WO2020143740A1 - 环二核苷酸前药分子及其制备方法和应用 - Google Patents

环二核苷酸前药分子及其制备方法和应用 Download PDF

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WO2020143740A1
WO2020143740A1 PCT/CN2020/071329 CN2020071329W WO2020143740A1 WO 2020143740 A1 WO2020143740 A1 WO 2020143740A1 CN 2020071329 W CN2020071329 W CN 2020071329W WO 2020143740 A1 WO2020143740 A1 WO 2020143740A1
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formula
cyclic dinucleotide
prodrug molecule
group
compound
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席真
王正华
王丹
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Nankai University
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Nankai University
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Priority to US17/422,137 priority Critical patent/US12257265B2/en
Priority to KR1020217025118A priority patent/KR102620495B1/ko
Priority to EP20739193.9A priority patent/EP3901161B1/en
Priority to JP2021540248A priority patent/JP7224688B2/ja
Publication of WO2020143740A1 publication Critical patent/WO2020143740A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/213—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids containing cyclic phosphate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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, in particular to a cyclic dinucleotide prodrug molecule, a method for preparing a cyclic dinucleotide prodrug molecule, and the application of a cyclic dinucleotide prodrug molecule.
  • Cyclic dinucleotide is a new type of second messenger molecule found in bacteria and mammals. In mammalian cells, cyclic dinucleosides can bind to immunostimulatory factors (STING) to stimulate the immune response. It can cause the expression of cytokines such as interferon beta and NF- ⁇ B, and promote the proliferation, differentiation and maturation of CD8 + T cells.
  • STING immunostimulatory factors
  • cyclic dinucleotide has a strong immune stimulating effect, it can be used in the treatment of viral and bacterial infections and cancer.
  • anti-cancer combined with traditional immunotherapy can show good results.
  • Aduro Corporation and Novartis' ADU-S100 have entered clinical phase II, and MSD's MK-1454 has entered clinical phase I.
  • auxiliary materials are mainly used for cyclic dinucleotide drug delivery systems.
  • lentivirus or adenovirus packaging cyclic dinucleotide synthetase after entering the cell by enzyme catalyzed synthesis of cyclic dinucleotide to promote immune response, but this method has a greater risk of not being used widely.
  • liposomes or lipid nanoparticles, cell penetrating peptides, protein gels containing polycationic amino acids, etc. as carriers to achieve the delivery of cyclic dinucleotide drugs has been reported, but the use of such carriers will Reduce the drug loading, and the introduction of carrier may cause some potential cytotoxicity.
  • the clinically adopted method for solving the effective uptake of cyclic dinucleoside by cells is intratumoral injection.
  • the cells can take up cyclic dinucleotides, but this uptake rate is low, so Larger doses are required for administration. Such large doses may cause excessive activation of the immune system, which may cause systemic or local inflammation.
  • the purpose of the present invention is to overcome the defect that cyclic dinucleotides in the prior art are difficult to be effectively taken up by cells.
  • the first aspect of the present invention provides a cyclic dinucleotide prodrug molecule or its stereoisomer, tautomer, nitrogen oxide, solvate, metabolite, pharmaceutically acceptable Salt, the cyclic dinucleotide prodrug molecule has the structure shown in Formula I, Formula II or Formula III:
  • Each of B 1 and B 2 is independently a natural base or an artificially modified base;
  • the natural base is selected from guanine, adenine, cytosine, thymine, and uracil;
  • the artificial The modified base is a base obtained by modifying a functional group by thio, halo or methylation, and the functional group is a nitroindole group, an aminoindole group, a xanthine group Or hypoxanthine groups;
  • Each of X 1 and X 2 is independently -H, -OCH 3 or -F;
  • Each Y 1 and Y 2 are independently selected from
  • R 1 , R 2 , R 3 and R 4 are each independently selected from substituted or unsubstituted C 1 -C 10 aliphatic hydrocarbon groups, substituted or unsubstituted C 6 -C 11 aromatic hydrocarbon groups, five-membered or six-membered hetero Cyclic groups; and the substituents optionally present in R 1 , R 2 , R 3 and R 4 are each independently selected from C 1 -C 5 alkyl groups, C 1 -C 5 alkoxy groups and halogens;
  • n 1 , n 2 , n 3 and n 4 are each independently an integer of 1-5.
  • the second aspect of the present invention provides a method for preparing a cyclic dinucleotide prodrug molecule as described above.
  • the method includes:
  • the first nucleoside monomer compound, the second nucleoside monomer compound and the first condensing agent are subjected to a first contact reaction in a first liquid reaction medium to obtain a linear dinucleotide intermediate;
  • the linear dinucleotide intermediate is subjected to a decyanation ethyl reaction, and then the obtained reaction product and a second condensing agent are subjected to a second contact reaction in a second liquid reaction medium;
  • the first nucleoside monomer compound has the structure represented by formula (13) or formula (14):
  • the second nucleoside monomer compound has the structure represented by formula (15) or formula (16):
  • Q represents a protective group on the extracyclic amino group in the bases represented by B 1 and B 2 , and each of the Q in formulas (13) to (16) is independently an acyl group.
  • the third aspect of the present invention provides a cyclic dinucleotide prodrug molecule or its stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, and pharmaceutically acceptable salts as described above Application in the preparation of medicines.
  • the cyclic dinucleotide prodrug molecule provided by the present invention can autonomously cross the cell membrane, release cyclic dinucleotide, and has high cell activity.
  • Figure 1 is the result of the dual fluorescence test in Test Example 2;
  • Figure 3 is the EC 50 value of the compound of Test Example 3 causing the expression of type I interferon in THP-1 cells;
  • a is the tumor imaging image of the day 0 administration of the hollow negative control mice of Test Example 4
  • b is the tumor imaging image of the day 0 administration of the mice of the experimental group administered ADU-S100 in Test Example 4
  • c is the test
  • the administration of compound 8 in the experimental group was given on day 0 of the tumor imaging of the mice in the experimental group
  • d is the tumor imaging of day 9 in the negative control group of mice in test example 4
  • e is the administration of ADU- in test example 4.
  • a is the tumor volume of mice in different experimental groups in Test Example 4
  • b is the survival rate of mice in different experimental groups in Test Example 4.
  • the first aspect of the present invention provides a cyclic dinucleotide prodrug molecule or its stereoisomer, tautomer, nitrogen oxide, solvate, metabolite, pharmaceutically acceptable salt.
  • the inventor of the present invention unexpectedly discovered in the study that by forming a phosphate protecting group containing a thioester structure and/or a disulfide bond structure as shown in Y 1 and/or Y 2 into a cyclic dinucleotide molecule, Phosphotriester cyclic dinucleotide prodrug molecules can eliminate the negative charge of phosphate, remove the protective group before entering the cytoplasm, and release biologically active cyclic dinucleotide molecules to overcome cyclic dinucleosides The defect that acid is difficult to be effectively taken up by cells.
  • the "C 1 -C 10 aliphatic hydrocarbon group” means an alkyl or alkenyl group having a total number of carbon atoms of 1-10, for example, methyl, ethyl, n-propyl, isopropyl, n- Butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, iso Octyl, vinyl, propylene, etc.
  • C 1 -C 6 aliphatic hydrocarbon group means an alkane group or alkene group having a total of 1-6 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl Group, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, isohexyl, vinyl, propenyl, etc.
  • the "C 6 -C 11 aromatic hydrocarbon group” means an aromatic (group) ring that has lost one or several hydrogen atoms, including an aromatic ring connected with other functional groups or substituents, for example, it can be Wait.
  • the five-membered or six-membered heterocyclic group represents a saturated or unsaturated five-membered heterocyclic group, or a saturated or unsaturated six-membered heterocyclic group, for example, may be Wait.
  • the "C 1 -C 5 alkyl group” represents an alkyl group having a total number of carbon atoms of 1-5, and may be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, Sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl.
  • the "C 1 -C 5 alkoxy group” means an alkoxy group having a total number of carbon atoms of 1-5, for example, it may be methoxy, ethoxy, propoxy, butoxy, Pentoxy etc.
  • halogen may be fluorine, chlorine, bromine, or iodine.
  • R 1 , R 2 , R 3 and R 4 are each independently selected from substituted or unsubstituted C 1 -C 6 aliphatic hydrocarbon groups, substituted or unsubstituted C 6 -C 11 aromatic hydrocarbon groups, five-membered or Six-membered heterocyclic group; and the substituents optionally present in R 1 , R 2 , R 3 and R 4 are each independently selected from C 1 -C 5 alkyl, C 1 -C 5 alkoxy and halogen .
  • Y 1 and Y 2 are the same; B 1 and B 2 are the same; X 1 and X 2 are the same.
  • n 1 , n 2 , n 3 and n 4 are the same and are integers of 1-3.
  • the cyclic dinucleotide prodrug molecule has any structure represented by formula (1) to formula (10):
  • the cyclic dinucleotide prodrug molecule has any of the structures shown in formula (1)-formula (10), and in formula (1)-(10),
  • B 1 and B 2 are the same and are selected from guanine, adenine, cytosine, thymine, and uracil;
  • X 1 and X 2 are both -H;
  • R 1 , R 2 , R 3 and R 4 are the same and are selected from substituted or unsubstituted C 1 -C 6 aliphatic hydrocarbon groups, substituted or unsubstituted C 6 -C 11 aromatic hydrocarbon groups, five-membered or six-membered hetero Cyclic group; and the substituents optionally present in R 1 , R 2 , R 3 and R 4 are selected from C 1 -C 5 alkyl groups.
  • the cyclic dinucleotide has a structure represented by formula (11),
  • the cyclic dinucleotide has a structure represented by formula (12),
  • the second aspect of the present invention provides a method for preparing the cyclic dinucleotide prodrug molecule of the present invention.
  • the protecting group used in the preparation of the cyclic dinucleotide prodrug molecule is an acyl group, such as benzoyl, isobutyryl, acetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl Wait.
  • the protecting group is selected from phenoxyacetyl or 4-isopropylphenoxyacetyl.
  • DMTr- is 4,4'-dimethoxytriphenylmethyl.
  • the amount of each substance in the method is not particularly limited, and those skilled in the art can select according to the needs of each reaction.
