Cyclic dinucleotide prodrug molecule and preparation method and application thereof

Abstract

The present invention relates to the field of medicines, and discloses a cyclic dinucleotide prodrug molecule and a preparation method and application thereof. The cyclic dinucleotide prodrug molecule has a structure as shown in Formula I, Formula II or Formula III, can autonomously cross the cell membrane, release the cyclic dinucleotide, and has high cell activity.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a cyclic dinucleotide prodrug molecule, a preparation method of the cyclic dinucleotide prodrug molecule, and applications of the cyclic dinucleotide prodrug molecule. Background Art

[0002] Cyclic dinucleotides are a new type of second messenger molecule found in bacteria and mammals. In mammalian cells, cyclic dinucleotides can bind to the immune stimulatory factor (STING) to stimulate immune response. It can induce the expression of cytokines such as interferon-β and NF-κB, promote CD8 + Proliferation, differentiation and maturation of T cells.

[0003] Because cyclic dinucleotides have a strong immunostimulatory effect, they can be used in the treatment of viral and bacterial infections and cancer. In particular, they can show good results when used in combination with traditional immunotherapy. Currently, Aduro and Novartis' ADU-S100 have entered Phase II clinical trials, and MSD's MK-1454 has entered Phase I clinical trials.

[0004] There are two phosphodiester bonds in the molecular structure of cyclic dinucleotides. The negatively charged phosphodiester bonds will first cause the drug to have poor lipid solubility, hindering the effective crossing of the cell membrane by cyclic dinucleotides. In addition, the phosphodiester bonds are also easily hydrolyzed, causing instability of the drug molecules in the circulatory system.

[0005] At present, auxiliary materials are mainly used for cyclic dinucleotide drug delivery systems. For example, early literature reported the use of lentivirus or adenovirus to package cyclic dinucleotide synthase, which enters the cell and synthesizes cyclic dinucleotides through enzyme catalysis to promote immune response, but this method has great risks and is not widely used.

[0006] There have been reports on the delivery of cyclic dinucleotide drugs by using liposomes or lipid nanoparticles, cell-penetrating peptides, protein gels containing polycationic amino acids, etc. as carriers, but the use of such carriers will reduce the drug loading capacity, and the introduction of carriers may cause some potential cytotoxicity.

[0007] The current clinical method to ensure the effective cellular uptake of cyclic dinucleotides is intratumoral injection. Although some articles have reported that cells can take up cyclic dinucleotides through direct intratumoral injection, the uptake rate is low, so larger doses are required. Such large doses may cause over-activation of the immune system, thereby causing systemic or local inflammatory responses.

[0008] Therefore, there are still some problems in how to effectively ensure the uptake of cyclic dinucleosides by cells. Summary of the invention

[0009] The purpose of the present invention is to overcome the defect in the prior art that cyclic dinucleotides are difficult to be effectively taken up by cells.

[0010] In order to achieve the above-mentioned object, the first aspect of the present invention provides a cyclic dinucleotide prodrug molecule or its stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, and pharmaceutically acceptable salts, wherein the cyclic dinucleotide prodrug molecule has a structure as shown in Formula I, Formula II or Formula III:

[0011]

[0012] In Formula I to Formula III,

[0013] Each of B1 and B2 is independently a natural base or an artificially modified base; the natural base is selected from guanine, adenine, cytosine, thymine and uracil; the artificially modified base is a base obtained by modifying the functional group by thiolation, halogenation or methylation, and the functional group is a nitroindole group, an aminoindole group, a xanthine group or a hypoxanthine group;

[0014] Each X1 and X2 are independently -H, -OCH3 or -F;

[0015] Each Y1 and Y2 are independently selected from

[0016] in,

[0017] R1, R2, R3 and R4 are each independently selected from substituted or unsubstituted C1-C 10 Aliphatic, substituted or unsubstituted C6-C 11 An aromatic hydrocarbon group, a five-membered or six-membered heterocyclic group; and the substituents optionally present in R1, R2, R3 and R4 are each independently selected from a C1-C5 alkyl group, a C1-C5 alkoxy group and a halogen;

[0018] n1, n2, n3 and n4 are each independently an integer of 1-5.

[0019] The second aspect of the present invention provides a method for preparing the cyclic dinucleotide prodrug molecule as described above, the method comprising:

[0020] a) subjecting a first nucleoside monomer compound, a second nucleoside monomer compound and a first condensing agent to a first contact reaction in a first liquid reaction medium to obtain a linear dinucleotide intermediate;

[0021] b) subjecting the linear dinucleotide intermediate to a decyanoethylation reaction under alkaline conditions, and then subjecting the obtained reaction product to a second contact reaction with a second condensing agent in a second liquid reaction medium;

[0022] c) performing a deprotection reaction on the cyclic dinucleotide prodrug molecule with a protecting group obtained after the second contact reaction;

[0023] Wherein, the first nucleoside monomer compound has a structure shown in formula (13) or formula (14):

[0024]

[0025] The second nucleoside monomer compound has a structure shown in formula (15) or formula (16):

[0026]

[0027] Wherein, Q represents a protecting group on the exocyclic amino group in the bases represented by B1 and B2, and the Q in formula (13) to formula (16) are each independently an acyl group.

[0028] The third aspect of the present invention provides a use of the cyclic dinucleotide prodrug molecule as described above or its stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, and pharmaceutically acceptable salts in the preparation of drugs.

