WO2021228284A1 - 新型sting激动剂及其制备方法和应用 - Google Patents

新型sting激动剂及其制备方法和应用 Download PDF

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WO2021228284A1
WO2021228284A1 PCT/CN2021/105135 CN2021105135W WO2021228284A1 WO 2021228284 A1 WO2021228284 A1 WO 2021228284A1 CN 2021105135 W CN2021105135 W CN 2021105135W WO 2021228284 A1 WO2021228284 A1 WO 2021228284A1
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compound
formula
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李艳梅
吴军军
赵朗
陈永湘
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清华大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/6574—Esters of oxyacids of phosphorus
    • C07F9/65746—Esters of oxyacids of phosphorus the molecule containing more than one cyclic phosphorus atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of medicine. Specifically, the present invention relates to a novel STING agonist cyclic dinucleotide and its preparation method and application.
  • STING interferon genes
  • cyclic dinucleotides can be used as an ideal choice for therapeutic agents or vaccine adjuvants.
  • the negative charge and easy hydrolysis of CDNs phosphodiester greatly hinders its clinical application value. Therefore, designing and developing appropriate methods to improve the stability and transmembrane efficiency of cyclic dinucleotides has important research value.
  • the present invention aims to solve at least one of the technical problems existing in the prior art, and provides a novel STING agonist and its preparation method and application.
  • the inventors designed and synthesized a new type of chemically modified cyclic dinucleotides (CDNs) by the liquid-phase one-bottle method, the structural formula of which is shown in SF.
  • the bases can be various natural and unnatural bases such as A and G.
  • the mentioned liquid-phase one-bottle method means that the multi-step reaction is carried out in a round-bottom flask.
  • the present invention provides the following technical solutions:
  • the present invention provides a compound represented by formula SF,
  • B 1 or B 2 are each independently selected from natural bases A, G, C, T, U and unnatural bases;
  • X is selected from -H, -OH, -F;
  • Y is selected from -OH and -SH.
  • the compound provided by the present invention undergoes phosphorylation modification and hydroxyl fluorination modification on cyclic dinucleotides.
  • Phosphorylation of cyclic dinucleotides can effectively increase the resistance of the compound against phosphatase hydrolysis.
  • the strong electron-attracting new type of cyclic dinucleotides can enhance the liposolubility and stability of cyclic dinucleotides. .
  • the compound is selected from at least one of the following compounds represented by formula SF1 or formula SF2:
  • B 1 or B 2 are each independently selected from natural bases A, G, C, T, U and unnatural bases.
  • the non-natural base is selected from modified natural bases, preferably I, mC, and artificial bases.
  • the present invention provides a method for preparing the compound according to any one of the embodiments of the first aspect of the present invention, including:
  • the compound represented by formula S1 the compound represented by formula S2, the compound represented by formula S3, the compound represented by formula S4, the compound represented by formula S5, and the compound represented by formula S6 are respectively as follows:
  • L1 and L2 in each compound are respectively Base protecting group;
  • Z is selected from silanol, F;
  • the method for preparing a compound described above may further include the following technical features:
  • the silyl hydroxyl group is selected from the group consisting of trimethylsilyl ether hydroxyl group, tert-butyldimethylsilyl ether hydroxyl group, and tert-butyldiphenylsilyl ether hydroxyl group.
  • the cyclization reagent is 5,5-dimethyl-2-chloro-1,3,2-dioxphosphacaprolactone phosphate
  • the oxidant is at least one selected from iodine or 3H-1,2-benzodithiol-3-one 1,1-dioxide.
  • the deprotection reaction in step (1) is carried out at room temperature.
  • the phosphorylation reaction in step (2) is carried out under anhydrous conditions
  • the oxidation and deprotection reactions in step (3) are carried out at room temperature
  • the nucleophilic substitution and oxidation reactions in step (4) are carried out at room temperature
  • the nucleophilic substitution reaction in step (5) is carried out at room temperature
  • the deprotection reaction in step (6) is carried out at 50°C in an oil bath.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound and pharmaceutically acceptable excipients, carriers, excipients, vehicles, or combinations thereof, and the compound is the first aspect of the present invention
  • the compound described in any embodiment may be a compound prepared by the method described in any embodiment of the second aspect of the present invention.
  • the present invention provides the use of a compound in the preparation of a medicament for the treatment of immune diseases, and the compound is the compound according to the first aspect of the present invention or is according to the present invention.
  • the medicine is used to treat tumors or used for antiviral and antibacterial purposes.
  • the drug is used to activate interferon gene stimulating protein (STING).
  • STING interferon gene stimulating protein
  • the present invention provides a drug combination comprising the compound according to the first aspect of the present invention or the compound prepared according to the method according to the second aspect of the present invention, and at least one compound for treatment Drugs for immune diseases.
  • the at least one drug used to treat immune diseases is selected from immune checkpoint blocking antibodies (anti-PD1, anti-PD-L1, anti-CTLA-4, etc.), immunostimulants ( At least one of CpG, imidazoquinoline and monophosphatidyl A, etc.), vaccines, chimeric antigen receptor T cells, radiotherapy and chemotherapy (doxorubicin, paclitaxel, cisplatin, etc.).
  • immune checkpoint blocking antibodies anti-PD1, anti-PD-L1, anti-CTLA-4, etc.
  • immunostimulants At least one of CpG, imidazoquinoline and monophosphatidyl A, etc.
  • vaccines chimeric antigen receptor T cells
  • radiotherapy and chemotherapy doxorubicin, paclitaxel, cisplatin, etc.
  • Fig. 1 is a flow cytometry result diagram of cells treated with different compounds and transfection reagents according to an embodiment of the present invention.
  • Fig. 2 is the results of tumor volume changes of mice in different treatment groups according to an embodiment of the present invention.
  • Fig. 3 is a diagram showing the results of enzyme-linked immunosorbent spot assays for different treatment groups according to an embodiment of the present invention.
  • Fig. 4 is the results of antigen-specific antibody titers in serum of different treatment groups provided according to an embodiment of the present invention.
  • Figure 5 is a graph showing the typing results of different antibodies in different treatment groups according to an embodiment of the present invention.
  • Fig. 6 is a graph showing the results of changes in tumor volume of mice in different treatment groups according to an embodiment of the present invention.
  • Stereoisomers refer to compounds that have the same chemical structure but differ in the arrangement of the atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometrical isomers (cis/trans) isomers, atropisomers, etc. .
  • Chiral refers to a molecule that can not overlap with its mirror image; and “achiral” refers to a molecule that can overlap with its mirror image.
  • Enantiomers refer to two isomers of a compound that cannot overlap but are mirror images of each other.
  • Diastereoisomers refer to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties and reactivity. Mixtures of diastereomers can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, such as HPLC.
  • optically active compounds that is, they have the ability to rotate the plane of plane-polarized light.
  • the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to one or more of its chiral centers.
  • the prefixes d and l or (+) and (-) are symbols used to specify the rotation of plane-polarized light caused by the compound, where (-) or l indicates that the compound is levorotatory.
  • Compounds prefixed with (+) or d are dextrorotatory.
  • a specific stereoisomer is an enantiomer, and a mixture of such isomers is called an enantiomeric mixture.
  • a 50:50 mixture of enantiomers is called a racemic mixture or a racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.
  • any asymmetric atom (for example, carbon, etc.) of the compound disclosed in the present invention may exist in a racemic or enantiomerically enriched form, such as (R)-, (S)- or (R,S)-configuration form exist.
  • each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess.
  • the compound of the present invention can be one of the possible isomers or a mixture of them, such as racemates and diastereomer mixtures (depending on the number of asymmetric carbon atoms) The form exists.
  • Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituent may have an E or Z configuration; if the compound contains a disubstituted cycloalkyl, the substituent of the cycloalkyl may have a cis or trans configuration.
  • Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physical and chemical properties of the components, for example, by chromatography Method and/or fractional crystallization method.
  • racemate of any final product or intermediate obtained can be resolved into optical enantiomers by a method familiar to those skilled in the art using known methods, for example, by performing diastereomeric salts of the obtained diastereomers. Separate.
  • the racemic product can also be separated by chiral chromatography, such as high performance liquid chromatography (HPLC) using a chiral adsorbent.