  • the conditions for performing the first contact reaction include: a temperature of 0-50°C and a time of 2-8h.
  • the conditions for performing the second contact reaction include: a temperature of 0-50°C and a time of 2-8h.
  • the first condensing agent and the second condensing agent are each independently selected from 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole, 2 ,4,6-triisopropylphenylsulfone-3-nitro1,2,4-triazole, 2,4,6-triisopropylbenzenesulfonyl chloride, 2,4,6-trimethyl At least one of benzenesulfonyl chloride and 1H-tetrazole and N-methylimidazole.
  • the first liquid reaction medium and the second liquid reaction medium are common reaction media in the art, for example, pyridine, methylene chloride, acetonitrile, dioxane, tetrahydrofuran, etc. Choose the type of reaction.
  • the amount of the first condensing agent is 2-3 mol relative to 1 mol of the first nucleoside monomer compound.
  • the amount of the second condensing agent is 4-5 mol relative to 1 mol of the linear dinucleotide intermediate.
  • the basic conditions may be provided by, for example, triethylamine, tert-butylamine, diethylamine, diisopropylamine, etc. Particularly preferably, in order to obtain a target product with higher yield, The basic conditions are provided by a solution of tert-butylamine in acetonitrile. Particularly preferably, the alkaline conditions are provided by a mixed solution of tert-butylamine and acetonitrile in a volume ratio of 1: (1-5).
  • the deprotection reaction is carried out in the presence of a mixed solution of diisopropylamine and methanol with a volume ratio of 1: (5-20).
  • the method for preparing the cyclic dinucleotide prodrug molecule of the present invention includes:
  • step 2) Add the linear dinucleotide intermediate obtained in step 1) to the mixed solution of tert-butylamine and acetonitrile and react for 10-30 min. After evaporating the solvent, add 1-(mesityl-2-sulfone)-3 -Nitro-1,2,4-triazole, dissolved in pyridine, reacted at 0-50°C for 2-8h to obtain a cyclic dinucleotide prodrug molecule with a protective group; wherein, the tert-butylamine and acetonitrile In the mixed solution, the volume ratio of tert-butylamine and acetonitrile is 1: (1-5);
  • the synthesis of the first nucleoside monomer compound and the second nucleoside monomer compound may be a method commonly used in the prior art, and a person skilled in the art may use the formula (13) provided by the present invention -The structure shown in equation (16) designs the synthetic route by itself.
  • the present invention exemplarily provides several specific synthesis methods here:
  • R may be R 1 or R 2
  • B may be B 1 or B 2
  • X may be X 1 or X 2
  • n may be n 1 or n 2 .
  • step (Ii) Add nucleoside phosphoramidite monomer, 5-ethylmercaptotetrazolium, and the thioester-substituted alcohol obtained in step (i) to anhydrous acetonitrile, and add tert-butyl after reacting for 1-3h under an inert gas atmosphere Base peroxyacid, after 30-60min, add sodium sulfite aqueous solution to quench, extract with organic solvent, and separate the nucleic acid monomer with thioester protecting group by column chromatography;
  • step (Iii) Dissolve the nucleic acid monomer with thioester protecting group obtained in step (ii) with dichloromethane, then add dichloroacetic acid, react at 20-30°C for 1-3h, and separate the product by column chromatography The first nucleoside monomer compound;
  • step (IV) Dissolve the thioester-protecting nucleic acid monomer obtained in step (ii) with acetonitrile, then add tert-butylamine, react at 20-30°C for 1-3h, and spin-dry the solvent to obtain the second nucleoside monohydrate Body compound.
  • R may be R 3 or R 4
  • B may be B 1 or B 2
  • X may be X 1 or X 2
  • n may be n 3 or n 4 .
  • Method two includes:
  • step (III) After dissolving the dicyanoethyl protected nucleotide monomer obtained in step (ii) with dichloromethane, add tert-butylamine, stir at 20-30°C for 10-30 min, spin dry the solvent, add 1-( Mesityl-2-sulfone)-3-nitro-1,2,4-triazole and the alcohol containing disulfide bond substitution obtained in step (i), dissolved in anhydrous pyridine, inert gas protection reaction After 4 hours, evaporate the solvent, extract with organic solvent, and separate by column chromatography to obtain the nucleotide monomer with disulfide bond substituted alcohol protected phosphate;
  • step (IV) Dissolve the nucleotide monomer with disulfide bond-substituted alcohol-protected phosphate obtained in step (iii) with dichloromethane, add dichloroacetic acid to react at 20-30°C for 1-3h, and perform column chromatography Separating the first nucleoside monomer compound;
  • step (V) After dissolving the nucleotide monomer with disulfide bond-substituted alcohol-protected phosphate obtained in step (iii) with acetonitrile, add tert-butylamine, stir at 20-30°C for 10-30min, and spin-dry the solvent to obtain The second nucleoside monomer compound.
  • the amount of each substance is not particularly limited, and those skilled in the art can select according to the needs of the reaction.
  • the third aspect of the present invention provides a cyclic dinucleotide prodrug molecule or its stereoisomers, tautomers, nitrogen oxides, solvates,
  • the medicine is an antiviral medicine, an antibacterial infection medicine or an anticancer medicine.
  • the cyclic dinucleotide prodrug molecule of the present invention can autonomously cross the cell membrane to release the cyclic dinucleotide, and the released cyclic dinucleotide can be combined with an immunostimulating factor (STING) to stimulate the immune response, It can be used in the preparation of antiviral, antibacterial and anticancer drugs.
  • STING immunostimulating factor
  • various post-processing methods conventionally used in the art can be used to post-process the obtained products.
  • the post-treatment methods include, but are not limited to: extraction, recrystallization, washing, drying, filtration, and the like.
  • the present invention is not repeated here, and the post-processing methods involved in the embodiments are only used for exemplary enumeration, and do not mean that it is an operation that must be adopted. Those skilled in the art can use conventional other means to replace it.
  • the anhydrous solvents required in the experiment are dried according to conventional methods.
  • Thin layer chromatography silica gel GF254 is produced by Tianjin Silida Company.
  • TLC is 254nm UV analysis, column chromatography silica gel is produced by Qingdao Ocean Chemical Company.
  • the NMR spectrometer uses Bruker AVANCE 400M Hz, with TMS as the internal standard.
  • High resolution mass spectrometry uses Varian 7.0T FTMS Fourier transform plasma cyclotron resonance high resolution mass spectrometry.
  • Normal temperature and room temperature refer to (25 ⁇ 3)°C.
  • reaction was quenched by adding an appropriate amount of water, extracted, the aqueous phase was extracted twice with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 2.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • MALDI-TOF-HRMS The relative molecular mass of C34H50N10O11P2S2 is: 900.2577, and a peak of [M+H] + 901.2654 is found.
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • reaction was quenched by adding an appropriate amount of water, most of the solvent was removed by rotary evaporation, 30 mL of dichloromethane was added to re-dissolve, the pH value of the 5 wt% oxalic acid aqueous solution was adjusted to 3, and the organic phase was separated. After the organic phase was washed with saturated saline, it was anhydrous Dry with sodium sulfate. After filtration, concentration and column chromatography, compound 11 (1.05 g) was obtained.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • the data indicates that the compound was synthesized correctly.
  • the nuclear magnetic data are:
  • MALDI-TOF-HRMS The relative molecular mass of C34H38N12O10P2S4 is: 964.1192, and a peak of [M+H] + 965.1271 is found.
  • the data indicates that the compound was synthesized correctly.
  • the cddA35 NMR data is:
  • the data indicates that the compound was synthesized correctly.
  • Compound solution preparation Weigh a certain amount of compound 8, compound 16, and compound cddA35 were dissolved in DMSO solution and formulated into a mother liquor with a mass concentration of 1 mg/mL.
  • Liquid phase conditions the instrument uses Agilent 1260 HPLC, chromatographic column: Agilent ZORABX SB-C18 5 ⁇ m [4.6x150mm], column temperature 25°C, injection 10uL, detection wavelength 254nM, chromatographic gradient method as shown in Table 1 below.
  • Dual-fluorescence reporter gene experiment After the cyclic dinucleotide prodrug molecule enters the cell, it will pass through the thioesterase or reducing environment in the cytoplasm of the cell, so that the thioester bond is broken or the disulfide bond is broken, and the resulting intermediate In the body, the sulfur atom in the ⁇ position will nucleophilically attack the carbon atom in the ⁇ position, thereby forming ethylene sulfide to leave, releasing the cyclic dinucleoside of the parent drug with a negatively charged phosphodiester structure, binding to activate the intracellular STING protein, phosphate Interferon regulatory factor 3 (IRF3), phosphorylated IRF3 enters the nucleus and binds to the interferon gene promoter, causing interferon expression. Based on this, the interferon gene promoter is introduced into the firefly luciferase promoter In this way, the dual-fluorescence reporter gene method can be used to measure the cell activity of
  • Compound solution preparation Compound 8 and Compound 16 were dissolved in DMSO, respectively, and formulated to a final concentration of 1 mM.
  • cddA35 is dissolved in water and is also formulated to a concentration of 1 mM.
  • pcDNA3.1-hSTING-wt plasmid is configured at a concentration of 400ng/ ⁇ L
  • pGL3-IFN ⁇ plasmid is configured at a concentration of 400ng/ ⁇ L
  • pGL4.74-Rluc plasmid is configured at a concentration of 100ng/ ⁇ L.
  • HEK293T cells reached about 70%, and the original medium was replaced with serum-free medium opti-DMEM 500mL to be transferred.
  • Prepare the above component solutions including pcDNA3.1-hsting-wt 2 ⁇ L, pGL3-IFN ⁇ 1 ⁇ L, pGL4.74-Rluc 1 ⁇ L, opti-DMEM medium to make up 50uL/well, four wells in parallel.
  • the above solution mixture was added to 50 ⁇ L of 2/50Lipo2000 transfection solution and mixed. The mixture was allowed to stand at room temperature for 15min and mixed evenly. 100 ⁇ L/well was transfected. After 4h, 1mL of 37°C DMEM medium was added.
  • THP-1-Lucia cells reached about 80%.
