term "pharmaceutical combination" means that the compounds of the present invention can be used in combination with other active agents for the purpose of the present invention. The additional active agent may be one or more additional compounds of the invention, or may be a second or additional (e.g., third) compound that is compatible with, i.e., does not adversely affect, each other, or has complementary activity. Such active agents are suitably present in combination in an effective amount to achieve the intended purpose. The additional active agents may be co-administered with the compounds of the present invention in a single pharmaceutical composition, or separately administered in separate discrete units from the compounds of the present invention, either simultaneously or sequentially when administered separately.

The term "pharmaceutically acceptable" refers to the attributes of materials that may be used in preparing a pharmaceutical composition, which are generally safe, non-toxic, neither biologically nor otherwise undesirable, and acceptable for veterinary as well as human pharmaceutical use.

The terms "pharmaceutically acceptable excipient", "pharmaceutically acceptable carrier" and "therapeutically inert excipient" are used interchangeably and refer to any pharmaceutically acceptable ingredient in a pharmaceutical composition that is not therapeutically active and is non-toxic to the subject to which it is administered, such as disintegrants, binders, fillers, solvents, buffers, tonicity agents, stabilizers, antioxidants, surfactants, carriers, diluents or lubricants used to formulate pharmaceutical products.

The term "pharmaceutically acceptable salt" as used herein means a salt of a compound of the invention which is pharmaceutically acceptable and which possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and may be inorganic or organic acid addition salts and base addition salts, including but not limited to: (1) Acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or an acid addition salt formed with an organic acid such as acetic acid, propionic acid, hexanoic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc.; or (2) a salt formed when an acidic proton present in the parent compound is replaced with a metal ion such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion, or coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, or the like. The general principles and techniques for preparing pharmaceutically acceptable salts are known to those skilled in the art, for example, as described in Berge et al, pharm ScL,66,1-19 (1977).

The term "pharmaceutically acceptable prodrug" as used herein means a compound of the invention having a cleavable group and which becomes pharmaceutically active in vivo by solvolysis or under physiological conditions, and specifically prodrugs include those compounds which can be oxidized, reduced, aminated, deammonified, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated or dephosphorylated to produce the active compound, including derivatives of the compounds of the invention. Prodrugs in various forms are well known in the art, and suitable prodrug moieties are described, for example, in "produgs and Targeted Delivery", j.

Prodrugs of CDN compounds described herein may generally increase the activity, bioavailability, or stability of the compounds. In general, alkylation, acylation or other lipophilic modification of the phosphate moiety or the use of other analogs of the nucleotide will help to increase the stability of the nucleotide.

The term "solvate" as used herein refers to a form of addition of a solvent comprising a stoichiometric or non-stoichiometric amount of solvent, including for example solvates with water, such as hydrates, or with organic solvents, such as methanol, ethanol or acetonitrile, i.e. as methanolate, ethanolate or acetonitrilate, respectively; or in the form of any polymorph. It will be understood that such solvates of the compounds of the invention also include solvates of pharmaceutically acceptable salts of the compounds of the invention.

The term "isotopic variant" as used herein refers to a compound wherein one or more atoms constituting the compound are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into one or more atoms of the compounds of the invention include, for example ² H、 ³ H、 ¹³ C、 ¹⁴ C、 ¹⁵ N、 ¹⁷ O、 ¹⁸ O、 ³¹ P、 ³² P、 ³⁵ S and ¹⁸ f, thereby forming the bookIsotopic variations of the compounds of the invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. In some embodiments, the incorporated isotope is 2H (deuterium); in other embodiments, the incorporated isotope is 3H (tritium).

The term "stereoisomer" as used herein denotes an isomer formed as a result of at least one asymmetric center. In compounds having one or more (e.g., 1,2, 3, or 4) asymmetric centers, they can result in racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Similarly, the compounds of the invention may exist as a mixture of two or more different structural forms in rapid equilibrium (commonly referred to as tautomers). It is to be understood that the scope of this application encompasses all such isomers or mixtures thereof in any ratio (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

The compounds of the invention may have one or more asymmetric centers and may thus be prepared as (R) -or (S) -stereoisomers, respectively, or as mixtures thereof. Compounds of formula (I) or structural fragments

Denotes the configuration of an asymmetric center, i.e. a chiral center. Accordingly, in the nomenclature of the compounds or intermediates provided herein, the configuration about the chiral center is denoted by R and S. One skilled in the art will appreciate that the phosphorothioate linkages in the compounds of the invention are inherently chiral, and may each exist in either the R or S configuration, and thus two phosphorothioate linkages are possible in the R, S, R and R, S forms. The present invention covers the compounds of the invention and embodiments thereof in substantially pure form or in the form of mixtures, the compounds containing two phosphorothioate linkages preferably being the R, S, R and R, S stereoisomers in substantially pure form, particularly preferably the R, R stereoisomers are substantially pure, i.e. both phosphorus atoms have the R configuration. Their absolute configuration assignments may be in accordance withLiterature methods are performed (Zhao et al, nucleotides and Nucleic Acids 2009, 289, 352-378. It is to be noted that the actual configuration of the compounds of the invention is not affected by the misassignment of configuration due to errors in literature procedures.

The term "substantially pure" as used herein with respect to CDNs means that a certain stereoconfigurational form is at least 75% pure with respect to other possible stereochemical configurations at the chiral centers indicated in the above figures. In preferred embodiments, a substantially pure CDN is at least 85% pure, at least 90% pure, at least 95% pure, at least 97% pure, and at least 99% pure. The substantially pure CDN preparations of the present invention are "stereochemically pure" in the sense that all CDNs within the preparation have a particular stereochemical configuration at these chiral centers, and is not intended to indicate that all CDNs within the preparation having a particular stereochemical configuration at these chiral centers are otherwise identical. For example, a substantially pure CDN R, R cGAMP phosphorothioate formulation may comprise a combination of R, R c-di-GMP phosphorothioate and R, R c-di-AMP phosphorothioate and still be a substantially pure cyclic purine dinucleotide formulation.

The term "protecting group" as used herein denotes a group that selectively blocks a reactive site in a polyfunctional compound, such that a chemical reaction can be selectively carried out at another unprotected reactive site in the sense normally associated therewith in synthetic chemistry. The protecting group may be removed at an appropriate point in time. Exemplary protecting groups include amino protecting groups including, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis (4-methoxyphenyl) benzyl), bz (benzoyl), i-BuCO (isobutyryl), benzyl, benzyloxycarbonyl (carbonylbenzyloxy, CBZ), fmoc (9-fluorenylmethoxycarbonyl), p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, tert-Butoxycarbonyl (BOC), and trifluoroacetyl; and hydroxy protecting groups including, but not limited to, ester and ether forming groups, particularly tetrahydropyranyloxy, bz (benzoyl), ^i- BuCO (isobutyryl), DMTr (bis (4-methoxyphenyl) benzyl), acetoxy, carbamoyloxy, benzyl and silyl ethers such asTBS (tert-butyldimethylsilyl), TBDPS (tert-butyldiphenylsilyl). Other examples of such groups are found in t.w.greene and p.g.m.wuts, "Greene's protective groups in organic synthesis", 5 th edition. John Wiley&Sons.，Inc.，Hoboken，New Jersey，2014。

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

The term "alkyl" as used herein means a straight or branched chain aliphatic hydrocarbon group having the indicated number of carbon atoms. Specifically, the alkyl group can have 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 7, 1 to 6,1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. Examples of suitable C1-14 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, dimethylmethyl, dipropylmethyl, ethylbutylmethyl, diethylmethyl, methylethylmethyl, ethylpropylmethyl, diethylethyl, diethylpropyl, dipropylethyl, and the like. Particular alkyl groups have 1 to 7 carbon atoms, for example 1 to 6 carbon atoms, 1 to 4 carbon atoms.

The term "halo" or "halogen" as used herein means fluorine (F), chlorine (Cl), bromine (Br) and iodine (I). Particular halo groups are fluoro or chloro.

For clarity, the term "guanine" is used herein

Can also be expressed as

The phosphorothioate group used in the structural formula of the compounds of the present invention can be drawn as

Compounds of the invention

The terms "compound described herein", "inventive compound" and the like, as used throughout this application, unless otherwise indicated, encompass the compounds of formula (X), formula (Y), and the specific embodiments formula (I), (II), (III), and (IV), each of the sub-general embodiments thereof and the specific or preferred embodiments thereof, their stereoisomers, tautomers, racemates, stable isotopic variations, pharmaceutically acceptable salts or solvates, and pharmaceutically acceptable prodrugs, each as described in the definitions section above. The compounds of the invention can be isolated as mixtures of isomers or as individual isomers which can be prepared synthetically, for example, by resolution of racemates by chromatography or fractional crystallization or from optically active starting materials. Similarly, references herein to "intermediates" are intended to encompass the free form thereof as well as each of the derivatives described above, whether or not claimed per se, if the context permits.

Preferably, the compounds of the invention are in free form or a pharmaceutically acceptable salt or solvate thereof; most preferably in free form or a pharmaceutically acceptable salt thereof.

Certain compounds of the present invention may exist in polymorphic or amorphous forms, which also fall within the scope of the present invention. When in solid crystalline form, the compounds of the invention may be in the form of a co-crystal with another chemical entity, and the specification includes all such co-crystals.

In particular, in one aspect, the present invention provides a cyclic dinucleotide compound of the formula:

for example, in

Wherein X ¹ And X ² Each independently selected from-OH and-SH; and at least one of the two nucleosides of the two nucleotides forming the ring has a cytotoxic or antiviral effect, preferably those antimetabolites, i.e., nucleoside-type anticancer drugs or derivatives thereof, including, but not limited to, clinically used enocitabine (enocitabine), fludarabine (fludarabine), cytarabine (cytarabine), nelarabine (nelarabine), deoxyfluorouracil (doxifluridine), capecitabine (capecitabine), azacitidine (azacitidine), pentostatin (pentostatin), cladribine (cladribine), gemcitabine (gemcitabine) and clofarabine (clofarabine), more preferably cladribine, gemcitabine and clofarabine or derivatives thereof. The nucleoside anticancer drug derivatives referred to herein are compounds derived by substituting hydrogen atoms or atomic groups in the structure of the compounds with other atoms or atomic groups, and retaining anticancer cytotoxicity.

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

wherein X ¹ And X ² Each independently selected from-OH and-SH;

b1 is adenine substituted by X

Wherein X is selected from Cl, F or-NHC _1-6 An alkyl group; or optionally substituted with R ^a Substituted cytosines

Wherein R is ^a Selected from H or-C (O) -C _1-14 An alkyl group;

R ₁ and R ₁ ' are each independently selected from H, F, or-OH;

B ₂ selected from adenine optionally substituted by X

Wherein X is selected from H,F or Cl; optionally substituted with R ^a Substituted cytosines

Wherein R is ^a Selected from H or-C (O) -C _1-14 An alkyl group; or guanine

Wherein OH is optionally substituted by C _1-6 Alkyl substitution;

means that the phosphate bond may be attached to the 2 'or 3' position of the pentose, the site of both not forming a ring with phosphate being R ₂ And R ₂ ' substitution; and

R ₂ and R ₂ ' are each independently selected from H, -OH or F;

with the proviso that when B ₁ Or B ₂ One of which is optionally substituted with R ^a Substituted cytosine where the carbon atom adjacent to the pentose ring to which it is attached is substituted with two F;

stereoisomers, tautomers, stable isotopic variations, pharmaceutically acceptable salts, prodrugs or solvates thereof.

In a particular embodiment, the compound of formula (Y) may be

Wherein B is ₁ 、B ₂ 、R ₁ 、R ₁ ’、R ₂ 、R ₂ ' has the meaning as defined above for the compound of formula (Y).

In a particular embodiment, the two phosphorothioate linkages in the compounds of the invention (when present) are in the R, S, R or R, S configuration or mixtures thereof. In a preferred embodiment, the two phosphorothioate linkages in the compounds of the invention (when present) are present in substantially pure form in the R, S, R or R, S configuration, with substantially pure R, R configuration being particularly preferred.

In a particular embodiment, the single phosphorothioate linkage present in the compounds of the invention may be present in the R or S configuration, preferably in the R configuration.

In one embodiment of a compound of formula (I), (II), (III), or (IV), B ₁ Is adenine substituted by X

Wherein X is selected from Cl or F, preferably Cl, or optionally R ^a Substituted cytosines

Wherein R is ^a Selected from H or-C (O) -C _1-14 An alkyl group.

In an embodiment of a compound of formula (I), (II), (III) or (IV), B ₁ Is adenine substituted by X

Wherein X is-NHC _1-6 Alkyl radicals, e.g. -NHCH ₃ 、-NHCH ₂ CH ₃ 、- NHCH ₂ CH ₂ CH ₃ preferably-NHCH ₃ 。

In an embodiment of a compound of formula (I), (II), (III) or (IV), B ₁ Is optionally substituted by R ^a Substituted cytosines

Wherein R is ^a Selected from H or-C (O) -C _1-14 An alkyl group.

In an embodiment of a compound of formula (I), (II), (III) or (IV), R ₁ And R ₁ ' are both H, or one of them is H and the other is F, or both are F.

In an embodiment of a compound of formula (I), (II), (III) or (IV), B ₂ Is adenine optionally substituted by X

Wherein X is selected from H, F orCl, preferably H or Cl.

In an embodiment of a compound of formula (I), (II), (III) or (IV), B ₂ Is optionally substituted by R ^a Substituted cytosines

Wherein R is ^a Selected from H or-C (O) -C _1-14 An alkyl group.

In one embodiment of a compound of formula (I), (II), (III), or (IV), B ₂ Is guanine

In an embodiment of a compound of formula (I), (II), (III) or (IV), R ₂ And R ₂ ' are both H, or one of them is H and the other is F, or one of them is H and the other is OH, or both are F.

In an embodiment of a compound of formula (I), (II), (III) or (IV), R ^a is-C (O) C _1-14 The alkyl group 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 radical) ₂ 、-C(O)CH(C _1-3 Alkyl radical) ₂ 、-C(O)CH ₂ CH(C _1-4 Alkyl radical) ₂ 、- C(O)CH ₂ CH(C _1-3 Alkyl radical) ₂ 。

The compounds of formula (I), (II), (III) or (IV) of the present invention also encompass any combination between the various embodiments described above, as well as preferred or exemplary embodiments thereof.

In a further specific embodiment, the present invention provides a cyclic dinucleotide compound of formula (Y) having the following sub-formula,

wherein B is ₁ 、B ₂ 、R ₁ 、R ₁ ’、R ₂ 、R ₂ ' has the meaning as defined above for the compounds of formula (I), (II), (III) or (IV) and the various embodiments thereof.

In one embodiment of the compounds of the formula (II-a) or (II-B), in particular of the formula (II-a), B ₁ Is composed of

And B ₂ Is composed of

In a particular such embodiment, R ₁ And R ₁ ' are both H; in another specific such embodiment, R ₁ And R ₁ One is H and the other is F. Further, in each of the specific embodiments, R ₂ And R ₂ ' are both H, or one is H and the other is F, or one is H and the other is OH.

In one embodiment of the compounds of the formula (II-a) or (II-B), in particular of the formula (II-a), B ₁ And B ₂ Are all optionally substituted by R ^a Substituted cytosines

In a particular such embodiment, R ₁ 、R ₁ ’、 R ₂ 、R ₂ ' both are F.

In one embodiment of the compounds of the formula (II-a) or (II-B), in particular of the formula (II-B), B ₁ Is composed of

And B ₂ Is composed of

In a particular such embodiment, R ₁ And R ₁ ' are both H; in another specific such embodiment, R ₁ And R ₁ One is H and the other is F. Further, in each of the embodiments, R ₂ And R ₂ ' are both H, or one is H and the other is F, or one is H and the other is OH, preferably one is H and the other is OH.

In one embodiment of the compounds of the formula (II-a) or (II-B), in particular of the formula (II-B), B ₁ Is optionally substituted by R ^a Substituted cytosines

And B ₂ Is composed of

In a particular such embodiment, R ₁ And R ₁ ' both are F. Further, in each of the embodiments, R ₂ And R ₂ ' are both H, or one is H and the other is F, or one is H and the other is OH, preferably one is H and the other is OH.

In one embodiment of the compound of formula (II-a) or (II-b), R ^a Is H, or is-C (O) C _1-10 Alkyl radicals, e.g. 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 radical) ₂ 、 -C(O)CH(C _1-3 Alkyl radical) ₂ 、-C(O)CH ₂ CH(C _1-4 Alkyl radical) ₂ 、-C(O)CH ₂ CH(C _1-3 Alkyl radical) ₂ 。

In one embodiment of the compounds of formula (II-a) or (II-B), B ₁ Is composed of

R ₁ And R ₁ Both are H.

R ₁ And R ₁ ' one is H and the other is F; in particular toIn the embodiment, F and B ₁ Located on the same side of the ribose.

Preference is given to

And R is ₁ And R ₁ ' are both H, or R ₁ And R ₁ ' one is H and the other is F, preferably R ₁ And R ₁ One is H and the other is F.

In one embodiment of the compound of formula (II-a) or (II-B), B ₁ Is optionally substituted by R ^a Substituted cytosines

Wherein R is ^a Selected from H, R ₁ And R ₁ ' both are F.

In one embodiment of the compounds of formula (II-a) or (II-B), B ₁ Is optionally substituted by R ^a Substituted cytosines

Wherein R is ^a is-C (O) C _1-10 Alkyl, preferably-C (O) CH (C) _1-4 Alkyl radical) ₂ ，R ₁ And R ₁ ' both are F.

In exemplary embodiments, R ^a is-C (O) CH (CH) ₃ ) ₂ 、-C(O)CH(CH ₂ CH ₃ ) ₂ 、- C(O)CH(CH ₃ )(CH ₂ CH ₃ )、-C(O)CH(CH ₂ CH ₂ CH ₃ ) ₂ 、- C(O)CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )、-C(O)CH ₂ CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )、 C(O)CH ₂ CH(CH ₂ CH ₂ CH ₃ ) ₂ Preferably R ^a Is C (O) CH (CH) ₂ CH ₂ CH ₃ ) ₂ 。

In one embodiment of the compound of formula (II-a) or (II-B), B ₂ Is guanine

Or adenine

And R is ₂ And R ₂ ' one is H and the other is selected from-OH or F.

In one embodiment of the compound of formula (II-a), B ₂ Is guanine

R ₂ And R ₂ ' one is H and the other is selected from-OH or F.

In one embodiment of the compound of formula (II-a), B ₂ Is adenine

R ₂ And R ₂ ' one is H and the other is selected from-OH or F.

In one embodiment of the compound of formula (II-a), B ₂ Is adenine substituted by halogen X

Wherein X is Cl; r ₂ And R ₂ ' one is H and the other is F, preferably F and B ₂ Located on the same side as the ribose.

In one embodiment of the compounds of formula (II-a), B ₂ Is adenine substituted by halogen X

Wherein X is C1; r ₂ And R ₂ ' are both H.

In one embodiment of the compounds of formula (II-a), B ₂ Is optionally substituted by R ^a Substituted cytosines

Wherein R is ^a Selected from H, R ₁ And R ₁ ' both are F.

In one embodiment of the compounds of formula (II-a), B ₂ Is represented by R ^a Substituted cytosines

In exemplary embodiments, R ^a is-C (O) C (CH) ₃ ) ₂ 、-C(O)CH(CH ₂ CH ₃ ) ₂ 、- C(O)CH(CH ₃ )(CH ₂ CH ₃ )、-C(O)CH(CH ₂ CH ₂ CH ₃ ) ₂ 、- C(O)CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )、-C(O)CH ₂ CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )、 C(O)CH ₂ CH(CH ₂ CH ₂ CH ₃ ) ₂ Preferably R ^a Is C (O) CH (CH) ₂ CH ₂ CH ₃ ) ₂ 。

In one embodiment of the compound of formula (II-B), B ₂ Is guanine

Or adenine

R ₂ And R ₂ One of' is H and the other is-OH.

In the compounds of formula (II-a) or formula (II-b) above and in various embodiments thereof, the two phosphorothioate linkages are in the R, S, R or R, S configuration or mixtures thereof; preferably in substantially pure form, and particularly preferably in substantially pure R, S, R or R, S configuration.

The embodiments given above for the compounds of the formula (II-a) or (II-b) and their preferred or exemplary embodiments, respectively, also apply to the compounds of the formula (I-a) or (I-b), the formula (III-a) or (III-b), the formula (IV-a) or (IV-b), respectively, i.e. the invention also encompasses compounds of the formula (I-a) or (I-b), the formula (III-a) or (III-b), the formula (IV-a) or (IV-b), wherein the particular substituents each take the particular definitions given above for the compounds of the formula (II-a) or (II-b) or embodiments thereof, respectively.

For example, in an embodiment of a compound of formula (I-a) or formula (I-B), a compound of formula (III-a) or formula (III-B), or a compound of formula (IV-a) or formula (IV-B), especially a compound of formula (I-B), formula (III-B) or formula (IV-B), B ₁ Is composed of

And B ₂ Is composed of

Preference is given to

In a particular such embodiment, R ₁ And R ₁ One is H and the other is F. Further, in each of the embodiments, R ₂ And R ₂ One of' is H and the other is OH.

The compounds of formula (I-a)/(I-b), (II-a)/(II-b), (III-a)/(III-b) or (IV-a)/(IV-b) according to the invention also encompass any combination between the various embodiments described above and preferred or exemplified embodiments thereof.

In yet a further embodiment, the present invention provides a cyclic dinucleotide compound of the following sub-formula,

wherein B is ₁ 、B ₂ 、R ₁ 、R ₁ ’、R ₂ 、R ₂ ' has the meaning as defined above for compounds of formula (I), (II), (III) or (IV) or each of the embodiments thereof; more particularly, having the meaning defined above for the compounds of formula (I-a)/(I-b), (II-a)/(II-b), (III-a)/(III-b) or (IV-a)/(IV-b) or each embodiment thereof.

In an embodiment of a compound of the formula (II-a ') or (II-B '), in particular of the formula (II-a '), B ₁ Is composed of

And B ₂ Is composed of

In a particular such embodiment, R ₁ And R ₁ ' are both H; in another specific such embodiment, R ₁ Is F, R ₁ ' is H, i.e., F is ipsilateral to B1 at the ribose. Further, in each of the specific embodiments, R ₂ And R ₂ ' are both H, or R ₂ Is H and R ₂ ' is F, or R ₂ Is F and R ₂ ' is H, or R ₂ Is OH and R ₂ ' is H.