  • HPLC high performance liquid chromatography
  • enantiomers can be prepared by asymmetric synthesis, for example, refer to Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2 nd Ed. Robert E.
  • tautomer or "tautomeric form” refers to structural isomers with different energies that can be converted into each other through a low energy barrier. If tautomerism is possible (as in solution), the chemical equilibrium of tautomers can be reached.
  • proton tautomers also called prototropic tautomers
  • keto-enol tautomerism include mutual transformations through the recombination of some bond-forming electrons.
  • keto-enol tautomerism are the tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers.
  • tautomerism is phenol-ketone tautomerism.
  • a specific example of phenol-ketone tautomerism is the interconversion of pyridine-4-ol and pyridine-4(1H)-one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.
  • C 1 ⁇ 6 alkyl group refers particularly each independently methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, and C 6 alkyl.
  • linking substituents are described.
  • the Markush variables listed for the group should be understood as the linking group.
  • alkyl represents the attached alkylene group or arylene group, respectively group.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable when administered to humans and generally do not produce allergies or similar inappropriate reactions, such as gastrointestinal discomfort, dizziness, and the like.
  • pharmaceutically acceptable refers to those approved by a federal regulatory agency or a national government or listed in the US Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient or base with which the compound is administered.
  • These pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • Water and aqueous solutions Saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, especially injectable solutions. Suitable drug carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • stereochemistry in the present invention usually refer to the following documents: SPParker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S ., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.
  • the compounds of the present invention may contain asymmetric centers or chiral centers, so there are different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including but not limited to, diastereomers, enantiomers, atropisomers, and their mixtures, such as racemic mixtures, constitute the present invention Part.
  • optically active compounds that is, they have the ability to rotate the plane of plane-polarized light.
  • the prefixes D, L or R, S are used to indicate the absolute configuration of the chiral center of the molecule.
  • the prefix d, l or (+), (-) is used to name the symbol of the plane-polarized light rotation of the compound, (-) or l means the compound is levorotatory, and the prefix (+) or d means the compound is dextrorotatory.
  • the chemical structures of these stereoisomers are the same, but their three-dimensional structures are different.
  • a specific stereoisomer may be an enantiomer, and a mixture of isomers is usually called an enantiomeric mixture.
  • racemic mixture A 50:50 mixture of enantiomers is called a racemic mixture or a racemate, which may result in no stereoselectivity or stereospecificity during the chemical reaction.
  • racemic mixture and “racemate” refer to an equimolar mixture of two enantiomers, lacking optical activity.
  • “Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way their atoms are arranged in space. The term “isomer” as used herein includes any and all geometric isomers and stereoisomers. For example, “isomers” include cis and trans isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (d) isomers , (1)-isomers, racemic mixtures thereof, and other mixtures thereof falling within the scope of this specification.
  • the "hydrate” of the present invention refers to the compound or its salt provided by the present invention, which also includes chemical or non-chemical equivalent of water bound by non-covalent intermolecular force, it can also be said that the solvent molecule is formed by water Associate.
  • solvate of the present invention refers to an association formed by one or more solvent molecules and the compound of the present invention.
  • Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.
  • the "nitrogen oxide” in the present invention means that when the compound contains several amine functional groups, one or more nitrogen atoms can be oxidized to form an N-oxide.
  • N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen-containing heterocyclic nitrogen atoms.
  • the corresponding amine can be treated with an oxidizing agent such as hydrogen peroxide or peracid (such as peroxycarboxylic acid) to form N-oxide (see Advanced Organic Chemistry, Wiley Interscience, 4th edition, Jerry March, pages).
  • N-oxides can be prepared by the method of LWDeady (Syn.Comm.1977, 7,509-514), in which, for example, in an inert solvent such as dichloromethane, the amine compound is combined with m-chloroperoxybenzoic acid (MCPBA) reaction.
  • LWDeady Syn.Comm.1977, 7,509-514
  • MCPBA m-chloroperoxybenzoic acid
  • prodrug represents the conversion of a compound into the compound shown in the present invention in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or the enzymatic conversion of the prodrug into the maternal structure in the blood or tissues.
  • Metal refers to the product obtained by the metabolism of a specific compound or its salt in the body.
  • the metabolites of a compound can be identified by techniques well known in the art, and its activity can be characterized by experimental methods as described in the present invention. Such products can be obtained by oxidizing, reducing, hydrolyzing, amidating, deamidating, esterifying, degreasing, enzymatic cleavage, etc. of the administered compound.
  • the present invention includes the metabolites of the compound, including the metabolites produced by fully contacting the compound of the present invention with a mammal for a period of time.
  • compositions can be prepared by the active ingredient and a pharmaceutically acceptable carrier.
  • the "pharmaceutically acceptable salt” used in the present invention refers to the organic and inorganic salts of the compound of the present invention.
  • Pharmaceutically acceptable salts are well known to us in the field, as described in the literature: S.M. Berge et al., describe pharmaceutical acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19, 1977.
  • non-toxic acid salts include, but are not limited to, inorganic acid salts formed by reaction with amino groups include hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, Nitrate, etc., and organic acid salts such as acetate, propionate, glycolate, oxalate, maleate, malonate, succinate, fumarate, tartrate, citric acid Salt, benzoate, mandelate, methanesulfonate, ethanesulfonate, toluenesulfonate, sulfosalicylate, etc., or obtained by other methods described in books and literature such as ion exchange These salts.
  • salts include adipate, malate, 2-hydroxypropionic acid, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, Borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate Salt, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, caproate, hydroiodide, 2-hydroxy-ethanesulfonate, lacturonate, lactate , Laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, Pectinate, persul
  • the present invention also intends to contemplate any quaternary ammonium salts formed by compounds containing N groups.
  • Water-soluble or oil-soluble or dispersed products can be obtained by quaternization.
  • Alkali metal or alkaline earth metal salts include sodium salt, lithium salt, potassium salt, calcium salt, magnesium salt, iron salt, zinc salt, copper salt, manganese salt, aluminum salt and the like.
  • Pharmaceutically acceptable salts further comprise suitable amine cation nontoxic ammonium, quaternary ammonium, and the counterion, such as halide, hydroxide, carboxylate, sulfated, phosphorylated compounds, nitrate compounds, C 1 -8 Sulfonates and aromatic sulfonates.
  • Amine salts such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methyl reduced glucose Amine, procaine, N-benzyl phenethylamine, 1-p-chlorobenzyl-2-pyrrolidine-1'-ylmethyl-benzimidazole, diethylamine and other alkyl amines, piperazine And tris(hydroxymethyl)aminomethane; alkaline earth metal salts such as but not limited to barium, calcium and magnesium; transition metal salts such as but not limited to zinc.
  • alkaline earth metal salts such as but not limited to barium, calcium and magnesium
  • transition metal salts such as but not limited to zinc.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable auxiliary materials, carriers, excipients, vehicles, or combinations thereof.
  • a mammal such as a human in the form of a medicine
  • it may be administered in the form of the compound itself or may contain, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient and a pharmaceutically acceptable
  • the carrier is administered in the form of a pharmaceutical composition.
  • “Combination” means a fixed combination in a single dosage unit form or a kit of parts for combined administration, wherein the compound disclosed in the present invention and the combination partner (medicine for the treatment of tumor diseases, AIDS, inflammatory response and immunodeficiency diseases) can be used in It can be administered independently at the same time or can be administered separately within a certain time interval, especially to make the joint partner show cooperation, such as synergy.
  • pharmaceutical composition as used herein means a product obtained by mixing or combining more than one active ingredient, and includes both fixed and non-fixed combinations of active ingredients.
  • the term “fixed combination” means that the active ingredients such as the compound disclosed in the present invention and the combination partner are simultaneously administered to the patient in the form of a single entity or dosage.
  • non-fixed combination means that the active ingredients such as the compound disclosed in the present invention and the combination partner are both administered to the patient simultaneously, jointly or successively without a specific time limit as separate entities.
  • pharmaceutically acceptable carrier includes pharmaceutically acceptable materials, compositions or carriers suitable for administering the compound of the present invention to mammals.
  • the carrier includes liquid or solid fillers, diluents, excipients, solvents or encapsulating materials that participate in carrying the subject matter or transferring it from one organ or part of the body to another organ or another part of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient.
  • materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, Ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil , Olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffer Agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ring,
  • wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate may also be present in the composition, as well as coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents and fragrances. , Preservatives and antioxidants.
  • antioxidants examples include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxybenzene Methyl ether (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, etc.; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid , Phosphoric acid, etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxybenzene Methyl ether (BHA), butylated hydroxyto
  • compositions of the present invention include those suitable for oral, nasal, topical, buccal, sublingual, rectal and/or parenteral administration.
  • the formulation can conveniently be presented in unit dosage form and can be prepared by any method well known in the pharmaceutical art.
  • the amount of active ingredient that can be combined with a carrier material to prepare a single dosage form is generally the amount of the compound that produces a therapeutic effect. Generally speaking, in units of one percent, the amount is about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10 to about 30%.
  • treatment is used to refer to obtaining the desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease.
  • Treatment encompasses diseases of mammals, especially humans, including: (a) preventing the occurrence of diseases or disorders in individuals who are susceptible to but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the development of the disease; Or (c) Alleviate the disease, for example, alleviate the symptoms associated with the disease.
  • Treatment encompasses any medication that administers a drug or compound to an individual to treat, cure, alleviate, ameliorate, alleviate, or inhibit the individual's disease, including but not limited to administering a drug containing the compound described herein to an individual in need.
  • SFG1 is an ipsilateral monosulfide monofluoro modified cyclic dinucleotide
  • SFG2 is a disulfide difluoro modified cyclic dinucleotide.
  • SFG1 and SFG2 can significantly activate antigen-presenting cells.
  • SFG1 and SFG2 as immunotherapy reagents and vaccine adjuvants can increase the titer of antigen-specific antibodies and inhibit tumor growth.
  • SFG1 and SFG2 compounds can be widely used in various immunotherapy such as tumor therapy, antiviral, antibacterial and other fields.
  • Example 1 guanylic acid was used as an example to synthesize SFG1 and SFG2 respectively, where SFG1 was a cyclic dinucleotide modified with monosulfur and monofluorine on the same side, and SFG2 was a cyclic dinucleotide modified with disulfide and fluorine.
  • SFG1 was a cyclic dinucleotide modified with monosulfur and monofluorine on the same side
  • SFG2 was a cyclic dinucleotide modified with disulfide and fluorine.
  • the preparation method is as follows, the specific steps can refer to the following two reaction formulas:
  • the conditions for synthesizing SFG1 are: adding 0.32 mL of water and 165 mg of iodine, stirring and reacting for 5 minutes, and sampling in the middle for ESI-MS detection to determine the completion of the oxidation. After the reaction is completed, pour the mixture into 70 mL aqueous solution containing 0.1 g of sodium bisulfite, stir for 5 min to consume excess oxidant, and then slowly add 2 g of sodium bicarbonate to neutralize the reaction system, and continue to stir for 5 min;
  • the conditions for synthesizing SFG2 are: add 0.32mL water and 130mg Beaucage reagent (3H-1,2-benzodithiol-3-one-1,1-dioxide) (0.75mmol, 1.5 equivalents), stir and react for 5min , Sampling halfway for ESI-MS detection to determine the completion of oxidation. After the reaction was completed, the reaction system was poured into 75 mL of water containing 2 g of sodium bicarbonate, and stirring was continued for 5 min.
  • Beaucage reagent 3H-1,2-benzodithiol-3-one-1,1-dioxide
  • reaction solution is slowly dropped into two 50 mL centrifuge tubes containing 30 mL of chromatographically pure acetone and being stirred while it is hot, and a white solid can be observed to slowly precipitate out.
  • the magnets are taken out and centrifuged at 7000 rpm for 10 min. Carefully remove the supernatant, add new acetone to wash and centrifuge, repeat twice, and drain the solvent under negative pressure to obtain the final product SFG1 with a yield of 37%.
  • Measured molecular weight (ESI-MS): [MH] - 707.2.
  • Example 2 evaluated the murine macrophage activation effect of the SFG1 and SFG2 compounds obtained in Example 1.
  • the experiment uses mouse-derived macrophages J774A.1 as the evaluation cell line.
  • the use concentration of SFG1/SFG2 is 10 ⁇ M
  • the incubation time is 14h
  • the PE-anti CD86 (activation marker) antibody staining label is used for flow cytometric analysis to evaluate the stimulation effect of the compound.
  • the ordinate in Figure 1 represents the relative expression of the activation marker CD86
  • the control in the abscissa refers to phosphate buffered saline PBS
  • SFG1 refers to the use of SFG1 to activate murine macrophages
  • SFG2 represents the use of SFG2 activates murine macrophages.
  • SFG1+Lipo means that lipo3000 transfects SFG1 to continue to activate murine macrophages.
  • SFG2+Lipo means lipo3000 transfects SFG2.
  • Example 3 the anti-tumor effect of the SFG1 compound prepared in Example 1 was evaluated.
  • the experiment used melanoma (B16-F10) tumor-bearing C57BL/6 mouse model as the evaluation platform for the anti-tumor effect of SFG1.
  • melanoma B16-F10 tumor-bearing C57BL/6 mouse model as the evaluation platform for the anti-tumor effect of SFG1.
  • the results are shown in Figure 2.
  • the control in Figure 2 refers to HEPES buffer.
  • the arrow in Figure 2 shows the administration time.
  • the results show that subcutaneous injection of SFG1 alone can significantly inhibit the growth of melanoma.
  • Example 4 the SFG2 compound prepared in Example 1 was used as a vaccine adjuvant to evaluate the effect of enhancing the antigen immune response and the anti-tumor effect.
  • the first step is to take spleen cells for enzyme-linked immunosorbent spot assay (ELISPOT) to characterize the cellular immunity of the vaccine.
  • ELISPOT enzyme-linked immunosorbent spot assay
  • Figure 3 the ordinate represents the number of spots per 1 million spleen cells
  • the abscissa represents phosphate buffer solution
  • OVA represents chicken ovalbumin
  • OVA+SFG2 represents the combination vaccine of OVA antigen and SFG2 adjuvant.
  • SFG2 can significantly enhance the antigen-specific cellular immune response of the OVA antigen.
  • the second is to determine the titer and typing of antigen-specific antibodies in the serum after immunization, using enzyme-linked immunosorbent assay, the results are shown in Figure 4 and Figure 5.
  • the ordinate represents the titers of antigen-specific antibodies in the serum after immunization
  • the abscissa represents chicken ovalbumin
  • OVA+SFG2 represents the combination vaccine of OVA antigen and SFG2 adjuvant.
  • SFG2 can significantly increase the specific antibody titer of OVA antigen.
  • the ordinate represents the relative content of antibody typing
  • the abscissa represents various antibody typing.
  • mice Female C57BL/6 mice were planted with B16-OVA cells. After the tumor diameter reached 4mm, the mice were subcutaneously immunized with injections once a day for a total of three injections.
  • the injection dosage is OVA 20 ⁇ g/piece, SFG2 6 ⁇ g/piece. Regularly monitor the tumor volume. The results are shown in Figure 6, and the results show that SFG2 as a vaccine adjuvant can significantly inhibit the growth of melanoma.