[0029] The cyclic dinucleotide prodrug molecule provided by the present invention can autonomously cross the cell membrane, release the cyclic dinucleotide, and has high cell activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the double fluorescence test result in Test Example 2;

[0031] Figure 2 is the result of the type I interferon test in the monocytic cell line THP-1 in Test Example 3;

[0032] Figure 3 The EC of the compound in Test Example 3 that induces type I interferon expression in THP-1 cells 50 value;

[0033] Figure 4 a is the tumor imaging image of the empty negative control group mice on day 0 of administration in Test Example 4, b is the tumor imaging image of the experimental group mice administered with ADU-S100 on day 0 of administration in Test Example 4, c is the tumor imaging image of the experimental group mice administered with compound 8 on day 0 of administration in Test Example 4, d is the tumor imaging image of the negative control group mice on day 9 of administration in Test Example 4, e is the tumor imaging image of the experimental group mice administered with ADU-S100 on day 9 of administration in Test Example 4, f is the tumor imaging image of the experimental group mice administered with compound 8 on day 9 of administration in Test Example 4, g is the statistical graph of the total fluorescence amount of tumor imaging of the mice in each experimental group on day 0 of administration in Test Example 4, and h is the statistical graph of the total fluorescence amount of tumor imaging of the mice in each experimental group on day 9 of administration in Test Example 4;

[0034] Figure 5 In the figure, a is the tumor volume of mice in different experimental groups in Test Example 4, and b is the survival rate of mice in different experimental groups in Test Example 4. DETAILED DESCRIPTION

[0035] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0036] As mentioned above, the first aspect of the present invention provides a cyclic dinucleotide prodrug molecule or its stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, and pharmaceutically acceptable salts.

[0037] The inventors of the present invention unexpectedly discovered during research that by introducing a phosphate protecting group containing a thioester structure and / or a disulfide bond structure as shown in Y1 and / or Y2 into a cyclic dinucleotide molecule, a phosphotriester cyclic dinucleotide prodrug molecule is formed, which can eliminate the influence of the negative charge of phosphate, remove the protecting group after entering the cytoplasm, and release the biologically active cyclic dinucleotide molecule, thereby overcoming the defect that cyclic dinucleotides are difficult to be effectively taken up by cells.

[0038] In the present invention, the "C1-C 10 The term "aliphatic group" refers to an alkyl or alkenyl group having 1 to 10 carbon atoms, 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, isooctyl, vinyl, propenyl, etc.

[0039] The “C1-C6 aliphatic group” refers to an alkane group or an alkene group having a total of 1 to 6 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, isohexyl, vinyl, propenyl, etc.

[0040] The "C6-C 11 The term "aromatic hydrocarbon group" refers to an aromatic ring that has lost one or more hydrogen atoms, including an aromatic ring connected to other functional groups or substituents, such as wait.

[0041] In the present invention, 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, wait.

[0042] In the present invention, the "C1-C5 alkyl group" refers to an alkyl group with a total carbon atom number of 1-5, for example, it can be methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, and neopentyl.

[0043] In the present invention, the "C1-C5 alkoxy group" refers to an alkoxy group having 1 to 5 carbon atoms, for example, methoxy, ethoxy, propoxy, butoxy, pentyloxy, etc.

[0044] In the present invention, the halogen may be fluorine, chlorine, bromine or iodine.

[0045] Preferably, R1, R2, R3 and R4 are each independently selected from substituted or unsubstituted C1-C6 aliphatic groups, substituted or unsubstituted C6-C 11 and the substituents optionally present in R1, R2, R3 and R4 are each independently selected from C1-C5 alkyl, C1-C5 alkoxy and halogen.

[0046] According to a preferred embodiment, in the structure represented by Formula I, Formula II or Formula III, Y1 and Y2 are the same; B1 and B2 are the same; X1 and X2 are the same.

[0047] According to another preferred embodiment, in Y1 and Y2, n1, n2, n3 and n4 are the same and are integers of 1-3.

[0048] According to another preferred embodiment, the cyclic dinucleotide prodrug molecule has any one of the structures shown in formula (1) to formula (10):

[0049]

[0050] The definitions of the groups in formula (1) to formula (10) are the same as those described above.

[0051] In a particularly preferred case, the cyclic dinucleotide prodrug molecule has any one of the structures shown in formula (1) to formula (10), and in formula (1) to (10),

[0052] B1 and B2 are the same and are selected from guanine, adenine, cytosine, thymine and uracil;

[0053] X1 and X2 are both -H;

[0054] R1, R2, R3 and R4 are the same and are selected from substituted or unsubstituted C1-C6 aliphatic groups, substituted or unsubstituted C6-C 11an aromatic hydrocarbon group, a five-membered or six-membered heterocyclic group; and the substituents optionally present in R1, R2, R3 and R4 are selected from C1-C5 alkyl groups.

[0055] The present invention provides a preferred embodiment, wherein the cyclic dinucleotide has a structure shown in formula (11),

[0056]

[0057] According to another preferred embodiment of the present invention, the cyclic dinucleotide has a structure shown in formula (12),

[0058]

[0059] As mentioned above, the second aspect of the present invention provides a method for preparing the cyclic dinucleotide prodrug molecule of the present invention.

[0060] According to the present invention, the protecting group used in the preparation of the cyclic dinucleotide prodrug molecule is an acyl group, for example, benzoyl, isobutyryl, acetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl, etc.

[0061] Particularly preferably, the protecting group is selected from phenoxyacetyl or 4-isopropylphenoxyacetyl.

[0062] In formula (13) and formula (14), DMTr- is 4,4'-dimethoxytriphenylmethyl.

[0063] In the present invention, there is no particular limitation on the amount of each substance used in the method, and those skilled in the art can select the amount according to the needs of each reaction.

[0064] Preferably, the conditions for carrying out the first contact reaction include: temperature of 0-50° C. and time of 2-8 h.

[0065] Preferably, the conditions for carrying out the second contact reaction include: temperature of 0-50° C. and time of 2-8 h.

[0066] Preferably, the first condensing agent and the second condensing agent are each independently selected from at least one of 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole, 2,4,6-triisopropylphenylsulfone-3-nitro-1,2,4-triazole, 2,4,6-triisopropylbenzenesulfonyl chloride, 2,4,6-trimethylbenzenesulfonyl chloride, 1H-tetrazole and N-methylimidazole.