In one embodiment of the compounds of the formula (II-a ') or (II-B '), in particular of the formula (II-a '), B ₁ And B ₂ Are all optionally substituted by R ^a Substituted cytosines

In an embodiment of a compound of the formula (II-a ') or (II-B '), in particular of the formula (II-B '), B ₁ Is composed of

And B ₂ Is composed of

In a particular such embodiment, R ₁ And R ₁ ' are both H; in another specific such embodiment, R ₁ Is F, R ₁ ' is H, i.e., F is ipsilateral to B1 at the ribose. Further, in each of the embodiments, R ₂ And R ₂ ' are both H, or R ₂ Is H and R ₂ ' is F, or R ₂ Is F and R ₂ ' is H, or R ₂ Is OH and R ₂ ' is H; preferably R ₂ Is OH and R ₂ ' is H.

In an embodiment of a compound of the formula (II-a ') or (II-B '), in particular of the formula (II-B '), B ₁ Is optionally substituted by R ^a Substituted cytosines

And B ₂ Is composed of

In a particular such embodiment, R ₁ And R ₁ ' both are F. Further, in each of the embodiments, R ₂ And R ₂ ' are both H, or R ₂ Is H and R ₂ ' is F, or R ₂ Is F and R ₂ ' is H, or R ₂ Is OH and R ₂ ' is H; preferably R ₂ Is OH and R ₂ ' is H.

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 radicals, e.g. 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 radical) ₂ 、- C(O)CH(C _1-3 Alkyl radical) ₂ 、-C(O)CH ₂ CH(C _1-4 Alkyl radical) ₂ 、-C(O)CH ₂ CH(C _1-3 Alkyl radical) ₂ 。

In an embodiment of a compound of formula (II-a ') or formula (II-B'), B ₁ Is composed of

R ₁ And R ₁ ' are both H, whereby the B ₁ Together with the ribose to which it is attached, form cladribine, a nucleoside antineoplastic agent.

In one embodiment of the compound of formula (II-a ') or (II-B'), B ₁ Is composed of

R ₁ Is F, R ₁ ' is H, i.e. F and B ₁ On the same side as the ribose sugar, whereby the B ₁ Together with the ribose to which it is attached, form the nucleoside antineoplastic agent clofarabine.

Preference is given to

And R is ₁ And R ₁ ' are both H, or R ₁ Is F and R ₁ ' is H, preferably R ₁ Is F and R ₁ ' is H.

In an embodiment of a compound of formula (II-a ') or formula (II-B'), B ₁ Is optionally substituted by R ^a Substituted cytosines

Wherein R is ^a Selected from H, R ₁ And R ₁ ' are both F, whereby the B ₁ Together with the ribose to which it is attached, form the nucleoside antineoplastic agent gemcitabine.

In one embodiment of the compound of formula (II-a ') or (II-B'), B ₁ Is optionally substituted by R ^a Substituted cytosines

Wherein R is ^a is-C (O) C _1-10 Alkyl, preferably-C (O) CH (C) _1-4 Alkyl radical) ₂ ，R ₁ And R ₁ ' are both F, whereby the B ₁ Together with the ribose to which they are attached, form an alkanoylated derivative of the nucleoside antitumor agent gemcitabine. In exemplary embodiments, R ^a is-C (O) CH (CH) ₃ ) ₂ 、-C(O)CH(CH ₂ CH ₃ ) ₂ 、- C(O)CH(CH ₃ )(CH ₂ CH ₃ )、-C(O)CH(CH ₂ CH ₂ CH ₃ ) ₂ 、- C(O)CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )、-C(O)CH ₂ CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )、 C(O)CH ₂ CH(CH ₂ CH ₂ CH ₃ ) ₂ Preferably R ^a Is C (O) CH (CH) ₂ CH ₂ CH ₃ ) ₂ 。

In an embodiment of a compound of formula (II-a'), B ₂ Is guanine

R ₂ ' is H, R ₂ Is selected from-OH or F.

In an embodiment of a compound of formula (II-a'), B ₂ Is adenine

R ₂ ' is H, R ₂ Is selected from-OH or F.

In an embodiment of a compound of formula (II-a'), B ₂ Is adenine substituted by halogen X

Wherein X is Cl, R ₂ Is H, R ₂ ' is F, and F and B ₂ On the same side as the ribose sugar, whereby the B ₂ Together with the ribose to which it is attached, form the nucleoside antineoplastic agent clofarabine.

Wherein X is Cl, R ₂ And R ₂ ' are both H, whereby the B ₂ Together with the ribose to which it is attached, form cladribine, a nucleoside antineoplastic agent.

In an embodiment of a compound of formula (II-a'), B ₂ Is optionally substituted by R ^a Substituted cytosines

Wherein R is ^a Selected from H, R ₁ And R ₁ ' are both F, whereby the B ₂ Together with the ribose to which it is attached, form the nucleoside antineoplastic agent gemcitabine.

In an embodiment of the compounds of the formula (II-a'), B ₂ Is represented by R ^a Substituted cytosines

Wherein R is ^a is-C (O) C _1-10 Alkyl, preferably-C (O) CH (C) _1-4 Alkyl radical) ₂ ，R ₁ And R ₁ ' are both F, whereby the B ₂ Together with the ribose to which they are attached, form an alkanoylated derivative of the nucleoside antitumor agent gemcitabine. In exemplary embodiments, R ^a is-C (O) CH (CH) ₃ ) ₂ 、-C(O)CH(CH ₂ CH ₃ ) ₂ 、- C(O)CH(CH ₃ )(CH ₂ CH ₃ )、-C(O)CH(CH ₂ CH ₂ CH ₃ ) ₂ 、- C(O)CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )、-C(O)CH ₂ CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )、 C(O)CH ₂ CH(CH ₂ CH ₂ CH ₃ ) ₂ Preferably R ^a Is C (O) CH (CH) ₂ CH ₂ CH ₃ ) ₂ 。

In an embodiment of the compounds of the formula (II-B'), B ₂ Is guanine

Or adenine

R ₂ ' is H, R ₂ is-OH.

The embodiments given above for the compounds of the formula (II-a ') or of the formula (II-b') and their preferred or exemplified embodiments, respectively, also apply to the compounds of the formula (I-a ') or of the formula (I-b'), of the formula (III-a ') or of the formula (III-b'), of the formula (IV-a ') or of the formula (IV-b'), respectively, i.e.the invention also encompasses compounds of the formula (I-a ') or of the formula (I-b'), of the formula (III-a ') or of the formula (III-b'), of the formula (IV-a ') or of the formula (IV-b'), in which the particular substituents in each case take the particular definitions given above for the compounds of the formula (II-a ') or of the formula (II-b') or of their embodiments, respectively.

For example, in an embodiment of a compound of formula (I-a ') or formula (I-B '), a compound of formula (III-a ') or formula (III-B '), or a compound of formula (IV-a ') or formula (IV-B '), especially a compound of formula (I-B '), formula (III-B ') or formula (IV-B '), B ₁ Is composed of

And B ₂ Is composed of

Preference is given to

In a particular such embodiment, R ₁ Is F and R ₁ ' is H. Further, in each of the embodiments, R ₂ Is OH and R ₂ ' is H.

It is intended that the compounds of the present invention encompass each of the embodiments above, as well as embodiments comprised of any combination or subcombination of the various embodiments described above, as well as embodiments comprised of any combination of any of the foregoing preferences or illustrations.

Preferred embodiments of the compounds of the present invention include the following compounds, stereoisomers, tautomers, stable isotopic variations, pharmaceutically acceptable salts, or solvates thereof,

pharmacological Activity and beneficial Effect of the Compounds of the invention

The cyclic dinucleotide compound provided by the invention has the following pharmacological activities and beneficial effects through research and discovery:

● Effectively stimulates THP-1 cells to secrete IFN-beta, thereby showing that the THP-1 cells have high affinity to STING receptors, can effectively activate STING and induce I-type interferon to generate;

● Effectively inhibiting the in vitro growth of the mouse colorectal cancer cell line CT26, showing that the compound has toxic effect on tumor cells and prevents the division and the propagation of the tumor cells;

● The anti-tumor activity in the immune healthy CT26 isogenic mouse bilateral transplantation tumor model is superior to that in the immune deficient mouse model, and the anti-tumor activity and the anti-tumor cytotoxicity function in the STING-activated mouse model are shown, so that the anti-tumor activity and the cytotoxicity function in the STING-activated mouse are simultaneously realized, and the anti-tumor effect in the STING-activated mouse bilateral transplantation tumor model is additive and even synergistic;

● The immunological memory function is shown in a CT26 syngeneic mouse transplantation tumor model, and the tumor recurrence can be effectively prevented;

● Shows good pharmacokinetic properties in liver cell metabolism studies, e.g. with longer t _1/2 And a low clearance rate, thereby allowing increased dosing intervals and better patient compliance; and

● The medicine is applied to the focus part locally, has accurate effect, can reduce the dosage, is difficult to diffuse outside the focus due to high molecular polarity, has limited toxicity and good safety.

Pharmaceutical composition

Another aspect of the invention provides pharmaceutical compositions comprising a compound of the invention and one or more pharmaceutically acceptable excipients, and methods of using the compounds of the invention for preparing such compositions.

The compositions or dosage forms are formulated, administered and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to medical practitioners.

Typical pharmaceutical compositions or dosage forms are prepared by mixing a compound of the invention with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, in Gennaro a.r. et al, remington: the Science and Practice of Pharmacy (2000) Lippincott, williams & Wilkins, philadelphia. The formulations may also include one or more buffering agents, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifying agents, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, fragrances, flavoring agents, diluents, and other known additives to provide an elegant appearance of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or to aid in the preparation of the pharmaceutical product (i.e., a pharmaceutical agent).

The compounds of the invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal administration, and if desired topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

In a preferred embodiment, there is provided a pharmaceutical composition comprising one or more compounds of the invention, wherein the pharmaceutical composition is suitable for intravenous, intratumoral, peritumoral or subcutaneous administration. The intratumoral (directly entering a tumor mass) or peritumoral (surrounding the tumor mass) administration of the compound can directly activate local hydrophilic dendritic cells, directly promote apoptosis of tumor cells or make the tumor cells sensitive to cytotoxic agents.

The compounds of the invention may be administered in any convenient form of administration, for example, tablets, powders, capsules, sterile injectable preparations, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches and the like. Such compositions may contain ingredients conventional in pharmaceutical formulations, such as diluents, carriers, pH adjusting agents, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents, antioxidants and other active agents. They may also contain other therapeutically valuable substances. Various formulation forms can be prepared according to conventional methods in the formulation art, see, for example, gennaro a.r. et al, remington: the Science and Practice of Pharmacy (2000) Lippincott, williams & Wilkins, philadelphia or pharmacopoeia of various countries.

In a preferred embodiment, there is provided a pharmaceutical composition comprising one or more compounds of the invention in the form of a parenteral formulation, especially a sterile injectable formulation, such as a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, or as a lyophilized powder. Among the acceptable vehicles or solvents that may be employed are, for example, water, 1, 3-butanediol, ringer's solution or isotonic sodium chloride solution; in addition, sterile fixed oils may be conventionally employed as a solvent or suspending medium for which any bland fixed oil may be employed including, for example, synthetic mono-or diglycerides, fatty acids and the like.

The dosage at which the compounds of the invention are administered may vary within wide limits and may, of course, be adjusted in each particular case to the individual requirements. Typically, an effective amount for systemic administration of a compound of the invention is from about 0.1 μ g/kg/day to about 50 mg/kg/day, e.g., from 0.5 μ g/kg/day to about 10 mg/kg/day, 1 μ g/kg/day to about 1 mg/kg/day. Each dosage unit may conveniently contain from 0.001. Mu.g to 10mg, for example from 0.01. Mu.g to 1mg, for example from 50. Mu.g to 500. Mu.g. An effective amount may be given in one or more doses, i.e., may be administered 1,2 or more times, and may be administered multiple times at equal or unequal intervals, including daily-daily or multiple times, weekly or on a regimen of administration every several days/weeks.

Use and method

In view of the ability of the compounds of the invention to activate STING, induce expression of type I interferons and proinflammatory cytokines such as IL-6, TNF- α and IFN- γ, etc., while possessing cytotoxic activity, another aspect of the invention provides therapeutic uses and methods for the compounds of the invention.

In one aspect, the compounds or pharmaceutical compositions described herein may be used as therapeutic substances for the treatment or prevention of diseases associated with or mediated by an immune response, in particular for the treatment or prevention of diseases associated with or mediated by STING, including inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, or as vaccine adjuvants.

In a preferred embodiment, the compounds or compositions of the invention are used as cytotoxic agents for the treatment or prevention of hyperproliferative diseases, especially tumors. In another preferred embodiment, the compounds or compositions of the invention are used for the treatment of recurrent tumors, or for the prevention of tumor recurrence.

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

In another aspect, the invention thus accordingly provides a method of inducing, stimulating or assisting an immune response in an individual comprising administering to the individual a compound or pharmaceutical composition of the invention. In one embodiment, the compounds of the invention are administered to an individual as immunotherapy to induce the in vivo production of a plethora of cytokines that are therapeutically useful in humans or animals, including type I interferons and proinflammatory cytokines such as IL-6, TNF- α, and IFN- γ, to modulate the immune system of the human or animal to achieve certain therapeutic benefits.

In another aspect, the invention thus accordingly provides a method of treating or preventing a disease associated with or mediated by an immune response, in particular a disease associated with or mediated by STING, including inflammatory, allergic or autoimmune diseases, infectious diseases or cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or pharmaceutical composition of the invention.

In a preferred embodiment, the present invention provides a method of treating or preventing a hyperproliferative disease, particularly a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or pharmaceutical composition of the present invention. In another preferred embodiment, the present invention provides a method of treating a recurrent tumor or preventing tumor recurrence comprising administering to a subject in need thereof a therapeutically effective amount of a compound or pharmaceutical composition of the present invention.

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

In another aspect, the invention accordingly provides the use of a compound or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of a disease associated with or mediated by an immune response, in particular a disease associated with or mediated by STING, including inflammatory, allergic or autoimmune diseases, infectious diseases or cancer.

In another aspect, the invention thus also provides the use of a compound or pharmaceutical composition of the invention in the preparation of a vaccine adjuvant.

In a preferred embodiment, the present invention provides the use of a compound or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of a hyperproliferative disease, in particular a tumour. In a further preferred embodiment, the invention provides the use of a compound or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a recurrent tumour or for the prevention of tumour recurrence.

In a preferred embodiment, the present invention provides the use of a compound or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of a viral infection.

Inflammation for the above uses and methods may be acute or chronic, involving inflammation of any organ or tissue of the body, including musculoskeletal, vascular, neurological, digestive, ocular, reproductive or other inflammation, and of course autoimmune disorders with inflammatory properties, as well as allergic disorders such as contact dermatitis, urticaria and respiratory allergies.

Autoimmune diseases for the above uses and methods refer to diseases in which the body has an immune response to an autoantigen that results in damage to its tissues, including, but not limited to, systemic lupus erythematosus, psoriasis, insulin-dependent diabetes mellitus, dermatomyositis, sjogren's syndrome, chronic fatigue syndrome, aplastic anemia, autoimmune hepatitis, multiple sclerosis, optic neuritis, pemphigus, rheumatoid arthritis, ulcerative colitis, crohn's disease, scleroderma, and the like.

Hyperproliferative diseases for the above uses and methods refer to physiological conditions characterized by uncontrolled or unregulated cell growth or death in a subject, particularly tumors or cancers, including solid and blood-borne tumors, including but not limited to brain, skin, bladder, ovarian, breast, stomach, pancreatic, prostate, colon, blood, lung and bone cancers. Examples of such cancer types include neuroblastoma, intestinal cancers such as rectal, colon, familial adenomatous polyposis and hereditary non-lymphoid colorectal cancers, esophageal, lip, larynx, nasopharynx, oral cavity, salivary gland, peritoneal, soft tissue sarcomas, urothelial, sweat gland, stomach, adenocarcinoma, medullary thyroid, papillary thyroid, kidney, renal parenchymal, ovarian, cervix, uterine corpus, endometrial, pancreas, prostate, testicular, breast (including HER2 negative breast), urinary, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, neuroblastoma and peripheral neuroectodermal tumors, hodgkin lymphoma, non-hodgkin lymphoma, burkitt lymphoma, acute Lymphocytic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), chronic myelogenous leukemia (CLL) and lymphocytic lymphoma, acute myelogenous leukemia (CLL), chronic myelogenous leukemia (chronic myeloid leukemia (CLL), myeloid leukemia (chronic myeloid leukemia), myeloid leukemia (chronic myelogenous leukemia), myeloid leukemia (dll), myeloid leukemia (chronic myelogenous leukemia), myeloproliferative lymphoma, myelogenous leukemia (dll), myeloproliferative lymphoma, myelogenous leukemia (CML), myeloproliferative lymphoma, myelogenous leukemia (AML) and other forms of the above mentioned neoplastic cancers.

In a preferred embodiment, the hyperproliferative diseases for the above uses and methods are small cell lung cancer, non-small cell lung cancer, colorectal cancer, liver cancer, breast cancer, ovarian cancer, gastric cancer, prostate cancer, melanoma, renal cell carcinoma, head and neck cancer, pancreatic cancer, hodgkin's lymphoma, leukemia or bladder cancer.

Viral infection for the above uses and methods refers to the process by which viruses including but not limited to double-and single-stranded DNA viruses, single plus-and double-stranded RNA viruses, single minus-and double-stranded RNA viruses, and retroviruses invade the body through a variety of routes and propagate in susceptible host cells, examples being hepatitis b, TTV, adenovirus, papilloma, herpes zoster, smallpox and vaccinia, influenza, hog cholera, hepatitis a, hepatitis c, hepatitis d, hepatitis e, hepatitis g, rabies, ebola, enterovirus, human immunodeficiency virus, and the like. The therapeutic uses and methods provided by the present invention are useful for the viral infections and diseases caused thereby.

By the use of a compound or composition of the invention as a vaccine adjuvant and in the preparation of a vaccine adjuvant as described above, it is meant that a compound or composition of the invention may be used as an adjuvant in therapeutic or prophylactic strategies employing vaccines comprising inactivated or attenuated bacteria or viruses, e.g. comprising inactivated tumor cells expressing and secreting one or more of GM-CSF, CCL-20, CCL3, IL-12p70, FLT-3 ligand, cytokine, together with one or more vaccines selected to stimulate an immune response to one or more predetermined antigens.

Pharmaceutical combination

In view of the advantageous pharmacological activity of the compounds of the invention, they may be used in combination with at least one other therapeutic agent or therapy to provide further therapeutic benefits, in addition to being useful for the above-mentioned therapeutic uses or methods alone.

Thus, in another aspect, the present invention also provides a pharmaceutical combination comprising or consisting of a cyclic dinucleotide compound, a stereoisomer, a tautomer, a stable isotopic variant, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, as described herein, and at least one other therapeutic agent.

In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutical combination as described herein and one or more pharmaceutically acceptable excipients.

In another aspect, the invention provides the use of a pharmaceutical combination comprising or a pharmaceutical composition comprising the herein described pharmaceutical combination for, or in the manufacture of a medicament for, the treatment or prevention of a disease, including a hyperproliferative disease, a viral infection or a disease associated with or mediated by STING, more particularly an inflammatory, allergic or autoimmune disease, an infectious disease or a cancer. In a preferred embodiment, the disease to which the use relates is a tumour or a viral infection.

In another aspect, the invention also provides methods of treatment wherein a compound of the invention is administered with a plethora of other therapeutic agents.

Inflammation, autoimmune diseases, hyperproliferative diseases and viral infections for the use of the pharmaceutical combinations of the invention and pharmaceutical compositions comprising the same are as described above for the uses and methods of the invention.

The compounds of the present invention may also be administered in combination with surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunooncology agents, and the like.

The other therapeutic agents used in combination with the present invention may be administered simultaneously, separately or sequentially with the compound of the present invention by the same or different route of administration. The other therapeutic agents may be co-administered with the compound of the invention in a single pharmaceutical composition or separately administered in separate discrete units from the compound of the invention, e.g. a combination product, preferably in kit form, which when administered separately may be simultaneous or sequential, which sequential administration may be close in time or remote in time. They may be prepared and/or formulated by the same or different manufacturers. Furthermore, the compound of the invention and the other therapeutic agent may be administered (i) prior to delivery of the combination product to a physician (e.g., in the case of a kit comprising a compound of the invention and an additional pharmaceutical agent); (ii) By the physician himself (or under the direction of the physician) just before administration; (iii) The combination therapy is added by the patient himself, for example together during the sequential administration of the compounds of the invention and the other therapeutic agents.

Thus, in another aspect, the invention also provides a kit comprising two or more separate pharmaceutical compositions, wherein at least one comprises a cyclic dinucleotide compound of the invention, a stereoisomer, a tautomer, a stable isotopic variant, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, and the remaining co-existing pharmaceutical compositions comprise at least one further therapeutic agent, and means for separately containing the compositions, such as a container, a portion bottle or a discrete foil package, e.g. a blister package for packaging tablets, capsules or the like. The kit of the invention is particularly suitable for administration of different dosage forms, such as oral dosage forms and parenteral dosage forms, or for administration of different compositions at different dosage intervals.

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

In particular embodiments, other therapeutic agents that may be used in combination with the compounds of the present invention include, but are not limited to, vaccines, adjuvants, immune checkpoint inhibitors, T cell receptor agonists, TLR agonists, therapeutic antibodies, lipids, liposomes, chemotherapeutic agents, immune regulatory cell lines, and the like.

In a particular embodiment, adjuvants used in combination with the compounds of the present invention, by their nature, can be used to stimulate or otherwise utilize the immune system to react to cancer antigens present on tumor cells, including but not limited to lipids, liposomes, fire fighting bacteria that induce innate immunity, compounds that mediate innate immune activation, and the like.

In a specific embodiment, the immune checkpoint inhibitor used in combination with a compound of the invention is for example selected from 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.

In a specific embodiment, T cell receptor agonists for use in combination with the compounds of the invention include, but are not limited to, CD28 agonists, OX40 agonists, GITR agonists, CD137 agonists, CD27 agonists, or HVEM agonists.