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Abstract

本发明涉及一种新型STING激动剂及其制备方法和应用。该化合物为式SF所示化合物或者式SF所示化合物的立体异构体、互变异构体、氮氧化合物、水合物、溶剂化物、代谢产物、药物学上接受的盐或前药。

Description

新型STING激动剂及其制备方法和应用 技术领域
本发明涉及药物领域，具体地，本发明涉及一种新型STING激动剂环二核苷酸及其制备方法和应用。
背景技术
肿瘤免疫治疗目前已逐渐发展成为癌症治疗的重要且极具潜力的方向。在免疫治疗中，如何有效增强肿瘤微环境免疫反应、缓解免疫耐受是关乎治疗效果的核心问题。作为近期发现的全新模式识别受体，干扰素基因刺激蛋白(stimulator of interferon genes，STING)通路已经吸引了众多大型药企的研发兴趣。诺华、默克和百时美施贵宝等公司相继投资开发STING通路激动剂应用于肿瘤临床免疫治疗。STING蛋白位于内质网上且高表达于T细胞和抗原提呈细胞，可以被其天然激动剂环二核苷酸(cyclic dinucleotides,CDNs)激活，从而促进下游宿主防御基因的转录翻译包括I型干扰素和其他促炎细胞因子。基于此，环二核苷酸可以作为治疗试剂或疫苗佐剂的理想选择。然而CDNs磷酸二酯的负电荷和易被酯酶水解的特性极大地阻碍其临床应用价值。因此设计和开发合适的方法提高环二核苷酸稳定性及跨膜效率具有重要的研究价值。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一，提供了一种新型STING激动剂及其制备方法和应用。
对环二核苷酸进行化学修饰提高其稳定性和脂溶性是当前改善其药物特性的重点研发方向之一。其中，磷硫酰化和羟基氟代是最具代表性的化学修饰类型。本发明的发明人通过研究发现，对环二核苷酸进行磷硫酰化可有效提高其抵抗磷酸酯酶的水解；同时进行氟代修饰，氟原子强烈的吸电子特性则可增强环二核苷酸的脂溶性及稳定性。
为了获得经过化学修饰的环二核苷酸，发明人利用液相一瓶法设计和合成了一类新型化学修饰的环二核苷酸(cyclic dinucleotides,CDNs)，其结构式如SF所示。其中碱基可为如A、G等各种天然和非天然类型碱基。所提到的液相一瓶法是指多步反应均在一个圆底烧瓶中进行。
具体而言，本发明提供了如下技术方案：
在本发明的第一方面，本发明提供了一种化合物，所述化合物如式SF所示，
Figure PCTCN2021105135-appb-000001
其中，B 1或B 2各自独立地选自于天然碱基A、G、C、T、U及非天然碱基；
X选自于-H，-OH、-F；
Y选自于-OH、-SH。
本发明所提供的化合物在环二核苷酸进行磷硫酰化修饰和羟基氟代修饰。通过对环二核苷酸进行磷硫酰化可以有效提高化合物抵抗磷酸酯酶的水解，而且由于氟代修饰，氟院子强烈的吸电子特新型可以增强环二核苷酸的脂溶性以及稳定性。
根据本发明的实施例，所述化合物选自下列式SF1所示化合物或者式SF2所示化合物中的至少一种：
Figure PCTCN2021105135-appb-000002
其中B 1或B 2各自独立地选自于天然碱基A、G、C、T、U及非天然碱基。
根据本发明的实施例，所述非天然碱基选自于经修饰的天然碱基，优选I、mC以及人造碱基。
在本发明的第二方面，本发明提供了一种制备本发明第一方面任一实施例所述的化合物的方法，包括：
(1)使式S1所示化合物和吡啶-三氟乙酸盐、叔丁胺、二氯乙酸发生脱保护反应，以便获得式S2所示化合物；
(2)使式S2所示化合物和式S3所示化合物发生磷酰化反应，以便获得式S4所示化合物；
(3)使式S4所示化合物和DDTT、二氯乙酸发生氧化和脱保护反应，以便获得式S5所示化合物；
(4)使式S5所示化合物与环化试剂、氧化剂发生亲核取代和氧化反应，以便获得S6所示化合物；
(5)使式S6所示化合物与叔丁胺发生亲核取代反应，以便获得S7所示化合物；
(6)使式S7所示化合物和甲胺和氢氟酸三乙胺盐发生脱保护反应，以便获得SF所示化合物；
其中，式S1所示化合物、式S2所示化合物、式S3所示化合物、式S4所示化合物、式S5所示化合物、式S6所示化合物分别如下所示：
Figure PCTCN2021105135-appb-000003
其中各化合物中L1，L2分别为
Figure PCTCN2021105135-appb-000004
碱基保护基；
Z选自硅羟基、F；
根据本发明的实施例，以上所述制备化合物的方法可以进一步包括如下技术特征：
根据本发明的实施例，所述硅羟基为选自三甲基硅醚羟基、叔丁基二甲基硅醚羟基、叔 丁基二苯基硅醚羟基。
根据本发明的实施例，所述环化试剂为5,5-二甲基-2-氯-1,3,2-二氧磷杂己内酰磷酸酯；
所述氧化剂为选自碘或3H-1,2-苯并二硫醇-3-酮1,1-二氧化物中的至少一种。
根据本发明的实施例，步骤(1)中所述脱保护反应在室温条件下进行。
根据本发明的实施例，步骤(2)中所述磷酰化反应在无水条件下进行；
根据本发明的实施例，步骤(3)中所述氧化和脱保护反应在室温条件下进行；
根据本发明的实施例，步骤(4)中所述亲核取代和氧化反应在室温条件下进行；
根据本发明的实施例，步骤(5)中所述亲核取代反应在室温条件下进行；
根据本发明的实施例，步骤(6)中所述脱保护反应在油浴50℃条件下进行。
在本发明的第三方面，本发明提供了一种药物组合物，包括化合物和药学上可接受的辅料、载体、赋形剂、溶媒或它们的组合物，所述化合物为本发明第一方面任一实施例所述的化合物或者为本发明第二方面任一实施例所述的方法制备的化合物。
在本发明的第四方面，本发明提供了一种化合物在制备药物中的用途，所述药物用于免疫疾病的治疗，所述化合物为本发明第一方面所述的化合物或者为根据本发明第二方面所述的方法制备的化合物。
根据本发明的实施例，所述药物用于治疗肿瘤或者用于抗病毒、抗菌。
根据本发明的实施例，所述药物用于激活干扰素基因刺激蛋白(STING)。
在本发明的第五方面，本发明提供了一种药物联合，包括本发明第一方面所述的化合物或者为根据本发明第二方面所述的方法制备的化合物，和至少一种用于治疗免疫疾病的药物。
根据本发明的实施例，所述至少一种用于治疗免疫疾病的药物选自免疫检查点阻断抗体(anti-PD1、anti-PD-L1和anti-CTLA-4等)、免疫刺激剂(CpG、咪唑喹啉和单磷脂酰A等)、疫苗、嵌合抗原受体T细胞、放射性疗法和化疗(阿霉素、紫杉醇和顺铂等)中的至少一种。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
图1是根据本发明的实施例提供的经过不同化合物和转染试剂处理后细胞的流式检测结果图。
图2是根据本发明的实施例提供的不同处理组小鼠的肿瘤体积变化结果。
图3是根据本发明的实施例提供的不同处理组酶联免疫吸附斑点测定结果图。
图4是根据本发明的实施例提供的不同处理组血清中抗原特异性抗体滴度结果。
图5是根据本发明的实施例提供的不同处理组不同抗体的分型结果图。
图6是根据本发明的实施例提供的不同处理组小鼠的肿瘤体积变化结果图。
具体实施方式
现在详细描述本发明的某些实施方案，其实例由随附的结构式和化学式说明。本发明意图涵盖所有的替代、修改和等同技术方案，它们均包括在如权利要求定义的本发明范围内。本领域技术人员应认识到，许多与本文所述类似或等同的方法和材料能够用于实践本发明。本发明绝不限于本文所述的方法和材料。在所结合的文献、专利和类似材料的一篇或多篇与本申请不同或相矛盾的情况下(包括但不限于所定义的术语、术语应用、所描述的技术，等等)，以本申请为准。
应进一步认识到，本发明的某些特征，为清楚可见，在多个独立的实施方案中进行了描述，但也可以在单个实施例中以组合形式提供。反之，本发明的各种特征，为简洁起见，在单个实施方案中进行了描述，但也可以单独或以任意适合的子组合提供。
除非另外说明，本发明所使用的所有科技术语具有与本发明所属领域技术人员的通常理解相同的含义。本发明涉及的所有专利和公开出版物通过引用方式整体并入本发明。