  • Compound 8, compound 16 and drug ADU-S100 were directly dissolved in the medium at a certain concentration and added to the 24-well plate, four wells in parallel. Incubate at 37°C and 5% CO 2 for 24h. After the cultivation, the luciferase activity was tested.
  • the EC 50 values of Compound 8 and Compound 16 causing type I interferon expression in the THP-1 cell line were 4.4 nM and 355 nM, respectively, compared to the control drug ADU-S100 (reference compound 2 in patent CN108430503A '3'-RR- (a) ( a)) of the EC 50 value of 41.5 ⁇ M, compound 16 and compound EC 50 values were increased by 8 9430-fold and 117-fold, increase the activity considerably.
  • mice BABL/C female mice weighing 18-22g. Source Guangdong Animal Experiment Center. Mice were fed with pellet feed, free to eat and drink.
  • Tumor cell line CT-26-Luc cells
  • a mouse tumor model cell culture, passage, collecting cells in the logarithmic phase of the cell, making a cell suspension with a concentration of (1.0 ⁇ 10 7 ) per milliliter, and injecting 0.1 mL of cell suspension (cell The number is 1.0 ⁇ 10 6 per mouse).
  • the tumor grows to a diameter of about 5 mm in about 10 days, and the cancer is successfully divided into three groups randomly.
  • Negative control group physiological saline solution
  • Positive control group ADU-S100 (clinical phase II drug), dose 1mg/kg
  • the tumor is administered after 10 days of tumor inoculation, intratumoral injection, 50 microliters/head, once every two days, three times in total.
  • IVIS Spectrum Imaging System small animal live imaging system
  • luciferin small molecule preparation prepare appropriate amount of luciferin sodium salt 15mg/mL with PBS solution, and order fluorescein sodium from Shanghai Yisheng Biological Co., Ltd. Prepare oxygen and isoflurane in advance. Inject 100 ⁇ L of fluorescein intraperitoneally and put the mouse into an anesthesia box. After the animal is anesthetized, transfer the mouse to the imaging chamber in the imager. Adjust the posture of the mouse as needed.
  • the imaging parameters are: automatic exposure, binning value is 2, and F value is 8.
  • the imaging time is 10 minutes.
  • the fluorescence value is converted into the number of photons, and the total number of fluorescent photons of each experimental group is counted.
  • the imager is PerkinElmer's IVIS Spectrum imaging platform. The result is shown in Figure 4.
  • the numbers in Figures 4a, 4b, 4c, 4d, 4e, and 4f represent the total number of fluorescent photons in the circular region (ROI, region of interest), and Figures 4g and 4h are the statistics of the total fluorescent photons in each experimental group.
  • the tumor volume and survival rate of mice in each group are shown in Figure 5.

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Abstract

本发明涉及药物领域，公开了一种环二核苷酸前药分子及其制备方法和应用，该环二核苷酸前药分子具有如式Ⅰ、式Ⅱ或式Ⅲ所示的结构，能够自主跨过细胞膜，释放出环二核苷酸，具有较高的细胞活性。

Description

环二核苷酸前药分子及其制备方法和应用 技术领域
本发明涉及药物领域，具体涉及一种环二核苷酸前药分子、一种环二核苷酸前药分子的制备方法，以及环二核苷酸前药分子的应用。
背景技术
环二核苷酸，是在细菌和哺乳动物中发现的一种新型的第二信使分子。在哺乳动物细胞中，环二核苷可以与免疫刺激因子(STING)结合，刺激免疫反应。其可以引起细胞因子比如β干扰素、NF-κB的表达，促进CD8 +T细胞的增殖分化成熟。
因为环二核苷酸具有强大的免疫刺激作用，可以将其用于病毒和细菌感染以及癌症的治疗中。特别是与传统免疫疗法联用抗癌能够表现出较好的结果。目前Aduro公司联合诺华的ADU-S100进入临床二期，MSD的MK-1454已经进入临床一期。
环二核苷酸分子结构中带有两个磷酸二酯键，这种带有负电荷的磷酸二酯键首先会引起药物脂溶性差，阻碍环二核苷酸有效的跨过细胞膜，再者这种磷酸二酯键也容易水解，从而造成药物分子在循环系统中的不稳定。
目前针对环二核苷酸药物递送体系主要采用辅助性材料。比如早期文献报道的利用慢病毒或者腺病毒包装环二核苷酸的合成酶，进入细胞后通过酶催化合成环二核苷酸以促进免疫反应，但这种方法存在较大的风险而不被广泛运用。
通过利用脂质体或者脂质纳米颗粒、细胞穿透肽、含有多聚阳离子氨基酸的蛋白质凝胶等作为载体以实现环二核苷酸药物的递送都已有报道，但这类载体的使用会降低药物的载药量，同时载体的引入可能会引起一些潜在的细胞毒性。
目前临床采用的解决环二核苷有效被细胞摄取的方法是瘤内注射，虽然已有文章报道这种直接瘤内注射，细胞能够摄取环二核苷酸，但这种摄取率较低，因此需要更大剂量的给药，这种大剂量的给药可能会造成免疫系统的过度激活，从而引起周身性或者局部的炎症反应。
因此，如何解决环二核苷有效被细胞摄取仍存在一些问题。
发明内容
本发明的目的是为了克服现有技术中环二核苷酸难以有效地被细胞摄取的缺陷。
为了实现上述目的，本发明第一方面提供了一种环二核苷酸前药分子或其立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐，该环二核苷酸前药分子具有如式I、式II或式III所示的结构：
Figure PCTCN2020071329-appb-000001
在式I-式III中，
各个B 1和B 2各自独立地为天然碱基或人工修饰的碱基；所述天然碱基选自鸟嘌呤基、腺嘌呤基、胞嘧啶基、胸腺嘧啶基和尿嘧啶基；所述人工修饰的碱基为将功能基团进行硫代、卤代或甲基化方法修饰而得到的碱基，所述功能基团为硝基吲哚基团、氨基吲哚基团、黄嘌呤基团或次黄嘌呤基团；
各个X 1和X 2各自独立地为-H、-OCH 3或-F；
各个Y 1和Y 2各自独立地选自
Figure PCTCN2020071329-appb-000002
其中，
R 1、R 2、R 3和R 4各自独立地选自取代或未取代的C 1-C 10的脂烃基、取代或未取代的C 6-C 11的芳烃基、五元或六元杂环基；且R 1、R 2、R 3和R 4中任选存在的取代基各自独立地选自C 1-C 5的烷基、C 1-C 5的烷氧基和卤素；
n 1、n 2、n 3和n 4各自独立地为1-5的整数。
本发明第二方面提供了一种制备如前文所述的环二核苷酸前药分子的方法，该方法包括：
a)将第一核苷单体化合物、第二核苷单体化合物和第一缩合剂在第一液态反应介质中进行第一接触反应，得到线性二核苷酸中间体；
b)在碱性条件下，将所述线性二核苷酸中间体进行脱氰乙基反应，然后将所得反应产物与第二缩合剂在第二液态反应介质中进行第二接触反应；
c)将进行所述第二接触反应后获得的带有保护基的环二核苷酸前药分子进行脱保护基反应；
其中，所述第一核苷单体化合物具有式(13)或式(14)所示的结构：
Figure PCTCN2020071329-appb-000003
所述第二核苷单体化合物具有式(15)或式(16)所示的结构：
Figure PCTCN2020071329-appb-000004
其中，Q表示B 1和B 2所示的碱基中的环外氨基上的保护基，且式(13)-式(16)中的所述Q各自独立地为酰基。
本发明第三方面提供一种如前文所述的环二核苷酸前药分子或其立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐在制备药物中的应用。
本发明提供的环二核苷酸前药分子能够自主跨过细胞膜，释放出环二核苷酸，具有较高的细胞活性。
附图说明
图1是测试例2中的双荧光测试结果；
图2是测试例3中在单核细胞系THP-1中的I型干扰素测试结果；
图3是测试例3中化合物在THP-1细胞中引起I型干扰素表达的EC 50值；
图4中a是测试例4中空阴性对照组小鼠给药第0天肿瘤成像图，b是测试例4中给药ADU-S100实验组小鼠给药第0天肿瘤成像图，c是测试例4中给药化合物8实验组小鼠给药第0天肿瘤成像图，d是测试例4中 阴性对照组小鼠给药第9天肿瘤成像图，e是测试例4中给药ADU-S100实验组小鼠给药第9天肿瘤成像图，f是测试例4中给药化合物8实验组小鼠给药第9天肿瘤成像图，g是测试例4中各实验组小鼠给药第0天肿瘤成像总荧光量统计图，h是测试例4中各实验组小鼠给药第9天肿瘤成像总荧光量统计图；