[0067] In the present invention, the first liquid reaction medium and the second liquid reaction medium are commonly used reaction media in the art, for example, pyridine, dichloromethane, acetonitrile, dioxane, tetrahydrofuran, etc. Those skilled in the art can select them according to the type of reaction.

[0068] Preferably, in step a), the amount of the first condensing agent used is 2-3 mol relative to 1 mol of the first nucleoside monomer compound.

[0069] Preferably, in step b), the amount of the second condensing agent used is 4-5 mol relative to 1 mol of the linear dinucleotide intermediate.

[0070] In step b), the alkaline condition can be provided by triethylamine, tert-butylamine, diethylamine, diisopropylamine, etc., and preferably, in order to obtain a higher yield of the target product, the alkaline condition of the present invention is provided by an acetonitrile solution of tert-butylamine. Particularly preferably, the alkaline condition is provided by a mixed solution of tert-butylamine and acetonitrile in a volume ratio of 1:(1-5).

[0071] Preferably, the deprotection reaction is carried out in the presence of a mixed solution of diisopropylamine and methanol in a volume ratio of 1:(5-20).

[0072] According to a preferred embodiment of the present invention, the method for preparing the cyclic dinucleotide prodrug molecule of the present invention comprises:

[0073] 1) adding the first nucleoside monomer compound, the second nucleoside monomer compound and 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole to pyridine, reacting at 0-50° C. for 2-8 hours under an inert gas atmosphere to obtain a linear dinucleotide intermediate;

[0074] 2) adding the linear dinucleotide intermediate obtained in step 1) to a mixed solution of tert-butylamine and acetonitrile to react for 10-30 minutes, evaporating the solvent, adding 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole, dissolving it with pyridine, and reacting it at 0-50° C. for 2-8 hours to obtain a cyclic dinucleotide prodrug molecule with a protective group; wherein, in the mixed solution of tert-butylamine and acetonitrile, the volume ratio of tert-butyl group to acetonitrile is 1:(1-5);

[0075] 3) dissolving the cyclic dinucleotide prodrug molecule with a protecting group in a mixed solution of diisopropylamine and methanol, and reacting at 20-30° C. for 3-5 hours to obtain the cyclic dinucleotide prodrug molecule; wherein the volume ratio of diisopropylamine to methanol in the mixed solution of diisopropylamine and methanol is 1:(5-20).

[0076] In the present invention, the synthesis of the first nucleoside monomer compound and the second nucleoside monomer compound can be a commonly used method in the prior art, and those skilled in the art can design a synthesis route based on the structures shown in formula (13) to formula (16) provided by the present invention. The present invention exemplarily provides several specific synthesis methods:

[0077] Method 1:

[0078]

[0079] In method one, R may be R1 or R2, B may be B1 or B2, X may be X1 or X2, and n may be n1 or n2. Method one includes:

[0080] (i) dissolving the thiol-substituted alcohol and triethylamine in dichloromethane, adding a dichloromethane solution of the acyl chloride dropwise at -80°C to -70°C, heating the temperature to 20-30°C to react for 1-3 hours, quenching with water, extracting with an organic solvent, and separating by column chromatography to obtain the thioester-substituted alcohol;

[0081] (ii) adding a nucleoside phosphoramidite monomer, 5-ethylmercaptotetrazole, and the thioester-substituted alcohol obtained in step (i) to anhydrous acetonitrile, reacting for 1-3 hours under an inert gas atmosphere, adding tert-butyl peroxyacid, and quenching by adding a sodium sulfite aqueous solution after 30-60 minutes, extracting with an organic solvent, and separating by column chromatography to obtain a nucleic acid monomer having a thioester protecting group;

[0082] (iii) dissolving the nucleic acid monomer having a thioester protecting group obtained in step (ii) in dichloromethane, then adding dichloroacetic acid, reacting at 20-30° C. for 1-3 hours, and separating by column chromatography to obtain the first nucleoside monomer compound;

[0083] (iv) dissolving the nucleic acid monomer having a thioester protecting group obtained in step (ii) in acetonitrile, then adding tert-butylamine, reacting at 20-30° C. for 1-3 hours, and then spinning off the solvent to obtain the second nucleoside monomer compound.

[0084] Method 2:

[0085]

[0086] In method 2, R may be R3 or R4, B may be B1 or B2, X may be X1 or X2, and n may be n3 or n4. Method 2 includes:

[0087] (i) adding N-chlorosuccinimidyl chloride and thiol (or thiophenol) to dichloromethane, reacting at 10-35° C. for 1-3 hours, adding thiol-substituted alcohol, continuing the reaction for 20-30 hours, then quenching with water, extracting with an organic solvent, and separating by column chromatography to obtain an alcohol containing a disulfide bond structure;

[0088] (ii) adding 3-hydroxypropionitrile, nucleoside phosphoramidite monomer and 5-ethylmercaptotetrazole to anhydrous acetonitrile, reacting for 1-3 hours under an inert gas atmosphere, adding tert-butyl peroxyacid, continuing the reaction for 30-60 minutes, then adding sodium bisulfite aqueous solution to quench, extracting with an organic solvent, and separating by column chromatography to obtain a dicyanoethyl-protected nucleic acid monomer;

[0089] (iii) dissolving the dicyanoethyl protected nucleotide monomer obtained in step (ii) in dichloromethane, adding tert-butylamine, stirring at 20-30° C. for 10-30 min, and then spinning the solvent to dryness, adding 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole and the alcohol containing disulfide bond structure substitution obtained in step (i), dissolving in anhydrous pyridine, reacting under inert gas protection for 4 hours, evaporating the solvent, extracting with an organic solvent, and separating by column chromatography to obtain a nucleotide monomer having disulfide bond substituted alcohol protected phosphate;

[0090] (iv) dissolving the nucleotide monomer having disulfide bond substituted alcohol protected phosphate obtained in step (iii) in dichloromethane, adding dichloroacetic acid and reacting at 20-30° C. for 1-3 hours, and separating by column chromatography to obtain the first nucleoside monomer compound;

[0091] (v) dissolving the nucleotide monomer having disulfide-substituted alcohol-protected phosphate obtained in step (iii) in acetonitrile, adding tert-butylamine, stirring at 20-30° C. for 10-30 min, and then drying the solvent to obtain the second nucleoside monomer compound.