定义或一般术语
术语“包含”、“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。
“立体异构体”是指具有相同化学构造，但原子或基团在空间上排列方式不同的化合物。立体异构体包括对映异构体、非对映异构体、构象异构体(旋转异构体)、几何异构体(顺/反)异构体、阻转异构体，等等。
“手性”是具有与其镜像不能重叠性质的分子；而“非手性”是指与其镜像可以重叠的分子。
“对映异构体”是指一个化合物的两个不能重叠但互成镜像关系的异构体。
“非对映异构体”是指有两个或多个手性中心并且其分子不互为镜像的立体异构体。非对映异构体具有不同的物理性质，如熔点、沸点、光谱性质和反应性。非对映异构体混合物可通过高分辨分析操作如电泳和色谱，例如HPLC来分离。
本发明所使用的立体化学定义和规则一般遵循S.P.Parker，Ed.，McGraw-Hill Dictionary of Chemical Terms(1984)McGraw-Hill Book Company，New York；and Eliel，E.and Wilen，S.，“Stereochemistry of Organic Compounds”，John Wiley&Sons，Inc.，New York，1994。
许多有机化合物以光学活性形式存在，即它们具有使平面偏振光的平面发生旋转的能 力。在描述光学活性化合物时，使用前缀D和L或R和S来表示分子关于其一个或多个手性中心的绝对构型。前缀d和l或(+)和(-)是用于指定化合物所致平面偏振光旋转的符号，其中(-)或l表示化合物是左旋的。前缀为(+)或d的化合物是右旋的。一种具体的立体异构体是对映异构体，这种异构体的混合物称作对映异构体混合物。对映异构体的50:50混合物称为外消旋混合物或外消旋体，当在化学反应或过程中没有立体选择性或立体特异性时，可出现这种情况。
本发明公开化合物的任何不对称原子(例如，碳等)都可以以外消旋或对映体富集的形式存在，例如(R)-、(S)-或(R,S)-构型形式存在。在某些实施方案中，各不对称原子在(R)-或(S)-构型方面具有至少50％对映体过量，至少60％对映体过量，至少70％对映体过量，至少80％对映体过量，至少90％对映体过量，至少95％对映体过量，或至少99％对映体过量。
依据起始物料和方法的选择，本发明化合物可以以可能的异构体中的一个或它们的混合物，例如外消旋体和非对应异构体混合物(这取决于不对称碳原子的数量)的形式存在。光学活性的(R)-或(S)-异构体可使用手性合成子或手性试剂制备，或使用常规技术拆分。如果化合物含有一个双键，取代基可能为E或Z构型；如果化合物中含有二取代的环烷基，环烷基的取代基可能有顺式或反式构型。
所得的任何立体异构体的混合物可以依据组分物理化学性质上的差异被分离成纯的或基本纯的几何异构体，对映异构体，非对映异构体，例如，通过色谱法和/或分步结晶法。
可以用已知的方法将任何所得终产物或中间体的外消旋体通过本领域技术人员熟悉的方法拆分成光学对映体，如，通过对获得的其非对映异构的盐进行分离。外消旋的产物也可以通过手性色谱来分离，如，使用手性吸附剂的高效液相色谱(HPLC)。特别地，对映异构体可以通过不对称合成制备，例如，可参考Jacques,et al.,Enantiomers,Racemates and Resolutions(Wiley Interscience,New York,1981)；Principles of Asymmetric Synthesis(2 nd Ed.Robert E.Gawley,Jeffrey Aubé,Elsevier,Oxford,UK,2012)；Eliel,E.L.Stereochemistry of Carbon Compounds(McGraw-Hill,NY,1962)；Wilen,S.H.Tables of Resolving Agents and Optical Resolutions p.268(E.L.Eliel,Ed.,Univ.of Notre Dame Press,Notre Dame,IN 1972)；Chiral Separation Techniques:A Practical Approach(Subramanian,G.Ed.,Wiley-VCH Verlag GmbH&Co.KGaA,Weinheim,Germany,2007)。
术语“互变异构体”或“互变异构形式”是指具有不同能量的可通过低能垒(low energy barrier)互相转化的结构异构体。若互变异构是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(protontautomer)(也称为质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键互变异构体(valence tautomer)包括通过一些成键电子的重组来进行的互相 转化。酮-烯醇互变异构的具体实例是戊烷-2,4-二酮和4-羟基戊-3-烯-2-酮互变异构体的互变。互变异构的另一个实例是酚-酮互变异构。酚-酮互变异构的一个具体实例是吡啶-4-醇和吡啶-4(1H)-酮互变异构体的互变。除非另外指出，本发明化合物的所有互变异构体形式都在本发明的范围之内。
在本说明书的各部分，本发明公开化合物的取代基按照基团种类或范围公开。特别指出，本发明包括这些基团种类和范围的各个成员的每一个独立的次级组合。例如，术语“C 1～6烷基”特别指各自独立的甲基、乙基、C 3烷基、C 4烷基、C 5烷基和C 6烷基。
在本发明的各部分，描述了连接取代基。当该结构清楚地需要连接基团时，针对该基团所列举的马库什变量应理解为连接基团。例如，如果该结构需要连接基团并且针对该变量的马库什基团定义列举了“烷基”，则应该理解，该“烷基”分别代表连接的亚烷基基团或亚芳基基团。
另外，需要说明的是，除非以其他方式明确指出，在本文中通篇采用的描述方式“各…和…独立地为”、“…和…各自独立地为”和“…和…分别独立地为”可以互换，应做广义理解，其既可以是指在不同基团中，相同符号之间所表达的具体选项之间互相不影响，也可以表示在相同的基团中，相同符号之间所表达的具体选项之间互相不影响。
术语“药学上可接受的”是指当给人施用时生理上可耐受的并且一般不产生过敏或相似不适当的反应，例如肠胃不适、眩晕等的分子实体和组合物。优选地，本文所用的术语“药学上可接受的”是指联邦监管机构或国家政府批准的或美国药典或其他一般认可的药典上列举的在动物中、更特别在人体中使用的。
术语“载体”指与所述化合物一同施用的稀释剂、辅剂、赋形剂或基质。这些药物载体可以是无菌液体，例如水和油类，包括石油、动物、植物或合成来源的，例如花生油、大豆油、矿物油、芝麻油等。水和水性溶液盐水溶液和水性葡萄糖与甘油溶液优选用作载体、特别是可注射溶液。适宜的药物载体描述于E.W.Martin的“Remington′s Pharmaceutical Sciences”中。
本发明中立体化学的定义和惯例的使用通常参考以下文献：S.P.Parker,Ed.,McGraw-Hill Dictionary of Chemical Terms(1984)McGraw-Hill Book Company,New York；and Eliel,E.and Wilen,S.,"Stereochemistry of Organic Compounds",John Wiley&Sons,Inc.,New York,1994.本发明的化合物可以包含不对称中心或手性中心，因此存在不同的立体异构体。本发明的化合物所有的立体异构形式，包括但绝不限于，非对映体，对映异构体，阻转异构体，和它们的混合物，如外消旋混合物，组成了本发明的一部分。很多有机化合物都以光学活性形式存在，即它们有能力旋转平面偏振光的平面。在描述光学活性化合物时，前缀D、L或R、S用来表示分子手性中心的绝对构型。前缀d、l或(+)、(-)用来命 名化合物平面偏振光旋转的符号，(-)或l是指化合物是左旋的，前缀(+)或d是指化合物是右旋的。这些立体异构体的化学结构是相同的，但是它们的立体结构不一样。特定的立体异构体可以是对映体，异构体的混合物通常称为对映异构体混合物。50:50的对映体混合物被称为外消旋混合物或外消旋体，这可能导致化学反应过程中没有立体选择性或立体定向性。术语“外消旋混合物”和“外消旋体”是指等摩尔的两个对映异构体的混合物，缺乏光学活性。
