Guanidyl-containing CDN (Content Delivery Network) analogue as well as preparation method and application thereof

Abstract

The invention belongs to the field of organic chemical synthesis and medicinal chemistry, and particularly relates to a guanidyl-containing CDN analogue as well as a preparation method and application thereof. According to the invention, a bimolecular synergetic cyclization strategy is adopted, a thiourea functional group and amine react in the presence of iodine and tetramethylpiperidine to synthesize the guanidyl connection-containing CDN analogue, and the method has the advantages of simple operation, mild reaction conditions, few byproducts and high atom economy, and is suitable for industrial production. The CDN analogue has relatively good biofilm formation inhibition activity and has a potential application prospect. The guanidyl-containing CDN analogue provided by the invention is a brand new CDN analogue, enriches a cyclic dinucleotide analogue library, and promotes the biological research of cyclic dinucleotide and the development of immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of organic chemical synthesis and medicinal chemistry, and particularly relates to a guanidine-containing CDN analogue as well as a preparation method and application thereof. BACKGROUND

[0002] Cyclic di-nucleotides (CDNs) are key second messenger molecules widely existing in prokaryotes and eukaryotes. They are initially discovered in bacteria and can regulate core physiological processes such as cell wall homeostasis maintenance, virulence factor production and biofilm formation. Bacterial-derived CDNs can act as specific ligands of stimulator of interferon genes (STING), and then decode the cyclic guanosine-adenosine synthase (cGAS)-STING pathway, which is one of the core regulatory mechanisms of human innate immune response.

[0003] More and more evidence shows that 2',3'-cGAMP and CDN analogues have shown potential as vaccine adjuvants and prospects for anti-tumor immunotherapy applications, and natural 3',3'-c-di-GMP has both STING agonistic activity and bacterial biofilm inhibition function. However, natural CDNs and traditional analogues have significant defects: the phosphodiester bond in the molecule has a negative charge, and is easily degraded by nucleases in vivo, resulting in low bioavailability and short duration of action.

[0004] To solve the above problems, the field attempts to modify the CDN molecular structure, and the optimization of the inter-nucleotide linkage is a key direction. Research has found that oligonucleotides containing guanidine inter-nucleotide linkages can significantly improve the stability and biological activity of the molecules--based on this, the present application designs a simple double-molecule synergistic cyclization strategy, which uses the reaction of thiourea functional groups and amines in the presence of iodine and 2,2,6,6-tetramethylpiperidine (TMP) to synthesize guanidine-containing CDN analogues, and verifies their biofilm inhibition activity, thereby providing a new direction for the biological research and immunotherapy application of CDN molecules. SUMMARY

[0005] Therefore, the present application aims to provide a guanidine-containing CDN analogue as well as a preparation method and application thereof.

[0006] To achieve the above-mentioned purpose, the technical solutions of the present application are as follows:

[0007] The present application provides a guanidine-containing CDN analogue, which has the following structure:

[0008]

[0009] Among them, X is any one of a base, a base analogue or a base derivative.

[0010] Preferably, X is any one of adenine, guanine, cytosine, thymine, or uracil.

[0011] The application also provides a preparation method of the guanidyl-containing CDN analogue, which comprises the following steps: taking 5'-amino-3'-FmocNCS-protected furan nucleoside monomer 1 as a substrate, performing a double-molecule synergistic cyclization reaction under nitrogen protection in an oxidizing agent and alkaline condition to obtain compound 2, and deprotecting the compound 2 to obtain the guanidyl-containing CDN analogue TM.

[0012]

[0013] In the formula, X is any one of a base, a base analogue, or a base derivative; the oxidizing agent is any one or more of iodine, hydroxyl (toluene sulfonyloxy) iodobenzene, and N-iodosuccinimide; and the base is any one or more of tetramethylpiperidine, triethylamine, N-methylimidazole, and 4-dimethylaminopyridine.