[0092] In method 1 and method 2, there is no particular limitation on the amount of each substance used, and those skilled in the art can select the amount according to the needs of the reaction.

[0093] The third aspect of the present invention provides a use of the cyclic dinucleotide prodrug molecule of the present invention or its stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, and pharmaceutically acceptable salts in the preparation of drugs.

[0094] Preferably, the drug is an antiviral drug, an antibacterial infection drug or an anticancer drug.

[0095] The cyclic dinucleotide prodrug molecule of the present invention can autonomously cross the cell membrane and release the cyclic dinucleotide. The released cyclic dinucleotide can bind to the immunostimulatory factor (STING) to stimulate the immune response and can be used in the preparation of antiviral, antibacterial infection and anticancer drugs.

[0096] The second aspect of the present invention relates to the reaction in which various post-treatment methods conventionally used in the art can be used to post-treat the obtained product. The post-treatment methods include, but are not limited to, extraction, recrystallization, washing, drying, filtering, etc. The present invention will not be repeated here, and the post-treatment methods involved in the embodiments are only used for illustrative purposes and do not represent operations that must be adopted. Those skilled in the art may use other conventional means to replace them.

[0097] The present invention will be described in detail below by way of examples. In the following examples, the solvents, raw materials and reagents used are all commercially available and are analytically pure or chemically pure unless otherwise specified.

[0098] The anhydrous solvents required in the experiment were dried according to conventional methods.

[0099] Instruments and methods for product separation and identification:

[0100] Thin layer chromatography silica gel GF254 was produced by Tianjin Silida Company.

[0101] TLC was analyzed at 254 nm UV, and the column chromatography silica gel was produced by Qingdao Ocean Chemical Company.

[0102] The NMR spectrometer used was a Bruker AVANCE 400 MHz, with TMS as the internal standard.

[0103] High-resolution mass spectrometry was performed using a Varian 7.0T FTMS Fourier transform plasma cyclotron resonance high-resolution mass spectrometer.

[0104] Normal temperature and room temperature both refer to (25±3)℃.

[0105] Example 1

[0106] Synthesis of cyclic dinucleotide prodrug molecule (Compound 8) with thiothioylethyl ester protection.

[0107]

[0108] 1) In a 100 mL flask, add 0.9 mL of 2-mercaptoethanol, 1.8 mL of triethylamine and 10 mL of redistilled dichloromethane to dissolve, add valeryl chloride (1.6 mL, dissolved in 10 mL of dichloromethane) dropwise at -78 ° C, add dropwise for half an hour, continue to react for 1 hour, slowly warm to room temperature, and continue to stir for 1 hour. Add appropriate amount of water to quench the reaction, extract, extract the aqueous phase with dichloromethane twice, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography to obtain compound 2.

[0109] The NMR data are:

[0110] 1H NMR (400MHz, CDCl3) δ3.73 (t, J = 6.1 Hz, 9H), 3.04 (t, J = 6.1 Hz, 9H), 1.23 (s, 41H).

[0111] 13C NMR (101MHz, CDCl3) δ207.36, 61.91, 46.57, 31.49, 27.40.

[0112] The data showed that the compound was synthesized correctly.

[0113] 2) Compound 2 (1.12 mmol), 5-ethylmercaptotetrazole (3.36 mmol) and compound 1 (1.12 mmol) were added to a flask, 10 mL of anhydrous acetonitrile was added, stirred at room temperature for 1 h, and then 1 mL of 5.5 M tert-butyl peroxyacid-decane solution was added. After stirring at room temperature for 40 minutes, sodium bisulfite aqueous solution was added and stirred, and after multiple extractions 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 3 (950 mg).

[0114] The NMR data are:

[0115] 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),7.0 6(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=1.3 Hz,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).

[0116] 13C NMR (101MHz, CDCl3) δ205.84,205.76,166.70,158.57,158.37,156.92,152.07,150.57,149.10,147.35,143.09,139.47,136.15,13 0.04,129.86,129.16,129.13,128.15,127.82,127.76,127.75,127.05,124.07,122.49,116.44,116.39,114.91,113.20,113.12,11 3.10,113.09,113.01,87.79,87.74,87.40,81.38,80.69,80.66,77.37,77.26,77.05,76.73,68.07,66.69,66.67,66.62,63.04,62.38,62.33,61.89,55.24,55.22,55.19,46.63,39.06,31.50,30.95,28.32,28.28,28.25,27.45,27.39,27.30,27.27,19.84,19.77.

[0117] The data showed that the compound was synthesized correctly.

[0118] 3) Add compound 3 (0.5 mmol) to a flask, add 10 mL of dichloromethane to dissolve, and then add 10 mL of 6% by volume dichloroacetic acid-dichloromethane solution under ice bath. After reacting for 5 min, add a small amount of methanol to quench the reaction. Add saturated aqueous sodium bicarbonate solution to neutralize, extract, dry, concentrate, and quickly precipitate by column chromatography to obtain compound 5 (330 mg).

[0119] The NMR data are:

[0120] 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=8.0H z,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=1.0 Hz,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).

[0121] 13C NMR (101MHz, CDCl3) δ205.88,205.79,166.81,156.94,152.09,150.58,149.05,143.11,12 9.87,123.91,122.49,116.46,116.41,114.91,87.78,87.73,87.37,80.62,80.59,77.37,7 7.26,77.06,76.74,68.08,66.71,66.69,66.65,66.63,63.00,62.40,62.35,61.89,46.63,39.14,39.10,31.49,28.35,28.32,28.28,28.25,27.39,27.31,19.87,19.85,19.80,19.78

[0122] The data showed that the compound was synthesized correctly.