“异构体”为具有相同分子式的不同化合物。“立体异构体”为仅仅原子的空间排列方式不同的异构体。如本文使用的术语“异构体”包括任何和所有的几何异构体和立体异构体。例如，“异构体”包括顺式和反式异构体、E-和Z-异构体、R-和S-对映异构体、非对映异构体、(d)异构体、(l)-异构体、其外消旋混合物、及落入本说明书范围的其它其混合物。
本发明的“水合物”是指本发明所提供的化合物或其盐，其还包括化学量或非化学当量通过非共价分子间力结合的水，也可说是溶剂分子是水所形成的缔合物。
本发明的“溶剂化物”是指一个或多个溶剂分子与本发明的化合物所形成的缔合物。形成溶剂化物的溶剂包括，但并不限于，水，异丙醇，乙醇，甲醇，二甲亚砜，乙酸乙酯，乙酸，氨基乙醇。
本发明的“氮氧化物”是指当化合物含几个胺官能团时，可将1个或大于1个的氮原子氧化形成N-氧化物。N-氧化物的特殊实例是叔胺的N-氧化物或含氮杂环氮原子的N-氧化物。可用氧化剂例如过氧化氢或过酸(例如过氧羧酸)处理相应的胺形成N-氧化物(参见Advanced Organic Chemistry,Wiley Interscience,第4版,Jerry March,pages)。尤其是，N-氧化物可用L.W.Deady的方法制备(Syn.Comm.1977,7,509-514)，其中例如在惰性溶剂例如二氯甲烷中，使胺化合物与间-氯过氧苯甲酸(MCPBA)反应。
化合物可存在多种不同几何异构体和互变异构体，所述式(I)-式(III)化合物包括所有此类形式。为避免疑惑，当化合物以几种几何异构体或互变异构体之一存在并且只具体描述或显示一种时，显然所有其它形式包括在式(I)-式(III)中。
本发明所使用的术语“前药”，代表一个化合物在体内转化为本发明所示的化合物。这样的转化受前体药物在血液中水解或在血液或组织中经酶转化为母体结构的影响。
除非其他方面表明，本发明的化合物的所有互变异构形式都包含在本发明的范围之内。
“代谢产物”是指具体的化合物或其盐在体内通过代谢作用所得到的产物。一个化合物的代谢产物可以通过所属领域公知的技术来进行鉴定，其活性可以通过如本发明所描述的那样采用试验的方法进行表征。这样的产物可以是通过给药化合物经过氧化，还原，水解，酰氨化，脱酰氨作用，酯化，脱脂作用，酶裂解等等方法得到。相应地，本发明包括化合物的代谢产物，包括将本发明的化合物与哺乳动物充分接触一段时间所产生的代谢产物。
本发明化合物的各种药学上可接受的盐形式都是有用的。术语“药学上可接受的盐”是指那些盐形式对于制药化学家而言是显而易见的，即它们基本上无毒并能提供所需的药代动力学性质、适口性、吸收、分布、代谢或排泄。其他因素，在性质上更加实用，对于选择也很重要，这些是：原材料的成本、结晶的容易、产率、稳定性、吸湿性和结果原料药的流动性。简单地讲，药物组合物可以通过有效成分与药学上可接受的载体制备得到。
本发明所使用的“药学上可接受的盐”是指本发明的化合物的有机盐和无机盐。药学上可接受的盐在所属领域是为我们所熟知的，如文献：S.M.Berge et al.,describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences,66:1-19,1977.所记载的。药学上可接受的无毒的酸形成的盐包括，但并不限于，与氨基基团反应形成的无机酸盐有盐酸盐，氢溴酸盐，磷酸盐，硫酸盐，高氯酸盐，硝酸盐等，和有机酸盐如乙酸盐，丙酸盐，乙醇酸盐，草酸盐，马来酸盐，丙二酸盐，琥珀酸盐，富马酸盐，酒石酸盐，枸橼酸盐，苯甲酸盐，扁桃酸盐，甲磺酸盐，乙磺酸盐，甲苯磺酸盐，磺基水杨酸盐等，或通过书籍文献上所记载的其他方法如离子交换法来得到这些盐。
其他药学上可接受的盐包括己二酸盐、苹果酸盐、2-羟基丙酸、藻酸盐、抗坏血酸盐、天冬氨酸盐、苯磺酸盐、苯甲酸盐、重硫酸盐、硼酸盐、丁酸盐、樟脑酸盐、樟脑磺酸盐、环戊基丙酸盐、二葡萄糖酸盐、十二烷基硫酸盐、乙磺酸盐、甲酸盐、反丁烯二酸盐、葡庚糖酸盐、甘油磷酸盐、葡萄糖酸盐、半硫酸盐、庚酸盐、己酸盐、氢碘酸盐、2-羟基-乙磺酸盐、乳糖醛酸盐、乳酸盐、月桂酸盐、月桂基硫酸盐、苹果酸盐、丙二酸盐、甲磺酸盐、2-萘磺酸盐、烟酸盐、硝酸盐、油酸盐、棕榈酸盐、扑酸盐、果胶酸盐、过硫酸盐、3-苯基丙酸盐、苦味酸盐、特戊酸盐、丙酸盐、硬脂酸盐、硫氰酸盐、对甲苯磺酸盐、十一酸盐、戊酸盐、等等。通过适当的碱得到的盐包括碱金属，碱土金属，铵和N +(C 1-4烷基) 4的盐。
本发明也拟构思了任何所包含N的基团的化合物所形成的季铵盐。水溶性或油溶性或分散产物可以通过季铵化作用得到。碱金属或碱土金属盐包括钠盐、锂盐、钾盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等等。药学上可接受的盐进一步包括适当的、无毒的铵，季铵盐和抗平衡离子形成的胺阳离子，如卤化物，氢氧化物，羧化物，硫酸化物，磷酸化物，硝酸化物，C 1-8磺酸化物和芳香磺酸化物。胺盐，例如但不限于N,N’-二苄基乙二胺，氯普鲁卡因，胆碱，氨，二乙醇胺和其它羟烷基胺，乙二胺，N-甲基还原葡糖胺，普鲁卡因，N-苄基苯乙胺，1-对-氯苄基-2-吡咯烷-1’-基甲基-苯并咪唑，二乙胺和其它烷基胺，哌嗪和三(羟甲基)氨基甲烷；碱土金属盐，例如但不限于钡，钙和镁；过渡金属盐，例如但不限于锌。
在本说明书中，如果在化学名称和化学结构间存在任何差异，结构是占优的。
本发明所使用的任何保护基团、氨基酸和其它化合物的缩写，除非另有说明，都以它们通常使用的、公认的缩写为准，或参照IUPAC-IUB Commission on Biochemical Nomenclature(参见Biochem.1972，11：942-944)。
本发明提供药物组合物，包含治疗有效量的式(I)所示化合物或其药学上可接受的盐和药学上可接受的辅料、载体、赋形剂、溶媒或它们的组合。当本发明的化合物以药物的形式施用于哺乳动物例如人时，其可以以化合物本身的形式被给予或者可以以含有例如0.1至99.5％(更优选0.5至90％)活性成分以及药学可接受的载体的药物组合物的形式被给予。
“联合”表示在单个剂量单位形式中的固定组合或用于组合施用的部分的药盒，其中本发明公开化合物和组合伴侣(治疗肿瘤疾病、艾滋病、炎症反应以及免疫缺陷疾病的药物)可以在同一时间独立施用或者可以在一定的时间间隔内分别施用，特别是使联合合伴侣表现出合作、例如协同作用。如本文所用的术语“药物组合物”表示将一种以上活性成分混合或组合所得到的产品，并且既包括活性成分的固定组合也包括非固定组合。术语“固定联合”表示活性成分如本发明公开化合物和组合伙伴以单一实体或剂量的形式同时施用于患者。术语“非固定联合”表示活性成分如本发明公开化合物和组合伙伴均作为单独实体同时、共同或无特定时间限制地先后施用于患者。
措辞“药学可接受的载体”在本领域中是公认的，包括适于将本发明的化合物施用于哺乳动物的药学可接受的材料、组合物或载体。所述载体包括参与携带主题物质或将其从一个器官或机体的一部分转移到另一个器官或机体的另一部分的液体或固体填充剂、稀释剂、赋形剂、溶剂或包封材料。各载体在与制剂中的其它成分相容和对患者无害的意义上必须是“可接受的”。可用作药学可接受的载体的材料的一些实例包括：糖类，如乳糖、葡萄糖和蔗糖；淀粉类，如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和醋酸纤维素；粉状西黄蓍胶；麦芽；明胶；滑石粉；赋形剂，如可可脂和栓剂蜡类；油类，如花生油、棉子油、红花油、芝麻油、橄榄油、玉米油和豆油；二醇类，如丙二醇；多元醇类，如甘油、山梨醇、甘露醇和聚乙二醇；酯类，如油酸乙酯和月桂酸乙酯；琼脂；缓冲剂，如氢氧化镁和氢氧化铝；海藻酸；无热原的水；等张盐水；林格氏溶液；乙醇；磷酸盐缓冲液；和药物制剂中所用的其它无毒的可相容的物质。