图5中a是测试例4中不同实验组小鼠肿瘤体积大小，b是测试例4中不同实验组小鼠的存活率。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
如前所述，本发明第一方面提供了一种环二核苷酸前药分子或其立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐。
本发明的发明人在研究中意外发现，通过在环二核苷酸分子中引入如Y 1和/或Y 2所示的含有硫酯结构和/或二硫键结构的磷酸酯保护基，形成磷酸三酯类环二核苷酸前药分子，能够消除磷酸负电荷的影响，在进入细胞质内进行保护基脱除，释放出具有生物活性的环二核苷酸分子，从而克服环二核苷酸难以有效地被细胞摄取的缺陷。
在本发明中，所述“C 1-C 10的脂烃基”表示碳原子总数为1-10的烷基或烯基，例如可以为甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基、正戊基、异戊基、叔戊基、新戊基、正己基、异己基、正庚基、异庚基、正辛基、异辛基、乙烯基、丙烯基等。
所述“C 1-C 6的脂烃基”表示碳原子总数为1-6的烷烃基或烯烃基，例如可以为甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基、正戊基、异戊基、叔戊基、新戊基、正己基、异己基、乙烯基、丙烯基等。
所述“C 6-C 11的芳烃基”表示失去一个或几个氢原子的芳香(族)环，包括连接有其它官能团或取代基的芳香环，例如可以为
Figure PCTCN2020071329-appb-000005
等。
在本发明中，所述五元或六元杂环基表示饱和或不饱和的五元杂环基，或者饱和或不饱和的六元杂环基，例如可以为
Figure PCTCN2020071329-appb-000006
等。
在本发明中，所述“C 1-C 5的烷基”表示碳原子总数为1-5的烷基，例如可以为甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基、正戊基、异戊基、叔戊基、新戊基。
在本发明中，所述“C 1-C 5的烷氧基”表示碳原子总数为1-5的烷氧基，例如可以为甲氧基、乙氧基、丙氧基、丁氧基、戊氧基等。
在本发明中，卤素可以为氟、氯、溴、碘。
优选地，R 1、R 2、R 3和R 4各自独立地选自取代或未取代的C 1-C 6的脂烃基、取代或未取代的C 6-C 11的芳烃基、五元或六元杂环基；且R 1、R 2、R 3和R 4中任选存在的取代基各自独立地选自C 1-C 5的烷基、C 1-C 5的烷氧基和卤素。
根据一种优选的具体实施方式，在式I、式II或式III所示的结构中，Y 1和Y 2相同；B 1和B 2相同；X 1和X 2相同。
根据另一种优选的具体实施方式，Y 1和Y 2中，n 1、n 2、n 3和n 4相同，且为1-3的整数。
根据另一种优选的具体实施方式，所述环二核苷酸前药分子具有式(1)-式(10)所示的任意一种结构：
Figure PCTCN2020071329-appb-000007
其中，式(1)-式(10)中的基团的定义与前文所述的定义对应相同。
特别优选情况下，所述环二核苷酸前药分子具有式(1)-式(10)所示的任意一种结构，且在式(1)-(10)中，
B 1和B 2相同，且选自鸟嘌呤基、腺嘌呤基、胞嘧啶基、胸腺嘧啶基和尿嘧啶基；
X 1和X 2均为-H；
R 1、R 2、R 3和R 4相同，且选自取代或未取代的C 1-C 6的脂烃基、取代或未取代的C 6-C 11的芳烃基、五元或六元杂环基；且R 1、R 2、R 3和R 4中任选存在的取代基选自C 1-C 5的烷基。
本发明提供一种优选的实施方式，所述环二核苷酸具有式(11)所示的结构，
Figure PCTCN2020071329-appb-000008
根据本发明另一种优选的实施方式，所述环二核苷酸具有式(12)所示的结构，
Figure PCTCN2020071329-appb-000009
如前所述，本发明的第二方面提供了一种制备本发明所述的环二核苷酸前药分子的方法。
根据本发明，环二核苷酸前药分子制备过程中所用的保护基为酰基，例如可以为苯甲酰基、异丁酰基、乙酰基、苯氧乙酰基、4-异丙基苯氧乙酰基等。
特别优选地，所述保护基选自苯氧乙酰基或4-异丙基苯氧乙酰基。
式(13)和式(14)，DMTr-为4，4’-二甲氧基三苯基甲基。
在本发明中，对所述方法中各物质的用量并没有特别的限定，本领域技术人员可以根据各个反应的需要进行选择。
优选地，进行所述第一接触反应的条件包括：温度为0-50℃，时间为2-8h。
优选地，进行所述第二接触反应的条件包括：温度为0-50℃，时间为2-8h。
优选地，所述第一缩合剂和所述第二缩合剂各自独立地选自1-(均三甲苯基-2-砜基)-3-硝基-1,2,4-三唑、2,4,6-三异丙基苯基砜基-3-硝基1,2,4-三唑、2,4,6-三异丙基苯磺酰氯、2,4,6-三甲基苯磺酰氯和1H-四唑和N-甲基咪唑中的至少一种。
在本发明中，所述第一液态反应介质和第二液态反应介质为本领域常用反应介质，例如可以为吡啶、二氯甲烷、乙腈、二氧六环、四氢呋喃等，本领域技术人员可以根据反应的类型进行选择。
优选地，在步骤a)中，相对于1mol的第一核苷单体化合物，所述第一缩合剂的用量为2-3mol。
优选地，在步骤b)中，相对于1mol的线性二核苷酸中间体，所述第二缩合剂的用量为4-5mol。
在步骤b)中，所述碱性条件可以由例如三乙胺、叔丁胺、二乙胺，二异丙胺等提供，特别优选情况下，为了获得收率更高的目标产物，本发明所述的碱性条件由叔丁胺的乙腈溶液提供。特别优选地，所述碱性条件由体积比为1：(1-5)的叔丁胺与乙腈形成的混合溶液提供。
优选地，所述脱保护基反应在体积比为1：(5-20)的二异丙胺和甲醇形成的混合溶液存在下进行。
根据本发明一种优选的实施方式，制备本发明所述的环二核苷酸前药分子的方法包括：
1)将所述第一核苷单体化合物、所述第二核苷单体化合物和1-(均三甲苯基-2-砜基)-3-硝基-1,2,4-三唑加入吡啶中，惰性气体氛围下，在0-50℃下反应2-8h，得到线性二核苷酸中间体；
2)将步骤1)得到的线性二核苷酸中间体加入到叔丁胺和乙腈的混合溶液中反应10-30min，蒸干溶剂后，加入1-(均三甲苯基-2-砜基)-3-硝基-1,2,4-三唑，用吡啶溶解，在0-50℃下反应2-8h得到带有保护基的环二核苷酸前药分子；其中，所述叔丁胺和乙腈的混合溶液中，叔丁胺和乙腈的体积比为1：(1-5)；
3)将带有保护基的环二核苷酸前药分子溶于二异丙胺和甲醇的混合溶液中，在20-30℃下反应3-5h得到环二核苷酸前药分子；其中，所述二异丙胺和甲醇的混合溶液中，二异丙胺和甲醇的体积比为1：(5-20)。
在本发明中，所述第一核苷单体化合物和所述第二核苷单体化合物的合成可以为现有技术中常用的方法，本领域技术人员可以根据本发明提供的式(13)-式(16)所示的结构自行设计合成路线。本发明在此示例性地提供几种具体的合成方法：
方法一：
Figure PCTCN2020071329-appb-000010
在方法一中，R可以为R 1或者R 2，B可以为B 1或者B 2，X可以为X 1或X 2，n可以为n 1或n 2。方法一包括：
(ⅰ)将巯基取代醇和三乙胺溶解在二氯甲烷中，在-80℃～-70℃下滴加酰氯的二氯甲烷溶液，升至20-30℃反应1-3h后，加水淬灭，用有机溶剂进行萃取，经柱层析分离得到硫酯取代的醇；
(ⅱ)将核苷亚磷酰胺单体、5-乙巯基四氮唑、步骤(ⅰ)所得的硫酯取代的醇加入无水乙腈中，在惰性气体氛围下反应1-3h后加入叔丁基过氧酸，30-60min后加入亚硫酸钠水溶液淬灭，用有机溶剂萃取，经柱层析分离得到具有硫酯保护基的核酸单体；
(ⅲ)将步骤(ⅱ)得到的具有硫酯保护基的核酸单体用二氯甲烷溶解，然后加入二氯乙酸，在20-30℃下反应1-3h后，经柱层析分离得到所述第一核苷单体化合物；
(ⅳ)将步骤(ⅱ)得到的具有硫酯保护基的核酸单体用乙腈溶解，然后加入叔丁胺，在20-30℃下反应1-3h后，旋干溶剂得到所述第二核苷单体化合物。
方法二：
Figure PCTCN2020071329-appb-000011
在方法二中，R可以为R 3或者R 4，B可以为B 1或者B 2，X可以为X 1或X 2，n可以为n 3或n 4。方法二包括：
(ⅰ)将N-氯代丁二酰亚胺酰氯和硫醇(或硫酚)加入二氯甲烷中，在10-35℃下反应1-3h后，加入巯基取代醇，继续反应20-30h，然后加水淬灭，用有机溶剂萃取，经柱层析分离得到含有二硫键结构取代的醇；
(ⅱ)将3-羟基丙腈、核苷亚磷酰胺单体和5-乙巯基四氮唑加入无水乙腈中，在惰性气体氛围下反应1-3h后加入叔丁基过氧酸，继续反应30-60min，然后加入亚硫酸氢钠水溶液淬灭，用有机溶剂萃取，经柱层析分离得到双氰乙基保护的核酸单体；
(ⅲ)将步骤(ⅱ)得到的双氰乙基保护的核苷酸单体用二氯甲烷溶解后，加入叔丁胺，在20-30℃下搅 拌10-30min后旋干溶剂，加入1-(均三甲苯基-2-砜基)-3-硝基-1,2,4-三唑和步骤(ⅰ)中所得的含有二硫键结构取代的醇，无水吡啶溶解，惰性气体保护反应4小时后，蒸干溶剂，有机溶剂萃取、柱层析分离得到具有二硫键取代醇保护磷酸酯的核苷酸单体；
(ⅳ)将步骤(ⅲ)所得的具有二硫键取代醇保护磷酸酯的核苷酸单体用二氯甲烷溶解，加入二氯乙酸在20-30℃下反应1-3h，经柱层析分离得到所述第一核苷单体化合物；
(ⅴ)将步骤(ⅲ)得到的具有二硫键取代醇保护磷酸酯的核苷酸单体用乙腈溶解后，加入叔丁胺，在20-30℃下搅拌10-30min后，旋干溶剂得到所述第二核苷单体化合物。
在方法一和方法二中，对各物质的用量并没有特别的限定，本领域技术人员可以根据反应的需要进行选择。
本发明第三方面提供了一种本发明所述的环二核苷酸前药分子或其立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐在制备药物中的应用。
优选情况下，所述药物为抗病毒药物、抗细菌感染药物或抗癌药物。
本发明所述的环二核苷酸前药分子在能够自主跨过细胞膜，释放出环二核苷酸，释放出的环二核苷酸能够与免疫刺激因子(STING)结合，刺激免疫反应，能够应用于制备抗病毒、抗细菌感染以及抗癌药物中。
本发明的第二方面涉及的反应中均可以采用本领域内常规使用的各种后处理方法对所得到的产物进行后处理。所述后处理的方法包括但不限于：萃取、重结晶、洗涤、干燥、过滤等。本发明在此不再赘述，并且实施例中涉及的后处理方法仅是用于示例性地列举，并不表示那是必须采用的操作，本领域技术人员可以采用常规的其它手段进行替代。
以下将通过实例对本发明进行详细描述。以下实例中，所用的溶剂、原料和试剂如不特别指明，均来自商购，均为分析纯或化学纯。
实验中所需的无水溶剂均按常规方法进行干燥。
产物分离、鉴别的仪器和方法：
薄层层析硅胶GF254为天津思利达公司生产。
TLC是在254nm紫外分析，柱层析硅胶为青岛海洋化工公司生产。
核磁共振谱仪使用的是Bruker AVANCE 400M Hz，以TMS为内标。
高分辨质谱使用的是Varian 7.0T FTMS傅里叶变换等离子回旋共振高分辨质谱。
常温和室温均指(25±3)℃。
实施例1
具有季戊酰硫代乙酯保护的环二核苷酸前药分子(化合物8)的合成。
Figure PCTCN2020071329-appb-000012
1)100mL烧瓶中，加入2-巯基乙醇0.9mL、三乙胺1.8mL和10mL重蒸二氯甲烷溶解，在-78℃条件下滴加季戊酰氯(1.6mL，用10mL二氯甲烷溶解)，半小时滴加完毕，继续反应1小时后，缓慢升至室温，继续搅拌1小时。加适量水淬灭反应，萃取，水相二氯甲烷萃取两次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，过滤，浓缩，柱层析分离得到化合物2。
核磁数据为：
1H NMR(400MHz,CDCl 3)δ3.73(t,J＝6.1Hz,9H),3.04(t,J＝6.1Hz,9H),1.23(s,41H).