[0123] 4) Compound 3 (0.27 mmol) and 10 mL of tert-butylamine / acetonitrile (1:3, v / v) were added to the flask, stirred at room temperature for 20 minutes, and the solvent was removed by rotary evaporation to obtain compound 4. Compound 2 (0.27 mmol) and 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole (1.63 mmol) were added to the flask, and 10 mL of pyridine was added and stirred at room temperature for 2 hours, and then a small amount of water was added to quench the reaction. After the solvent was removed by rotary evaporation, 20 mL of dichloromethane was added to dissolve, and an appropriate amount of 5% by mass oxalic acid aqueous solution was added. The organic phase was separated and dried, filtered and concentrated, and a 3% by volume dichloroacetic acid dichloromethane solution was added. After stirring for 5 minutes, an appropriate amount of methanol was added, and the mixture was neutralized with a saturated sodium bicarbonate aqueous solution, extracted, and the organic phase was dried, filtered, concentrated, and separated by column chromatography to obtain compound 6 (320 mg).

[0124] The NMR data are:

[0125] 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(d dd,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).

[0126] 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, 148.57,148.55,143.66,143.62,142.44,129.73,129.69,123.57,12 3.00,122.15,116.76,116.73,114.81,114.77,114.75,87.44,87.40, 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.

[0127] The data showed that the compound was synthesized correctly.

[0128] 5) Compound 6 (0.22 mmol) was added to 10 mL of tert-butylamine / acetonitrile (1:3, v / v), stirred at room temperature for 20 min, and then the solvent was removed by rotary evaporation. 100 mL of pyridine was added to dissolve the mixture, and 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole (1.35 mmol) was added. After reacting at room temperature for 4 hours, the solvent was removed and 20 mL of dichloromethane was added to dissolve the mixture. An appropriate amount of 5% by mass aqueous oxalic acid solution was added, the organic phase was separated and dried, filtered and concentrated, and then separated by column chromatography to obtain compound 7 (160 mg).

[0129] The NMR data are:

[0130] 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).

[0131] 13C NMR (101MHz, CDCl3) δ205.76,205.74,205.60,205.53,172.28,167.03,157.76, 157.05,157.02,156.60,152.63,152.57,152.48,152.38,152.23,151.49,151.4 1,151.31,151.20,148.70,148.66,148.58,143.05,142.75,142.55,142.32,129.95,129.81,129.58,129.54,123.57,123.44,122.75,122.38,122.35,122.33, 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.

[0132] The data showed that the compound was synthesized correctly.

[0133] 6) Compound 7 (150 mg) was added to a flask, and 5 mL of methanol was added to dissolve it. 500 μL of diisopropylamine was added, and the mixture was stirred at room temperature for 4 hours. The solvent was dried by spin drying, and compound 8 (80 mg) was directly separated by column chromatography.

[0134] The NMR data are:

[0135] 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).

[0136] 13C NMR (101MHz, CDCl3) δ205.98,205.90,155.69,155.61,152.81,149.41,1 49.31,139.68,139.53,120.21,120.08,85.29,82.93,82.55,79.07,77. 91,77.49,77.38,77.17,76.85,67.12,67.05,67.00,65.48,65.09,50.4 1,46.67,46.63,36.67,36.61,28.51,28.44,28.35,28.28,27.31,27.27.

[0137] MALDI-TOF-HRMS: The relative molecular mass of C34H50N10O11P2S2 is 900.2577, and [M+H] + Peak of 901.2654.

[0138] The data showed that the compound was synthesized correctly.

[0139] Example 2

[0140] Synthesis of cyclic dinucleotide prodrug molecule (Compound 16) with pyridyldithio substituted ethyl ester protection.

[0141]

[0142] 1) Add 3.42g of N-chlorosuccinimide to a flask, add 10mL of dry dichloromethane, add 2.85g of 2-mercaptopyridine (dissolved in 5mL of dichloromethane) dropwise under an ice bath, drip completely in half an hour, continue to stir at low temperature for 1 hour, dissolve 2g of 2-mercaptoethanol in 5mL of dichloromethane, add dropwise to the flask under an ice bath, and stir at room temperature for 24 hours after the addition is complete. After the reaction is completed, rapid column chromatography is performed to obtain compound 10.

[0143] The NMR data are:

[0144] 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).

[0145] 13C NMR (101MHz, DMSO) δ159.40,148.84,139.51,122.01,120.57,59.48,41.77.

[0146] The data showed that the compound was synthesized correctly.

[0147] 2) Add compound 1 (1.68 mmol), 5-ethylmercaptotetrazole (5.07 mmol), 3-hydroxypropionitrile (1.68 mmol), and 15 mL of acetonitrile to a flask. After stirring at room temperature for 1 hour, add 1 mL of 5.5 M tert-butyl hydroperoxide-decane solution. Continue stirring at room temperature for 40 minutes, then add an appropriate amount of sodium bisulfite aqueous solution. Extract with dichloromethane several times, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography to obtain compound 9 (1.27 g).

[0148] The NMR data are:

[0149] 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).

[0150] 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.

[0151] The data showed that the compound was synthesized correctly.

[0152] 3) Compound 9 (1.37 mmol) was dissolved in 20 mL of acetonitrile, 5 mL of tert-butylamine was added, stirred at room temperature for 20 min, and then dried. Compound 10 (1.38 mmol), 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole (6.86 mmol) were added, and dissolved in 20 mL of pyridine, and stirred at room temperature for 2 hours. An appropriate amount of water was added to quench the reaction, most of the solvent was removed by rotary evaporation, 30 mL of dichloromethane was added to redissolve, and the pH value was adjusted to 3 with 5 wt% oxalic acid aqueous solution, and the organic phase was separated, washed with saturated brine, and dried over anhydrous sodium sulfate. Filtered, concentrated, and separated by column chromatography to obtain compound 11 (1.05 g).