In a particular embodiment, TLR agonists for use in combination with the compounds of the invention include, but are not limited to, pam3Cys, CFA, MALP2, pam2Cys, FSL-1, hib-OMPC, poly adenosine-poly uridylate (poly AU), LPS, bacterial flagellin, monophosphoryl lipid a (MPL), imiquimod, rehmold, loxoribine, and the like.

In a particular embodiment, chemotherapeutic agents used in combination with the compounds of the present invention include, but are not limited to, alkylating antineoplastic agents, platinum-based antineoplastic agents, antimetabolites, antimicrotubule agents, antimitotic agents, topoisomerase inhibitors, and antitumor antibiotics, among others.

In a specific embodiment, therapeutic antibodies used in combination with the compounds of the invention include, but are not limited to, muromonab-CD3, infliximab

And adalimumab

Omalizumab

Daclizumab

Rituximab(trade

)、Ibritumomab(trade

)、Tositumomab

Cetuximab

Trastuzumab

Alemtuzumab

Lym-1

Ipilimumab

Vitaxin、Bevacizumab

Abciximab

Other therapeutic antibodies that may 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-CD 47 antibodies.

In other embodiments, the compounds of the invention may also be used in combination with a PKC inhibitor, a BCR-ABL inhibitor, an HSP90 inhibitor, an inhibitor of PI3K and/or mTOR, an FGFR inhibitor, an inhibitor of cytochrome P450 HDM2, an aromatase inhibitor, an inhibitor of the P53 and/or P53/Mdm2 interaction, or an inhibitor of CSF-1R tyrosine kinase.

Examples of the above classes of therapeutic agents, as well as examples of other therapeutic agents that may be used in combination with the compounds of the present invention, are found in WO2016/145102 and WO2018/060323, the respective contents of which are incorporated herein.

As for the above-described compounds, pharmaceutical compositions, methods, uses, pharmaceutical combinations and kits of the present invention, the cyclic dinucleotide compounds of the present invention, stereoisomers, tautomers, stable isotopic variations, pharmaceutically acceptable salts, prodrugs or solvates thereof in each of the preferred embodiments described above, or the pharmaceutical compositions thereof are preferred, the compounds defined in the specific embodiments, i.e., the compounds of examples 1 to 26, are more preferred, and those compounds showing excellent activity in the active examples are most preferred.

For the above-described compounds, pharmaceutical compositions, methods, uses, pharmaceutical combinations and kits of the present invention, it is preferred to use free forms or pharmaceutically acceptable salts or prodrugs of the cyclic-dinucleotide compounds defined herein, preferably free forms or pharmaceutically acceptable salts or prodrugs of the substantially pure cyclic-dinucleotide compounds defined herein.

For the above-described therapeutic uses and methods of the invention, it is preferred that the subject to be used or treated is a mammal, preferably a human.

When a dose of a drug or a pharmaceutically acceptable salt thereof is described herein, it is understood that the dose is based on the weight of the free base, excluding any hydrates or solvates thereof, unless otherwise specified.

Process for the preparation of the compounds of the invention

General synthetic method

The compounds of the present invention, stereoisomers, tautomers, stable isotopic variations, pharmaceutically acceptable salts or solvates thereof can be prepared by a variety of methods well known in the art of organic synthesis, including the methods set forth below, in the examples, or by methods analogous thereto as understood by those of skill in the art.

The general synthetic scheme for the synthesis of the compounds of the invention is illustrated below. Suitable reaction conditions for each reaction step are known to those skilled in the art or can be determined routinely. In particular, the process steps for the synthesis of the compounds of the invention may be carried out under reaction conditions known per se (including those specifically mentioned), in the absence or generally in the presence of solvents or diluents (including, for example, solvents or diluents which are inert to and can dissolve the reagents used), in the absence or presence of catalysts, condensing agents or neutralizing agents (for example ion exchangers, such as cation exchangers, for example in the H + form), according to the nature of the reaction and/or the reactants, at reduced, normal or elevated temperatures (for example from about-100 ℃ to about 190 ℃, including, for example, from about-78 ℃ to about 150 ℃, for example from about 0 ℃ to about 125 ℃, room temperature, -20 to 40 ℃ or reflux temperature), under atmospheric pressure or in closed containers, when appropriate under pressure, and/or under an inert atmosphere, for example an oxygen or nitrogen atmosphere.

The starting materials and reagents used in preparing these compounds are generally commercially available or can be prepared by the methods described below, by methods analogous to those set forth below, or by methods known in the art. If desired, the starting materials and intermediates in the synthetic reaction schemes can be isolated and purified using conventional techniques, including, but not limited to, filtration, distillation, crystallization, chromatography, and the like. The materials may be characterized using conventional methods including physical constants and spectroscopic data.

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

One skilled in the art will recognize that stereocenters exist in the compounds of formula I. The mixture of isomers formed can be separated into the individual isomers, e.g. diastereoisomers or enantiomers, or into any desired mixture of isomers, e.g. racemates or mixtures of diastereomers, at all stages of the reaction, see e.g. E.L.Eliel, S.H.Wilen and L.N.Mander "stereoschemistry of Organic Compounds" (Wiley-Interscience, 1994).

Scheme 1 below illustrates one general synthetic route that may be used to prepare the compounds of formula II as defined herein and various embodiments thereof. Each variable of the general formulae in the following schemes has the same meaning as in the compounds defined herein or in each of their embodiments, unless otherwise indicated.

Scheme 1

Wherein P is ₁ And P ₂ Is a suitable hydroxy protecting group, P ₃ And P ₄ Are suitable hydroxy or amino protecting groups including, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis (4-methoxyphenyl) benzyl), bz (benzoyl), ^i- BuCO (isobutyryl group). The deprotection used in the synthetic scheme of the compounds of the present invention is carried out under acidic conditions (including but not limited to, such as acetic acid/water, trifluoroacetic acid/water, etc.), basic conditions (including but not limited to, such as aqueous ammonia, ammonia/methanol solution, etc.) or in the presence of fluorine-containing negative ion compounds (including but not limited to, such as tetrabutylammonium fluoride, triethylamine trihydrofluoride, etc.).

Scheme 2 illustrates the synthetic routes for compounds of formula a and formula C in scheme 1, as well as the synthesis of further intermediates used therein.

Scheme 2

Scheme 3 illustrates the synthetic route when the compound of formula H is a compound of formula H-1 (gemcitabine prodrug LY 2334737) as follows.

Scheme 3

Schemes 4-7 illustrate synthetic routes to the compounds of formula B in scheme 1, as well as the synthesis of intermediates used further therein.

Scheme 4

Scheme 5

Scheme 6

Scheme 7

Scheme 8 illustrates a synthetic route to the compound of formula D in scheme 1.

Scheme 8

Specifically, the present invention provides a method for preparing the above-mentioned compound of the present invention, which comprises:

reacting a compound of formula A

Wherein B is ₁ 、R ₁ 、R ₁ ' has the meaning as defined above for the compounds of formula (II) according to the invention or for each of its embodiments; p1 is a suitable hydroxy protecting group such as, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis (4-methoxyphenyl) benzyl), bz (benzoyl), ^i- BuCO (isobutyryl);

with a compound of formula B in the presence of a base, such as DBU,

wherein B is ₂ 、R ₂ 、R ₂ ' has the meaning as defined above for the compounds of formula (II) according to the invention or for each of its embodiments; p2 is a suitable hydroxy protecting group and P3 and P4 are each a suitable hydroxy or amino protecting group such as, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis (4-methoxyphenyl) benzyl), bz (benzoyl), ^i- BuCO (isobutyryl);

or a compound of formula C

Wherein B is ₁ 、R ₁ 、R ₁ ' and P1 are as defined above for compounds of formula A;

with a compound of formula D in the presence of a base, such as DBU,

wherein B is ₂ 、R ₂ 、R ₂ ', P2, P3 and P4 are as defined above for the compound of formula B;

to obtain the compound of the formula E,

selective deprotection of a compound of formula E above, for example under trifluoroacetic acid/water, tetrabutylammonium fluoride or triethylamine trihydrofluoride conditions, affords a compound of formula F

Wherein the various radicals have the meanings defined above;

a) When R is ₂ ' = -O (H), and when the protecting group P3 is benzoyl, the compound of formula F and (-) -PSI reagent are subjected to ring closing in the presence of base, such as DBU, to obtain the cyclic dinucleoside compound of formula G

Then debenzoyl protection is carried out in ammonia water or ammonia methanol solution to obtain the cyclic dinucleoside compound shown in the formula II,

wherein R is ₂ ' = -O (H), the remaining individual groups having the meanings defined above for compounds of formula II or individual embodiments thereof;

or b) when R is ₂ ' = -F or-H, the compound of formula F is cyclized with (-) -PSI reagent in the presence of a base, e.g., DBU, to give the cyclic dinucleoside the compound of formula II

Wherein R is ₂ ' = -F or-H, the remaining individual groups having the meanings defined above for the compound of formula II or each embodiment thereof.

The compounds of formula a and C may be prepared as follows:

selectively protecting primary alcohol in the presence of alkali such as imidazole,

to obtain a compound of formula C

Reacting the compound of formula C above with a (+) -PSI reagent in the presence of a base, such as DBU, to provide a compound of formula A,

wherein B is ₁ 、R ₁ 、R ₁ ' and P1 are as defined above for the compound of formula A.

Wherein when the compound of formula H is a compound of formula H-1 (gemcitabine prodrug LY 2334737),

which is prepared by a compound of a formula H-1-1 and 2-propylpentanoic acid,

the compound of formula B may be prepared as a form of formula B-1 as follows:

the compound of formula H is protected at both hydroxy groups in the presence of a base such as imidazole,

to give a compound of the formula B-1-1

Selective removal of the protecting group on a primary alcohol, e.g., under trifluoroacetic acid/water conditions, of a compound of formula B-1-1 affords a compound of formula B-1

Wherein B is ₂ 、R ₂ 、R ₂ ', P2 are as defined above for the compound of formula B.

The compound of formula B may also be prepared as a form of formula B-2 as follows:

the secondary alcohol is further protected by a compound of formula C in the presence of a base such as DBU/pyridine,

to give a compound of the formula B-2-1

Selectively removing the protecting group on the primary alcohol of the compound of formula B-2-1 under, for example, acetic acid/water conditions to obtain a compound of formula B-2,

wherein B is ₂ 、R ₂ 、R ₂ ', P1, P2 are as defined above for compounds of formula A or formula B.

The compound of formula B may also be prepared as a form of formula B-3 as follows:

the compound of formula B-3-1 selectively protects a primary alcohol and a secondary alcohol in the presence of a base such as imidazole,

to obtain the compound of the formula B-3-2,

reacting a compound of formula B-3-2 above with a protecting group such as benzoyl chloride in the presence of a base such as N-methylimidazole to give a compound of formula B-3-3

Selectively removing the protecting group on the primary alcohol from the compound of formula B-3-3 under, for example, trifluoroacetic acid/water conditions to obtain a compound of formula B-3,

wherein B is ₂ 、R ₂ 、R ₂ ', P1, P2 and P3 are as defined above for compounds of formula A or formula B.

The compound of formula B may also be prepared as a form of formula B-4 as follows:

reacting a compound of formula B-4-1 with an unprotected secondary alcohol therein using a protecting group such as benzoyl chloride in the presence of a base such as N-methylimidazole,

to obtain the compound of the formula B-4-2,

selective deprotection of a Compound of formula B-4-2 from a Primary alcohol, for example under acetic acid/Water conditions, to give a Compound of formula B-4

Wherein B is ₂ 、R ₂ 、R ₂ ', P1, P2 and P4 are as defined above for the compound of formula A or formula B.

The compounds of formula D can be prepared as follows:

reacting a compound of formula B with a (-) -PSI reagent in the presence of a base, such as DBU,

to give a compound of the formula D

Wherein B is ₂ 、R ₂ 、R ₂ ’、P ₂ 、P ₃ And P ₄ As defined above for the compound of formula B.

Scheme 9 below illustrates one general synthetic route that may be used to prepare the compounds of formulas (I-b), (II-b), (III-b), (IV-b), and various embodiments thereof, as defined herein. Each variable of the general formulae in the following schemes has the same meaning as in the compounds defined herein or in each of their embodiments, unless otherwise indicated.

Scheme 9

Wherein each X is independently a hydroxyl group or a thiol group; r ¹ 、R ¹ ’、B ₁ And B ₂ Each as defined above for formulae (I-b), (II-b), (III-b), (IV-b) and each of its sub-formulae and embodiments; p ₁ And P ₂ Is a suitable hydroxy protecting group, P ₃ And P ₄ Are suitable hydroxy or amino protecting groups including, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis (4-methoxyphenyl) benzyl), bz (benzoyl), ^i- BuCO (isobutyryl group). P ₅ Are suitable hydroxy or mercapto protecting groups on the phosphoric acid/phosphate ester, including but not limited to nitrile ethyl.

The deprotection used in the above synthetic scheme of the compounds of the present invention is carried out under acidic conditions (including but not limited to, e.g., acetic acid/water, trifluoroacetic acid/water, etc.), basic conditions (including but not limited to, e.g., aqueous ammonia, ammonia/methanol solution, methylamine/ethanol solution, lithium hydroxide, etc.), or in the presence of fluorine-containing negative ion compounds (including but not limited to, e.g., tetrabutylammonium fluoride, triethylamine trihydrofluoride, ammonium fluoride, etc.). The oxidation conditions used in the synthetic schemes for the compounds of the present invention are carried out in the presence of, but not limited to, iodine and the sulfidation conditions are carried out in the presence of, but not limited to, 3H-1, 2-benzodisul-col-3-one.

Scheme 10 illustrates a synthetic route to the compound of formula J in scheme 9, and the synthesis of further intermediates used therein.

Scheme 10

Scheme 11 illustrates the synthetic route to the compounds of formula K, and the synthesis of further intermediates used therein.

Scheme 11

reacting a compound of formula J

Wherein B is ₁ 、R ₁ 、R ₁ ' has the meaning as defined above for the compounds of the formula (I-b), (II-b), (III-b), (IV-b) or the individual embodiments thereof according to the invention; p3 is a suitable hydroxy protecting group such as, but not limited to, TBS (t-butyldimethylsilyl), DMTr (bis (4-methoxyphenyl) benzyl), bz (benzoyl), ^i- BuCO (isobutyryl group),

with a compound of formula K in the presence of a base (e.g. DBU),

wherein B is ₂ Have the meaning as defined above for the compounds of the formula (I-b), (II-b), (III-b), (IV-b) or the individual embodiments thereof according to the invention; p1, P2 are suitable hydroxy protecting groups and P4 is a suitable hydroxy or amino protecting group, such as, but not limited to, TBS (tert-butyldimethylsilyl), DMTr (bis (4-methoxyphenyl) benzyl), bz (benzoyl), ^i- BuCO (isobutyryl group),

or reacting a compound of formula J with a compound of formula L in the presence of tetrazole and oxidizing or sulfurizing, wherein the oxidizing conditions include, but are not limited to, the use of iodine, t-butyl hydroperoxide, and the like, and the sulfurizing conditions include, but are not limited to, the use of N, N-dimethyl-N' - (3-thio-3H-1, 2, 4-dithiazol-5-yl) cimetidine (DDTT), 3H-1, 2-benzodisul col-3-one, and the like; the introduction and removal of the protecting groups is carried out according to standard methods well known to the person skilled in the art,

wherein B is ₂ P1, P2 and P4 are as defined above for the compound of formula K;

to obtain the compound of the formula M,

wherein X is OH or SH, P ₅ Are suitable hydroxy or thiol protecting groups on the phosphoric acid/phosphate ester such as, but not limited to, nitriloethyl;

selective deprotection of a compound of formula M above, e.g., under lithium hydroxide conditions, affords a compound of formula N

Wherein the various radicals have the meanings defined above;

a) Reacting the compound of formula N with a (+) -PSI agent or (+) -PSI agent in the presence of a base, such as DBU, to provide a compound of formula O,

then deprotecting P1 (e.g., DMTr) in an acetic acid/water solution to give a compound of formula P,

(ii) ring closure of the compound of formula P in the presence of a base, e.g. DBU, to give a cyclic dinucleoside compound of formula Q,

then deprotecting P4 (e.g., TBS) in the presence of ammonium fluoride or the like to provide a cyclic dinucleoside compound of formula (I-b), (II-b), (III-b) or (IV-b), wherein R is ² Is OH and R ² ' is a hydrogen atom in the formula H,

wherein the various radicals have the meanings defined above;

b) Or reacting the compound of formula N with diphenyl phosphite in the presence of a base, such as DBU, to provide a compound of formula R,

then deprotecting P1 (e.g., DMTr) in an acetic acid/water solution to give a compound of formula S,

the compound of formula S is cyclized in the presence of an activating reagent, such as pivaloyl chloride, and oxidized or sulfurized to give the cyclic dinucleoside compound of formula Q

wherein the individual radicals have the meanings defined above.

The compound of formula J can be prepared as follows:

protecting the secondary alcohol with a compound of formula C in the presence of a base such as N-methylimidazole,

to obtain the compound of the formula J-1,

selectively deprotecting the compound of formula J-1 to obtain a compound of formula J,

wherein each group is as defined above.

The compounds of formula K can be prepared as follows:

reacting a compound of formula K-1 with a (+) -PSI reagent in the presence of a base, such as DBU,

to give a compound of the formula K

Wherein each group is as defined above.

The experimental materials and reagents used in the above synthetic methods and procedures, unless otherwise specified, are commercially available, prepared according to methods of the prior art, or prepared according to methods analogous to those disclosed herein. The synthesis conditions used in the above synthesis methods and schemes, if not otherwise specified, can be routinely determined by those skilled in the art.

The invention also relates to a preparation method which comprises the following steps: wherein a compound obtainable in any step of the various preparation processes and schemes described herein in intermediate form is used as starting material and the remaining process steps are carried out, or wherein the starting material is formed in situ under the reaction conditions or is used in the form of a derivative, e.g. in protected form or in salt form, or a compound obtainable according to the process of the invention is generated under the process conditions and is further processed in situ.

Examples

The present invention will be further described with reference to the following examples. It should be noted that the following examples should not be construed as limiting the scope of the present invention.

Unless a formula is clearly wrong, when the chemical name of any compound of the present invention is inconsistent with a given formula, the formula prevails.

Experimental procedures without specific conditions noted in the following examples are generally carried out according to conventional conditions for such reactions, or according to conditions recommended by the manufacturer. The experimental materials and reagents used in the following examples are commercially available, prepared according to prior art methods or prepared according to methods similar to those disclosed in the present application, unless otherwise specified.

Unless otherwise indicated, percentages and parts are weight percentages and parts; the liquid ratio is volume ratio; all temperatures are given in degrees celsius unless otherwise indicated.

In the following examples of the present invention, ¹ h NMR spectra ³¹ P NMR spectra were typically recorded using a Bruker 400MHz NMR and 500MHz NMR spectrometer with chemical shifts expressed in δ (ppm); mass spectra were recorded using an Agilent 1290 liquid chromatography +6120B Mass Spectrometry LCMS LC MS. Purifying the silica gel column by adopting Biotage SelektSEL-2SV or ISO-1 SV; preparative liquid chromatography purification was carried out using Gilson 281 (column: waters Xbridge 19X250mm,5 μm or WELCH C18, 21.2X250mm,10 μm. Mobile phase: A: water (10 mM NH. Sub.10 mM. Sub.M.) as column ₄ HCO ₃ Or 0.05% formic acid), B: acetonitrile (or 0.05% formic acid). Flow rate: 20-30mL/min. Detection wavelength: 214nm/254 nm), or otherwise.

The following abbreviations are used in the following synthetic examples, and each of the abbreviations not listed has a meaning commonly understood by those skilled in the art.

List of abbreviations

CDCl ₃ DeuteriumChloroform substitute

DMSO-d ₆ Deuterated dimethyl sulfoxide

MHz

MS-ESI electrospray mass spectrum

DBU 1, 8-diazabicycloundec-7-ene

DMF N, N-dimethylformamide

CDI N, N' -carbonyldiimidazole

TMSCl trimethylchlorosilane

TFA trifluoroacetic acid

THF tetrahydrofuran

H ₂ O water

HOAc acetic acid

BzCl benzoyl chloride

IFN interferon

FBS fetal bovine serum

PBS phosphate buffer solution

ELISA enzyme-linked immunosorbent assay

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

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

Synthetic examples

Preparation of intermediate 1: 2-chloro-5 ' -O-tert-butyldimethylsilyl-2 ' -deoxy-2 ' -fluoro-beta-adenosine

To a solution of 2-chloro-2 '-deoxy-2' -fluoro-beta-adenosine (2.50g, 8.25mmol) in DMF (15 mL) was added imidazole (1.12g, 16.5 mmol) and tert-butyldimethylchlorosilane (1.31g, 8.66mmol) in that order, and the resulting mixture was stirred at 20-25 ℃ for 16 hours. The reaction mixture was poured into water (150 mL) 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 eluting with petroleum ether/ethyl acetate (2: 3) to give a white solid (2.53 g).

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

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

To a0 deg.C solution of intermediate 1 (1.00g, 2.40mmol) and (+) -PSI reagent (1.39g, 3.12mmol) in tetrahydrofuran (10 mL) was added 1, 8-diazabicycloundec-7-ene (474mg, 3.12mmol) and the resulting mixture was stirred at 0-5 deg.C for 1 hour. The reaction mixture was diluted with ethyl acetate (50 mL). The organic phase was washed successively with a 10% aqueous solution of sodium dihydrogenphosphate (50 mL) and a saturated brine (50 mL), and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by a silica gel column, and eluted with petroleum ether/ethyl acetate (3: 7) to give a white solid (1.50 g).

¹ H NMR(400MHz，CDCl ₃ )δ8.09(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(162MHz，CDCl ₃ ) δ101.9。

Preparation of intermediate 3: 2-chloro-5 '-O-tert-butyldimethylsilyl-2' -deoxyadenosine

Intermediate 3 was obtained from 2' -deoxyadenosine using the route of intermediate 1.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.29(s，1H)，7.82(brs，2H)，6.26(t，J＝6.4Hz，1H)， 5.37(d，J＝4.0Hz，1H)，4.45-4.32(m，1H)，3.90-3.76(m，1H)，3.73-3.60(m，2H)， 2.75-2.61(m，1H)，2.37-2.25(m，1H)，0.84(s，9H)，0.02(s，6H)。

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

Intermediate 4 was obtained from intermediate 3 and the (+) -PSI reagent using the route of intermediate 2.