[0014] Preferably, the reaction solvent in the process of preparing compound 2 from compound 1 is a mixed solvent of dichloromethane and acetonitrile, the oxidizing agent is iodine, and the base is tetramethylpiperidine.

[0015] Preferably, the reaction solvent in the process of preparing compound 2 from compound 1 is a mixed solvent of dichloromethane and acetonitrile in a volume ratio of 1 / 1.

[0016] Preferably, the reaction temperature in the process of preparing compound 2 from compound 1 is room temperature.

[0017] Preferably, the material feeding ratio in the process of preparing compound 2 from compound 1 is that 5'-amino-3'-FmocNCS-protected furan nucleoside monomer 1 is 1.0 molar equivalent, the oxidizing agent is 0.6 molar equivalent, and the base is 1.8 molar equivalent.

[0018] Preferably, the reaction conditions for preparing TM from compound 2 are that compound 2 is dissolved in tetrahydrofuran, triethylamine trihydrofluoride is added, and the reaction is performed at room temperature, after which the reaction is extracted and concentrated, and then ammonia water is added and the reaction is performed under heating.

[0019] Preferably, the specific reaction conditions for preparing TM from compound 2 are that 1 molar equivalent of compound 2 is dissolved in tetrahydrofuran, 8-10 molar equivalents of triethylamine trihydrofluoride are added, and the reaction is performed at room temperature for 3 h, after which the reaction system is diluted with dichloromethane, the organic phase is washed with a sodium chloride aqueous solution, and then ammonia water is added and the reaction is performed at a temperature of 50℃ for 12 h.

[0020] The application also provides use of the guanidyl-containing CDN analogue in any of the above aspects in the preparation of a biological membrane inhibitor.

[0021] Further, the biological membrane inhibitor is a drug for inhibiting biological membranes of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

[0022] The present application has the following beneficial effects:

[0023] (1) The present application adopts a double-molecule synergistic cyclization strategy, and synthesizes a guanidyl group-containing CDN analogue by reacting a thiourea functional group with an amine in the presence of iodine and tetramethylpiperidine, and catalyzing and oxidizing itself under the condition of an oxidant iodine and tetramethylpiperidine to perform double-molecule synergistic cyclization to obtain the CDN analogue, wherein different bases can be replaced by glycosidation in the furan nucleoside monomer to realize the expansion of the cyclic dinucleotide analogue.

[0024] (2) The preparation method of the CDN analogue provided by the present application uses a furan nucleoside monomer containing a base as a substrate, and catalyzes and oxidizes itself under the condition of tetramethylpiperidine and an oxidant iodine to perform double-molecule synergistic cyclization, so that the guanidyl group-containing CDN analogue can be quickly prepared at room temperature, the method is simple in operation, mild in reaction condition, less in by-products, and high in atom economy, and is suitable for industrial production.

[0025] (3) The present application further provides a guanidyl group-containing CDN analogue, which has good biological membrane formation inhibition activity and potential application prospect.

[0026] (4) The guanidyl group-containing CDN analogue provided by the present application is a brand-new CDN analogue, which enriches the cyclic dinucleotide analogue library and promotes the biological research and immunotherapy development of the cyclic dinucleotide. DETAILED DESCRIPTION

[0027] The technical solutions of the present application will be further described in detail below with reference to specific embodiments. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above content of the present application is covered in the scope of protection intended by the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified; in the following examples, the instruments and equipment are conventional laboratory instruments and equipment unless otherwise specified; in the following examples, the test materials can be prepared or purchased according to the methods reported in the existing literature unless otherwise specified.