[0153] The NMR data are:

[0154] 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).

[0155] 13C NMR (101MHz, CDCl3) δ159.66,158.82,158.79,158.59,158.55,156.94,15 2.03,150.57,149.80,149.09,147.36,146.87,144.23,143.14,141.13,1 39.48,137.32,135.27,132.51,131.39,130.03,130.00,129.85,129.52, 129.13,128.70,128.32,128.07,127.92,127.81,127.76,127.03,124.06, 122.46,121.26,121.16,120.23,116.47,116.43,114.92,114.90,113.55,113.20,113.13,113.12,113.10,113.08,113.00,87.75,87.69,87.28,8 1.36,80.69,80.09,77.39,77.27,77.07,76.75,68.09,65.92,65.87,65.83,62.99,62.45,62.40,55.36,55.24,39.07,38.23,38.16,19.84,19.77.

[0156] The data showed that the compound was synthesized correctly.

[0157] 4) Compound 11 (0.8 mmol) was added to a flask, 10 mL of dichloromethane was added to dissolve, 10 mL of 6% by volume dichloroacetic acid in dichloromethane was added to an ice-water bath, stirring was continued for 5 minutes, a small amount of methanol was added to quench the reaction, saturated sodium bicarbonate aqueous solution was added to neutralize after the red color faded, extraction was followed by drying over anhydrous sodium sulfate, concentration, and column chromatography separation to obtain compound 13 (540 mg).

[0158] The NMR data are:

[0159] 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,2 H), 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).

[0160] 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.

[0161] The data showed that the compound was synthesized correctly.

[0162] 5) Compound 11 (0.45 mmol) was added to the flask, 6 mL of acetonitrile and 2 mL of tert-butylamine were added, and the mixture was stirred at room temperature for 30 minutes, and the solvent was removed by rotary evaporation to obtain compound 12. Compound 13 (0.37 mmol), 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole (1.11 mmol), 15 mL of ultra-dry pyridine were added, and the mixture was stirred at room temperature for 2 hours. A small amount of water was added to quench the reaction, and the solvent was removed by rotary evaporation. 20 mL of dichloromethane was added to dissolve the mixture, and an appropriate amount of 5% by mass oxalic acid aqueous solution was added. The organic phase was separated and dried, filtered and concentrated, and a 3% by volume dichloroacetic acid solution in dichloromethane was added. After stirring for 5 minutes, an appropriate amount of methanol was added, and the mixture was neutralized with a saturated sodium bicarbonate aqueous solution, extracted, and the organic phase was dried, filtered, concentrated, and separated by column chromatography to obtain compound 14 (350 mg).

[0163] The NMR data are:

[0164] 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,4 H),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).

[0165] 13C NMR (101MHz, CDCl3) δ165.07,158.84,158.77,158.70,152.58,151.94,151.49,150.64,150.1 8,149.83,149.76,143.02,141.81,137.32,133.41,133.36,132.85,132.77,128.77,128.75, 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.

[0166] The data showed that the compound was synthesized correctly.

[0167] 6) Compound 14 (0.23 mmol) was added to 10 mL of tert-butylamine / acetonitrile (1:3, v / v), stirred at room temperature for 20 min, and then the solvent was removed by rotary evaporation. 100 mL of pyridine was added to dissolve the mixture, and 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole (1.38 mmol) was added. After reacting at room temperature for 4 hours, the solvent was removed and 20 mL of dichloromethane was added to dissolve the mixture. An appropriate amount of 5% by mass aqueous oxalic acid solution was added, the organic phase was separated and dried, filtered and concentrated, and then separated by column chromatography to obtain compound 15 (181 mg).

[0168] The NMR data are:

[0169] 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).

[0170] 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.

[0171] The data showed that the compound was synthesized correctly.

[0172] 7) Compound 15 (100 mg) was added to a flask, and 5 mL of methanol was added to dissolve it. 500 μL of diisopropylamine was added, and the mixture was stirred at room temperature for 4 hours. The solvent was dried by spin drying, and compound 16 (65 mg) was directly separated by column chromatography.

[0173] The NMR data are:

[0174] 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).

[0175] MALDI-TOF-HRMS: The relative molecular mass of C34H38N12O10P2S4 is 964.1192, and [M+H] +Peak of 965.1271.

[0176] The data showed that the compound was synthesized correctly.

[0177] Comparative Example 1

[0178] cddA35 (structure as shown in Table 2) was prepared according to the method in the literature (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.).

[0179] The cddA35 NMR data are:

[0180] 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).

[0181] 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.

[0182] The data showed that the compound was synthesized correctly.

[0183] Test Example 1

[0184] Determination of the oil-water partition coefficient logP of two cyclic dinucleotide prodrug molecules.

[0185] 1) Preparation of compound solution: Weigh a certain amount of compound 8, compound 16, and compound cddA35, respectively, dissolve them in DMSO solution and prepare a mother solution with a mass concentration of 1 mg / mL.

[0186] 2) Take 20uL from the prepared mother liquor and add it to an EP tube containing 1mL of n-octanol and 1mL of pure water (10mM K2HPO4pH7.0), vortex for 2 hours, and stand at 4°C overnight to separate the two phases. Separate the two phases and take 400uL respectively. Centrifuge at 3000 rpm for 5 minutes, filter with the corresponding 0.22μM filter membrane, and measure the area of ​​the absorption peaks of the two phases under the same HPLC parameters. Repeat the experiment three times, and calculate the logP value of the target compound by taking the log of the peak area ratio in the n-octanol phase and the water phase, as shown in Table 2.

[0187] Liquid phase conditions: The instrument used was Agilent 1260HPLC, chromatographic column: Agilent ZORABX SB-C18 5μm [4.6x 150mm], column temperature 25°C, injection 10uL, detection wavelength 254nM, and the chromatographic gradient method is shown in Table 1 below.