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

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

Intermediate 5 was obtained from 2' -deoxy-2 ',2' -difluorocytidine using the route for intermediate 1.

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

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

Intermediate 6 was obtained from intermediate 5 and the (+) -PSI reagent using the route of intermediate 2.

¹ H NMR(400MHz，DMSO-d ₆ )δ7.73(d，J＝8.0Hz，1H)，6.34(t，J＝8.0Hz，1H)， 6.21(d，J＝8.0Hz，1H)，5.36-5.24(m，1H)，5.04(s，1H)，4.87(s，1H)，4.55-4.43(m， 1H)，4.17-4.06(m，1H)，4.05-3.86(m，2H)，2.66-2.52(m，1H)，2.30-2.05(m，2H)，2.02- 1.80(m，3H)，1.80-1.62(m，7H)，0.92(s，9H)，0.13(s，3H)，0.12(s，3H)。 ³¹ P NMR(162MHz，CDCl ₃ )δ103.0。MS-ESI[M+H] ⁺ ：624.2。

Preparation of intermediate 7: n4- (2-Propylpentanoyl) -2' -deoxy-2 ',2' -difluorocytidine

To a0 ℃ solution of 2' -deoxy-2 ',2' -difluorocytidine hydrochloride (2.00g, 6.67mmol) in pyridine (20 mL) was added dropwise chlorotrimethylsilane (3.61g, 33.4 mmol), and the resulting mixture was stirred at 0-5 ℃ for 2 hours. Meanwhile, carbonyldiimidazole (1.19g, 7.33mmol) was added in portions to an acetonitrile solution (20 mL) of 2-propylpentanoic acid (1.06g, 7.33mmol), and the resulting mixture was stirred at 25 ℃ for 2 hours. Then, the obtained acetonitrile mixture was added dropwise to the pyridine mixture at the previous 0 ℃ and the resulting mixture was stirred at 45 ℃ for 16 hours. Ethanol (20 mL) was added to the mixture, and the mixture was stirred at 45 ℃ for 0.5 hour. Then, water (20 mL) was added to the mixture, and the mixture was stirred at 45 ℃ for 1 hour. The resulting reaction solution was spun dry and diluted with water (50 mL). The pH of the mixture was adjusted to 2-3 with 2N aqueous hydrochloric acid, and the mixture was 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 by silica gel column eluting with petroleum ether/ethyl acetate (2: 3) to give a white solid (1.20 g).

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

Preparation of intermediate 8: n4- (2-Propylpentanoyl) -5' -O-tert-butylbisMethylsilyl-2 ' -deoxy-2 ',2' -difluorocytidine

Intermediate 8 was obtained from intermediate 7 using the route of intermediate 1.

¹ H NMR(400MHz，CDCl ₃ )δ9.07(brs，1H)，8.27(brs，1H)，7.51(d，J＝8.0Hz，1H)， 6.45-6.30(m，1H)，4.46-4.33(m，1H)，4.12-4.00(m，2H)，3.91(dd，J＝8.0，2.4Hz， 1H)，2.25-2.12(m，1H)，1.71-1.60(m，2H)，1.56-1.42(m，2H)，1.40-1.25(m，4H)，1.05- 0.85(m，15H)，0.13(s，6H)。

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

Intermediate 9 was obtained from intermediate 8 and the (+) -PSI reagent using the route of intermediate 2.

¹ H NMR(400MHz，CDCl ₃ )δ9.63(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＝4.0Hz，6H)。

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-glandGlycosides

To a solution of intermediate 1 (1.53g, 3.67mmol) in pyridine (10 mL) was added bis (4-methoxyphenyl) benzyl chloride (1.49g, 4.40mmol), and the resulting mixture was stirred at 25 ℃ for 2 hours. Then bis (4-methoxyphenyl) benzyl chloride (497mg, 1.47mmol) and 1, 8-diazabicycloundec-7-ene (669 mg,4.40 mmol) were added in that order and the resulting mixture was stirred at 20-25 ℃ for 16 h. Bis (4-methoxyphenyl) benzyl chloride (1.49g, 4.40mmol) and 1, 8-diazabicycloundec-7-ene (669mg, 4.40mmol) were then added in sequence and the resulting mixture was stirred at 20-25 ℃ for 24 h. 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 eluting with petroleum ether/ethyl acetate (5: 1) to give a white solid (2.60 g). ¹ H NMR(400MHz，DMSO-d ₆ )δ8.00(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)。

Preparation of intermediate 11: n6-bis (4-methoxyphenyl) benzyl-2-chloro-3 ' -bis (4-methoxyphenyl) benzyl-2 ' -deoxy-2 ' -fluoro-beta-adenosine

To a solution of intermediate 10 (2.60g, 2.55mmol) in tetrahydrofuran (15 mL) was added tetrabutylammonium fluoride (1.33g, 5.10 mmol), and the resulting mixture was stirred at 25 ℃ 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 silica gel column eluting with petroleum ether/ethyl acetate (2: 3) to give a white solid (1.20 g).

¹ H NMR(400MHz，DMSO-d ₆ )δ8.17(d，J＝2.4Hz，1H)，7.97(s，1H)，7.50-7.10(m， 18H)，6.90-6.78(m，8H)，6.30-6.18(m，1H)，4.86(t，J＝6.0Hz，1H)，4.30-4.10(m， 3H)，3.76-3.68(m，12H)，3.49-3.30(m，2H)。

Preparation of intermediate 12: n6-bis (4-methoxyphenyl) benzyl-2-chloro-5 ' -O-tert-butyldimethylsilyl 3' -bis (4-methoxyphenyl) benzyl-2 ' -deoxyadenosine

To a solution of intermediate 3 (1.80g, 4.51mmol) in pyridine (10 mL) was added bis (4-methoxyphenyl) benzyl chloride (3.81g, 11.3 mmol) followed by 1, 8-diazabicycloundec-7-ene (743mg, 11.3 mmol), and the resulting mixture was stirred at 20-25 ℃ for 16 h. The reaction mixture was concentrated, 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 a silica gel column, and eluted with petroleum ether/ethyl acetate (3: 7) to give a white solid (2.50 g).

¹ H NMR(400MHz，DMSO-d ₆ )δ8.19(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)。

Preparation of intermediate 13: n6-bis (4-methoxyphenyl) benzyl-2-chloro-3 '-bis (4-methoxyphenyl) benzyl-2' -deoxyadenosine

Intermediate 13 was obtained from intermediate 12 using the route of intermediate 11.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.32(s，1H)，7.87(s，1H)，7.50-7.10(m，18H)，6.95- 6.77(m，8H)，6.24(t，J＝6.8Hz，1H)，4.83(brs，1H)，4.39-4.28(m，1H)，3.85-3.65(m， 13H)，3.22-3.12(m，1H)，2.35-2.21(m，1H)，1.88-1.70(m，1H)。

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

To a solution of 2' -deoxy-2 ',2' -difluorocytidine hydrochloride (3.00g, 10.0 mmol) in DMF (50 mL) were added imidazole (3.41g, 50.0 mmol) and tert-butyldimethylchlorosilane (4.53g, 30.0 mmol) in this order, and the resulting mixture was stirred at 20-25 ℃ for 2 hours, and then at 60 ℃ 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 eluting with methylene chloride: methanol (9: 2) to give a white solid (2.70 g).

¹ H NMR(400MHz，DMSO-d ₆ )δ7.53(d，J＝7.6Hz，1H)，7.40(brs，2H)，6.17(t，J＝ 8.0Hz，1H)，5.78(d，J＝7.6Hz，1H)，4.40-4.22(m，1H)，3.98-3.83(m，2H)，3.78-3.70 (m，1H)，1.00-0.75(m，18H)，0.15-0.00(m，12H)。

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

Intermediate 14 (1.00g, 2.04mmol) was dissolved in a mixture of tetrahydrofuran solution (10 mL), trifluoroacetic acid (5 mL) and water (5 mL) at 0 deg.C and stirred at 0-5 deg.C for 2 hours. The pH of the reaction mixture was adjusted to about 9 with saturated aqueous sodium bicarbonate 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 eluting with ethyl acetate: methanol (17: 3) to give a white solid (620 mg).

¹ H NMR(400MHz，DMSO-d ₆ )δ7.67(d，J＝7.6Hz，1H)，7.50-7.35(m，2H)，6.14(t， J＝8.0Hz，1H)，5.79(d，J＝7.6Hz，1H)，5.26(d，J＝7.6Hz，1H)，4.40-4.22(m，1H)， 3.86-3.72(m，2H)，3.68-3.50(m，1H)，0.88(s，9H)，0.11(s，3H)，0.10(s，3H)。MS-ESI [M+H] ⁺ ：378.2。

Preparation of intermediate 16: n4- (2-propyl-pentanoyl) -3' -5' -di-O-tert-butyldimethylsilyl-2 ' -deoxy-2 ',2' -difluorocytidine

To a solution of 2-propylpentanoic acid (322mg, 2.24mmol) in acetonitrile (5 mL) at 25 ℃ was added carbonyldiimidazole (363mg, 2.24mmol), and the resulting mixture was stirred at 25 ℃ for 2 hours. The resulting mixture was added dropwise to a0 ℃ solution of intermediate 14 (1.0 g, 2.03mmol) in pyridine (10 ml). The resulting mixture was stirred at 45 ℃ for 16 hours, and the resulting reaction solution was spun dry, 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 using petroleum ether/ethyl acetate (1: 1) to give a white solid (430 mg).

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

Preparation of intermediate 17: n4- (2-propyl-pentanoyl) -3 '-O-tert-butyldimethylsilyl-2' -deoxy-2 ',2' -difluorocytidine

Intermediate 17 was obtained from intermediate 16 using the route of intermediate 15.

¹ H NMR(400MHz，CDCl ₃ )δ8.38(s，1H)，8.00(d，J＝7.6Hz，1H)，7.50(d，J＝7.6 Hz，1H)，6.25(t，J＝7.6Hz，1H)，4.49-4.40(m，1H)，4.08(d，J＝12.4Hz，1H)，4.02- 3.90(m，1H)，3.88-3.75(m，1H)，2.51(s，1H)，2.40-2.30(m，1H)，1.75-1.55(m，2H)，1.52-1.41(m，2H)，1.38-1.28(m，4H)，0.95-0.85(m，15H)，0.14(d，J＝2.8Hz，6H)。 MS-ESI[M+H] ⁺ ：504.2。

Intermediate 18:5 '-O-tert-butyldimethylsilyl-2' -tert-butyldimethylsilyl-adenosine, and

intermediate 19:5 '-O-tert-butyldimethylsilyl-3' -tert-butyldimethylsilyl-adenosine

To a tetrahydrofuran solution (40 mL) of adenosine (3.35g, 12.5 mmol) were added triethylenediamine (7.0 g,62.5 mmol) and silver nitrate (5.08g, 28.8 mmol) in this order at room temperature (10 ℃). The mixture was stirred at room temperature (10 ℃) for 30min, then tert-butyldimethylsilyl chloride (4.71g, 30mmol) was added and the resulting mixture was stirred at 10 ℃ for 16 h. The reaction mixture was filtered through celite, and 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 eluting with petroleum ether/ethyl acetate (1: 1) to give intermediate 18 (1.0 g, white solid). Elution with ethyl acetate afforded intermediate 19 (4.4 g, white solid).

Intermediate 18:

¹ H NMR(400MHz，CDCl ₃ )δ8.35(s，1H)，8.24(s，1H)，6.11(d，J＝4.8Hz，1H)，5.72 (brs，2H)，4.64(t，J＝4.8Hz，1H)，4.31-4.26(m，1H)，4.24-4.20(m，1H)，4.02(dd，J＝ 11.6，2.8Hz，1H)，3.87(dd，J＝11.6，2.8Hz，1H)，2.76(d，J＝3.6Hz，1H)，0.96(s， 9H)，0.84(s，9H)，0.18-0.11(m，6H)，-0.05(s，3H)，-0.12(s，3H)。

intermediate 19:

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

preparation of intermediate 20: n4-benzoyl-2 ' -5' -di-O-tert-butyldimethylsilyl-3 ' -benzoyl-adenosine

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

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

Preparation of intermediate 21: n4-benzoyl-2 '-O-tert-butyldimethylsilyl-3' -benzoyl-adenosine

Intermediate 21 was obtained from intermediate 20 using the route for intermediate 15.

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

Preparation of intermediate 22: n4-benzoyl-5 ' -O-tert-butyldimethylsilyl-3 ' -tert-butyldimethylsilyl-2 ' -benzoyl-adenosine

Intermediate 22 was obtained from intermediate 19 and benzoyl chloride using the route of intermediate 20.

¹ H NMR(400MHz，CDCl ₃ )δ9.02(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＝6.0Hz，1H)，5.81(t，J＝5.6 Hz，1H)，4.84(dd，J＝4.8，3.6Hz，1H)，4.26(dd，J＝6.0，3.2Hz，1H)，4.02(dd，J＝ 11.6，3.2Hz，1H)，3.85(dd，J＝11.6，2.8Hz，1H)，0.95(s，9H)，0.84(s，9H)，0.18-0.11 (m，6H)，0.03--0.05(m，6H)。

Preparation of intermediate 23: n4-benzoyl-3 '-tert-butyldimethylsilyl-2' -benzoyl-adenosine

Intermediate 23 was obtained from intermediate 22 using the route of intermediate 15.

¹ H NMR(400MHz，CDCl ₃ )δ9.08(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＝7.6Hz，1H)，6.01(dd，J＝7.6，5.2Hz，1H)，4.94 (d，J＝5.2Hz，1H)，4.35(d，J＝0.8Hz，1H)，4.07(dd，J＝13.2，1.6Hz，1H)，3.84(d， J＝12.4Hz，1H)，0.89(s，9H)，0.11(s，3H)，0.00(s，3H)。

Preparation of intermediate 24: n2-isobutyryl-5 ' -O-bis (4-methoxyphenyl) benzyl-3 ' -O-benzoyl-2 ' -O-tert-butyldimethylsilyl-guanosine

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

Preparation of intermediate 25: n2-isobutyryl-3 '-O-benzoyl-2' -O-tert-butyldimethylsilyl-guanosine

Intermediate 24 (5.50g, 6.30mmol) was dissolved in a mixture of glacial acetic acid (60 mL) and water (10 mL) and stirred at 25 ℃ for 2 h. The reaction mixture was spun dry and then evaporated twice with ethyl acetate (25 mL each). The crude product was purified on a silica gel column and eluted with ethyl acetate to give a white solid (2.2 g).

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

Preparation of intermediate 26: n2-isobutyryl-5 ' -O-bis (4-methoxyphenyl) benzyl-3 ' -tert-butyldimethylsilyl-2 ' -benzoyl-guanosine

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

Preparation of intermediate 27: n2-isobutyryl-3 '-tert-butyldimethylsilyl-2' -benzoyl-guanosine

Intermediate 27 was obtained from intermediate 26 using the route of intermediate 25.

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

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

Intermediate 28 was obtained from guanosine using the route of intermediate 18. MS-ESI [ M + H ]] ⁺ ：513.3。

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

Intermediate 29 was obtained from intermediate 28 using the route for intermediate 20.

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

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

Intermediate 30 was obtained from intermediate 29 using the route for intermediate 15.

¹ H NMR(400MHz，DMSO-d ₆ )δ10.71(brs，1H)，8.05(d，J＝7.2Hz，3H)，7.75-7.68 (m，1H)，7.59(t，J＝7.6Hz，2H)，6.51(s，2H)，5.84(d，J＝7.6Hz，1H)，5.58(dd，J＝ 5.6，1.6Hz，1H)，5.42(t，J＝5.6Hz，1H)，4.96(dd，J＝7.6，5.2Hz，1H)，4.39-4.25(m， 1H)，4.20-4.00(m，1H)，0.57(s，9H)，-0.12(s，3H)，-0.29(s，3H)。MS-ESI[M- H] ^- ：501.3。

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

To a solution of 2 '-deoxy-2' -fluoroadenosine (1.10g, 4.09mmol) in DMF (10 mL) were added imidazole (1.81 g,26.6 mmol) and 4-dimethylaminopyridine (50mg, 0.41mmol), followed by tert-butyldimethylchlorosilane (2.16g, 14.3mmol), and the resulting mixture was stirred at 15-20 ℃ 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 eluting with petroleum ether/ethyl acetate (3: 2) to give a white solid (1.90 g).

¹ H NMR(400MHz，CDCl ₃ )δ8.35(s，1H)，8.12(s，1H)，6.25(dd，J＝16.0，2.4Hz， 1H)，5.66(brs，2H)，5.48-5.20(m，1H)，4.79-4.62(m，1H)，4.18-3.95(m，2H)，3.85- 3.72(m，1H)，1.00-0.80(m，18H)，-0.05-0.18(m，12H).MS-ESI[M+H] ⁺ ：498.4。

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

Intermediate 31 was obtained from intermediate 31 using the route for intermediate 15.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.37(s，1H)，8.14(s，1H)，7.38(brs，2H)，6.22(dd， J＝16.4，3.6Hz，1H)，5.70-5.40(m，1H)，5.29(t，J＝5.6Hz，1H)，4.80-4.62(m，1H)， 4.02-3.90(m，1H)，3.79-3.68(m，1H)，3.63-3.48(m，1H)，0.90(s，9H)，0.14(s，3H)， 0.13(s，3H)。

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

Intermediate 33 was obtained from intermediate 32 and the (-) -PSI reagent using the route for intermediate 2.

¹ H NMR(400MHz，CDCl ₃ )δ8.36(s，1H)，8.05(s，1H)，6.30-6.18(m，1H)，5.58(brs， 2H)，5.50-5.22(m，1H)，4.80-4.62(m，3H)，4.50-4.25(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)。

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

Intermediate 34 was obtained from 2 '-deoxy-2' -fluoroguanosine using the route of intermediate 31.

1H NMR(400MHz，DMSO-d ₆ )δ10.69(brs，1H)，7.84(s，1H)，6.52(brs，2H)，6.00 (d，J＝16.8Hz，1H)，5.36(d，J＝52.0Hz，1H)，4.62-4.45(m，1H)，3.98-3.86(m，2H)， 3.74(d，J＝11.6Hz，1H)，0.93-0.85(m，18H)，0.20-0.00(m，12H)。MS-ESI [M+H] ⁺ ：515.3。

Preparation of intermediate 35:3' -O-tert-butyldimethylsilyl-2 ' -deoxy-2 ' -fluoro-guanosine

Intermediate 35 was obtained from intermediate 34 using the route of intermediate 15.

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

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

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

Preparation of intermediate 37: [3 '-O-Thiophosphodiester-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]

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

¹ H NMR(400MHz，DMSO-d ₆ )δ8.26(s，1H)，8.08(s，1H)，7.91(brs，3H)，7.45-7.10 (m，18H)，6.95-6.76(m，8H)，6.32-6.15(m，2H)，5.40-5.18(m，2H)，5.00-4.80(m，2H)， 4.40-4.02(m，3H)，3.85-3.62(m，15H)，0.84(s，9H)，0.01(s，6H)。

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

Intermediate 38 was obtained from intermediate 4 and intermediate 13 in a mixed solution of tetrahydrofuran and acetonitrile using the route for intermediate 37.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.68(s，1H)，8.22(s，1H)，7.82(brs，2H)，7.75(s， 1H)，7.42(d，J＝8.0Hz，2H)，7.39-7.10(m，16H)，6.95-6.78(m，8H)，6.32-6.10(m， 2H)，4.90-4.80(m，1H)，4.43-4.36(m，1H)，4.15-4.08(m，1H)，3.85-3.40(m，17H)， 2.50-2.20(m，4H)，0.81(s，9H)，0.00(s，3H)，-0.02(s，3H)。

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

Intermediate 39 was obtained from intermediate 6 and intermediate 15 in a mixed solution of tetrahydrofuran and acetonitrile using the route for intermediate 37.

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

Preparation of intermediate 40: [3' -O-Thiophosphoric 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]

Intermediate 40 was obtained from intermediate 17 and intermediate 9 using the route for intermediate 37.

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

Preparation of intermediate 41: [3' -O-Thiophosphoric 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]

Intermediate 41 was obtained from intermediate 21 and intermediate 2 using the route for intermediate 37.

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

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

Intermediate 42 was obtained from intermediate 21 and intermediate 4 using the route for intermediate 37. MS-ESI [ (M + H)] ⁺ ：1067.2。

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

Intermediate 43 was obtained from intermediate 23 and intermediate 2 in N, N' -dimethylformamide using the route for intermediate 37.

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

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

Intermediate 44 was obtained from intermediate 2 and intermediate 25 in DMF solution using the route for intermediate 37.

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

Intermediate product45 preparation of: [3' -O-Thiophosphodiester-2-chloro-5 ' -O-tert-butyldimethylsilyl-2 ' -deoxyadenosine]- (3 ', 5') - [ N2-isobutyryl-3 '-O-benzoyl-2' -O-tert-butyldimethylsilyl-guanosine]

Intermediate 45 was obtained from intermediate 4 and intermediate 25 in DMF solution using the route for intermediate 37.

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

Preparation of intermediate 46: [3' -O-Thiophosphodiester-5 ' -O-t-butyldimethylsilyl-2 ' -deoxy-2 ',2' -difluorocytidine]- (3 ', 5') - [ N2-isobutyryl-3 '-O-benzoyl-2' -O-tert-butyldimethylsilyl-guanosine]

Intermediate 46 was obtained from intermediate 6 and intermediate 25 in DMF solution using the route for intermediate 37.

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

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

Intermediate 47 was obtained from intermediate 30 and intermediate 9 in DMF solution using the route for intermediate 37.

¹ H NMR(400MHz，DMSO-d ₆ )δ11.10(brs，1H)，10.60(brs，1H)，8.22(d，J＝7.6Hz， 1H)，8.16(s，1H)，8.02(d，J＝7.6Hz，2H)，7.68(t，J＝7.6Hz，1H)，7.56(t，J＝7.6Hz， 2H)，7.35(d，J＝7.6Hz，1H)，6.51(brs，2H)，6.23(t，J＝7.6Hz，1H)，5.82(d，J＝7.6 Hz，1H)，5.67(d，J＝5.2Hz，1H)，5.09(dd，J＝7.6，5.2Hz，1H)，5.02-4.95(m，1H)， 4.41(d，J＝3.6Hz，1H)，4.18-3.92(m，4H)，2.70-2.55(m，1H)，1.65-1.50(m，2H)， 1.45-1.30(m，2H)，1.28-1.15(m，4H)，0.96-0.88(m，12H)，0.87-0.80(m，9H)，0.53(s， 9H)，-0.11(s，3H)，-0.31(s，3H)。MS-ESI[(M-2H)/2] ^- ：540.3。

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

Intermediate 48 was obtained from intermediate 27 and intermediate 2 in DMF solution using the route for intermediate 37.