[0028] Example 1: Synthesis of (2R, 3R, 3aS, 7aR, 9R, 10S, 10aR, 14aR)-2, 9-bis(6-amino-9H-purin-9-yl)-5, 12-dioxahexacontanhydrodifluoro[3, 2-d: 3', 2'-j] [1, 3, 7, 9] tetraazacyclododecane-3, 10-diol TM-a

[0029] (1) Synthesis of N,N'-(((2R,3R,3aR,5Z,7aR,9R,10S,10aS,12E,14aR)-3,10-bis((tert-butyldimethyl- dimethyl)oxy)-5,12-bis(((9H-fluoro-9-yl)methyl)imino)hexadeca-hydrodifuran[3,2-d:3',2'- j][l,3,7,9]tetraazacyclododec-2,9-diyl)bis(9H-purine-9,6-diyl))dibenzamide 2a

[0030]

[0031] In a 25 mL reaction tube, a stirring magnet was added, compound 1a (0.48 mmol) was dissolved in a mixed solution of dichloromethane / acetonitrile = 1 / 1 (v / v, 5 mL), followed by the addition of iodine (73 mg, 0.29 mmol) and tetramethylpiperidine (0.15 mL, 0.87 mmol), and reacted at room temperature for 10 min under a nitrogen atmosphere. After the reaction was completed, dichloromethane was added for dilution, and the organic phase was washed with an aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with dichloromethane / methanol (gradient 1-2% methanol) to obtain 2a (183 mg, 26%) as a white solid.

[0032] 1 H NMR (600 MHz, CDC13): δ 9.27 (br, 1H), 8.97 (br, 1H), 8.72 (s, 2H), 8.03 (d, J = 7.2 Hz, 4H), 7.93 (s, 2H), 7.72 (t, J = 7.8 Hz, 4H), 7.64-7.58 (m, 6H), 7.54 (t, J = 7.8 Hz, 4H), 7.37-7.32 (m, 4H), 7.28 (t, J = 7.2 Hz, 4H), 6.06 (s, 2H), 4.85 (d, J = 4.2 Hz, 2H), 4.42 (dd, J = 10.8, 7.2 Hz, 2H), 4.33-4.29 (m, 2H), 4.24 (br, 2H), 4.18 (t, J = 6.6 Hz, 2H), 3.85 (dd, J = 8.4, 3.0 Hz, 2H), 3.70 (dd, J = 10.8, 5.4 Hz, 2H), 3.31 (t, J = 10.8 Hz, 2H), 0.94 (s, 18H), 0.21 (s, 6H), 0.18 (s, 6H);

[0033] 13C NMR (150 MHz, CDC13): δ 163.5, 160.9, 153.0, 151.1, 149.9, 144.3, 144.2, 141.4, 141.1, 133.7, 133.1, 129.1, 128.0, 127.7, 127.1, 127.0, 125.2, 125.1, 123.8, 120.2, 95.0, 74.5, 73.0, 66.3, 56.3, 47.3, 44.2, 25.8, 18.2, -4.4, -4.7;

[0034] MALDI-TOF-HRMS calcd for C 78 H 86 N 16 O 10 Si2[M+2H] 2+ : 731.3120, found 731.3135.

[0035] (2) Synthesis of (2R, 3R, 3aS, 7aR, 9R, 10S, 10aR, 14aR)-2, 9-bis(6-amino-9H-purin-9-yl)- 5, 12-diazenium hexadecanehydrodifluoro[3, 2-d: 3', 2'-j] [1, 3, 7, 9]tetraazacyclododecane-3, 10-diol TM-a

[0036]

[0037] In a 25 mL round bottom flask was added a stirring magnet, a solution of compound 2a (100 mg, 0.07 mmol) in tetrahydrofuran (2 mL), triethylamine trifluorohydrofluoride (0.1 mL, 0.7 mmol), and stirred at room temperature for 3 h. The mixture was diluted with dichloromethane, the organic phase was washed with aqueous sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Ammonia (28-30% ammonia water, 2 mL) was then added, and stirred at 50 °C for 12 h. Diluted with water, extracted with dichloromethane, and then concentrated under reduced pressure. The crude was purified by RP-HPLC to give TM-a (17 mg, 42%, retention time: 20.2-23.1 min) as a white foamy solid.