13C NMR(101MHz,CDCl 3)δ207.36,61.91,46.57,31.49,27.40.
数据表明化合物合成正确。
2)将化合物2(1.12mmol)、5-乙巯基四氮唑(3.36mmol)和化合物1(1.12mmol)加入到烧瓶中，加入10mL无水乙腈室温搅拌1h后加入1mL 5.5M叔丁基过氧酸-癸烷溶液。继续室温搅拌40分钟后，加入亚硫酸氢钠水溶液搅拌，二氯甲烷多次萃取后，有机相合并，饱和食盐水洗涤，无水硫酸钠干燥后，过滤，浓缩，柱层析分离得到化合物3(950mg)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ9.59(s,1H),8.77(s,1H),8.17(d,J＝7.2Hz,1H),7.46–7.23(m,10H),7.21–7.14(m,3H),7.06(t,J＝8.1Hz,3H),6.89–6.76(m,4H),6.46(dd,J＝9.5,5.3Hz,1H),5.41–5.31(m,1H),4.88(s,2H),4.49(d,J＝1.3Hz,1H),4.38–4.29(m,2H),4.18(dd,J＝15.0,6.9Hz,2H),3.99(dd,J＝13.0,1.3Hz,1H),3.89(d,J＝13.1Hz,1H),3.83–3.71(m,7H),3.18(t,J＝6.9Hz,4H),3.05(s,1H),2.81(t,J＝6.0Hz,2H),2.77–2.66(m,1H),1.29–1.17(m,12H).
13C NMR(101MHz,CDCl3)δ205.84,205.76,166.70,158.57,158.37,156.92,152.07,150.57,149.10,147.35,143.09,139.47,136.15,130.04,129.86,129.16,129.13,128.15,127.82,127.76,127.75,127.05,124.07,122.49,116.44,116.39,114.91,113.20,113.12,113.10,113.09,113.01,87.79,87.74,87.40,81.38,80.69,80.66,77.37,77.26,77.05,76.73,68.07,66.69,66.67,66.62,63.04,62.38,62.33,61.89,55.24,55.22,55.19,46.63,39.06,31.50,30.95,28.32,28.28,28.25,27.45,27.39,27.30,27.27,19.84,19.77.
数据表明化合物合成正确。
3)向烧瓶中加入化合物3(0.5mmol)，加入10mL二氯甲烷溶解后，冰浴下加入6体积％的二氯乙酸- 二氯甲烷溶液10mL。反应5min后加入少量甲醇淬灭反应。加入碳酸氢钠饱和水溶液中和，萃取后干燥，浓缩，柱层析快速分子得到化合物5(330mg)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ8.78(s,1H),8.24(d,J＝6.1Hz,1H),7.43–7.31(m,2H),7.06(t,J＝8.0Hz,3H),6.48(dd,J＝9.4,5.3Hz,1H),5.35(dd,J＝8.9,5.3Hz,1H),4.89(s,2H),4.50(d,J＝1.0Hz,1H),4.44–4.27(m,2H),4.18(dd,J＝15.0,6.9Hz,2H),3.95(ddd,J＝13.2,12.1,1.9Hz,2H),3.18(t,J＝6.8Hz,3H),2.83(t,J＝6.0Hz,2H),2.72(dt,J＝13.9,5.4Hz,1H),1.25(s,10H).
13C NMR(101MHz,CDCl3)δ205.88,205.79,166.81,156.94,152.09,150.58,149.05,143.11,129.87,123.91,122.49,116.46,116.41,114.91,87.78,87.73,87.37,80.62,80.59,77.37,77.26,77.06,76.74,68.08,66.71,66.69,66.65,66.63,63.00,62.40,62.35,61.89,46.63,39.14,39.10,31.49,28.35,28.32,28.28,28.25,27.39,27.31,19.87,19.85,19.80,19.78
数据表明化合物合成正确。
4)向烧瓶中加入化合物3(0.27mmol)、叔丁胺/乙腈(1：3，v/v)10mL，室温搅拌20分钟后，旋蒸除去溶剂得到化合物4。烧瓶中继续加入化合物2(0.27mmol)，1-(均三甲苯基-2-砜基)-3-硝基-1,2,4-三唑(1.63mmol)，加入10mL吡啶室温下搅拌2小时后加少量水淬灭反应，旋蒸除去溶剂后，加入20mL二氯甲烷溶解，加入适量5质量％草酸水溶液，分出有机相干燥，过滤浓缩，加入3体积％的二氯乙酸的二氯甲烷溶液，搅拌5min后加入适量甲醇，饱和碳酸氢钠水溶液中和，萃取，有机相干燥，过滤，浓缩，柱层析分离得到化合物6(320mg)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ9.96–9.72(m,2H),8.66(dt,J＝7.8,5.3Hz,2H),8.35(dd,J＝13.1,6.2Hz,2H),7.24(ddd,J＝15.0,10.1,2.5Hz,5H),7.05–6.81(m,7H),6.49(ddd,J＝25.0,17.3,8.7Hz,2H),5.72(s,1H),5.37(s,1H),5.24(q,J＝5.9Hz,2H),4.90(s,4H),4.51(s,1H),4.46–4.21(m,6H),4.20–3.99(m,5H),3.84(t,J＝14.1Hz,2H),3.09(ddd,J＝52.8,29.8,23.2Hz,7H),2.81(d,J＝5.2Hz,4H),2.62(td,J＝13.2,4.7Hz,1H),1.18(dt,J＝9.7,6.8Hz,22H).
13C NMR(101MHz,CDCl3)δ205.70,205.69,205.64,167.56,157.15,157.10,157.06,152.47,151.75,151.44,151.35,150.60,150.56,148.97,148.57,148.55,143.66,143.62,142.44,129.73,129.69,123.57,123.00,122.15,116.76,116.73,114.81,114.77,114.75,87.44,87.40,86.79,84.57,83.67,80.24,78.10,77.85,77.58,77.46,77.26,76.94,68.29,68.17,66.88,66.82,66.61,66.55,62.76,62.63,62.58,53.57,46.56,46.52,38.90,38.05,37.86,31.87,29.64,29.31,28.38,28.34,28.30,28.27,28.20,27.41,27.25,22.65,19.79,19.72,14.14.
数据表明化合物合成正确。
5)将化合物6(0.22mmol)加入叔丁胺/乙腈(1：3，v/v)10mL，室温搅拌20min后，旋蒸除去溶剂，加入100mL吡啶溶解，加入1-(均三甲苯基-2-砜基)-3-硝基-1,2,4-三唑(1.35mmol)，室温反应4小时后除去溶剂，加入20mL二氯甲烷溶解，加入适量5质量％草酸水溶液，分出有机相干燥，过滤浓缩，柱层析分离得到化合物7(160mg)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ9.69(s,2H),8.80(d,J＝3.2Hz,2H),8.43–8.15(m,2H),7.30(td,J＝15.8,8.5Hz,7H),7.14–6.85(m,9H),6.49(ddd,J＝17.3,11.7,5.4Hz,2H),5.67–5.33(m,2H),4.91(s,4H),4.78(s,1H),4.65(d,J＝4.9Hz,1H),4.61–4.30(m,6H),4.26–4.05(m,6H),3.67–3.36(m,2H),3.16(dt,J＝9.8,7.1Hz,5H),2.78(td,J＝14.4,5.6Hz,2H),1.27–1.17(m,23H).
13C NMR(101MHz,CDCl3)δ205.76,205.74,205.60,205.53,172.28,167.03,157.76,157.05,157.02,156.60, 152.63,152.57,152.48,152.38,152.23,151.49,151.41,151.31,151.20,148.70,148.66,148.58,143.05,142.75,142.55,142.32,129.95,129.81,129.58,129.54,123.57,123.44,122.75,122.38,122.35,122.33,121.78,121.58,114.88,114.77,114.57,114.55,85.60,85.18,83.04,82.55,78.87,77.30,77.10,76.78,68.12,67.19,67.02,66.96,66.92,66.88,66.83,66.74,65.38,65.24,65.10,52.28,46.59,46.56,36.60,36.34,31.90,29.67,29.64,29.34,28.43,28.36,28.29,28.22,28.16,27.29,27.24,27.23,22.68,14.13.
数据表明化合物合成正确。
6)向烧瓶中加入化合物7(150mg)，加入甲醇5mL溶解，加入500μL二异丙胺，室温下搅拌4小时，旋干溶剂，直接柱层析分离得到化合物8(80mg)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ8.33(d,J＝8.4Hz,2H),7.94(d,J＝8.1Hz,2H),6.41(dd,J＝13.9,7.4Hz,2H),6.20(d,J＝30.1Hz,4H),5.45(d,J＝59.0Hz,2H),4.51(dd,J＝16.6,8.1Hz,3H),4.39(t,J＝8.3Hz,2H),4.16(tt,J＝9.5,4.9Hz,5H),3.17(dd,J＝11.4,6.6Hz,4H),2.82–2.66(m,2H),2.26(s,6H),1.22(d,J＝13.3Hz,18H).