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

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

Intermediate 49 was obtained from intermediate 27 and intermediate 4 in DMF solution using the route for intermediate 37.

¹ H NMR(400MHz，DMSO-d ₆ )δ12.55(brs，1H)，12.14(brs，1H)，8.37(s，1H)，8.28 (s，1H)，7.94-7.76(m，4H)，7.65(t，J＝7.6Hz，1H)，7.49(t，J＝8.0Hz，2H)，6.33-6.21 (m，2H)，5.95-5.85(m，1H)，5.10-5.02(m，1H)，4.75(d，J＝4.8Hz，1H)，4.31-4.23(m， 2H)，4.22-4.15(m，1H)，4.14-4.09(m，1H)，3.91-3.78(m，2H)，2.93-2.86(m，1H)，2.76- 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)。

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

Intermediate 50 was obtained from intermediate 32 and intermediate 2 in DMF solution using the route for intermediate 37.

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

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

Intermediate 51 was obtained from intermediate 3 and intermediate 33 using the route for intermediate 37.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.51(s，1H)，8.25(s，1H)，8.13(s，1H)，7.82(brs， 2H)，7.31(brs，2H)，6.30-6.15(m，2H)，5.69(dt，J＝52.4，4.0Hz，1H)，5.00-4.86(m， 1H)，4.80-4.65(m，1H)，4.19-4.00(m，3H)，3.99-3.85(m，1H)，3.78-3.62(m，2H)，2.75- 2.62(m，1H)，2.60-2.50(m，1H)，0.88(s，9H)，0.81(s，9H)，0.14(s，3H)，0.11(s，3H)， 0.00(s，3H)，-0.01(s，3H)。

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

Intermediate 52 was obtained from intermediate 1 and intermediate 36 in DMF solution using the route for intermediate 37.

¹ H NMR(400MHz，DMSO-d ₆ )δ10.64(brs，1H)，8.12(s，1H)，8.05(s，1H)，7.93-7.82 (m，3H)，6.54(brs，2H)，6.36-6.25(m，1H)，6.00(dd，J＝14.8，4.8Hz，1H)，5.58-5.28 (m，2H)，5.15-4.98(m，1H)，4.60-4.50(m，1H)，4.15-4.00(m，3H)，3.98-3.88(m，3H)， 0.86(s，18H)，0.10-0.04(m，12H)。

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

Intermediate 53 was obtained from intermediate 3 and intermediate 36 in DMF solution using the route for intermediate 37.

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

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

Intermediate 37 (260mg, 0.188mmol) was dissolved in a mixture of tetrahydrofuran (2 mL), trifluoroacetic acid (2 mL) and water (1 mL) and stirred at 25 ℃ for 2 h. The reaction mixture was spin dried, diluted with methanol (5 mL), and the pH of the mixture was adjusted to about 8-9 with saturated aqueous sodium bicarbonate. After spin-drying, the solution was diluted again with methanol (5 mL) and filtered. The filtrate was concentrated, and the resulting crude product was purified by silica gel column eluting with ethyl acetate/methanol (7: 3) to give a white solid (120 mg).

¹ H NMR(400MHz，DMSO-d ₆ )δ8.30(s，1H)，8.23(s，1H)，7.88(brs，4H)，6.40-6.18 (m，2H)，6.06(d，J＝5.2Hz，1H)，5.45-5.12(m，2H)，5.04(t，J＝6.0Hz，1H)，5.00- 4.85(m，1H)，4.50-4.40(m，1H)，4.18-3.90(m，4H)，3.75-3.58(m，2H)。MS-ESI[M- H] ^- ：682.8。

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

Tetrabutylammonium fluoride (34mg, 0.132mmol) was added to a tetrahydrofuran solution (5 mL) of intermediate 38 (150mg, 0.110mmol), and the resulting mixture was stirred at 25 ℃ for 3 hours. The reaction mixture was spin-dried, dissolved in a mixed solution of glacial acetic acid (2 mL) and water (0.5 mL), and stirred at 25 ℃ for 0.5 hour. The reaction mixture was spin-dried, and the resulting crude product was purified by silica gel column eluting with ethyl acetate/methanol (3: 1) to give a white solid (45 mg). MS-ESI [ M-H ]] ^- ：647.0。

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

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

¹ H NMR(400MHz，DMSO-d ₆ )δ7.77(d，J＝7.2Hz，1H)，7.65(d，J＝7.6Hz，1H)， 7.45-7.25(m，4H)，6.30(d，J＝6.4Hz，1H)，6.20-6.05(m，2H)，5.89-5.70(m，2H)，5.13 (t，J＝6.0Hz，1H)，4.90-4.75(m，1H)，4.20-4.00(m，2H)，4.00-3.60(m，5H)。

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

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

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

Intermediate 58 was obtained from intermediate 41 using the route for intermediate 56. MS-ESI [ M + H ]] ⁺ ：857.0。

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

Intermediate 59 was obtained from intermediate 42 using the route for intermediate 56. MS-ESI [ M + H ]] ⁺ ：839.0。

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

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

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

Intermediate 61 was obtained from intermediate 44 using the route for intermediate 60.

¹ H NMR(400MHz，DMSO-d ₆ )δ12.56(brs，1H)，12.11(brs，1H)，8.35-8.20(m，2H)， 8.10-8.00(m，2H)，7.91(brs，2H)，7.75-7.45(m，3H)，6.38-6.18(m，1H)，6.00-5.75(m， 2H)，5.70-5.21(m，2H)，5.16-4.90(m，2H)，4.50-4.36(m，1H)，4.28-4.00(m，2H)，3.90- 3.58(m，2H)，3.00-2.82(m，1H)，1.36-1.05(m，6H)。MS-ESI[M-H] ^- ：837.0。

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

Intermediate 62 was obtained from intermediate 45 using the route of intermediate 60.

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

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

Intermediate 63 was obtained from intermediate 46 using the route for intermediate 60.

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

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

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

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

Intermediate 65 was obtained from intermediate 48 using the route for intermediate 56. MS-ESI [ M-H ]] ^- ：837.0。

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

Intermediate 66 was obtained from intermediate 49 using the route followed for intermediate 56. MS + ESI [ M + H ]] ⁺ ：821.1。

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

Intermediate 67 was obtained from intermediate 50 using the route for intermediate 60.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.47(s，1H)，8.24(s，1H)，8.15(s，1H)，7.91(brs， 2H)，7.35(brs，2H)，6.32-6.20(m，2H)，5.88(brs，1H)，5.55-5.30(m，2H)，5.18-4.87 (m，2H)，4.53(d，J＝16.4Hz，1H)，4.18-4.08(m，2H)，4.10-4.02(m，1H)，4.01-3.92 (m，1H)，3.75-3.59(m，2H)。MS-ESI[M+H] ⁺ ：651.0。

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

Intermediate 68 was obtained from intermediate 51 using the route for intermediate 56.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.50(s，1H)，8.34(s，1H)，8.15(s，1H)，7.84(brs， 2H)，7.31(brs，2H)，6.30-6.18(m，2H)，5.90(d，J＝5.6Hz，1H)，5.45(dt，J＝52.0，3.6 Hz，1H)，5.12-4.90(m，2H)，4.60-4.45(m，1H)，4.18-4.00(m，3H)，3.99-3.85(m，1H)， 3.65-3.50(m，2H)，2.70-2.55(m，1H)，2.50-2.40(m，1H)。

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

Intermediate 69 was obtained from intermediate 52 using the route of intermediate 60.

¹ H NMR(400MHz，DMSO-d ₆ )δ10.64(s，1H)，8.24(s，1H)，8.00(s，1H)，7.88(brs，2H)，6.54(brs，2H)，6.28(dd，J＝18.0，3.6Hz，1H)，6.02(dd，J＝16.0，3.6Hz，1H)， 5.80(d，J＝5.4Hz，1H)，5.44(d，J＝4.4Hz，1H)，5.30(d，J＝7.6Hz，1H)，5.04(d，J ＝6.0Hz，1H)，4.95(t，J＝14.8Hz，1H)，4.42(dt，J＝10.4，5.6Hz，1H)，4.11-3.99(m， 3H)，3.98-3.89(m，1H)，3.77-3.60(m，2H)。MS-ESI[M+H] ⁺ ：667.2。

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

Intermediate 69 was obtained from intermediate 53 using the route for intermediate 60.

¹ H NMR(400MHz，DMSO-d ₆ )δ10.67(brs，1H)，8.37(s，1H)，8.04(s，1H)，7.85(brs，2H)，6.58(brs，2H)，6.26(t，J＝7.2Hz，1H)，6.03(d，J＝15.6Hz，1H)，5.85(d，J＝5.6 Hz，1H)，5.38(d，J＝52.8Hz，1H)，5.10(s，1H)，4.97(s，1H)，4.46(d，J＝14.8Hz， 1H)，4.20-4.00(m，4H)，4.00-3.90(m，1H)，3.70-3.50(m，2H)，2.78-2.60(m，1H)。 MS-ESI[M-H] ^- ：647.2。

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]

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

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]

To intermediate 59 (210 m)g,0.25 mmol) and (-) -PSI reagent (335mg, 0.75mmol) in DMF (10 mL) 1, 8-diazabicycloundecen-7-ene (570mg, 3.75mmol) was added and the resulting mixture was stirred at 25 ℃ for 1 hour. After the reaction mixture was spin-dried, the reaction mixture was reprecipitated with ethyl acetate (20 mL), and the obtained solid was slurried with ethyl acetate (20 mL), filtered and dried to obtain a crude product. The crude product was purified by preparative liquid chromatography (10 mM ammonium bicarbonate as additive) to give a white solid (15.8 mg) after lyophilization, which was used directly as the next reaction. MS-ESI [ M + H ]] ⁺ ：917.0。

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]

Intermediate 73 was obtained from intermediate 60 and the (-) -PSI reagent using the route of intermediate 71. MS-ESI [ M-H ]] ^- ：932.8。

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

Intermediate 74 and intermediate 75 were obtained from intermediate 61 and the (-) -PSI reagent using the route of intermediate 72.

Intermediate 74: MS-ESI [ M-H ]] ^- :844.8, retention time: 4.96min;

intermediate 75: MS-ESI [ M-H ]] ^- :844.9, retention time: 5.20min. Analysis of LCMS: agilent 1100+ G1946D LCMS,4.6x150mm Waters Xbridge C18.5 μm analytical column, mobile phase A10 mM NH ₄ HCO ₃ Aqueous solution, B acetonitrile, flow rate 1.0ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-0.1min,5% by weight B;0.1-8min,5-95% by weight B;8-15min,95% B.

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

Intermediate 76 and intermediate 77 were obtained from intermediate 62 and the (-) -PSI reagent using the route for intermediate 72.

Intermediate 76: MS-ESI [ M-H ]] ^- :897.2, retention time: 1.69min;

intermediate 77: MS-ESI [ M-H ]] ^- :897.2, retention time: 1.74min. Analysis of LCMS: agilent 1100+ G1946D LCMS,4.6x50mm Waters Xbridge C18.5 μm analytical column, mobile phase A is 10mM NH ₄ HCO ₃ Water solution, B is acetonitrile, the flow rate is 1.8min/min, the double-wavelength ultraviolet absorption monitoring of 214nm and 254nm, gradient elution: 0-0.1min,5% by weight of B;0.1-2.5min,5-95% by weight B;2.5-5min, 95% B.

Intermediate 78The preparation of (1): (2 ', 3') -cyclo- [2 '-O-phosphodiester-N2-isobutyryl-3' -O-benzoyl-guanosine]- [3 '-O-phosphorothioate diester-2' -deoxy-2 ',2' -difluorocytidine]

Intermediate 78 taken the route of intermediate 72, starting from intermediate 63 and the (-) -PSI reagentThus obtaining the product. Intermediate 78: MS-ESI [ (M-2H)/2)] ^- ：437.0。

Intermediate 79The preparation of (1): (2 ', 3') -cyclo- [2 '-O-phosphorothioate diester-3' -O-benzoyl-guanosine]- [3 '-O-phosphorothioate diester-N4- (2-propylpentanoyl) -2' -deoxy-2 ',2' -difluorocytidine]

Intermediate 79 was obtained from intermediate 64 and the (-) -PSI reagent using the route of intermediate 72. MS-ESI [ M-H ]] ^- ：931.0。

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

Intermediates 80 and 81 were obtained from intermediate 65 and the (-) -PSI reagent using the route of intermediate 72. Intermediate 80: MS-ESI [ M-H ]] ^- :915.0, retention time: 1.50min;

intermediate 81: MS-ESI [ M-H ]] ^- :915.0, retention time: 1.53min. Analysis of LCMS: agilent 1100+ G1946D LCMS,4.6x50mm Waters Xbridge C18.5 μm analytical column, mobile phase A is 10mM NH ₄ HCO ₃ Aqueous solution, B acetonitrile, flow rate 1.8ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-0.2min,5% by weight of B;0.2-1.5min,5-95% by weight B;1.5-3min,95% B.

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

Intermediate 82 was obtained from intermediate 66 and the (-) -PSI reagent using the route for intermediate 72. MS-ESI [ M-H ]] ^- ：896.8。

Preparation of intermediate 83: 2-chloro-5 '-O-tert-butyldimethylsilyl-3' -O-benzoyl-2 '-deoxy-2' -fluoro-beta-adenosine

Intermediate 83 was obtained from intermediate 1 using the route of intermediate 20.

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

Preparation of intermediate 84: 2-chloro-3 ' -O-benzoyl-2 ' -deoxy-2 ' -fluoro-beta-adenosine

Intermediate 84 was obtained from intermediate 83 using the route for intermediate 60.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.40(d，J＝2.4Hz，1H)，8.12(d，J＝7.6Hz，2H)， 7.99(brs，2H)，7.74(t，J＝7.2Hz，1H)，7.60(t，J＝7.6Hz，2H)，6.55(dd，J＝18.2，3.6 Hz，1H)，5.81-5.78(m，1H)，5.77-5.66(m，1H)，4.35(q，J＝4.4Hz，1H)，3.89-3.80(m， 2H)。

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) -3 a-methyl-6- (prop-1-en-2-yl) -2-thiohexahydrobenzo [ d ] c][1，3，2]Oxathiolan-2-yl) oxy) tetrahydrofuran-2-yl) -6-oxy-6, 9-dihydro-1H-purin-2-yl) isobutyramide

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

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

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

Intermediate 86 was obtained from intermediate 84 and intermediate 85 in DMF using the route for intermediate 37.

MS-ESI[M+H] ⁺ ：1255.0。

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

Intermediate 86 (1.0 g,0.8 mmol) was added to a mixed solution (10 mL) of tetrahydrofuran/water/methanol (8: 4: 1), cooled to 0 deg.C, and lithium hydroxide monohydrate (54mg, 1.6 mmol) was added, and the resulting mixture was stirred at 0 deg.C for 20min. 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 eluting with ethyl acetate/methanol (10: 1) to give a white solid (733 mg).

¹ H NMR(400MHz，DMSO-d ₆ )δ12.02(s，1H)，11.67(s，1H)，8.21(t，J＝5.2Hz，1H)， 8.14(s，1H)，7.84(s，2H)，7.37(d，J＝7.6Hz，2H)，7.29-7.14(m，7H)，6.87-6.77(m， 4H)，6.23(dd，J＝13.9，4.5Hz，1H)，6.08-5.98(m，2H)，5.49-5.38(m，1H)，5.23-5.04 (m，1H)，4.66-4.59(m，1H)，4.35-4.24(m，1H)，3.99-3.95(m，1H)，3.77-3.71(m，2H)， 3.71-3.63(m，7H)，3.22-3.00(m，2H)，2.79-2.68(m，1H)，1.12-1.07(m，6H)，0.82(s， 9H)，0.13(d，J＝13.4Hz，6H)。

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

Intermediate 88 was obtained as crude product from intermediate 87 and (-) -PSI reagent in tetrahydrofuran using the route of intermediate 2 and was used directly in the next reaction. MS-ESI [ M + H-DMTr ]] ⁺ ：1095.0。

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

Intermediate 89 was obtained from intermediate 88 using the route of intermediate 25.

MS-ESI[M+H] ⁺ ：1095.0。

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]

Intermediate 89 (90mg, 0.082mmol) was dissolved in anhydrous DMF (7 mL) and 4A molecular sieves (90 mg) were added and dried under stirring at room temperature (28 ℃) under nitrogen for 0.5 h. DBU (75.0 mg, 0.492mmol) was then added via syringe and after stirring at room temperature (28 ℃ C.) for 0.5 h, celite was added and the filtrate was rotary dried to give the crude intermediate 90 (100 mg, crude) which was used directly in the next reaction.

MS-ESI[M+H] ⁺ ：926.8。

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

Intermediate 84 (500mg, 1.23mmol) and (2R, 3R,4R, 5R) -5- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4- ((tert-butyldimethylsilyl) oxy) -2- (2-isobutylamino-6-oxy-1, 6-dihydro-9H-purin-9-yl) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramide (1.78g, 1.84mmol) were dissolved in a mixed solution of acetonitrile/tetrahydrofuran (8 mL/8 mL), 4A molecular sieves (500 mg) were added, and stirring was carried out at room temperature (28 ℃ C.) for 0.5 hour under nitrogen. Then adding acetonitrile solution of tetrazole (8.17mL, 0.45M) by using a syringe; after stirring at room temperature (28 ℃ C.) for 2 hours, 70% t-butanol peroxide (520.9mg, 4.05mmol, 0.544 mL) was added, and after stirring at room temperature (28 ℃ C.) for 20 minutes, the mixture was quenched with 50% aqueous sodium thiosulfate pentahydrate (5 mL); celite was added and the filtrate was diluted with ethyl acetate (30 mL) and 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 eluting with petroleum ether/ethyl acetate (10: 1-1: 10) to give a mixture (1.9 g) which was used directly in the next step.

MS-ESI[M+H] ⁺ ：1296.0。

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]

To intermediate 91 (1g, 0.775mmol) was added 33% methylamine ethanol solution (8 mL), and stirred at room temperature for 3 hours. The reaction solution was spin-dried and purified by C18 reverse phase column, eluted with water (containing 0.1% ammonium bicarbonate) and acetonitrile (3: 2), and lyophilized to give a pink solid (550 mg).

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

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]

Diphenyl phosphite (137mg, 0.47mmol) was added to a solution of intermediate 92 (250 mg, 0.235mmol), DBU (143mg, 0.94mmol) and 4A molecular sieves in pyridine (8 mL) at 0 deg.C under nitrogen, and the mixture was stirred at room temperature for 2 hours. The reaction solution was filtered and then spin-dried, and was used as it was in the next reaction.

MS-ESI[M+H] ⁺ ：1129.8。

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]

Intermediate 94 was obtained from intermediate 93 using the route for intermediate 25.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.26(s，1H)，7.99(s，1H)，7.88(s，2H)，6.55(s，2H)， 6.27(dd，J＝17.2，4.0Hz，1H)，6.00-5.70(m，3H)，5.44-5.21(m，1H)，5.09-4.98(m， 1H)，4.87-4.71(m，1H)，4.47(d，J＝4.0Hz，1H)，4.07-3.97(m，1H)，3.91-3.73(m，3H)， 3.56-3.49(m，5H)，0.89(s，9H)，0.14(s，6H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ1.72， -1.58。MS-ESI[M+H] ⁺ ：826.8。

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]

Pivaloyl chloride (48mg, 0.40mmol) was added to a pyridine solution (15 mL) of intermediate 94 (55mg, 0.067 mmol) at 0 ℃ and the resulting reaction mixture was stirred at room temperature for 3 hours. Then, a mixed solution of elemental iodine (26mg, 0.10 mmol) in acetonitrile (0.5 mL) and water (0.1 mL) was added thereto, and the resulting reaction solution was stirred at room temperature for 16 hours. The reaction was quenched with 10% aqueous sodium thiosulfate (5 mL), spin dried, purified using a C18 reverse phase column, eluted with water (containing 0.1% ammonium bicarbonate) and acetonitrile (4: 1), and lyophilized to give a white solid (20 mg). MS-ESI [ M + H ]] ⁺ ：825.0。

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]

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

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

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

Intermediate 97 is obtained by a synthetic route using intermediate 95, by reacting intermediate 92 with (+) -PSI reagent, deprotecting and cyclizing reaction, and purifying by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Waters XBridge C18 10 μm preparative column, mobile phase A10 mM ammonium bicarbonate aqueous solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength ultraviolet absorption monitoring, gradient elution: 0-2min, 20B; 2-14min, 20-40B 14-14.2min, 40-95B 14.2-18min, 95B. The compound retention time 12.5 min).

¹ H NMR(400MHz，DMSO-d ₆ )δ10.59(s，1H)，8.18(d，J＝2.4Hz，1H)，8.11(s，1H)， 7.45-6.98(m，5H)，6.33-6.23(m，1H)，5.82(d，J＝8.6Hz，1H)，5.42(s，1H)，5.32- 5.23(m，2H)，4.33(d，J＝4.0Hz，1H)，4.19-3.70(m，9H)，0.91(s，9H)，0.15(s，6H)。 ¹⁹ F NMR(376MHz，DMSO)δ-196.51(s)。 ³¹ P NMR(162MHz，DMSO)δ53.76(s)， -0.49(s)。MS-ESI[M+H] ⁺ ：841.0。

Preparation of intermediate 98: (2 ',3 ') -cyclo- (Rp, rp) - [2' -O-phosphorothioic acid bis (phospho-phosphoric acid)ester-N2-isobutyryl-3' -O-tert-butyldimethylsilyl-guanosine]- [3' -O-phosphorothioate diester-2-chloro-2 ' -deoxy-2 ' -fluoro-beta-adenosine]

Intermediate 98 is obtained by reaction of intermediate 87 with (+) -PSI reagent, deprotection and cyclization reaction using the synthetic route of intermediate 90.