[0038] 1 H NMR (400 MHz, D20): δ 8.26 (s, 2H), 8.22 (s, 2H), 6.31 (s, 2H), 4.88 (d, J = 5.2 Hz, 2H), 4.42-4.35 (m, 2H), 3.96 (dd, J = 10.0, 4.8 Hz, 2H), 3.90 (dd, J = 10.8, 6.0 Hz, 2H), 3.60 (t, J = 10.4 Hz, 2H).

[0039] 13 C NMR (150MHz, D2O): δ155.7,155.5,152.7,148.4,140.0,118.9,93.4,72.3,71.9,54.8,43.4;

[0040] MALDI-TOF-HRMS calcd for C 22 H 30 N 16 O4[M+2H] 2+ :291.1312,found 291.1317.

[0041] Example 2: Synthesis of 1,1′-((2R,3R,3aS,7aR,9R,10S,10aR,14aR)-3,10-dihydroxy-5,12-diaminohexadecanedifluorofuran[3,2-d:3′,2′-j][1,3,7,9]tetraazacyclododecene-2,9-diyl)bis(5-methylpyrimidine-2,4(1H,3H)-dione)TM-b

[0042]

[0043] Compound 2b was prepared by the same method used to prepare 2a in Example 1.

[0044]

[0045] A magnetic stir bar was added to a 25 mL round-bottom flask, compound 2b (100 mg, 0.08 mmol) was added, followed by the addition of tetrahydrofuran (2 mL) and stirring to dissolve. Triethylamine trihydrofluoride (0.1 mL, 0.7 mmol) was then added, and the mixture was stirred at room temperature for 3 h. The mixture was diluted with dichloromethane, the organic phase was washed with aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Ammonia solution (28-30% ammonia, 2 mL) was then added, and the mixture was stirred at 50 °C for 12 h. The mixture was diluted with water, extracted with dichloromethane, and then concentrated under reduced pressure. The crude product was purified by RP-HPLC to give TM-b (12 mg, 27%, retention time: 16.0-18.5 min), a white, foamy solid.

[0046] 1 H NMR(600MHz,D2O)δ7.37(s,2H),5.88(s,2H),4.60(d,J=4.8Hz,2H),4.22-4.17(m,2H ),3.83(q,J=5.4Hz,2H),3.57(t,J=5.4Hz,2H),3.53(t,J=10.8Hz,2H),1.84(s,6H);

[0047] 13 C NMR (150MHz, D2O) δ166.5,155.5,151.2,137.6,111.3,95.9,71.8,71.5,54.5,43.1,11.4;

[0048] MALDI-TOF-HRMS calcd for C 22 H 32 N 10 O8[M+2H] 2+ :282.1197,found 282.1174.

[0049] Example 3: Synthesis of 1,1′-((2R,3R,3aS,7aR,9R,10S,10aR,14aR)-3,10-dihydroxy-5,12-diaminohexadecanedifluorofuran[3,2-d:3′,2′-j][1,3,7,9]tetraazacyclododecene-2,9-diyl)bis(4-aminopyrimidine-2(1H)-one)TM-c

[0050]

[0051] Compound 2c was prepared by referring to the method for preparing 2a in Example 1.

[0052]

[0053] A magnetic stir bar was added to a 25 mL round-bottom flask, followed by a solution of compound 2c (100 mg, 0.07 mmol) in tetrahydrofuran (2 mL). Triethylamine trihydrofluoride (0.1 mL, 0.7 mmol) was then added, and the mixture was stirred at room temperature for 3 h. The mixture was diluted with dichloromethane, the organic phase was washed with an aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Ammonia solution (28-30% ammonia, 2 mL) was then added, and the mixture was stirred at 50 °C for 12 h. The mixture was diluted with water, extracted with dichloromethane, and then concentrated under reduced pressure. The crude product was purified by RP-HPLC to give TM-c (18 mg, 47%, retention time: 16.0-18.1 min), a white, foamy solid.