在组合物中也可以存在润湿剂、乳化剂和润滑剂如十二烷基硫酸钠和硬脂酸镁，以及着色剂、释放剂、包衣剂、甜味剂、矫味剂和芳香剂、防腐剂和抗氧化剂。
药学可接受的抗氧化剂的实例包括：水溶性抗氧化剂，如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠等；油溶性抗氧化剂，如棕榈酸抗坏血酸酯、丁基化羟基苯甲醚(BHA)、丁化羟基甲苯(BHT)、卵磷脂、棓酸丙酯、α-生育酚等；和金属螯合剂，如柠檬酸、乙二胺四乙酸(EDTA)、山梨醇、酒石酸、磷酸等。
本发明的药物组合物包括适于口服、鼻、局部、口含、舌下、直肠和/或胃肠外施用的那些。制剂可以方便地以单位剂型形式存在并且可以通过药学领域公知的任何方法来制备。可以与载体物质组合来制备单剂量形式的活性成分的量一般是产生治疗作用的化合物的量。一般而言，以百分之一为单位，该量为约1％至约99％活性成分，优选约5％至约70％，最优选约10至约30％。
术语“治疗”用于指获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病或病症发生；(b)抑制疾病，例如阻滞疾病发展；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将药物或化合物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药，包括但不限于将含本文所述化合物的药物给予有需要的个体。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照本领域常用的技术手段进行。以下给出的实施例采用鸟苷酸进行验证。实验过程中合成了两类代表性结构分别为CDN(SFG) 1(缩写为SFG1)和CDN(SFG) 2(缩写为SFG2)(图2)。SFG1为同侧单硫单氟修饰环二核苷酸，SFG2为双硫双氟修饰环二核苷酸。并通过后续免疫实验表明SFG1和SFG2均可显著激活抗原提呈细胞。SFG1和SFG2作为免疫治疗试剂和疫苗佐剂均可提高抗原特异性抗体滴度和抑制肿瘤生长。SFG1和SFG2类化合物可广泛应用于各类免疫治疗当中如肿瘤治疗、抗病毒、抗菌等领域。
Figure PCTCN2021105135-appb-000005
实施例1
实施例1以鸟苷酸作为示例，分别合成了SFG1和SFG2，其中SFG1为同侧单硫单氟修饰的环二核苷酸，SFG2为双硫双氟修饰的环二苷酸。其制备方法如下，具体步骤可以参考下面的两个反应式：
化合物S2(D2/E2)
称取0.5mmol鸟苷酸亚磷酰胺单体(S1)(合成SFG1，Z＝OTBS；合成SFG2，Z＝F)和0.116g(0.6mmol)吡啶三氟乙酸盐放入50mL圆底烧瓶中，加入磁子和2.5mL乙腈搅拌溶解，再加入18μL水搅拌反应1min。之后加入2.5mL t-BuNH 2搅拌反应10min。反应结束后负压旋除溶剂至蓬松状，再加入5mL乙腈旋至蓬松状，重复操作两次除去t-BuNH 2。加入6mL二氯甲烷(DCM)溶解固体，之后加入90μL水和6mL 6％二氯乙酸(DCS)的DCM溶液(4.4mmol)搅拌反应10min，中途取样进行ESI-MS检测判断DMTr保护基脱除情况。加入0.7mL吡啶中和DCA，负压浓缩至2mL，再加入4mL无水乙腈溶解样品，负压旋除溶剂，重复操作三次，最后一次剩余1mL时停止旋蒸，使用橡胶塞塞住待用。
化合物S4(D4/E4)
另外称取0.557g(0.65mmol)氟代鸟苷酸亚磷酰胺单体(S3)放入10mL圆底烧瓶中，加入4mL无水乙腈溶解固体，负压旋除乙腈至蓬松状，重复操作四次，在最后一次剩余2mL时停止旋蒸，使用橡胶塞封闭。使用注射器快速吸取上述干燥好的单体加入到含有S2(D2/E2)的圆底烧瓶中，搅拌反应2min。
化合物S5(D5/E5)
向含有S4(D4/E4)的圆底烧瓶中加入0.113g DDTT(((二甲基氨基-亚甲基)氨基)-3H-1,2,4-二噻唑-3-硫酮，0.55mmol，1.1当量)硫化三价磷，搅拌反应30min，负压浓缩至油状。加入8mL DCM溶解油状物，在加入0.09mL水和8mL 6％DCA的DCM溶液(1.5mmol)搅拌反应10min，中途取样进行ESI-MS检测判断DMTr保护基脱除情况。反应完全后加入5mL吡啶，负压浓缩至2mL，再加入15mL吡啶浓缩至10mL，使用橡胶塞塞住。
化合物S6(D6/E6)
称取0.34g 5,5-二甲基-2-氧代-2-氯-1,3,2-二氧磷(DMOCP，1.75mmol)加入到含有S5(D5/E5)的圆底烧瓶中，搅拌反应10min。
合成SFG1的条件为：加入0.32mL水和165mg碘单质，搅拌反应5min，中途取样进行ESI-MS检测判断氧化完成情况。反应完成后，将混合物倒入含有0.1g亚硫酸氢钠的70mL水溶液中，搅拌反应5min消耗掉多余氧化剂，再缓慢加入2g碳酸氢钠中和反应体系，继续搅拌5min；
合成SFG2的条件为：加入0.32mL水和130mg Beaucage试剂(3H-1,2-苯并二硫醇-3-酮-1,1-二氧化物)(0.75mmol，1.5当量)，搅拌反应5min，中途取样进行ESI-MS检测判断氧化完成情况。反应完成后，将反应体系倒入含有2g碳酸氢钠的75mL水中， 继续搅拌5min。将反应体系倒入含有80mL乙醚/乙酸乙酯(体积1/1)混合溶剂的分液漏斗中，加入80mL水进行萃取分离操作，收集有机相之后负压旋除溶剂得到油状液体即为化合物S5。
化合物S7(D7/E7)
将S6转移至25mL圆底烧瓶中，加入2mL乙酸乙酯油泵负压旋除，重复操作三次以去除吡啶。加入2.5mL乙腈溶解油状物，再加入2.5mL t-BuNH 2搅拌反应10min。之后加入2.5mL乙腈负压旋干，重复操作三次去除t-BuNH 2。再加入2.5mL甲醇溶解样品，同样负压旋至泡沫状。
化合物SFG1
向含有S6(D6)的圆底烧瓶中加入10mL 33％甲胺无水乙醇溶液(质量比)，塞上橡胶塞搅拌反应1.5h，中途取样进行ESI-MS检测判断异丁酰基(iBu)保护基脱除情况。反应完成后，浓缩至油状，加入400μL吡啶和200μL三乙胺继续旋蒸至油状，重复操作三次将产物由t-BuNH 2盐形式转变为三乙胺盐形式。接着加入400μL吡啶溶解油状物，橡胶塞塞住瓶口，将圆底烧瓶至于50℃油浴锅中搅拌。使用注射器分别吸取1.4mL三乙胺和0.83mL三乙胺氢氟酸盐并将二者同步缓慢注射到圆底烧瓶中，持续1min。注射完成后搅拌反应1h，中途取样进行ESI-MS检测判断TBS保护基脱除情况。反应完成后，趁热将反应溶液缓慢滴到两个含有30mL色谱纯丙酮并且正在搅拌的50mL离心管中，可以观察到白色固体慢慢析出。沉淀完成之后，取出磁子，7000rpm离心10min。小心去掉上清，加入新的丙酮洗涤离心，重复两次，负压抽干溶剂即以37％的产率得到最终产物SFG1。理论分子量：C 20H 22FN 10O 12P 2S -，[M-H] -＝707.1。实测分子量(ESI-MS)：[M-H] -＝707.2。
化合物SFG2
向含有S6(E6)的圆底烧瓶中加入10mL 33％甲胺无水乙醇溶液(质量比)，塞上橡胶塞搅拌反应1.5h，中途取样进行ESI-MS检测判断异丁酰基(iBu)保护基脱除情况。反应完成后，浓缩至油状，加入400μL吡啶和200μL三乙胺继续旋蒸至油状，重复操作三次将产物由t-BuNH 2盐形式转变为三乙胺盐形式。接着加入400μL吡啶溶解油状物，橡胶塞塞住瓶口，将圆底烧瓶至于50℃油浴锅中搅拌。趁热将反应溶液缓慢滴到两个含有30mL色谱纯丙酮并且正在搅拌的50mL离心管中，可以观察到白色固体慢慢析出。沉淀完成之后，取出磁子，7000rpm离心10min。小心去掉上清，加入新的丙酮洗涤离心，重复两次，负压抽干溶剂即以30％的产率得到最终产物SFG2。理论分子量：C 20H 21F 2N 10O 10P 2S 2 -，[M-H] -＝725.0。实测分子量(ESI-MS)：[M-H] -＝725.1。