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

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-diazabicycloundec-7-ene-salts

To a solution of intermediate 54 (120mg, 0.150mmol) and (-) -PSI reagent (201mg, 0.450mmol) in DMF (8 mL) was added 1, 8-diazabicycloundec-7-ene (342mg, 2.25mmol) and the resulting mixture was stirred at 25 ℃ for 1 h. The reaction mixture was spin dried and reprecipitated with ethyl acetate (20 mL). The solids were collected and the resulting crude product was purified by preparative liquid chromatography (Gilson 281 preparative HPLC,19X250mm Welch 10 μm preparative column, mobile phase A10 mM ammonium bicarbonate aqueous solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength ultraviolet absorption monitoring, gradient elution: 0-3min,0-3% B3-14min, 3-30% B;14-14.3min, 30-95B 14.3-20min,95% B. The compound retention time was 13.5 min) to give a white solid (20 mg) after lyophilization.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.37(s，1H)，8.22(s，1H)，7.90(brs，4H)，7.12(brs， 6H)，6.33-6.20(m，2H)，5.56-5.30(m，2H)，5.20-5.00(m，2H)，4.22-3.85(m，6H)，3.55- 3.40(m，2H)，3.35-3.15(m，1H)，2.70-2.55(m，1H)，1.96-1.85(m，1H)，1.73-1.52(m， 3H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ53.47。MS-ESI[M-H] ^- ：760.9。

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

Example 2 was obtained by following the reaction of intermediate 55 with (-) -PSI reagent using the route of example 1, purification by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Welch 10 μm preparative column, mobile phase A0.05% aqueous formic acid, B0.05% aqueous formic acid in acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorbance monitoring, gradient elution: 0-3min, 0-5B 3-14min, 5-50B 14-14.3min, 50-95B 14.3-20min, 95B. The compound retention time 12 min).

¹ H NMR(400MHz，DMSO-d ₆ )δ8.53(s，1H)，8.38(s，1H)，7.84(brs，4H)，6.33-6.18 (m，2H)，5.20-4.75(m，2H)，4.30-3.75(m，6H)，3.00-2.70(m，2H)，2.70-2.50(m，2H). ³¹ P NMR(162MHz，DMSO-d ₆ )δ53.68，52.69。MS-ESI[M-H] ^- ：724.9。

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

Example 3 was obtained by following the reaction of intermediate 56 with (-) -PSI reagent using the route of example 1, purification by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Welch 10 μm preparative column, mobile phase A0.05% aqueous formic acid, B0.05% aqueous formic acid in acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorbance monitoring, gradient elution: 0-3min, 0-5B 3-14min, 5-30B 14-14.3min, 30-95B 14.3-20min, 95B. The compound retention time was 18 min).

¹ H NMR(400MHz，DMSO-d ₆ )δ9.20-8.80(m，2H)，8.31(d，J＝6.4Hz，1H)，8.28- 8.00(m，2H)，7.90(d，J＝6.0Hz，1H)，6.20-6.05(m，3H)，6.00(d，J＝6.4Hz，1H)， 4.95-4.68(m，2H)，4.30-4.05(m，4H)，3.90-3.70(m，2H)。 ³¹ P NMR(162MHz，DMSO- d ₆ )δ53.27，52.67。MS-ESI[M+H] ⁺ ：683.0。

Example 4: (3 ', 3') -cyclo- (Rp, rp) -bis- [3 '-O-phosphorothioate diester-N4- (2-propylpentanoyl) -2' -deoxy-2 ',2' -difluorocytidine]Diammonium salts

Example 4 was obtained by purification using the route of example 1, after reaction from intermediate 57 and (-) -PSI reagent, by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Welch 10 μm preparative column, mobile phase A10 mM ammonium bicarbonate aqueous solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength ultraviolet absorption monitoring, gradient elution: 0-3min,0-35% B3-14min, 35-70% B14-14.3 min, 70-95% B14.3-20min, 95B, the compound retention time 14 min.).

1H NMR(400MHz，DMSO-d ₆ )δ11.05(s，1H)，11.00(s，1H)，8.39(d，J＝7.6Hz， 1H)，8.16(d，J＝7.6Hz，1H)，7.40(d，J＝7.6Hz，1H)，7.34(d，J＝7.6Hz，1H)，7.12 (t，J＝52.0Hz，8H)，6.30-6.18(m，2H)，5.02-4.75(m，2H)，4.35-4.10(m，4H)，3.83(t， J＝11.2Hz，2H)，2.70-2.52(m，2H)，1.62-1.45(m，4H)，1.40-1.17(m，12H)，0.85(t，J ＝7.2Hz，12H)。31P NMR(162MHz，DMSO)δ54.54，53.29。MS-ESI[M+H] ⁺ ： 935.1。

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 salts

To intermediate 71 (200mg, 0.214mmol) was added 7M methanolic ammonia (2 mL) and the resulting mixture was stirred at 25 ℃ for 6 hours. And (4) spin-drying the reaction mixed solution to obtain a crude product. The resulting crude product was purified by preparative liquid chromatography (Gilson 281 preparative HPLC,19X250mm Welch 10 μm preparative column, mobile phase A0.05% aqueous formic acid, B0.05% aqueous formic acid in acetonitrile, flow rate 25ml/min, dual wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-3min,0-5% B3-14min, 5-30% B14-14.3 min,30-95% B14.3-20min, 95% B.the compound retention time was 14 min), and lyophilized to give a white solid (4.0 mg).

¹ H NMR(500MHz，DMSO-d ₆ )δ8.63(s，1H)，8.23(s，1H)，8.17(s，1H)，7.92(brs， 2H)，7.29(brs，2H)，6.33-6.28(m，1H)，6.12(d，J＝8.5Hz，1H)，5.40-5.15(m，2H)， 5.05-4.95(m，1H)，4.55(t，J＝3.5Hz，1H)，4.36-4.24(m，2H)，4.19(s，1H)，4.10-3.95 (m，2H)，3.90-3.78(m，1H)，3.72(d，J＝12.0Hz，1H)。 ³¹ P NMR(162MHz，DMSO- d ₆ )δ53.08，48.81。MS-ESI[M-H] ^- ：724.8。

Example 6: (2 ',3 ') -cyclo- (Rp, rp) - [2' -O-phosphorothioate diester-adenosine]- [3 '-O-phosphorothioate diester-2-chloro-2' -deoxyadenosine]Diammonium salts

Example 6 was obtained following the procedure of example 5 from intermediate 72 and 28% ammonia followed by purification by preparative liquid chromatography (10 mM ammonium bicarbonate as additive). Analysis of LCMS: agilent 1100+ G1946D LCMS,4.6x50mm Waters Xbridge C18.5 μm analytical column, mobile phase A is 10mM ammonium bicarbonate water solution, B is acetonitrile, flow rate is 1.8ml/min,214 and 254nm dual-wavelength ultraviolet absorption monitoring, gradient elution: 0-1.5min, 5-95%; 1.5-3min,95% by weight B. The compound retention time was 0.35min.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.61(s，1H)，8.40(s，1H)，8.17(s，1H)，7.86(brs， 2H)，7.41-7.06(m，10H)，6.30-6.18(m，1H)，6.09(d，J＝8.4Hz，1H)，5.42-5.30(m， 2H)，5.30-5.20(m，1H)，4.27(d，J＝4.4Hz，1H)，4.20-4.05(m，2H)，4.04-3.90(m，2H)， 3.72-3.62(m，2H)，2.85-2.55(m，2H)。31P NMR(162MHz，DMSO-d ₆ )δ56.62， 53.65。MS-ESI[M-H] ^- ：707.0。

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 salts

Example 7 was prepared using the route of example 5 from intermediate 73 and 28% ammonia and was purified by preparative liquid chromatography (10 mM ammonium bicarbonate as additive). Analysis of LCMS: agilent 1100+ G1946D LCMS,4.6x150mm Waters Xbridge C18.5 μm analytical column, mobile phase A is 10mM ammonium bicarbonate water solution, B is acetonitrile, flow rate is 1ml/min,214 and 254nm double-wavelength ultraviolet absorption monitoring, gradient elution: 0-8min,5-95% by weight B;8-15min,95% by volume B. The compound retention time was 4.1min.

¹ H NMR(400MHz，DMSO-d ₆ )δ8.62(s，1H)，8.23(s，1H)，8.19(s，1H)，7.95(brs， 2H)，7.65(brs，2H)，7.14(t，J＝52.0Hz，8H)，6.30-6.22(m，1H)，6.12(d，J＝8.4Hz， 1H)，5.50-5.32(m，2H)，5.30-5.15(m，1H)，5.08-4.92(m，1H)，4.30-4.00(m，5H)，3.95- 3.86(m，1H)，3.68(d，J＝12.0Hz，1H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ57.14，54.13。MS-ESI[(M-2H)/2] ^- ：362.0。

Example 8: (2 ',3 ') -cyclo- (Sp, rp) - [2' -O-phosphorothioate diester-guanosine]- [3' -O-phosphorothioate diester-2-chloro-2 ' -deoxy-2 ' -fluoro-beta-adenosine]A 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 salts

Examples 8 and 9 were obtained by purification using preparative liquid chromatography (Gilson 281 preparative HPLC,19X250mm Welch 10 μm preparative column, mobile phase A10 mM aqueous ammonium bicarbonate solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-3min,0-2% B3-14min, 2-37% B14-14.3 min,37-95% B14.3-20min, 95% B, compound 8 retention time 13min, compound 9 retention time 15 min) after reaction from intermediate 74 and 28% aqueous ammonia using the route of example 5.

Example 8:

¹ H NMR(400MHz，DMSO-d ₆ )δ10.62(brs，1H)，8.19(s，1H)，8.11(s，1H)，7.94(brs， 2H)，7.10(t，J＝50.8Hz，8H)，6.63(brs，2H)，6.25(dd，J＝24.0，2.8Hz，1H)，5.85(d， J＝8.8Hz，1H)，5.50-5.15(m，4H)，4.30-4.13(m，2H)，4.12-3.92(m，4H)，3.72(d，J＝ 12.0Hz，1H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ56.84，54.05。MS-ESI[M-H] ^- ：741.0。

example 9:

¹ H NMR(400MHz，DMSO-d ₆ )δ10.67(brs，1H)，8.23(s，1H)，8.08(s，1H)，7.93(brs， 2H)，7.10(t，J＝51.2Hz，8H)，6.67(brs，2H)，6.30(dd，J＝18.8，3.6Hz，1H)，5.77(d， J＝8.0Hz，1H)，5.58-5.38(m，1H)，5.02-4.92(m，1H)，4.86-4.78(m，1H)，4.69-4.60 (m，1H)，4.33-3.75(m，7H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ59.03，54.74。MS-ESI [M-H] ^- ：740.9。

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 salts

Example 10 was purified by preparative liquid chromatography (Gilson 281 preparative HPLC,19X250mm Welch 10 μm preparative column, mobile phase A10 mM aqueous ammonium bicarbonate solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-3min,0-3% B3-14min, 3-38% B14-14.3 min, 38-95% B14.3-20min, 95% B; compound retention time 13 min) following reaction from intermediate 75 and 28% aqueous ammonia using the procedure of example 5. Example 10 was also obtained following the route of example 25, deprotection of intermediate 98, and purification by preparative liquid chromatography. Two different synthetic routes give the exact same product.

¹ H NMR(400MHz，DMSO-d ₆ )δ10.63(brs，1H)，8.22(s，1H)，8.18(s，1H)，7.93(brs， 2H)，7.12(t，J＝51.2Hz，8H)，6.69(brs，2H)，6.28(dd，J＝24.0，2.4Hz，1H)，5.88(d， J＝8.4Hz，1H)，5.42-4.80(m，4H)，4.45(d，J＝4.0Hz，1H)，4.39-4.10(m，3H)，4.08- 3.94(m，1H)，3.93-3.70(m，2H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ54.07，50.13。 MS-ESI[M-H] ^- ：741.0。

Example 11: (2 ',3 ') -cyclo- (Sp, rp) - [2' -O-phosphorothioate diester-guanosine]- [3 '-O-phosphorothioate diester-2-chloro-2' -deoxy-adenosine]Diammonium salts

Example 11 was prepared using the route of example 5 from intermediate 76 and 7M ammonia in methanol and purified by preparative liquid chromatography (10 mM ammonium bicarbonate as additive). Analysis of LCNS: agilent 1100+ G1946D LCMS,4.6x150mm Waters XBridge C18.5 μm analytical column, mobile phase A is 10mM ammonium bicarbonate water solution, B is acetonitrile, flow rate is 1ml/min,214 and 254nm dual-wavelength ultraviolet absorption monitoring, gradient elution: 0-8min,5-95% by weight B;8-15min,95% by volume B. The compound retention time was 3.3min.

¹ H NMR(400MHz，DMSO-d ₆ )δ10.63(brs，1H)，8.39(s，1H)，8.21(s，1H)，7.83(brs， 2H)，7.10(t，J＝50.8Hz，8H)，6.67(brs，2H)，6.30-6.18(m，1H)，5.87(d，J＝8.4Hz， 1H)，5.35-5.20(m，2H)，4.28(d，J＝4.0Hz，1H)，4.21-3.92(m，4H)，3.80-3.60(m，2H)， 2.92-2.70(m，1H)，2.69-2.55(m，1H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ56.96，54.34。 MS-ESI[M-H] ^- ：722.9。

Example 12: (2 ',3 ') -cyclo- (Rp, rp) - [2' -O-phosphorothioate diester-guanosine]- [3 '-O-phosphorothioate diester-2-chloro-2' -deoxy-adenosine]Diammonium salts

Example 12 was prepared using the route of example 5 from intermediate 77 and 7M ammonia in methanol and purified by preparative liquid chromatography (10 mM ammonium bicarbonate as additive). Analysis of LCMS: agilent 1100+ G1946D LCMS,4.6x150mm Waters Xbridge C18.5 μm analytical column, mobile phase A is 10mM ammonium bicarbonate water solution, B is acetonitrile, flow rate is 1ml/min,214 and 254nm double-wavelength ultraviolet absorption monitoring, gradient elution: 0-8min, 5-95%; 8-15min,95% by volume B. The compound retention time was 3.3min.

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

Example 13: (2 ',3 ') -cyclo- (Rp, rp) - [2' -O-phosphorothioate diester-guanosine]- [3 '-O-phosphorothioate diester-2' -deoxy-2 ',2' -difluorocytidine]Diammonium salts

Example 13 was obtained by following the reaction of intermediate 78 with 28% aqueous ammonia using the route of example 5, purification by preparative liquid chromatography (Gilson 281 preparative HPLC,19X250mm Welch 10 μm preparative column, mobile phase A10 mM aqueous ammonium bicarbonate solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-3min,0-3% B3-14min, 3-25% B14-14.3 min,25-95% B14.3-20min, 95% B, compound retention time 2 min).

¹ H NMR(400MHz，DMSO-d ₆ )δ8.16(s，1H)，7.60-6.70(m，11H)，6.58(brs，2H)， 6.35-6.20(m，1H)，5.90-5.70(m，2H)，5.22-5.10(m，1H)，5.00-4.80(m，2H)，4.52(s， 1H)，4.32-4.15(m，2H)，4.11(s，1H)，4.00-3.82(m，2H)，3.78-3.65(m，1H)。 ³¹ P NMR (162MHz，DMSO-d ₆ )δ53.24，48.05。MS-ESI[(M-2H)/2] ^- ：350.0。

Example 14: (2 ',3 ') -cyclo- (Rp, rp) - [2' -O-phosphorothioate diester-guanosine]- [3' -O-Thiophosphoric diester-N4- (2-propylpentanoyl) -2' -deoxy-2 ',2' -difluorocytidine]Diammonium salts

Example 14 and was obtained by purification using preparative liquid chromatography (Gilson 281 preparative HPLC,19X250mm Welch 10 μ M preparative column, mobile phase A10 mM aqueous ammonium bicarbonate solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-3min,0-10% B3-14min, 10-55% B14-14.3min, 55-95% B14.3-20min, 95% B. The compound retention time 11 min.) after reaction from intermediate 79 and 7M methanolic ammonia using the route of example 5.

¹ H NMR(400MHz，DMSO-d ₆ )δ11.07(brs，1H)，10.62(s，1H)，8.68-7.97(m，2H)， 7.40-6.97(m，9H)，6.59(brs，2H)，6.34-6.20(m，1H)，5.90-5.70(m，1H)，5.47-5.09(m， 2H)，5.05-4.74(m，1H)，4.29(d，J＝4.4Hz，1H)，4.20(d，J＝9.6Hz，1H)，4.13-3.99 (m，2H)，3.95-3.80(m，lH)，3.85-3.65(m，1H)，2.70-2.55(m，1H)，1.60-1.46(m，2H)， 1.45-1.10(m，6H)，0.86(t，J＝7.2Hz，6H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ56.16， 53.39。MS-ESI[M-H] ^- ：827.0。

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 salts

Example 15 was prepared by the procedure of example 5, using preparative liquid chromatography (Gilson 281 preparative HPLC,19X250mm Welch 10 μ M preparative column, mobile phase A10 mM aqueous ammonium bicarbonate solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-3min,0-3% B3-14min, 3-33% B14-14, after reaction from intermediate 80 and 7M methanolic ammonia.3min,33-95% by weight of B;14.3-20min,95% by weight of B. The compound has a retention time of 11 min). ¹ H NMR(400MHz，DMSO-d ₆ )δ8.20(d，J＝2.8Hz，1H)，8.04(s，1H)，8.03-6.87(m， 10H)，6.60(brs，2H)，6.22(dd，J＝24.0，2.0Hz，1H)，5.85(d，J＝8.8Hz，1H)，5.51- 5.15(m，3H)，4.35-3.89(m，6H)，3.72(d，J＝12.4Hz，1H)。 ³¹ P NMR(162MHz， DMSO-d ₆ )δ56.65，54.03。MS-ESI[M-H] ^- ：741.0。

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 salts

Example 16 was obtained by following the reaction of the intermediate 81 with 7M methanolic ammonia using the route of example 5, and purifying by preparative liquid chromatography (Gilson 281 preparative HPLC,19X250mm Welch 10 μ M preparative column, mobile phase A10 mM aqueous ammonium bicarbonate, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-3min, 0-3B 3-14min, 3-33B 14-14.3min, 33-95B 14.3-20min, 95B. The compound retention time 13 min). ¹ H NMR(400MHz，DMSO-d ₆ )δ10.63(brs，1H)，8.22(s，1H)，8.09(s，1H)，7.94(brs， 2H)，7.38-6.94(m，8H)，6.65(brs，2H)，6.28(dd，J＝24.0，2.4Hz，1H)，5.87(d，J＝8.8 Hz，1H)，5.42-5.11(m，3H)，5.00(s，1H)，4.56-4.42(m，1H)，4.39-4.21(m，2H)，4.13 (s，1H)，4.05-3.94(m，1H)，3.89-3.71(m，2H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ 53.97，49.08。MS-ESI[M-H] ^- ：740.9。

Example 17: (3 ',3 ') -cyclo- (Rp, rp) - [3' -O-phosphorothioate diester-guanosine]- [3 '-O-phosphorothioate diester-2-chloro-2' -deoxy-adenosine]Diammonium salts

Example 17 following the procedure of example 5, from intermediate 82 and 7M methanolic ammonia, purification was performed by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Welch 10 μ M preparative column, mobile phase A: l 0mM aqueous ammonium bicarbonate, B: acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-3min,0-3% B3-14min, 3-33% B14-14.3 min,33-95% B14.3-20min, 95% B. The compound retention time was 11 min).

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

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

Example 18 was prepared using the procedure of example 1, following reaction between intermediate 67 and the (-) -PSI reagent, purification by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Welch 10 μm preparative column, mobile phase A10 mM aqueous ammonium bicarbonate, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorbance monitoring, gradient elution: 0-3min,0-5% B3-14min, 5-33% B14-14.3min, 33-95% B14.3-20min, 95B. Compound retention time 11 min).

¹ H NMR(400MHz，DMSO-d ₆ )δ9.62(brs，2H)，8.39(s，1H)，8.26(d，J＝2.0Hz，1H)， 8.18(s，1H)，7.36(brs，2H)，7.15(t，J＝52.0Hz，2H)，6.31-6.20(m，2H)，5.46-5.25(m， 2H)，5.15-5.00(m，1H)，4.95-4.83(m，1H)，4.42-4.25(m，1H)，4.10-3.98(m，2H)，3.80- 3.60(m，2H)，3.60-3.40(m，6H)，3.30-3.20(m，3H)，2.70-2.55(m，3H)，1.96-1.85(m， 3H)，1.73-1.50(m，9H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ53.37，52.10。MS-ESI [M+H] ⁺ ：729.0。

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-diazabicycloundec-7-ene-salts

Example 19 was obtained by following the reaction of intermediate 68 with (-) -PSI reagent using the route of example 1, purification by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Welch 10 μm preparative column, mobile phase A0.05% aqueous formic acid, B0.05% aqueous formic acid in acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorbance monitoring, gradient elution: 0-3min, 0-10B 3-14min, 10-50B; 14-14.3min, 50-95B 14.3-20min, 95B. Compound retention time 10 min).

¹ H NMR(400MHz，DMSO-d ₆ )δ9.56(s，1H)，8.60(s，1H)，8.50-8.35(m，1H)，8.28 (s，1H)，7.85(brs，2H)，6.40-6.20(m，2H)，5.70-5.40(m，1H)，5.18-4.80(m，2H)，4.60- 3.95(m，6H)，3.65-3.45(m，2H)，3.30-3.20(m，1H)，2.85-2.71(m，1H)，2.70-2.55(m， 2H)，1.96-1.85(m，1H)，1.73-1.50(m，3H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ54.78， 54.00。MS-ESI[M+H] ⁺ ：710.8。

Example 20: (3 ',3 ') -cyclo- (Rp, rp) - [3' -O-phosphorothioate diester-2 ' -deoxy-2 ' -fluoroguanosine]- [3' -O-Thiophosphodiester-2-chloro-2 ' -deoxy-2 ' -fluoro-beta adenosine]Diammonium salts

EXAMPLE 20 was obtained by following the reaction of intermediate 69 with (-) -PSI reagent using the route of example 1, purification by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Waters XBridge C18 μm preparative column, mobile phase A10 mM ammonium bicarbonate aqueous solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorbance monitoring, gradient elution: 0-2min, 5B 2-9min, 5-15B 9-19min, 15-25B 19-19.5min, 25-95B 19.5-22.5min, 95B. The compound retention time 9 min).