13C NMR(101MHz,CDCl3)δ205.98,205.90,155.69,155.61,152.81,149.41,149.31,139.68,139.53,120.21,120.08,85.29,82.93,82.55,79.07,77.91,77.49,77.38,77.17,76.85,67.12,67.05,67.00,65.48,65.09,50.41,46.67,46.63,36.67,36.61,28.51,28.44,28.35,28.28,27.31,27.27.
MALDI-TOF-HRMS：C34H50N10O11P2S2的相对分子质量为:900.2577，发现[M+H] +901.2654的峰。
数据表明化合物合成正确。
实施例2
具有吡啶二硫基取代乙酯保护的环二核苷酸前药分子(化合物16)的合成。
Figure PCTCN2020071329-appb-000013
1)向烧瓶中加入3.42g的N-氯代丁二酰亚胺，加入10mL干燥的二氯甲烷，冰浴下滴加2.85g的2-巯基吡啶(溶解在5mL二氯甲烷中)，半小时滴完，继续低温搅拌反应1小时，将2g的2-巯基乙醇溶解在5mL二氯甲烷中，冰浴下滴加至烧瓶中，滴加完后常温搅拌24小时。反应结束后之间快速柱层析分离得到化合物10。
核磁数据为：
1H NMR(400MHz,DMSO)δ9.32(s,2H),8.52(d,J＝4.6Hz,1H),8.00–7.89(m,2H),7.39–7.30(m,1H),3.63(t,J＝6.2Hz,2H),2.96(t,J＝6.2Hz,2H).
13C NMR(101MHz,DMSO)δ159.40,148.84,139.51,122.01,120.57,59.48,41.77.
数据表明化合物合成正确。
2)烧瓶中，加入化合物1(1.68mmol)，5-乙巯基四氮唑(5.07mmol)，3-羟基丙腈(1.68mmol)，15mL乙腈，常温搅拌1小时后加入5.5M叔丁基过氧化氢-癸烷溶液1mL，继续常温搅拌40分钟后，加入适量亚硫酸氢钠水溶液。二氯甲烷多次萃取，有机相合并后饱和食盐水洗涤，无水硫酸钠干燥，过滤，浓缩，柱层析分离得到化合物9(1.27g)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ8.71(dd,J＝31.2,1.2Hz,1H),8.21–8.10(m,1H),8.01(d,J＝7.2Hz,2H),7.60(t,J＝7.4Hz,1H),7.50(t,J＝7.5Hz,2H),7.37(d,J＝7.1Hz,1H),7.26(dd,J＝5.9,2.4Hz,6H),7.16(d,J＝8.7Hz,2H),6.87–6.75(m,4H),6.57–6.37(m,1H),5.36(dd,J＝19.5,14.0Hz,1H),4.46(d,J＝18.9Hz,1H),4.41–4.21(m,4H),3.93(dd,J＝43.4,12.6Hz,1H),3.78(d,J＝5.7Hz,6H),3.53–3.37(m,1H),3.18(ddd,J＝15.2,13.1,8.8Hz,1H),2.82(t,J＝5.8Hz,3H),2.79–2.65(m,2H).
13C NMR(101MHz,CDCl3)δ164.63,158.60,152.16,150.59,150.26,147.39,142.67,139.51,133.32,133.01,130.08,130.03,129.15,128.93,128.11,127.96,127.84,127.79,127.06,113.26,113.14,87.64,87.24,81.05,77.38,77.26,77.06,76.74,62.92,62.76,62.70,62.65,55.26,39.05,19.88,19.81.
数据表明化合物合成正确。
3)将化合物9(1.37mmol)，溶解在20mL乙腈中，加入叔丁胺5mL，常温搅拌20min后，旋干。加入化合物10(1.38mmol)，1-(均三甲苯基-2-砜基)-3-硝基-1,2,4-三唑(6.86mmol)，20mL吡啶溶解，常温搅拌2小时。加入适量水淬灭反应，旋蒸除去大部分溶剂，加入30mL二氯甲烷重新溶解，5重量％的草酸水溶液调节pH值为3，分出有机相，有机相用饱和食盐水洗涤后，无水硫酸钠干燥。过滤，浓缩后柱层析分离得到化合物11(1.05g)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ9.57(d,J＝50.7Hz,1H),8.78(s,1H),8.69(s,1H),8.52–8.44(m,1H),8.18(dd,J＝13.4,3.1Hz,1H),8.04(s,1H),7.71–7.61(m,2H),7.42–7.33(m,4H),7.32–7.23(m,6H),7.21–7.17(m,2H),7.11–7.02(m,4H),6.92–6.76(m,4H),6.54–6.40(m,1H),5.41–5.26(m,1H),4.89(d,J＝5.5Hz,2H),4.54–4.18(m,5H),3.99(d,J＝12.9Hz,1H),3.88(d,J＝12.9Hz,1H),3.84(s,1H),3.82–3.76(m,5H),3.46(dd,J＝17.1,3.0Hz,1H),3.25–3.01(m,3H),2.88–2.63(m,3H).
13C NMR(101MHz,CDCl3)δ159.66,158.82,158.79,158.59,158.55,156.94,152.03,150.57,149.80,149.09,147.36,146.87,144.23,143.14,141.13,139.48,137.32,135.27,132.51,131.39,130.03,130.00,129.85,129.52,129.13,128.70,128.32,128.07,127.92,127.81,127.76,127.03,124.06,122.46,121.26,121.16,120.23,116.47,116.43,114.92,114.90,113.55,113.20,113.13,113.12,113.10,113.08,113.00,87.75,87.69,87.28,81.36,80.69,80.09,77.39,77.27,77.07,76.75,68.09,65.92,65.87,65.83,62.99,62.45,62.40,55.36,55.24,39.07,38.23,38.16,19.84,19.77.
数据表明化合物合成正确。
4)将(0.8mmol)化合物11加入到烧瓶中，加10mL二氯甲烷溶解，冰水浴中加6体积％的二氯乙酸二氯甲烷溶液10mL。继续搅拌5分钟后，加少量甲醇淬灭反应，红色褪去后加饱和碳酸氢钠水溶液中和，萃取后无水硫酸钠干燥，浓缩，柱层析分离得到化合物13(540mg)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ8.74(s,1H),8.62(d,J＝73.6Hz,1H),8.45(dd,J＝4.7,3.5Hz,1H),8.25(d,J＝3.3Hz,1H),8.04–7.96(m,3H),7.64(dt,J＝4.6,1.5Hz,2H),7.53(ddd,J＝29.9,14.9,7.4Hz,4H),7.10(ddd,J＝5.2,4.1,2.4Hz,1H),6.44(dd,J＝9.1,5.5Hz,1H),5.34(t,J＝5.5Hz,1H),4.52–4.36(m,3H),4.31(dt,J＝12.3,6.0Hz,2H),3.90(dd,J＝38.9,12.8Hz,2H),3.10(t,J＝6.3Hz,2H),2.80(t,J＝6.0Hz,2H),2.69(dd,J＝14.1,5.4Hz,1H).
13C NMR(101MHz,CDCl3)δ166.62,164.98,158.92,152.14,152.10,150.81,150.07,149.86,145.25,144.40,142.96,137.29,133.60,133.26,133.10,132.14,129.17,128.95,127.95,127.84,124.24,121.29,120.32,116.49,116.45,87.63,87.58,87.05,80.51,77.28,66.03,62.85,62.54,62.50,39.15,39.11,38.34,38.27,19.87,19.80.
数据表明化合物合成正确。
5)烧瓶中加入化合物11(0.45mmol)，加入乙腈6mL，叔丁胺2mL，室温下搅拌30分钟后，旋蒸除去溶剂得到化合物12。加入化合物13(0.37mmol)，1-(均三甲苯基-2-砜基)-3-硝基-1,2,4-三唑(1.11mmol)，加入15mL超干吡啶，室温下搅拌2小时。加少量水淬灭反应，旋蒸除去溶剂后，加入20mL二氯甲烷溶解，加入适量5质量％的草酸水溶液，分出有机相干燥，过滤浓缩，加入3体积％的二氯乙酸的二氯甲烷溶液，搅拌5min后加入适量甲醇，饱和碳酸氢钠水溶液中和，萃取，有机相干燥，过滤，浓缩，柱层析分离得到化合物14(350mg)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ8.67(dd,J＝29.4,8.5Hz,2H),8.51–8.20(m,4H),7.96(t,J＝8.9Hz,4H),7.70–7.33(m,10H),7.05(dt,J＝11.0,7.2Hz,2H),6.51(dd,J＝13.0,6.4Hz,1H),6.45–6.33(m,1H),5.38(s,1H),5.24(dd,J＝17.5,5.5Hz,1H),4.52–4.20(m,10H),3.79(dt,J＝19.8,11.3Hz,3H),3.23–2.89(m,6H),2.78(d,J＝18.5Hz,3H),2.68–2.51(m,1H),1.22(s,3H).
13C NMR(101MHz,CDCl3)δ165.07,158.84,158.77,158.70,152.58,151.94,151.49,150.64,150.18,149.83,149.76,143.02,141.81,137.32,133.41,133.36,132.85,132.77,128.77,128.75,128.69,128.08,128.05,124.14,123.59,121.28,121.21,120.21,120.12,116.73,87.40,86.64,84.54,80.22,77.33,66.06,65.77,62.67,38.92,38.15,38.08,29.69,19.86,19.79,0.02.