[0188] Table 1. HPLC liquid phase gradient method

[0189] Time (min) Volume %A (10 mM TEAA buffer) Volume %B(MeCN) Flow rate (mL / min) 1 98 2 1 6 0 100 1 10 0 100 1 13 98 2 1 15 98 2 1

[0190] Table 2

[0191]

[0192] From the results in Table 2, it can be seen that after using substituted mercaptoethanol to protect the phosphate ester, its logP is greatly improved. Compared with cddA35 without exposed negative charge of phosphate, its logP value is -3.43, which is basically fat-insoluble. The logP values ​​of compound 8 protected by thioacetyl ester and compound 16 protected by dithioethanol are 1.93 and 0.85 respectively, which greatly improves the fat solubility and is conducive to the compound to pass through the cell membrane and enter the cell to exert an immunostimulatory effect.

[0193] Test Example 2

[0194] Activity determination of two cyclic dinucleotide prodrug molecules in HEK293T cells

[0195] Principle: 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 cell cytoplasm, thereby breaking the thioester bond or the disulfide bond, and then the intermediate, the sulfur atom at the β position will nucleophilically attack the carbon atom at the α position, thereby forming ethylene thioide and leaving, releasing the parent drug with a negatively charged phosphodiester structure of the cyclic dinucleoside, which binds and activates the intracellular STING protein and phosphorylates interferon regulatory factor 3 (IRF3). The phosphorylated IRF3 enters the cell nucleus and binds to the promoter of the β interferon gene, causing the expression of interferon. Based on this, the interferon gene promoter is introduced into the promoter of firefly luciferase, so that the cellular activity of the prodrug molecule can be measured using the dual fluorescence reporter gene method.

[0196] Experimental Materials:

[0197] (1) Preparation of compound solutions: Compound 8 and Compound 16 were dissolved in DMSO to a final concentration of 1 mM. cddA35 was dissolved in water to a final concentration of 1 mM.

[0198] (2) Preparation of plasmid solution: The concentration of pcDNA3.1-hSTING-wt plasmid was 400 ng / μL, the concentration of pGL3-IFNβ plasmid was 400 ng / μL, and the concentration of pGL4.74-Rluc plasmid was 100 ng / μL.

[0199] Test method:

[0200] When the confluence of HEK 293T cells reaches about 70%, the original culture medium is replaced with serum-free culture medium opti-DMEM 500mL for transfection. Prepare the above component solutions, including pcDNA3.1-hsting-wt 2μL, pGL3-IFNβ 1μL, pGL4.74-Rluc 1μL, and opti-DMEM culture medium to make up 50uL / well, four wells in parallel. Add 50μL 2 / 50Lipo2000 transfection solution to the above solution mixtures, let the mixture stand at room temperature for 15min, mix evenly, transfect 100μL / well, and add 1mL 37℃ DMEM culture medium 4h later. After 18 hours, the culture medium was further replaced with 500 mL of serum-free or opti-DMEM, 8 μL (5 μM) of compound 8, 16 and cddA35 mixture in 92 μL of opti-DMEM, and allowed to stand for 15 minutes. 100 μL / well was transfected, and 1 mL of 37°C DMEM culture medium was added 4 hours later, and culture was continued for 24 hours. After 24 hours, the dual fluorescence results were detected, such as Figure 1 shown.

[0201] from Figure 1It can be seen from the results that the unmodified highly negatively charged cyclic dinucleoside cddA is difficult to cross the cell membrane, while the two cyclic dinucleotide compounds 8 and 16 protected by phosphotriester provided by the present invention can cross the cell membrane autonomously without the action of any transfection reagent, and release the parent drug molecule cddA35 in the cytoplasm due to the action of thioesterase or reducing environment, thereby activating the STING pathway and causing the expression of interferon.

[0202] Test Example 3

[0203] Activity determination of two cyclic dinucleotide prodrug molecules in monocytic cell line THP-1 cells

[0204] Test method:

[0205] When the confluence of THP-1-Lucia cells reached about 80%, compound 8, compound 16 and drug ADU-S100 were directly dissolved in the culture medium at a certain concentration and added to a 24-well plate, with four wells in parallel. The culture was continued for 24 hours at 37°C and 5% CO2. After the culture, the luciferase activity was tested. The test results of type I interferon of compound 8, compound 16 and drug ADU-S100 in the monocytic cell line THP-1 at a concentration of 10 μM are shown in the following figure. Figure 2 As shown; Compound 8 and Compound 16 induce EC of type I interferon expression in THP-1 cell line 50 The value is determined by a dose-response curve of serial dilutions at multiple concentrations, such as Figure 3 shown.

[0206] from Figure 2 The results show that in the monocytic THP-1 cell line, in the absence of any transfection reagent, at a concentration of 10 μM, both compound 8 and compound 16 can induce strong expression of type I interferon, and their activity is much higher than that of ADU-S100, a drug that has entered the second phase of clinical trials. Figure 3 It can be seen that compounds 8 and 16 induce EC expression of type I interferon in THP-1 cell line 50 The values ​​were 4.4 nM and 355 nM, respectively, compared with the EC of the control drug ADU-S100 (reference compound 2'3'-RR-(A)(A) in patent CN108430503A) 50 The value was 41.5 μM, and the EC values ​​of compound 16 and compound 8 were 50 The values ​​increased by 9430 times and 117 times respectively, which is a considerable improvement in activity.

[0207] Test Example 4

[0208] Anticancer activity test of compound 8

[0209] Experimental Materials:

[0210] Mice: BABL / C female mice, weighing 18-22 g, from Guangdong Animal Experiment Center, fed with pelleted feed, with free access to food and water.