Figure PCTCN2021105135-appb-000006
Figure PCTCN2021105135-appb-000007
实施例2
实施例2对实施例1所获得的SFG1、SFG2化合物的鼠源巨噬细胞活化效果进行了评价。
实验采用鼠源巨噬细胞J774A.1作为评价细胞系。SFG1/SFG2的使用浓度均为10μM，孵育时间14h，之后用PE-anti CD86(活化标记物)抗体染色标记进行流式分析，评价化合物的刺激效果。鉴于环二核苷酸的跨膜效果比较差，我们同时测定了在有转染试剂(加lipo3000转染试剂)和无转染试剂条件下的活化效果。
如图1所示，图1中纵坐标代表活化标志物CD86的相对表达量，横坐标中对照是指磷酸盐缓冲液PBS，SFG1是指采用SFG1对鼠源巨噬细胞进行活化，SFG2代表采用SFG2对鼠源巨噬细胞进行活化，SFG1+Lipo代表采用lipo3000转染SFG1对鼠源巨噬细胞继续活化，SFG2+Lipo代表lipo3000转染SFG2结果表明SFG1和SFG2均能有效活化抗原提呈细胞，在有转染试剂促进跨膜的帮助下刺激效果更加显著，且两种条件下SFG2活性都好于SFG1。
实施例3
实施例3对实施例1所制备的SFG1化合物的抗肿瘤效果进行了评价。
实验采用黑色素瘤(B16-F10)荷瘤C57BL/6小鼠模型作为SFG1抗肿瘤效果的评价平台。每组7只小鼠，待肿瘤直径达到4mm，进行皮下注射，间隔一天给药一次，给药四次，给药剂量20μg/只。
其结果如图2所示，图2中对照是指HEPES缓冲液。图2中箭头示出的为给药时间。结果表明单独SFG1皮下注射可以显著抑制黑色素瘤生长。
实施例4
实施例4以实施例1所制备的SFG2化合物作为疫苗佐剂，对于增强抗原免疫反应效果和抗肿瘤效果进行了评价。
实验采用鸡卵清蛋白(ovalbumin,OVA)作为模型抗原，首先进行C57BL/6小鼠免疫评价。OVA蛋白抗原用量为20μg，SFG2用量为6μg，间隔两周免疫四次。免疫完成后隔一周对小鼠进行处理，进行免疫相关表征。
首先是取脾脏细胞进行酶联免疫吸附斑点测定(ELISPOT)表征疫苗的细胞免疫情况，其结果如图3所示。图3中纵坐标代表每100万脾脏细胞出现的斑点数，横坐标中PBS代表磷酸盐缓冲溶液，OVA代表鸡卵清白蛋白，OVA+SFG2代表OVA抗原与SFG2佐剂组合疫苗。结果表明SFG2可以显著增强OVA抗原的抗原特异性细胞免疫反应。
其次是测定免疫后血清中抗原特异性抗体滴度和分型情况，采用酶联免疫吸附测定，其结果如图4和图5所示。图4中纵坐标代免疫后血清中抗原特异性抗体滴度，横坐标中OVA代表鸡卵清白蛋白，OVA+SFG2代表OVA抗原与SFG2佐剂组合疫苗。结果表明SFG2可以显著提高OVA抗原的特异性抗体滴度。图5中纵坐标代表抗体分型的相对含量，横坐标代表各类抗体分型。
为了验证上述OVA疫苗的抗肿瘤效果，我们采用B16-OVA肿瘤细胞系作为评价模型。雌性C57BL/6小鼠种植B16-OVA细胞，待肿瘤直径达到4mm后，进行小鼠皮下免疫，间隔一天注射一次，共注射三次。注射用量OVA 20μg/只，SFG2 6μg/只。定时监测肿瘤体积。其结果如图6所示，结果表明SFG2作为疫苗佐剂可以显著抑制黑色素瘤生长。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对 的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 一种化合物，其特征在于，为式SF所示化合物或者式SF所示化合物的立体异构体、互变异构体、氮氧化合物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药：
    Figure PCTCN2021105135-appb-100001
    其中，B 1或B 2各自独立地选自于天然碱基A、G、C、T、U、I及非天然碱基；
    X选自于-H，-OH、-F；
    Y选自于-OH、-SH。
  2. 根据权利要求1所述的化合物，其特征在于，所述化合物选自下列式SF1所示化合物或者式SF2所示化合物中的至少一种：
    Figure PCTCN2021105135-appb-100002
    其中B 1或B 2各自独立地选自于天然碱基A、G、C、T、U及非天然碱基。
  3. 根据权利要求1或2所述的化合物，其特征在于，所述非天然碱基选自于经修饰的天然碱基，优选为I、mC以及人造碱基。
  4. 一种制备权利要求1～3中任一项所述的化合物的方法，其特征在于，包括：
    (1)使式S1所示化合物和吡啶-三氟乙酸盐、叔丁胺、二氯乙酸发生脱保护反应，以便获得式S2所示化合物；
    (2)使式S2所示化合物和式S3所示化合物发生磷酰化反应，以便获得式S4所示化合物；
    (3)使式S4所示化合物和DDTT、二氯乙酸发生氧化和脱保护反应，以便获得式S5所示化合物；
    (4)使式S5所示化合物与环化试剂、氧化剂发生亲核取代和氧化反应，以便获得式 S6所示化合物；
    (5)使式S6所示化合物与叔丁胺发生亲核取代反应，以便获得式S7所示化合物；
    (6)使式S7所示化合物和甲胺和氢氟酸三乙胺盐发生脱保护反应，以便获得式SF所示化合物；
    其中，式S1所示化合物、式S2所示化合物、式S3所示化合物、式S4所示化合物、式S5所示化合物、式S6所示化合物分别如下所示：
    Figure PCTCN2021105135-appb-100003
    其中各化合物中L1，L2分别为
    Figure PCTCN2021105135-appb-100004
    碱基保护基；
    Z选自硅羟基、F。
  5. 根据权利要求4所述的方法，其特征在于，所述硅羟基为选自三甲基硅醚羟基、叔丁基二甲基硅醚羟基、叔丁基二苯基硅醚羟基。
  6. 根据权利要求4所述的方法，其特征在于，所述环化试剂为5,5-二甲基-2-氯-1,3,2-二氧磷杂己内酰磷酸酯；
    所述氧化剂为选自碘或3H-1,2-苯并二硫醇-3-酮1,1-二氧化物中的至少一种。
  7. 根据权利要求4所述的方法，其特征在于，步骤(1)中所述脱保护反应在室温条件下进行；
    任选地，步骤(2)中所述磷酰化反应在无水条件下进行；
    任选地，步骤(3)中所述氧化和脱保护反应在室温条件下进行；
    任选地，步骤(4)中所述亲核取代和氧化反应在室温条件下进行；
    任选地，步骤(5)中所述亲核取代反应在室温条件下进行；
    任选地，步骤(6)中所述脱保护反应在油浴50℃条件下进行。
  8. 一种药物组合物，其特征在于，包括：
    化合物；和
    药学上可接受的辅料、载体、赋形剂、溶媒或它们的组合，
    所述化合物为权利要求1～3中任一项所述的化合物或者为根据权利要求4～7中任一项所述的方法制备的化合物。
  9. 化合物在制备药物中的用途，所述化合物为权利要求1～3中任一项所述的化合物或者为根据权利要求4～7中任一项所述的方法制备的化合物，所述药物用于免疫疾病的治疗；
    任选地，所述药物用于治疗肿瘤或者用于抗病毒、抗菌；
    任选地，所述药物用于激活干扰素基因刺激蛋白。
  10. 一种药物联合，其特征在于，包括：
    权利要求1～3中任一项所述的化合物或者为根据权利要求4～7中任一项所述的方法制备的化合物；和
    至少一种用于治疗免疫疾病的药物；
    任选地，所述至少一种用于治疗免疫疾病的药物选自免疫检查点阻断抗体、免疫刺激剂、疫苗、嵌合抗原受体T细胞、放射性疗法和化疗中的至少一种。
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