数据表明化合物合成正确。
6)将化合物14(0.23mmol)加入叔丁胺/乙腈(1：3，v/v)10mL，常温搅拌20min后，旋蒸除去溶剂，加入100mL吡啶溶解，加入1-(均三甲苯基-2-砜基)-3-硝基-1,2,4-三唑(1.38mmol)，常温反应4小时后除去溶剂，加入20mL二氯甲烷溶解，加入适量5质量％的草酸水溶液，分出有机相干燥，过滤浓缩，柱层析分离得到化合物15(181mg)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ9.76(s,2H),8.76(d,J＝9.7Hz,2H),8.53–8.36(m,2H),8.36–8.18(m,2H),7.68–7.55(m,3H),7.37–7.26(m,5H),7.14–6.98(m,8H),6.45(dd,J＝13.8,6.2Hz,2H),5.71–5.36(m,2H),4.93(s,4H),4.66–4.32(m,9H),4.12(d,J＝5.1Hz,1H),3.67–3.34(m,2H),3.11(t,J＝6.2Hz,4H),2.73(dd,J＝13.3,7.5Hz,2H),1.26(s,2H).
13C NMR(101MHz,CDCl3)δ167.30,158.86,158.68,158.57,157.10,157.06,152.49,152.42,152.35,151.38,151.33,151.24,151.16,149.81,148.67,148.60,143.12,142.92,142.76,142.55,137.22,137.18,129.77,123.49,123.39,123.28,122.28,122.26,121.19,120.10,120.08,114.85,114.55,85.58,85.23,83.03,82.59,79.03,78.17,77.84,77.48,77.36,77.16,76.84,68.21,66.14,66.09,65.93,65.88,65.44,64.99,53.52,38.29,38.23,38.12,38.05,38.00,36.29,31.88,29.65,29.62,29.32,22.66,14.13.
数据表明化合物合成正确。
7)向烧瓶中加入化合物15(100mg)，加入甲醇5mL溶解，加入500μL二异丙胺，常温搅拌4小时候，旋干溶剂，直接柱层析分离得到化合物16(65mg)。
核磁数据为：
1H NMR(400MHz,CDCl3)δ8.33(d,J＝8.4Hz,2H),7.94(d,J＝8.1Hz,2H),6.41(dd,J＝13.9,7.4Hz,2H),6.20(d,J＝30.1Hz,4H),5.45(d,J＝59.0Hz,2H),4.51(dd,J＝16.6,8.1Hz,3H),4.39(t,J＝8.3Hz,2H),4.16(tt,J＝ 9.5,4.9Hz,5H),3.17(dd,J＝11.4,6.6Hz,4H),2.82–2.66(m,2H),2.26(s,6H),1.22(d,J＝13.3Hz,18H).
MALDI-TOF-HRMS：C34H38N12O10P2S4的相对分子质量为:964.1192，发现[M+H] +965.1271的峰。
数据表明化合物合成正确。
对比例1
cddA35(结构如表2中所示)参照文献(Wang,B.；Wang,Z.；Javornik,U.；Xi,Z.；Plavec,J.,Computational and NMR spectroscopy insights into the conformation of cyclic di-nucleotides.Sci Rep 2017,7(1),16550.)中的方法进行制备。
cddA35核磁数据为：
1H NMR(400MHz,D2O)δ8.15(s,2H),7.80(s,2H),6.16(d,J＝6.4Hz,2H),5.22–5.04(m,2H),4.26(d,J＝9.8Hz,4H),4.02(dd,J＝11.2,4.1Hz,2H),3.05–2.98(m,2H),2.75–2.62(m,2H).
13C NMR(101MHz,D2O)δ154.39,151.78,146.71,139.32,118.08,84.87,83.30,83.19,70.16,62.25,46.71,39.01,8.26.
数据表明化合物合成正确。
测试例1
两种环二核苷酸前药分子的油水分配系数logP的测定。
1)化合物溶液配制：称取一定量的化合物8、化合物16、化合物cddA35分别用DMSO溶液溶解并配制成质量浓度为1mg/mL的母液。
2)从配好的母液中取20uL加入到装有1mL正辛醇和1mL纯净水(10mM K 2HPO 4pH7.0)混合液的EP管中，振荡涡旋2小时，4℃静置过夜分层。分离两相分别取400uL。3000转离心5分钟，以相应的0.22μM滤膜过滤，相同的HPLC参数条件下测定两相吸收峰的面积，重复实验三次，对正辛醇相和水相中的峰面积比值取log计算得到目标化合物的logP值，如表2所示。
液相条件：仪器使用Agilent 1260HPLC，色谱柱：Agilent ZORABX SB-C18 5μm[4.6x 150mm]，柱温25℃，进样10uL，检测波长254nM，色谱梯度方法如下表1。
表1、HPLC液相梯度方法
时间(min) 体积％A(10mM TEAA缓冲液) 体积％B(MeCN) 流速(mL/min)
1 98 2 1
6 0 100 1
10 0 100 1
13 98 2 1
15 98 2 1
表2
Figure PCTCN2020071329-appb-000014
Figure PCTCN2020071329-appb-000015
通过表2的结果可以看出，使用取代巯基乙醇保护磷酸酯后，其logP有很大的提高。相比没有磷酸负电荷裸露的cddA35来说，其logP值为-3.43，基本无脂溶性。而采用硫代酰乙酯保护的化合物8和二巯基乙醇保护的化合物16，其logP分别为1.93和0.85，极大提高了脂溶性，有利于化合物透过细胞膜，进入细胞内发挥免疫刺激作用。
测试例2
两种环二核苷酸前药分子在HEK293T细胞中的活性测定
原理：双荧光报告基因实验：环二核苷酸前药分子进入细胞后，会经过细胞胞质内的硫酯酶或者还原性环境，从而硫酯键断裂或者二硫键断裂，进而得到的中间体，β位的硫原子会亲核进攻α位碳原子，从而形成环硫乙烷离去，释放母药具有负电荷的磷酸二酯结构的环二核苷，结合激活细胞内STING蛋白，磷酸化干扰素调节因子3(IRF3)，磷酸化的IRF3进入细胞核内结合β干扰素基因的启动子，引起干扰素的表达，基于此，将干扰素基因启动子引入到萤火虫荧光素酶的启动子中，从而可以利用双荧光报告基因方法测定前药分子的细胞活性。
实验材料：
(1)化合物溶液配制：化合物8、化合物16分别用DMSO溶解，配制成终浓度为1mM。cddA35用水溶解，同样配制成浓度为1mM。
(2)质粒溶液配制：pcDNA3.1-hSTING-wt质粒配置为浓度400ng/μL，pGL3-IFNβ质粒配置为浓度400ng/μL，pGL4.74-Rluc质粒配置为浓度100ng/μL。
测试方法：
HEK 293T细胞汇合度达到约70％，将原培养基换成无血清培养基opti-DMEM 500mL待转。配制以上成分溶液，其中包括pcDNA3.1-hsting-wt 2μL,pGL3-IFNβ1μL,pGL4.74-Rluc 1μL,opti-DMEM培养基补足50uL/孔，四孔平行。以上溶液混合物分别加入50μL 2/50Lipo2000转染液混合，混合物室温静置15min之后混合均匀，100μL/孔转染，4h后加入1mL 37℃DMEM培养基。18h后将培养基进一步换成有或者无血清培养基opti-DMEM500mL，8μL(5μM)化合物8、16和cddA35混合液92μL opti-DMEM混匀，静置15min，100μL/孔转染，4h后加入1mL 37℃DMEM培养基，继续培养24h。24h之后检测双荧光结果，如图1所示。
从图1的结果可以看出，未经修饰的高负电性的环二核苷cddA很难跨过细胞膜，而本发明提供的两种采用磷酸三酯保护的环二核苷酸化合物8和化合物16能够在没有任何转染试剂的作用下，自主跨过细胞膜，在细胞质内由于硫酯酶或者还原性环境作用下，释放出母药分子cddA35，从而激活STING通路，引起干扰素的表达。
测试例3
两种环二核苷酸前药分子的在单核细胞系THP-1细胞中的活性测定
测试方法：
THP-1-Lucia细胞汇合度达到约80％，将化合物8、化合物16以及药物ADU-S100按照一定的浓度直接溶解到培养基中加入到24孔板中，四孔平行。在37℃、5％CO 2的条件下继续培养24h。培养结束后，测试荧光素酶活性。其中，化合物8、化合物16以及药物ADU-S100在10μM作用浓度下在单核细胞系THP-1中的I型干扰素测试结果如图2所示；化合物8和化合物16在THP-1细胞系中引起I型干扰素表达的EC 50值则通过多个浓度的连续稀释物的剂量-响应曲线来确定，如图3所示。
从图2结果可知，在单核细胞THP-1细胞系中，在无任何转染试剂作用下，在10μM作用浓度下，化合物8和化合物16都可以引起I型干扰素的强烈表达，其活性远高于目前已经进入临床二期的药物ADU-S100。从图3可知，化合物8和化合物16在THP-1细胞系中引起I型干扰素表达的EC 50值分别为4.4nM和355nM，相比对照药物ADU-S100(专利CN108430503A中的参照物化合物2’3’-RR-(A)(A))的EC 50值为41.5μM，化合物16和化合物8的EC 50值分别提高了9430倍和117倍，活性有相当大的提高。
测试例4
化合物8的抗癌活性测试
实验材料：
小鼠：BABL/C雌性小鼠，体重18-22g。来源广东省动物实验中心，小鼠以颗粒饲料喂养，自由摄食和饮水。
肿瘤细胞株：CT-26-Luc细胞
小鼠肿瘤模型建立：细胞培养、传代，在细胞对数期收集细胞，做成浓度为(1.0×10 7)每毫升的细胞悬液，小鼠右前肢腋下注射0.1mL细胞悬液(细胞数目为1.0×10 6个/只)，10天左右肿瘤长至直径约为5mm致癌成功，随机均分为三组。
阴性对照组：生理盐水溶液
阳性对照组：ADU-S100(临床II期药)，剂量1mg/kg
实验组：化合物8，剂量1mg/kg
给药方法：肿瘤接种生长10天后开始给药，瘤内注射，50微升/只，每两天给药一次，一共给药三次。通过化学发光方法，使用IVIS Spectrum Imaging System(小动物活体成像系统)观察小鼠内肿瘤生长状况。成像前，荧光素小分子准备：用PBS溶液配制适量荧光素钠盐15mg/mL，荧光素钠订购于上海翊圣生物有限公司。提前准备氧气、异氟烷。通过腹腔注射荧光素100μL，将小鼠放入麻醉盒中，待动物麻醉后，将小鼠转移至成像仪中的成像室内。根据需要，调整小鼠的姿势。成像参数为：自动曝光，Binning值为2，F值为8。成像时间选择10分钟。成像后，将荧光值转换为光子数，统计个实验组总的荧光光子数。成像仪为PerkinElmer公司的IVIS Spectrum成像平台。其结果如图4所示。其中图4a、4b、4c、4d、4e和4f中数字代表圆形区域(ROI，感兴趣区)内总的荧光光子数，图4g和4h则分别为各实验组的总荧光光子数的统计图。
各组小鼠的肿瘤体积大小和存活率结果如图5所示。其中，肿瘤体积大小通过游标卡尺测量肿瘤长、宽，按照公式(体积＝长×宽×宽/2)计算得到，存活率＝各实验组肿瘤体积小于2000立方毫米的小鼠数/各实验组小鼠总数。