[0211] Tumor cell line: CT-26-Luc cells

[0212] Establishment of mouse tumor model: Cell culture and passage, cells were collected in the logarithmic phase and made into a concentration of (1.0×10 7 ) per ml of cell suspension, 0.1 mL of cell suspension (cell number 1.0×10 6 After about 10 days, the tumor grew to a diameter of about 5 mm, carcinogenesis was successful, and the mice were randomly divided into three groups.

[0213] Negative control group: physiological saline solution

[0214] Positive control group: ADU-S100 (clinical phase II drug), dose 1 mg / kg

[0215] Experimental group: Compound 8, dose 1 mg / kg

[0216] Dosage method: Dosage began 10 days after the tumor was inoculated and grown, with intratumoral injection, 50 μL / mouse, once every two days, for a total of three doses. The chemiluminescence method was used to observe the growth of tumors in mice using the IVIS Spectrum Imaging System (small animal in vivo imaging system). Before imaging, the fluorescein small molecule was prepared: an appropriate amount of fluorescein sodium salt of 15 mg / mL was prepared with PBS solution, and fluorescein sodium was ordered from Shanghai Yishen Biological Co., Ltd. Prepare oxygen and isoflurane in advance. By intraperitoneal injection of 100 μL of fluorescein, the mouse was placed in the anesthesia box. After the animal was anesthetized, the mouse was transferred to the imaging chamber in the imager. Adjust the posture of the mouse as needed. The imaging parameters are: automatic exposure, Binning value of 2, and F value of 8. The imaging time is selected as 10 minutes. After imaging, the fluorescence value is converted into the number of photons, and the total number of fluorescence photons in each experimental group is counted. The imager is the IVIS Spectrum imaging platform of PerkinElmer. The results are as follows. Figure 4 As shown. Figure 4 The numbers in a, 4b, 4c, 4d, 4e, and 4f represent the total number of fluorescent photons in the circular region (ROI, region of interest). Figure 4 g and 4h are statistical graphs of the total fluorescence photon number of each experimental group.

[0217] The tumor volume and survival rate of mice in each group are shown in Figure 5The tumor volume was measured by vernier calipers using the length and width of the tumor and calculated according to the formula (volume = length × width × width / 2). The survival rate = the number of mice in each experimental group with a tumor volume less than 2000 cubic millimeters / the total number of mice in each experimental group.

[0218] from Figure 4 and Figure 5 The results show that in the CT-26 mouse tumor model, at a dose of 1 mg / kg, compared with the control drug ADU-S100 which can only control the growth of mouse tumors, the tumors of the mice in the experimental group using compound 8 of the present invention basically disappeared, which further indicates that compound 8 has good anti-tumor activity and its effect is better than the clinical drug ADU-S100.

Claims

1. A cyclic dinucleotide prodrug molecule or a pharmaceutically acceptable salt thereof, characterized in that: The cyclic dinucleotide prodrug molecule has the following structure: B1 and B2 are identical and are adenine based; X1 and X2 are both -H; R1, R2, R3 and R4 are the same and are selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C 11 An aromatic hydrocarbon group or a six-membered heterocyclic group; and the substituents optionally present in R1, R2, R3 and R4 are selected from C1-C5 alkyl groups; The six-membered heterocyclic group is 2. The cyclic dinucleotide prodrug molecule according to claim 1, wherein The cyclic dinucleotide prodrug molecule has a structure shown in formula (11), 3. The cyclic dinucleotide prodrug molecule according to claim 1, wherein The cyclic dinucleotide prodrug molecule has a structure shown in formula (12), 4. A method for preparing the cyclic dinucleotide prodrug molecule according to any one of claims 1 to 3, the method comprising: a) subjecting a first nucleoside monomer compound, a second nucleoside monomer compound and a first condensing agent to a first contact reaction in a first liquid reaction medium to obtain a linear dinucleotide intermediate; b) subjecting the linear dinucleotide intermediate to a decyanoethylation reaction under alkaline conditions, and then subjecting the obtained reaction product to a second contact reaction with a second condensing agent in a second liquid reaction medium; c) performing a deprotection reaction on the cyclic dinucleotide prodrug molecule with a protecting group obtained after the second contact reaction; Wherein, the first nucleoside monomer compound has a structure shown in formula (13): The second nucleoside monomer compound has a structure shown in formula (15): Wherein, Q represents a protecting group on the exocyclic amino group in the bases represented by B1 and B2, and the protecting group is a phenoxyacetyl group or a 4-isopropylphenoxyacetyl group; Y1 and Y2 are each independently selected from n1, n2, n3 and n4 are all 2.

5. The method according to claim 4, wherein: The conditions for carrying out the first contact reaction include: temperature of 0-50° C. and time of 2-8 h.

6. The method according to claim 4, wherein: The conditions for carrying out the second contact reaction include: temperature of 0-50° C. and time of 2-8 h.

7. The method according to claim 4, wherein: The first condensing agent and the second condensing agent are each independently selected from at least one of 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole, 2,4,6-triisopropylphenylsulfone-3-nitro-1,2,4-triazole, 2,4,6-triisopropylbenzenesulfonyl chloride, 2,4,6-trimethylbenzenesulfonyl chloride, 1H-tetrazole and N-methylimidazole.

8. The method according to claim 4, wherein: In step a), the amount of the first condensing agent used is 2-3 mol relative to 1 mol of the first nucleoside monomer compound.

9. The method according to claim 4, wherein: In step b), the amount of the second condensing agent used is 4-5 mol relative to 1 mol of the linear dinucleotide intermediate.

10. The method according to claim 4, wherein: In step b), the basic conditions are provided by a solution of tert-butylamine in acetonitrile.

11. The method according to claim 10, wherein: The alkaline condition is provided by a mixed solution of tert-butylamine and acetonitrile in a volume ratio of 1:(1-5).

12. The method according to claim 4, wherein: The deprotection reaction is carried out in the presence of a mixed solution of diisopropylamine and methanol in a volume ratio of 1:(5-20).

13. Use of the cyclic dinucleotide prodrug molecule or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of anticancer drugs.

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