[0054] 1H NMR(600MHz,D2O)δ7.62(d,J=7.2Hz,2H),6.03(d,J=7.8Hz,2H),5.91(s,2H),4.58(d,J=4.8Hz,2H ),4.30-4.25(m,2H),3.88(q,J=5.4Hz,2H),3.57(t,J=10.8Hz,2H),3.53(dd,J=10.2,4.8Hz,2H);

[0055] 13 C NMR (150MHz, D2O) δ166.4,157.1,155.8,141.7,96.6,96.0,72.2,71.8,54.7,43.3;

[0056] MALDI-TOF-HRMS calcd for C 20 H 30 N 12 O6[M+2H] 2+ :267.1200,found 267.1190.

[0057] Example 4: Synthesis of 9,9′-((2R,3R,3aS,7aR,9R,10S,10aR,14aR)-3,10-dihydroxy-5,12-diaminohexadecanedifluorofuran[3,2-d:3′,2′-j][1,3,7,9]tetraazacyclododecene-2,9-diyl)bis(2-amino-1,9-dihydro-6H-purine-6-one)TM-d

[0058]

[0059] Compound 2d was prepared by the same method used to prepare 2a in Example 1.

[0060]

[0061] A magnetic stir bar was added to a 25 mL round-bottom flask, followed by a solution of compound 2d (100 mg, 0.07 mmol) in tetrahydrofuran (2 mL). Triethylamine trifluoroate (0.1 mL, 0.7 mmol) was then added, and the mixture was stirred at room temperature for 3 h. The mixture was diluted with dichloromethane, the organic phase was washed with an aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Ammonia solution (28-30% ammonia, 2 mL) was then added, and the mixture was stirred at 50 °C for 12 h. The system was diluted with water, extracted with dichloromethane, and then concentrated under reduced pressure. The crude product was purified by RP-HPLC to give TM-d (17 mg, 39%, retention time: 18.0-19.7 min), a white, foamy solid.

[0062] 1 H NMR(600MHz,D2O)δ8.11(s,2H),6.41(s,2H),4.76-4.73(m,2H),4.39(td,J=10.2,5.4Hz ,2H),3.92(dd,J=10.2,5.4Hz,2H),3.75(dd,J=10.2,4.8Hz,2H),3.63(t,J=10.2Hz,2H).

[0063] Example 5: Synthesis of 1,1′-((2R,3R,3aS,7aR,9R,10S,10aR,14aR)-3,10-dihydroxy-5,12-diaminohexadecanedifluorofuran[3,2-d:3′,2′-j][1,3,7,9]tetraazacyclododecene-2,9-diyl)bis(pyrimidine-2,4(1H,3H)-dione)TM-e

[0064]

[0065] Compound 2e was prepared by referring to the method for preparing 2a in Example 1.

[0066]

[0067] A magnetic stir bar was added to a 25 mL round-bottom flask, followed by a solution of compound 2e (100 mg, 0.08 mmol) in tetrahydrofuran (2 mL). Triethylamine trihydrofluoride (0.1 mL, 0.7 mmol) was then added, and the mixture was stirred at room temperature for 3 h. The mixture was diluted with dichloromethane, the organic phase was washed with an aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Ammonia (28-30% ammonia, 2 mL) was then added, and the mixture was stirred at 50 °C for 12 h. The mixture was diluted with water, extracted with dichloromethane, and then concentrated under reduced pressure. The crude product was purified by RP-HPLC to give TM-e (15 mg, 33%, retention time: 17.5-20.9 min), a white, foamy solid.

[0068] 1 H NMR(600MHz,D2O)δ7.62(d,J=7.8Hz,2H),5.93(s,2H),5.86(d,J=7.8Hz,2H),4.65(d,J=4.8Hz,2H ),4.30-4.25(m,2H),3.89(dd,J=10.2,5.4Hz,2H),3.58(d,J=10.2Hz,2H),3.57(t,J=4.8Hz,2H);

[0069] 13C NMR (150MHz, D2O) δ155.6,141.8,102.1,96.2,71.9,71.7,54.4,43.2;

[0070] MALDI-TOF-HRMS calcd for C 20 H 28 N 10 O8[M+2H] 2+ :268.1040,found 268.1034.