¹ H NMR(400MHz，DMSO-d ₆ )δ10.69(brs，1H)，8.26(s，1H)，7.98(s，1H)，7.92(brs， 2H)，7.15(t，J＝52.0Hz，6H)，6.64(brs，2H)，6.26(dd，J＝17.2，3.6Hz，1H)，6.08(d，J＝16.8Hz，1H)，5.43-5.35(m，1H)，5.31-5.22(m，1H)，5.18-5.02(m，1H)，4.99-4.85 (m，1H)，4.42-4.30(m，1H)，4.26(d，J＝8.8Hz，1H)，4.15-4.00(m，2H)，3.90-3.70(m， 2H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ54.20，53.14。MS-ESI[M-H] ^- ：743.2。

Example 21: (3 ',3 ') -cyclo- (Rp, rp) - [3' -O-phosphorothioate diester-2 ' -deoxy-2 ' -fluoroguanosine]- [3 '-O-phosphorothioate diester-2-chloro-2' -deoxyadenosine]Diammonium salts

EXAMPLE 21 was obtained by following the reaction of intermediate 70 with (-) -PSI reagent using the route of example 1, purification by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Waters Xbridge C18. Mu.m preparative column, mobile phase A10 mM ammonium bicarbonate aqueous solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-2min, 5-10.6min, 20-95% B10.6-12.6min, 95-B, compound retention time 7.3 min).

¹ H NMR(400MHz，DMSO-d ₆ )δ10.67(brs，1H)，8.49(s，1H)，7.92(s，1H)，7.82(brs， 2H)，7.14(t，J＝52.0Hz，6H)，6.62(s，2H)，6.25(dd，J＝6.4，4.8Hz，1H)，6.05(d，J＝ 16.8Hz，1H)，5.44(dd，J＝52.0，4.0Hz，1H)，5.05-4.85(m，2H)，4.33(d，J＝12.4Hz，1H)，4.23(d，J＝9.2Hz，1H)，4.10-3.92(m，2H)，3.98-3.50(m，2H)，2.78-2.54(m， 2H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ52.56，52.51。MS-ESI[M-H] ^- ：725.2。

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

Example 22 was prepared by following the procedure of example 9, from intermediate 95 and ammonium fluoride in methanol, and purifying by preparative liquid chromatography (Gilson 281 preparative HPLC,19X250mm Waters XBridge C18. Mu.m preparative column, mobile phase A10 mM ammonium bicarbonate in water, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-2min, 5B 2-15.6min, 5-20B 15.6-15.8min, 20-95B 15.8-18min, 95B. The compound retention time 6.6 min).

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

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

To intermediate 96 (75mg, 0.074mmol) was added 33% methylamine ethanol solution (3 mL), and stirred at room temperature for 3 hours. The reaction mixture was spin-dried, and ammonium fluoride (82mg, 2.2mmol) and methanol (3 mL) were added thereto, followed by stirring at 60 ℃ for 16 hours. Purification by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Waters XBridge C18 μm preparative column, mobile phase A10 mM aqueous ammonium bicarbonate, B acetonitrile, flow rate 25ml/min, dual wavelength UV absorption monitoring at 214 and 254nm, gradient elution: 0-2min,5% B;2-18.9min,5-15% B18.9-19.4min, 15-95% B19.4-22.4min, 95% B. The compound retention time 8.5 min) and lyophilization gave a white solid (30.3 mg).

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

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

Example 24 is obtained by purification by preparative liquid chromatography (Gilson 281 preparative HPLC,19x250mm Waters XBridge C18. Mu.m 10 μm preparative column, mobile phase A10 mM aqueous ammonium bicarbonate solution, B acetonitrile, flow rate 25ml/min,214 and 254mM dual wavelength ultraviolet absorption monitoring, gradient elution: 0-2min, 20B 2-14min, 20-40B 14-14.2min, 40-95B 14.2-18min, 95B. The compound retention time 2 min), following the reaction between intermediate 97 and ammonium fluoride in methanol solution using the procedure of example 9.

¹ H NMR(400MHz，DMSO-d ₆ )δ10.58(s，1H)，8.19(d，J＝2.8Hz，1H)，8.04-7.82 (m，2H)，7.37-7.00(m，4H)，6.73-6.53(m，1H)，6.27(dd，J＝24.0，2.4Hz，1H)，5.83 (d，J＝8.3Hz，1H)，5.44-5.19(m，2H)，5.18-5.06(m，1H)，5.04-4.95(m，1H)，4.36 -4.27(m，2H)，4.12(s，1H)，4.09-3.96(m，2H)，3.79-3.65(m，2H). ³¹ P NMR(162 MHz，DMSO-d ₆ )δ53.89(s)，-1.20(s)。19F NMR(376MHz，DMSO-d ₆ )δ-195.45 (s)。MS-ESI[M+H] ⁺ ：726。

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 salts

Intermediate 90 (100 mg, crude) was dissolved in 7M methanolic ammonia (3 mL), stirred at room temperature (28 ℃ C.) for 4 hours and then spun dry. The resulting residue was dissolved in methanol (1 mL), ammonium fluoride (60.5mg, 1.63mmol) was added, the reaction was stirred with heating (60 ℃ C.) for 20 hours, and after completion of the reaction was monitored by LCMS and HPLC, the reaction solution was purified by preparative liquid chromatography (Gilson 281 preparative HPLC,19X250mm Waters XBridge C18 10 μm preparative column, mobile phase A was 10mM ammonium bicarbonate aqueous solution, B was acetonitrile, flow rate 25mL/min,214 and 254nm dual wavelength UV absorption monitoring, gradient elution: 0-2min, 5B 2-18.9min, 5-15B 18.9-19.4min, 15-95B 19.4-22.4min, 95B. The compound retention time 10 min), and lyophilized to give a white solid (21 mg).

¹ H NMR(400MHz，DMSO-d ₆ )δ10.71(s，1H)，8.44(s，1H)，8.23(s，1H)，7.92(s，2H)， 7.11(t，J＝52Hz，6H)，6.56(s，2H)，6.29(d，J＝28Hz，1H)，5.95(d，J＝8.4Hz，1H)， 5.35(d，J＝50.4Hz，1H)，5.20(t，J＝11.8Hz，1H)，5.11-5.05(m，1H)，4.48(d，J＝3.6 Hz，1H)，4.39-4.28(m，2H)，4.14(s，1H)，4.02-3.96(m，1H)，3.88-3.83(m，1H)，3.81- 3.76(m，1H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ53.30，50.07。 ¹⁹ F NMR(376MHz， DMSO-d ₆ )δ-196.87。MS-ESI[M+H] ⁺ ：742.8。

Example 26: (2 ',3 ') -cyclo- (Rp, rp) - [2' -O-phosphorothioate diester-guanosine]- [3' -O-Thiophosphodiester-2-methylamino-2 ' -deoxy-2 ' -fluoro-beta-adenosine]Diammonium salts

EXAMPLE 26 is a white solid obtained by purifying by the route of example 25, a by-product resulting from a deprotection reaction of the intermediate 98 with an ethanol solution of methylamine by preparative liquid chromatography using a preparative liquid chromatography reaction solution (Gilson 281 preparative HPLC,19x250mm Waters xBridge C18 10 μm preparative column, mobile phase A10 mM aqueous ammonium bicarbonate solution, B acetonitrile, flow rate 25ml/min,214 and 254nm dual wavelength ultraviolet absorption monitoring, gradient elution: 0-2min, 5B 2-15.6min, 5-15B 15.6-15.8min, 15-95B, 15.8-18min, 95B, compound retention time 14.1 min) and lyophilizing.

¹ H NMR(400MHz，DMSO-d6)δ10.69(brs，1H)，8.27(s，1H)，8.02(s，1H)，7.34-6.97 (m，3H)，6.31(brs，1H)，6.28(d，J＝2.4Hz，1H)，5.90(d，J＝0.8Hz，1H)，5.44-5.15 (m，3H)，4.44(d，J＝0.4Hz，1H)，4.35-4.27(m，1H)，4.24-4.12(m，2H)，4.03-3.94(m， 1H)，3.93-3.79(m，2H)，2.85(s，3H)。 ³¹ P NMR(162MHz，DMSO-d ₆ )δ54.02，51.06。 MS+ESI-.[M+H]：738.0。

Active examples

Example 1: the activation effect of the compound on IFN-beta secretion of THP-1 cells

THP-1 cell is a human monocytic leukemia cell line, and its STING phenotype is HAQ type, namely R71H-G230A-R293Q. In this experiment, human IFN-beta DuoSet ELISA Kit (R & D, cat # DY 814-05) and DuoSet ELISA Ancillary Reagent Kit 2 (R & D, cat # DY 008) from R & D were used to evaluate the activation of IFN-beta secretion from THP-1 cells by representative compounds of the invention.

Upon recovery of THP-1 cells (ATCC # TIB-202), the cells were thawed within 1min by rapid shaking of the cryovial in a 37 ℃ water bath. The cell suspension after thawing was mixed with RPMI1640 medium (Hyclone, cat # SH 30027.01) containing 10% FBS (Life technology, cat # 10099-141), centrifuged at 1000 rpm for 5min, and the supernatant was discarded. Resuspending the cell pellet in 5mL of complete medium (10% FBS-containing RPMI1640 medium), and placing in a bottom volume of 25cm ² Into a cell culture flask of (2), at 37 ℃ 5% ₂ And culturing in a cell culture box with 95% humidity. And carrying out cell passage when the cell confluence rate reaches about 80%. When the cells are passaged, all the cells in the culture bottle are transferred to a 15mL centrifuge tube, centrifuged for 5min at 1000 rpm, and the supernatant is discarded. 5mL of fresh complete medium was added to resuspend the cell pellet, and 1mL was placed in a bottom area of 25cm ² The cell culture flask of (4) was supplemented with 4mL of fresh complete medium to continue the culture. Plating is carried out when the cell confluence rate reaches about 80% again. When plating, 1/5 of the cell suspension is kept and the culture is continued, and the rest 4/5 of the cell suspension is put into a 15mL centrifuge tube according to the cell passage method. The cells were washed once with RPMI1640 medium (without serum addition) and centrifuged to remove the supernatant. Cells were resuspended in RPMI1640 medium (no serum added). Detecting the cell viability rate by trypan blue exclusion method, and plating when the cell viability rate is ensured to be more than 95%. The density was 1.1X 10 using RPMI1640 medium (without serum addition) ⁶ Viable cells/mL of cell suspension, 180. Mu.L of cell suspension was added to a 96-well cell culture plate well (NUNC, cat # 167008) so that the cell density in the plate was 2X 10 ⁵ Individual viable cells/well.

A 10mM compound DMSO stock solution was first serially diluted 3.16-fold with DMSO (Sigma, cat # D2650) to the 5 th concentration and a 6 th DMSO control containing no compound was set. Then, the DMSO solutions containing different concentrations of the compounds were diluted by 10-fold using PBS so that the DMSO content in each concentration of the compound solution was 10%. Finally, 20. Mu.L of the above solution was added to the corresponding cell culture plate to give an initial concentration of 100. Mu.M compound, a dilution factor of 3.16 times in the adjacent concentration, and a DMSO content of 1% in the cell culture plate. The cell plates were placed in a cell incubator for 24 hours.

ELISA detection process with reference to R & D System, cat # DY814-05 instructions.

Coating of ELISA plate: the capture antibody (mouse anti-human IFN- β capture antibody PART # 844508) was diluted with PBS (R & D System Cat # DY 006) to working concentration, 100 μ L of capture antibody working solution was added to a 96-well ELISA plate, which was sealed and placed at room temperature for overnight incubation. The capture antibody working solution was discarded, and the cell plate was washed 3 times with a washing buffer (0.05% Tween-20 in PBS pH 7.2-7.4, R &D System Cat # -WA126) using 400. Mu.L of the washing buffer per well, each washing sufficiently removing the washing buffer, and the final washing inverting the plate on clean paper to completely remove the washing buffer. mu.L of blocking buffer (1% BSA in PBS pH 7.2-7.4, R & D System Cat # #DY995) was added per well and incubated at room temperature for 1-2 hours. The above washing steps were repeated, preparing each plate for sample addition.

Sample detection: mu.L of sample or standard (recombinant human IFN-. Beta.standard, PART # 844510) was added to each well, plates were closed and incubated at room temperature for 2 hours, and the washing step of the above plate coating method was repeated. Then, 100 μ L of detection antibody (biotinylated mouse anti-human IFN- β detection antibody, PART # 844509) was added per well, the plates were sealed and left to incubate for 2 hours at room temperature, and the washing step of the above plate coating method was repeated. Subsequently, 100. Mu.L of Streptavidin-HRP (PART # 893975) working solution was added to each well, the plates were closed and left to incubate at room temperature for 20min, the process was protected from light, and the washing steps of the above plate coating method were repeated. Then, 100. Mu.L of substrate solution (Colar reagent tA (H) was added to each well ₂ O ₂ ) 1: 1 mixture with Color Reagent B (tetramethylbenzidine), R&D System Cat # DY 999), closed and left to incubate at room temperature for 20min, protected from light. Finally, 50. Mu.L of stop solution (2 NH) was added to each well ₂ SO ₄ ，R&D System Cat # DY 994), tap the cell plate to ensure thorough mixing.

The OD450 of each well was measured using a multifunctional microplate reader (Molecular Devices, spectramax M3). If a wavelength calibration function is available, set to 540nm or 570nm. If the wavelength calibration function is not available, OD450 minus OD540 or OD570 is used. This was completed within 30min after the addition of the stop solution.

2'3' -cGAMP (Invivogen, cat # tlrl-nacga 23) was used as a positive control compound and ADU-S100 (MCE, cat # HY 12885A) was used as a control compound:

data were analyzed using GraphPad Prism 7.0 software and a standard curve for ELISA IFN- β content was obtained using log-log plots. And substituting the OD value detected by each sample hole into a standard curve equation to obtain the corresponding IFN-beta concentration. The data were fitted by nonlinear sigmoidal regression to obtain a dose-effect curve of IFN- β concentration versus compound concentration, and EC50 values were calculated.

TABLE 1-activation of IFN-beta secretion by THP-1 cells by representative example Compounds (EC 50, μ M)

Examples	THP-1 Activity	Examples	THP-1 Activity
				5	B	16	A
8	B	17	A
				9	B	18	B
10	A	19	B
				11	B	21	B
12	A	2′，3′-cGAMP	B
				13	B	ADU-S100	A
15	B	26	A

Wherein: a: EC50 is 1-10 mu M; b: EC50 of 10.1-100 mu M

Experimental results show that the compound of the embodiment has an activating effect on the secretion of IFN-beta of THP-1 cells. As can be seen from table 1 and figure 1, the compounds of examples 10, 12, 16, 17 and 26 have, inter alia, significant STING agonist activity: their EC50 is stronger than 2',3' -cGAMP, both comparable to ADU-S100; of these, the compounds of examples 10, 16 and 17 stimulated IFN- β secretion from THP-1 cells at highest concentrations above ADU-S100.

Example 2: inhibitory Activity of the Compound of the present invention on proliferation of CT26 cells

CT26 cells are a mouse colorectal cancer cell line. This experiment was conducted by evaluating the inhibitory activity of a compound against the proliferation of CT26 cells using the CellTiter-Glo Luminescence Cell vitality Assay kit of Promega.

When CT26 cells (ATCC # CRL-2638) were recovered, the cells were thawed within 1min by rapidly shaking the frozen tubes in a water bath at 37 ℃. The cell suspension after thawing was mixed with DMEM medium (GE, cat # SH 30243.01) containing 10% FBS (GIBCO, cat # 10099-141), centrifuged at 1000 rpm for 5min, and the supernatant was discarded. 5mL of complete medium (10% FBS-containing DMEM medium) was taken to resuspend the cell pellet, and the pellet was placed in a bottom area of 25cm ² The cell culture flask of (3) is charged at 37 ℃ with 5% CO ₂ And culturing in a cell culture box with 95% humidity. And carrying out cell passage when the cell confluence rate reaches 70-80%. When the cells are passaged, all the cells in the culture bottle are transferred to a 15mL centrifuge tube, centrifuged for 5min at 1000 rpm, and the supernatant is discarded. 5mL of fresh complete medium was added to resuspend the cell pellet, and 1mL was placed in a bottom area of 25cm ² The cell culture flask of (4) was supplemented with 4mL of fresh complete medium to continue the culture. And plating when the cell confluence rate reaches 70-80% again. When plating, 1/5 of the cell suspension is kept and the culture is continued, and the rest 4/5 of the cell suspension is put into a 15mL centrifuge tube according to the cell passage method. The cells were washed once with DMEM medium (without serum) and centrifuged to remove the supernatant. Cells were resuspended in DMEM medium (without serum addition). Detecting the cell viability rate by trypan blue exclusion method, and plating when the cell viability rate is over 95 percent. DMEM medium (without addition)Serum) at a density of 1.1X 10 ⁴ Viable cells/mL of cell suspension, 90 μ L of cell suspension was added to a 96-well clear flat-bottom black-walled cell culture plate well (corning, cat # 3603) such that the cell density in the culture plate was 1000 viable cells/well. A control group containing no cells, no compound, and complete medium only (i.e., a culture medium control) was set, and a control group containing no compound, and cells (i.e., a cell control) was set. The cell plates were placed in a cell incubator overnight.

A 10mM compound DMSO stock solution was first serially diluted 3.16-fold with DMSO (Sigma, cat # D2650) to 9-fold concentration and a 10-fold concentration compound-free DMSO control was set. Then, DMSO solutions containing different concentrations of compounds were diluted 10-fold with PBS (Solarbio, cat # P1020) to give 10% DMSO content in each concentration of compound solution. Finally, 10. Mu.L of the above solution was added to the corresponding cell culture plate so that the starting concentration of the compound was 100. Mu.M, the dilution factor of the adjacent concentration was 3.16 times, and the DMSO content in the cell culture plate was 1%. The cell plate was placed in a cell incubator for an additional 120 hours.

After 120 hours, the CellTiter-Glo reagent (Promega, cat # G7572) was thawed and the cell plates were allowed to equilibrate to room temperature for 30min, 100uL of CellTiter-Glo was added to each well of the plate, the cells were lysed thoroughly by shaking on an orbital shaker for 5min, the plates were allowed to stand at room temperature for 20min to stabilize the signal, and the luminescence values of each well were scanned at full wavelength using a multifunctional microplate reader (Molecular Devices, spectramax M3).

The following compounds and ADU-S100 (MCE, cat # HY 12885A) were used as controls: clofarabine (Clofabine; wen lake Huaren technology, cat # HR-00701002), cladribine (Cladripine; CSNpharm, cat # CSN 10004), gemcitabine Hydrochloride (Gemcitabine Hydrochloride; shaoyuan, cat # SY 014538), gemcitabine prodrug LY2334737 (made by house, intermediate 7).

Cell viability was calculated for each concentration of compound using the following formula:

cell survival rate (%) = (Lum) _{Drug to be tested} -Lum _{Control of culture fluid} )/(Lum _{CellsControl of} -Lum _{Culture fluid control} )×100％。

Data were analyzed using GraphPad Prism 7.0 software, fitted to data using nonlinear S-curve regression to derive dose-response curves, and IC50 values were calculated therefrom.

TABLE 2 inhibition of CT26 cell proliferation (EC 50, μ M)

Wherein: a: IC50 < 1 μ M; b: IC 501-10 mu M; NA: IC50 > 10. Mu.M

Experimental results show that the compounds of representative examples are capable of inhibiting the in vitro growth of CT26 tumor cells; of these, the compounds of examples 3, 10 and 13 showed particularly significant inhibitory activity, whereas ADU-S100 showed no significant tumor cell inhibitory activity. In combination with the results of activity example 1, the results indicate that the compounds of the examples have multifunctional antitumor properties, i.e., tumor immunological activity of STING agonists and cytotoxic effects of antimetabolic anticancer drugs.

Example 3: antitumor activity of the compound of the invention in a bilateral transplantation tumor model of a CT26 syngeneic mouse

6-8 weeks BALB/c mice (purchased from Shanghai Ling Biotech Co., ltd.) were subcutaneously inoculated with 5X 10 cells on the left and right back sides ⁵ CT26 cells (supplied by Taicang Zexin Biotech Co., ltd., ATCC # CRL-2638) were inoculated in a volume of 0.1 mL/side. When the tumor grows to an average volume of 100mm ³ At that time, the tumor size and mouse body weight were randomly grouped and dosing was initiated. The day of the first administration was day 0. Two doses were administered, and on

days

0 and 4, 50ug of compound/mouse (i.e., 2.5 mg/kg) was administered intratumorally to the right, using the same volume of PBS (Hyclone, cat # SH 30258.01) as a control. The left tumor was not treated. Mice in the control group were sacrificed 13 days after the administration, and mice in the treatment group were sacrificed 21 days after the administration. During the experiment, the tumor volume and body weight were measured 3 times per week and calculated as V = D × D/2 (where D represents the major diameter of the tumor and D represents the minor diameter of the tumor).

Data at day 13 post-dose indicate that the compounds of the examples tested were all able to significantly inhibit bilateral tumor growth. The right-side group was more significant and most of the tumors disappeared (fig. 2-a). Among them, ADU-S100 treatment group showed 99.1% inhibition of tumor growth, example 17 showed 99.5% inhibition of tumor growth, and the remaining groups showed 100% inhibition of tumor growth, and no tumors were detected. At the same time, the untreated left side tumor growth slowed (fig. 2-B). On day 13 post-dose, the growth inhibition rates for the left tumor groups were: ADU-S100, 64.2%; example 10, 91.2%; example 12, 75.3%; example 16, 71.7%; example 17, 67.4%. In addition, in this experiment, it was observed that the body weight of individual mice decreased within 15% after administration, and the body weight recovered after the administration was stopped (fig. 2-C). The results show that the compound of the invention shows equivalent or better tumor inhibition capability than ADU-S100 in a CT26 isogenic double-sided tumor model.