从图4和图5结果可知，在CT-26小鼠肿瘤模型中，在给药1mg/kg剂量下，相比与对照药物ADU-S100仅能控制小鼠肿瘤的生长，而使用本发明中化合物8的实验组小鼠肿瘤基本消失，进而说明化合物8具有良好的抗肿瘤活性，其效果优于临床用药ADU-S100。

Claims (12)

  1. 一种环二核苷酸前药分子或其立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐，其特征在于，该环二核苷酸前药分子具有如式I、式II或式III所示的结构：
    Figure PCTCN2020071329-appb-100001
    在式I-式III中，
    各个B 1和B 2各自独立地为天然碱基或人工修饰的碱基；所述天然碱基选自鸟嘌呤基、腺嘌呤基、胞嘧啶基、胸腺嘧啶基和尿嘧啶基；所述人工修饰的碱基为将功能基团进行硫代、卤代或甲基化方法修饰而得到的碱基，所述功能基团为硝基吲哚基团、氨基吲哚基团、黄嘌呤基团或次黄嘌呤基团；
    各个X 1和X 2各自独立地为-H、-OCH 3或-F；
    各个Y 1和Y 2各自独立地选自
    Figure PCTCN2020071329-appb-100002
    R 1、R 2、R 3和R 4各自独立地选自取代或未取代的C 1-C 10的脂烃基、取代或未取代的C 6-C 11的芳烃基、五元或六元杂环基；且R 1、R 2、R 3和R 4中任选存在的取代基各自独立地选自C 1-C 5的烷基、C 1-C 5的烷氧基和卤素；
    n 1、n 2、n 3和n 4各自独立地为1-5的整数。
  2. 根据权利要求1所述的环二核苷酸前药分子，其中，Y 1和Y 2相同；B 1和B 2相同；X 1和X 2相同。
  3. 根据权利要求1或2所述的环二核苷酸前药分子，其中，所述环二核苷酸前药分子具有式(1)-式(10)所示的任意一种结构：
    Figure PCTCN2020071329-appb-100003
    Figure PCTCN2020071329-appb-100004
    其中，式(1)-式(10)中的基团的定义与权利要求1或2中的定义对应相同。
  4. 根据权利要求3所述的环二核苷酸前药分子，其中，在式(1)-式(10)中，
    B 1和B 2相同，且选自鸟嘌呤基、腺嘌呤基、胞嘧啶基、胸腺嘧啶基和尿嘧啶基；
    X 1和X 2均为-H；
    R 1、R 2、R 3和R 4相同，且选自取代或未取代的C 1-C 6的脂烃基、取代或未取代的C 6-C 11的芳烃基、五元或六元杂环基；且R 1、R 2、R 3和R 4中任选存在的取代基选自C 1-C 5的烷基。
  5. 根据权利要求1所述的环二核苷酸前药分子，其中，所述环二核苷酸前药分子具有式(11)所示的结构，
    Figure PCTCN2020071329-appb-100005
  6. 根据权利要求1所述的环二核苷酸前药分子，其中，所述环二核苷酸前药分子具有式(12)所示的结构，
    Figure PCTCN2020071329-appb-100006
  7. 一种制备权利要求1-6中任意一项所述的环二核苷酸前药分子的方法，该方法包括：
    a)将第一核苷单体化合物、第二核苷单体化合物和第一缩合剂在第一液态反应介质中进行第一接触反应，得到线性二核苷酸中间体；
    b)在碱性条件下，将所述线性二核苷酸中间体进行脱氰乙基反应，然后将所得反应产物与第二缩合剂在第二液态反应介质中进行第二接触反应；
    c)将进行所述第二接触反应后获得的带有保护基的环二核苷酸前药分子进行脱保护基反应；
    其中，所述第一核苷单体化合物具有式(13)或式(14)所示的结构：
    Figure PCTCN2020071329-appb-100007
    所述第二核苷单体化合物具有式(15)或式(16)所示的结构：
    Figure PCTCN2020071329-appb-100008
    其中，Q表示B 1和B 2所示的碱基中的环外氨基上的保护基，且式(13)-式(16)中的所述Q各自独立地为酰基；
    优选地，所述保护基为苯氧乙酰基或4-异丙基苯氧乙酰基。
  8. 根据权利要求7所述的方法，其中，进行所述第一接触反应的条件包括：温度为0-50℃，时间为2-8h；
    优选地，进行所述第二接触反应的条件包括：温度为0-50℃，时间为2-8h。
  9. 根据权利要求7所述的方法，其中，所述第一缩合剂和所述第二缩合剂各自独立地选自1-(均三甲苯基-2-砜基)-3-硝基-1,2,4-三唑、2,4,6-三异丙基苯基砜基-3-硝基1,2,4-三唑、2,4,6-三异丙基苯磺酰氯、2,4,6-三甲基苯磺酰氯和1H-四唑和N-甲基咪唑中的至少一种；
    优选地，在步骤a)中，相对于1mol的第一核苷单体化合物，所述第一缩合剂的用量为2-3mol；
    优选地，在步骤b)中，相对于1mol的线性二核苷酸中间体，所述第二缩合剂的用量为4-5mol。
  10. 根据权利要求7所述的方法，其中，在步骤b)中，所述碱性条件由叔丁胺的乙腈溶液提供；
    优选地，所述碱性条件由体积比为1：(1-5)的叔丁胺与乙腈形成的混合溶液提供。
  11. 根据权利要求7所述的方法，其中，所述脱保护基反应在体积比为1：(5-20)的二异丙胺和甲醇形成的混合溶液存在下进行。
  12. 权利要求1-6中任意一项所述的环二核苷酸前药分子或其立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐在制备药物中的应用；
    优选地，所述药物为抗病毒药物、抗细菌感染药物或抗癌药物。
PCT/CN2020/071329 2019-01-10 2020-01-10 环二核苷酸前药分子及其制备方法和应用 Ceased WO2020143740A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102596204A (zh) * 2009-07-06 2012-07-18 昂托瑞公司 新的核酸前药及其方法用途
CN107849084A (zh) * 2015-12-03 2018-03-27 葛兰素史密斯克莱知识产权发展有限公司 作为sting调节剂的环状嘌呤二核苷酸
CN108430503A (zh) 2015-10-28 2018-08-21 艾杜罗生物科技公司 用于激活“干扰素基因刺激物”依赖性信号传导的组合物和方法

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102199183B (zh) * 2010-03-26 2013-12-18 北京大学 环二鸟苷酸及其类似物和制备方法
PL2931738T3 (pl) * 2012-12-13 2019-07-31 Aduro Biotech, Inc. Kompozycje zawierające cykliczne dinukleotydy purynowe o zdefiniowanej stereochemii i sposoby ich otrzymywania i zastosowania
RS59500B1 (sr) * 2013-05-18 2019-12-31 Aduro Biotech Inc Sastavi i metode za aktiviranje signaliziranja koje je zavisno od „stimulatora gena za interferon“
JP2016538344A (ja) * 2013-11-19 2016-12-08 ザ・ユニバーシティ・オブ・シカゴＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｃｈｉｃａｇｏ 癌処置としてのｓｔｉｎｇアゴニストの使用
EP3546473B1 (en) * 2014-12-16 2025-12-10 Kayla Therapeutics Fluorinated cyclic [(2',5')p(3',5')p]-dinucleotides for cytokine induction
CR20180101A (es) * 2015-08-13 2018-04-12 Merck Sharp & Dohme Compuestos di-nucleóticos cíclicos como agonistas de sting
US11033569B2 (en) 2016-07-06 2021-06-15 Sperovie Biosciences, Inc. Compounds,Compositions, and methods for the treatment of disease
US9905856B1 (en) 2016-12-28 2018-02-27 Nanotek Instruments, Inc. Flexible and shape-conformal rope-shape alkali metal-sulfur batteries
US10903527B2 (en) 2017-05-08 2021-01-26 Global Graphene Group, Inc. Rolled 3D alkali metal batteries and production process

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102596204A (zh) * 2009-07-06 2012-07-18 昂托瑞公司 新的核酸前药及其方法用途
CN108430503A (zh) 2015-10-28 2018-08-21 艾杜罗生物科技公司 用于激活“干扰素基因刺激物”依赖性信号传导的组合物和方法
CN107849084A (zh) * 2015-12-03 2018-03-27 葛兰素史密斯克莱知识产权发展有限公司 作为sting调节剂的环状嘌呤二核苷酸

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BAIFAN WANG, ZHENGHUA WANG, UROŠ JAVORNIK, ZHEN XI ,JANEZ PLAVEC: "Computational and NMR Spectroscopy Insights into the Conformation of Cyclic di-Nucleotides", SCIENTIFIC REPORTS, vol. 7, no. 1, 16550, 29 November 2017 (2017-11-29), pages 1 - 11, XP055717235, ISSN: 2045-2322, DOI: 10.1038/s41598-017-16794-4 *
See also references of EP3901161A4
WANG, B.WANG, Z.JAVORNIK, U.XI, Z.PLAVEC, J.: "Computational and NMR spectroscopy insights into the conformation of cyclic dinucleotides", SCI REP, vol. 7, no. 1, 2017, pages 16550, XP055717235, DOI: 10.1038/s41598-017-16794-4

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