[0071] Example 6: Study on the biomembrane inhibitory activity of the compound

[0072] Three bacterial strains were used: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus.

[0073] Dilute the bacteria to 5×10⁻⁶ 5 CFU / mL, or absorbance OD 595 Approximately 0.4 to 0.6. Add 75 μL of the above-diluted bacterial solution to each well of a 96-well plate. Dissolve the compound in 25 μL of sterile physiological saline and add the compound to the wells at gradient concentrations of 78 μM, 52 μM, 39 μM, 19 μM, and 10 μM. Perform three replicates per group. After incubating at 37°C for 18 h, aspirate the bacterial culture and wash three times with PBS. (Note: Be careful not to puncture the biofilm during washing.) Invert the plate onto paper and allow it to dry for 30 min. Add 150 μL of crystal violet and let it stand for 15 min. Remove the crystal violet by washing with PBS to remove any residual crystal violet, and dry for 30 min. Add 30% ethanol and incubate at 37°C for 30 min to dissolve the biofilm. Add 100 μL to a new 96-well plate and measure the absorbance at 595 nm using a microplate reader.

[0074] Table 1. Biomembrane inhibition rate (%) of compounds at a concentration of 78 μM.

[0075]

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A CDN analog containing a guanidine group, the structure of which is as follows: in, X can be any one of a base, a base analog, or a base derivative.

2. The guanidine-containing CDN analog according to claim 1, characterized in that, X is any one of adenine, guanine, cytosine, thymine, and uracil.

3. A method for preparing a CDN analog containing a guanidine group, characterized in that, The method uses 5'-amino-3'-FmocNCS-protected furanoside monomer 1 as a substrate, and carries out a bimolecular concerted cyclization reaction under nitrogen protection and with an oxidant and basic condition to obtain compound 2. Compound 2 is then deprotected to obtain a guanidine-containing CDN analog TM. The reaction route is as follows: Wherein, X is any one of a base, a base analog, or a base derivative; the oxidizing agent is any one or more of iodine, hydroxy(toluenesulfonyloxy)iodobenzene, or N-iodosuccinimide; and the base is any one or more of tetramethylpiperidine, triethylamine, N-methylimidazole, or 4-dimethylaminopyridine.

4. The method according to claim 3, characterized in that, The reaction solvent for preparing compound 2 from compound 1 is a mixture of dichloromethane and acetonitrile, the oxidant is iodine, and the base is tetramethylpiperidine.

5. The method according to claim 4, characterized in that, The reaction temperature for preparing compound 2 from compound 1 was room temperature.

6. The method according to claim 5, characterized in that, The material feed ratio for the preparation of compound 2 from compound 1 is as follows: 1.0 molar equivalent of 5'-amino-3'-FmocNCS protected furan nucleoside monomer 1, 0.6 molar equivalent of oxidant, and 1.8 molar equivalent of base.

7. The method according to claim 3, characterized in that, The reaction conditions for preparing TM from compound 2 are as follows: compound 2 is dissolved in tetrahydrofuran, triethylamine trihydrofluoride is added and reacted at room temperature, after which the mixture is extracted and concentrated, and then ammonia is added and the temperature is raised to react.

8. The method according to claim 7, characterized in that, The specific reaction conditions for preparing TM from compound 2 are as follows: 1 molar equivalent of compound 2 is dissolved in tetrahydrofuran, 8-10 molar equivalents of triethylamine trihydrofluoride are added, the reaction is carried out at room temperature for 3 h, the reaction system is diluted with dichloromethane, the organic phase is washed with sodium chloride aqueous solution, the mixture is concentrated under reduced pressure, ammonia is added, and the reaction is carried out at 50℃ for 12 h.

9. The use of the guanidine-containing CDN analogue according to any one of claims 1-2 in the preparation of biofilm inhibitor drugs.

10. The application according to claim 9, characterized in that, The biofilm inhibitors mentioned are drugs that inhibit biofilms of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

Images