Example 4: antitumor activity of the compound of the invention in a CT26 nude mouse transplantation tumor model

6-8 weeks BALB/c nude mice (purchased from Shanghai Ling Biotech limited) were subcutaneously inoculated with 5X 10 cells on the right posterior side and the back side, respectively ⁵ CT26 cells (supplied by Taicangxin Biotech Co., ltd., ATCC # CRL-2638) were inoculated in a volume of 0.1mL. When the tumor grows to an average volume of 100mm ³ In time, the administration was performed at random in groups according to the tumor size and the mouse weight. The day of the first administration was day 0. Two doses were given, and on

days

0 and 4, 50ug of compound/mouse (i.e., 2.5 mg/kg) was intratumorally injected on the right side, with the same volume of PBS (Hyclone, cat # SH 30258.01) as a control. Mice in the control group were sacrificed 9 days after administration, and mice in the ADU-S100 and example 10 compound-treated groups were sacrificed successively 14 days and 16 days later. During the experiment, tumor volume and body weight were measured 3 times per week, and the tumor volume was calculated by the formula V = D × D/2 (same above).

Data at day 9 after dosing indicate that the tested compound of example 10 of the invention was able to significantly inhibit tumor growth in T cell immunodeficient nude mice at 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 residual immune activity or other unknown reasons. Regardless of whether example 10 or ADU-S100, they showed better tumor growth inhibition in immunocompromised healthy mice than in nude mice (FIG. 2D). This experiment verifies that ADU-S100 acts primarily by activating T cell-mediated immunity; the compound of the present example 10 has tumor-inhibiting activity due to its multifunctional mechanism, and in this case, it is proved that it has tumor-inhibiting ability superior to that of STING agonist ADU-S100.

Example 5: immunological memory function of compound of the invention in CT26 syngeneic mouse or nude mouse transplantation tumor model

6-8 weeks BALB/c immunized healthy mice or nude mice (purchased from Shanghai Ling Chang Biotech Co., ltd.) were inoculated with 5X 10 of the vaccine subcutaneously on the right dorsal side ⁵ CT26 cells (supplied by Taicangxin Biotech Co., ltd., ATCC # CRL-2638) were inoculated in a volume of 0.1mL. When the tumor grows to an average volume of 100mm ³ At this time, the administration was randomly divided and started according to the tumor size and the mouse body weight. The day of the first administration was day 0. Two administrations were given, intratumorally injecting 50ug of compound/mouse (i.e., 2.5 mg/kg) on

days

0, 4. PBS (Hyclone, cat # SH 30258.01) at the same volume was used as a control. Mice in the nude or BALB/c mice experiments were sacrificed 9 or 13 days after administration, respectively. After 21 days, the mice were again inoculated subcutaneously 5X 10 on the left posterior side of the back ⁵ CT26 cells were seeded in a volume of 0.1mL. Blank BALB/c or nude mice without any treatment were inoculated at the same time as controls. During the experiment, the tumor volume and body weight were measured 3 times per week and calculated as V = D × D/2 (same above).

The results show that the compound of example 10 of the present invention tested significantly inhibited CT26 tumor growth in both immunocompromised and immunodeficient mice. The drug effect of the immune healthy mice is more prominent (figure 3-A). On day 5 after the administration, the tumors of the mice of the treatment group of example 10 were completely disappeared. On day 21, the compound-treated group of example 10 was re-seeded with CT26 cells. After 7 days (day 30)) The mean tumor volume of unimmunized blank BALB/c mice was 75mm ³ While the average tumor volume of the mice previously treated in example 10 was 29mm ³ . By the end of the experiment on day 33, the average tumor volume of the mice in the control group and the mice in the immunized group in example 10 was 648mm ³ And 101mm ³ . This experiment demonstrates that, following treatment with the compound of example 10, immunocompromised mice develop an immunological memory and produce strong immunological rejection of the re-inoculated allogeneic cells, effectively preventing tumor recurrence.

On the other hand, in immunodeficient nude mice, after the administration was stopped, the tumor continued to grow in the individual mice (FIG. 3-B). Finally 3 mice with smaller tumor volumes were selected for re-inoculation with CT26 cells on day 21. The results show that the re-inoculated CT26 tumor cells have similar growth speed with the blank mouse tumor cells. The results indicate that no immunological memory was generated in the nude mouse experiment, and further verify that the compound of example 10 also has cytotoxic tumor-inhibiting ability without involvement of the immune system in the model.

Example 6: hepatocyte metabolic stability assay of Compounds of the invention

The tests for the metabolic Stability of hepatocytes of 5 species (mouse, rat, dog, monkey, human) were carried out analogously to the compounds of the invention according to standard Methods customary in the art for in vitro metabolic Stability studies, for example the Methods described in (Kerns, edward H. And Di Li (2008), drug-like Properties: concepts, structure Design and Methods: from ADME to sensitivity Optimization, san Diego: academic Press, di, li et al, optimization of a high Throughput micro statistical Screening analysis for Profiling Drug Discovery tests, J biomol. Screen.2003,8 (4), 453.).

The hepatocytes used in the experiment were: human hepatocytes (SHQY, lot # HEP 190006); dog hepatocytes (IVT, lot # ZMB); monkey hepatocytes (Xenotech, lot # 2010022); rat hepatocytes (SHQY, lot # HEP 134045); mouse hepatocytes (BioIVT, cat. # M005052, lot. # MEO).

The cryopreserved hepatocyte cells of the cryopreserved tubes were taken out of the liquid nitrogen tank and immediately placed in shaking water at 37. + -. 1 ℃In the bath, for 2 minutes ± 15 seconds. The hepatocytes were transferred to 50mL of hepatocyte thawing medium (composition: williams E medium, 35mL, invitrogen, cat # A1217601; isotonic Percoll solution, 13.5 mL, general-purpose company, cat #17-0891-01; du's phosphate buffer, 1.5mL, invitrogen, cat #14200-075, glutamax, 500. Mu.L, invitrogen, cat #35050061, 750. Mu.L HEPES, invitrogen, cat # 30115606; fetal bovine serum, 2.5mL, invitrogen, cat #10091130; recombinant human insulin, 50. Mu.L, invitrogen, cat #12585014; dexamethasone (prepared as a 10mM DMSO solution), 5. Mu.L, sigma, cat # D1756), mixed gently, and centrifuged at 500 rpm for 3min. After centrifugation, the supernatant was carefully aspirated (without disturbing the cell pellet), 10 Xvolume of pre-warmed KHB buffer (Krebs-Henseleit buffer, sigma, cat # K3753-10X 1L) and 5.6g/L HEPES was added, the cell pellet was resuspended, mixed gently, and centrifuged at 500 rpm for 3min. The supernatant was aspirated without touching the cell pellet, and cell viability and number were determined. The hepatocytes were counted, and then the cell suspension KHB buffer was diluted to an appropriate density (viable cell density =2 × 10) ⁶ Individual cells/mL). The hepatocyte solution was placed on ice until use.

A2 Xdosing solution was prepared in preheated KHB (1% dimethyl sulfoxide) and centrifuged at 5594g for 15min (Thermo Multifuge. Times.3R), where 200. Mu.M of the dosing solution: add 20. Mu.L of compound stock (10mM, DMSO solution) to 980. Mu.L of dimethyl sulfoxide; 2 × medicated solution: to 990. Mu.L of KHB was added 10. Mu.L of 200. Mu.M spiking solution (2. Mu.M after dilution).

To the indicated different time points wells 50 μ L of pre-heated 2x dosing solution was added. 50 μ L of the pre-warmed hepatocyte solution (2X 10) ⁶ Individual cells/mL) were added to the designated wells for 15min, 30min, 60min and 120min detection, and then timing was started and the reaction plate was placed in a 37 ℃ incubator.

Add 100. Mu.L of IS (Salicornide or imipramine) -containing acetonitrile (Merck, cat. # CN 34854-4L) to a well designed for 0min, mix gently, then add 50. Mu.L of pre-warmed hepatocyte solution (2X 10) ⁶ Individual cells/mL), the well plate is closed. At 15min, 30min, 60min and 120minSeparately, 100. Mu.L 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 10min (600 rpm). The plates were sonicated for 2min and then centrifuged at 5594g for 15min (Thermo Multifuge x 3R). 50 μ L of supernatant per well was transferred to a 96-well sample plate containing 50 μ L of ultrapure water (mileore, ZMDS 50F 01) for LC/MS analysis.

Taking the concentration (C0) of the compound to be detected at the time point of T0 as 100%, converting the concentrations of other incubation time points into percentage residual, performing linear regression on the incubation time by using the natural logarithm of the percentage residual of each time point to obtain a slope K, and then calculating the clearance rate (CI) of the hepatocytes according to the following formula _int ) And in vitro half-life (T1/2):

T1/2＝1n2/K＝0.693/K

Cl _int = (0.693/T1/2) × (1/hepatocyte density) × scale factor

Wherein, the density of the liver cells is the final concentration of the liver cells in the incubation system of the experiment: 1 x 10 ⁶ one/mL. Scaling factor = number of hepatocytes × liver weight (for 5 species of hepatocytes, mouse 11812.5 × 10, respectively) ⁶ Rat 4680X 10/kg ⁶ 6880 × 10 dog/kg ⁶ Each kg, monkey 3900X 10 ⁶ Person 2544.3X 10/kg ⁶ One/kg).

As can be seen from FIG. 4A, the compounds of the present invention, such as the compound of example 10, showed better metabolic stability in 5 species of hepatocytes; it has a long metabolic half-life and a low clearance rate.

Example 7: discovery and identification of metabolites of the compounds of the invention

The main metabolites of the compounds of the invention in 5 species (mouse, rat, dog, monkey, human) hepatocytes were similarly identified according to standard Methods of in vitro metabolic Stability studies conventional in the art, for example (Kerns, edward H. And Di Li (2008), drug-like Properties: concepts, structure Design and Methods: from ADME to accessibility Optimization, san Diego: academic Press, di, li et al, optimization of a high Throughput micro biological Performance Screening Assay for Profiling Drug Discovery assays, J biomol. Screen.2003,8 (4), 453.).

The hepatocytes used in the experiment were: human hepatocytes (SHQY, cat. # BQHPCH10, lot. # HEP 190006-TA 05); dog hepatocytes (BioIVT, cat. # M00205, lot. # ZMB); monkey hepatocytes (XENOTECH, cat. # PPCH2000, lot. # 2010022); rat hepatocytes (SHQY, cat. # BQR 1000. H15, lot. # HEP 134049); mouse hepatocytes (BioIVT, cat. # M005052, lot. # MEO).

HI hepatocyte maintenance medium (BIOIVT, cat. # Z99009; lot. # C02060C) was preheated to 37 ℃. The cryopreserved hepatocytes were removed from the liquid nitrogen tank and immediately placed in a 37 + -1 deg.C shaking water bath for 2min + -15 seconds. The hepatocytes were transferred to 50ml of HI medium, gently mixed, and centrifuged at 500 rpm for 3min. After centrifugation, the supernatant was carefully aspirated (without disturbing the cell pellet). Add 10 Xvolume of prewarmed HI medium, resuspend the cell pellet, gently mix, centrifuge at 500 rpm for 3min. The supernatant was aspirated without touching the cell pellet. The hepatocytes were counted, and then the cell suspension was diluted with HI medium to an appropriate density (viable cell density =2 × 10) ⁶ Individual cells/mL), the hepatocyte solution was placed on ice until use.

2 × dosing solution was prepared in pre-warmed HI medium. 2 × medicated solution (20 μ M) was prepared as follows: to 3992 μ LHI medium was added 8 μ L of 10mM compound stock (20 μ M, 0.2% dimethyl sulfoxide after dilution).

Preheating hepatocyte solution and 2 Xdosing solution, assigning for T ₂₄₀ And T _240-w/o 200 μ L of pre-heated 2 Xdosing solution was added to the wells of (1). For T0, 1200. Mu.L acetonitrile (Merck, cat. # CN 34854-4L) and 200. Mu.L hepatocyte solution (2X 10) were added to the wells ⁶ Individual cells/mL), then 200 μ L of pre-heated 2x dosing solution is added and the well plate is closed. 200 μ L of the pre-warmed hepatocyte solution (2X 10) ⁶ Individual cells/mL) to the indicated for T ₂₄₀ In the hole of (a); to designate for T _240-w/o Add 200 μ L of pre-warmed HI medium to the wells of (1) and start timing; the reaction plate was placed in CO at 37 deg.C ₂ An incubator.

At 240min, 1200 μ L acetonitrile was added to each of the designated wells, and the wells were then closed. After quenching, the plates were sonicated for 2min and then centrifuged at 1400 rpm for 5min. 1200. Mu.L of the supernatant was evaporated under a stream of nitrogen until dry. The dried extract was then redissolved with 200. Mu.L acetonitrile: water (1: 3 v/v), vortexed for 2min, and centrifuged at 14000 rpm for 5min. 2/5. Mu.L of the supernatant was injected into LC-UV-MS for analysis.

As can be seen from FIG. 4-B, the compound of the present invention, such as the compound of example 10, can detect the cytotoxic small molecule Clofarabine (Clofarabine) produced by its decomposition in 5 species of liver cells, and this is the main metabolite of the compound of example 10. In combination with the results of activity example 6, it is highly likely that both CDN molecules with STING agonistic activity and small cytotoxic molecules are present within the body for a period of time, thereby achieving a combination at the molecular level, providing an enhanced, even synergistic, anti-tumor effect. The results also explain the reason why it works well in mouse tumor suppression experiments.

Example 8: drug effect comparison of antitumor activity of the compound of the invention in combination with pure CDN STING agonist and cytotoxic drug in a bilateral transplantation tumor model of a CT26 syngeneic mouse

6-8 weeks BALB/c mice (purchased from Shanghai Ling Biotech Co., ltd.) were subcutaneously inoculated with 5X 10 cells on the left and right back sides ⁵ CT26 cells (supplied by Tecangxin Biotech, inc., ATCC # CRL-2638) were seeded in a volume of 0.1 mL/side. When the tumor grows to an average volume of 100mm ³ At the same time, the administration was started by randomly grouping the compounds according to the tumor size and the mouse weight (the compound group of the present invention; the single drug group of CDN STING agonist; the single drug group of cytotoxic drug; the single combination group of CDN STING agonist and cytotoxic drug). The day of the first administration was day 0. Three times in total, right intratumoral administration on

days

0, 4 and 7, with the same volume of PBS (Hyclone, cat # SH 30258.01) as a control. The left tumor was not treated. The control mice were sacrificed 13 days after dosing and the treated mice were sacrificed 21 days later. During the experiment, the tumor volume and body weight were measured 3 times per week and calculated as V = D × D/2 (same above).

The experimental result shows that the tumor growth inhibition rate of the compound group is obviously higher than the inhibition rate of a CDN STING single-drug group and a cytotoxic single-drug group, the activity of the compound group is equivalent to the activity of the latter two groups, even higher than the activity of the latter two groups, and the improved and even synergistic tumor inhibition activity is generated.

General or preferred definitions of a given feature in various enumerated embodiments of the present invention may be combined with general or preferred definitions of other given features to yield yet further embodiments of the present invention. As if such combinations were specifically and individually set forth herein, unless the context clearly indicates otherwise.

In this specification, several prior publications are referenced. These publications, while not considered to be relevant to the patentability of the invention, are incorporated herein by reference in their entirety. The reference in this specification to any prior publication (or information derived from it), is not, and should not be taken as, an acknowledgment or admission or any form of suggestion that the corresponding prior publication (or information derived from it) forms part of the common general knowledge in the field of technology to which this specification relates.

Claims

1. A cyclic dinucleotide compound of formula (II),

wherein

B ₁ Is adenine substituted by X

Wherein X is selected from Cl, F or NHC _1-6 An alkyl group; or optionally substituted with R ^a Substituted cytosines

Wherein R is ^a Selected from H or-C (O) -C _1-14 An alkyl group;

R ₁ and R ₁ ' each is independently selected from H, F or-OH;

B ₂ selected from adenine optionally substituted by X

Wherein X is selected from H, F or Cl; optionally substituted with R ^a Substituted cytosines

Wherein R is ^a Selected from H or-C (O) -C _1-14 An alkyl group; or guanine

Wherein OH is optionally substituted by C _1-6 Alkyl substitution;

means that the phosphate bond may be attached to the 2 'or 3' position of the pentose, the site of both not forming a ring with phosphate being R ₂ And R ₂ ' substituted; and

R ₂ and R ₂ ' are each independently selected from H, -OH or F;

provided that when B ₁ Or B ₂ One of which is optionally substituted with R ^a Substituted cytosine where the carbon atom adjacent to the pentose ring to which it is attached is substituted with two F;

a stereoisomer, tautomer, stable isotopic variant, pharmaceutically acceptable salt, prodrug or solvate thereof.

2. A compound of formula (II), a stereoisomer, a tautomer, a stable isotopic variation, a pharmaceutically acceptable salt, a prodrug or a solvate thereof according to claim 1, wherein

Representing a phosphate bond to the 3' position of the pentose, formula (II) having

3. A compound according to claim 2, wherein B ₁ Is composed of

R ₁ And R ₁ ' are both H, or R ₁ And R ₁ One is H and the other is F.

4. A compound according to claim 2, wherein B ₁ Is adenine substituted by X

Wherein X is selected from NHC _1-6 Alkyl, preferably NHCH ₃ ，R ₁ And R ₁ ' are both H, or R ₁ And R ₁ One is H and the other is F.

5. A compound according to claim 2, wherein B ₁ Is optionally substituted by R ^a Substituted cytosines

Wherein R is ^a Selected from H or-C (O) -C _1-14 Alkyl radical, R ₁ And R ₁ ' both are F.

6. A compound according to any one of claims 2-5, wherein B ₂ Is guanine

Or adenine

R ₂ And R ₂ ' one is H and the other is selected from-OH or F.

7. A compound according to any one of claims 2 to 5, wherein B ₂ Is adenine substituted by X

Wherein X is Cl, R ₂ And R ₂ One of' is H, the other is F, or R ₂ And R ₂ Both are H.

8. A compound according to any one of claims 2 to 5, wherein B ₂ Is optionally substituted by R ^a Substituted cytosines

Wherein R is ^a Selected from H or-C (O) -C _1-14 Alkyl radical, R ₂ And R ₂ ' both are F.

9. A compound of formula (II), a stereoisomer, a tautomer, a stable isotopic variation, a pharmaceutically acceptable salt, a prodrug or a solvate thereof according to claim 1, wherein

Representing a phosphate bond to the 2' position of the pentose, formula (II) having

10. A compound according to claim 9, wherein B ₁ Is composed of

R ₁ And R ₁ ' are both H, or R ₁ And R ₁ One is H and the other is F.

11. A compound according to claim 9In which B is ₁ Is adenine substituted by X

Wherein X is NHC _1-6 Alkyl, preferably NHCH ₃ ，R ₁ And R ₁ ' are both H, or R ₁ And R ₁ One is H and the other is F.

12. A compound according to claim 9, wherein B ₁ Is optionally substituted by R ^a Substituted cytosines

13. A compound according to any one of claims 9-12, wherein B ₂ Is guanine

Or adenine

R ₂ And R ₂ One of' is H and the other is-OH.

14. A compound according to claim 1,2 or 9 which is

Or a pharmaceutically acceptable salt or solvate thereof.

15. A compound according to claim 14, wherein B ₁ Is composed of

R ₁ And R ₁ ' are both H, or R ₁ Is F and R ₁ ' is H.

16. A compound according to claim 14, wherein B ₁ Is adenine substituted by X

Wherein X is NHC _1-6 Alkyl, preferably NHCH ₃ ，R ₁ And R ₁ ' are both H, or R ₁ Is F and R ₁ ' is H.

17. A compound according to claim 14, wherein B ₁ Is optionally substituted by R ^a Substituted cytosines

18. A compound according to any one of claims 14 to 17, wherein in formula (II-a'), B ₂ Is guanine

Or adenine

R ₂ ' is H, R ₂ Is selected from-OH or F.

19. A compound according to any one of claims 14 to 17, wherein in formula (II-a'), B ₂ Is adenine substituted by X

Wherein X is Cl, R ₂ Is H, another R ₂ ' is F, or R ₂ And R ₂ ' are both H.

20. A compound according to any one of claims 14 to 17, wherein in formula (II-B'), B ₂ Is guanine

Or adenine

R ₂ ' is H, R ₂ is-OH.

21. Cyclic dinucleotide compounds selected from

Or a pharmaceutically acceptable salt or solvate thereof.

22. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1-21 and a pharmaceutically acceptable excipient.

23. A pharmaceutical composition according to claim 22 in a form for topical administration.

24. A method for the prevention or treatment of a disease associated with or mediated by STING, in particular a viral infection or a tumour in a mammal, especially a human, which method comprises administering an effective amount of a compound according to any one of claims 1 to 21 or a pharmaceutical composition according to any one of claims 22-23.

25. Use of a compound according to any one of claims 1 to 21 or a pharmaceutical composition according to any one of claims 22-23 as a STING agonist for the treatment or prevention of a disease associated with or mediated by STING, in particular a viral infection or a tumor.

26. Use of a compound according to any one of claims 1 to 21 or a pharmaceutical composition according to any one of claims 22-23 as a cytotoxic agent for the treatment or prevention of a viral infection or a tumour.

27. Use of a compound according to any one of claims 1 to 21 or a pharmaceutical composition according to any one of claims 22-23 as multifunctional active agents for immunotherapy and cytotoxic treatment.

28. The use according to claim 27, wherein the multifunctional active agent is used to activate STING signaling pathway to activate immune system to function against tumor and viral replication, to cause tumor cell death or to prevent viral replication by releasing cytotoxic agents, to continue activating STING to kill tumor cells by releasing tumor DNA, and to provide "immunological memory" or the ability to sustain immunity to tumors by releasing tumor neoantigens to generate antibody-antigen responses.

29. Use of a compound according to any one of claims 1 to 21 or a pharmaceutical composition according to any one of claims 22-23 in the manufacture of a medicament for the treatment or prevention of a disease associated with or mediated by STING, in particular a viral infection or a tumour.

30. Use of a compound according to any one of claims 1 to 21 or a pharmaceutical composition according to any one of claims 22 to 23 in the manufacture of a cytotoxic agent for the treatment or prevention of a viral infection or a tumour.

31. A method or use according to any one of claims 24-26 and 28-30, wherein the tumour is selected from brain cancer, head and neck cancer, skin cancer, melanoma, bladder cancer, ovarian cancer, breast cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, blood cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bone cancer, colorectal cancer, liver cancer, renal cell carcinoma, pancreatic cancer, hodgkin's lymphoma or leukaemia.