Cyclic dinucleotide analogue containing triazole structural unit as well as preparation method and application of cyclic dinucleotide analogue

Abstract

The invention discloses a cyclic dinucleotide analogue containing a triazole structural unit as well as a preparation method and application of the cyclic dinucleotide analogue. The cyclic dinucleotide analogue containing the triazole structural unit is obtained by taking a macrocyclic ribose phosphate skeleton containing a diazido group as a substrate and expanding the macrocyclic ribose phosphate skeleton and an aromatic alkynyl compound through a click chemistry method, has relatively good biofilm formation inhibition activity, has a potential application prospect, and can be widely applied to biofilm biofilm formation inhibition. According to the invention, a cyclic dinucleotide analogue library is enriched, and biological research of cyclic dinucleotide and development of immunotherapy are promoted.

Description

Technical Field

[0001] The invention belongs to the field of organic chemical synthesis and medicinal chemistry, and specifically relates to a cyclic dinucleotide analog containing a triazole structural unit, and a preparation method and application thereof. Background Art

[0002] Cyclic dinucleotides are a class of cyclic compounds composed of two purine nucleotides connected by two phosphodiester bonds. As second messengers, they participate in the regulation of various important physiological processes of bacteria and play an important role in the nucleic acid immunity mechanism of higher organisms. At the same time, they are directly related to various human disease states. With the deepening of research, people have also found that cyclic dinucleotides are also important immune transmitters for bacterial antiviral immunity. The latest research also confirms that there is an evolutionary connection between the antiviral immune signaling mechanism of microorganisms and higher organisms using cyclic dinucleotides as second messengers.

[0003] There are two types of natural cyclic dinucleotides that have been discovered: one is cyclic dinucleotides derived from microorganisms, in which two purine nucleotides are connected by 3 ， -5 ， Phosphodiester bonds form a ring, including 3 ， , 3 ， -Cyclic diguanylate (3 ， , 3 ， -c-di-GMP), 3 ， , 3 ， -cyclic di-AMP (3 ， , 3 ， -c-di-AMP) and 3 ， , 3 ， -cyclic GMP (3 ， , 3 ， -cGAMP), such cyclic dinucleotides can be used as second messengers to make molecules ubiquitous in bacteria and participate in a variety of physiological processes, including cell differentiation, the transition from motile state to biofilm state, the production of pathogenic factors, the formation of biofilm, etc. One type is 2 ， , 3 ， -cyclic GMP (2 ， , 3 ， -cGAMP), whose two purine nucleotide bases are linked by a 2 ， -5 ， - and a 3 ， -5 ， -Two phosphodiester bonds are connected to form a ring. This cyclic dinucleotide is the endogenous ligand of STING. It binds to the interferon gene stimulator STING, activates the cascade reaction of downstream signals, and ultimately induces the production of type I interferon and other cytokines, initiating an immune response.

[0004] In view of this, the design and development of cyclic dinucleotide (CDNS) analogs is of great significance. Summary of the invention

[0005] The object of the present invention is to provide a cyclic dinucleotide analog containing a triazole structural unit and a preparation method and application thereof.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides a cyclic dinucleotide analog containing a triazole structural unit, and its structure is as follows:

[0008]

[0009] Wherein, X is any one of carbon or nitrogen, and R1 is any one or more of unsubstituted, monosubstituted or polysubstituted alkyl, alkoxy, and halogen.

[0010] Preferably, when X is carbon, R1 is any one of unsubstituted, 2-fluoro substituted, 3-fluoro substituted, 4-fluoro substituted, 3,5-difluoro substituted, 3,4-difluoro substituted, 4-bromo substituted, and 4-methoxy substituted; when X is nitrogen, R is unsubstituted.

[0011] The present invention also provides the use of the above-mentioned cyclic dinucleotide analogs containing triazole structural units in the preparation of biofilm inhibitor drugs.

[0012] The present invention also provides a method for synthesizing a cyclic dinucleotide analog containing a triazole structural unit by using a macrocyclic ribose-phosphate skeleton. The method uses a macrocyclic ribose-phosphate skeleton 1 containing a diazide group as a substrate and an aromatic cycloalkynyl compound 2 as a raw material reagent. Under alkali and metal ion catalysis conditions and nitrogen conditions, a compound 3 is reacted to obtain a cyclic dinucleotide analog TM containing a triazole structural unit. The reaction route is as follows:

[0013]

[0014] Wherein, X is any one of carbon or nitrogen, R1 is any one or more of unsubstituted, monosubstituted or polysubstituted alkyl, alkoxy, halogen, and the base is any one or more of triethylamine, N-methylimidazole, sodium carbonate, and dimethylamine.

[0015] Preferably, the reaction solvent in the process of preparing compound 3 from compound 1 is tetrahydrofuran, the base is triethylamine, and the metal ion catalyst is cuprous iodide.

[0016] Preferably, the reaction temperature in the process of preparing compound 3 from compound 1 is room temperature.

[0017] Preferably, the material feed ratio in the process of preparing compound 3 from compound 1 is: 1.0 molar equivalent of the diazide macrocyclic ribose phosphate compound 1, 4.0 molar equivalents of the aromatic cycloalkynyl compound 2, 2.0 molar equivalents of the base, and 0.4 molar equivalents of the metal ion catalyst.

[0018] Preferably, the reaction conditions for preparing TM from compound 3 are: first add pyridine, cis-pyridine-2-carbaldehyde oxime, and N,N,N,N-tetramethylguanidine to react at room temperature, and then add ammonia water to react at a higher temperature.

[0019] Preferably, the specific reaction conditions for preparing TM from compound 3 are: take 0.02 mmol of compound 3, add 2 mL of pyridine, add 0.8 mmol of cis-pyridine-2-carbaldehyde oxime, and 0.7 mmol of N,N,N,N-tetramethylguanidine, react at room temperature for 16 h, then add 2 mL of ammonia water, and react in an oil bath at 50 degrees Celsius for 2 days.

[0020] The beneficial effects of the present invention are:

[0021] (1) The present invention provides a method for synthesizing cyclic dinucleotide analogs using a macrocyclic ribose-phosphate backbone, wherein the method uses a macrocyclic ribose-phosphate backbone containing a diazide group as a substrate and reacts with a variety of electron-rich, electron-deficient and electrically neutral alkynyl group-containing compounds to achieve the expansion of cyclic dinucleotide analogs through a click chemistry method.

[0022] (2) The method of the present invention uses a macrocyclic ribose phosphate skeleton containing a diazide group as a substrate and an aromatic acetyl compound as a raw material reagent. Under alkali and metal ion catalysis conditions, a cyclic dinucleotide analog containing a triazole group can be prepared by reacting at room temperature under nitrogen conditions. The method is simple to operate, has mild reaction conditions, produces few by-products, has high atom economy, and is suitable for industrial production.

[0023] (3) The present invention also provides a cyclic dinucleotide analog containing a triazole structural unit. The cyclic dinucleotide analog containing a triazole structural unit has good biofilm formation inhibitory activity and has potential application prospects.

[0024] (4) The present invention provides a new class of cyclic dinucleotide analogs, enriches the cyclic dinucleotide analog library, and promotes the biological research of cyclic dinucleotides and the development of immunotherapy. DETAILED DESCRIPTION

[0025] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods; the instruments and equipment used in the following embodiments, unless otherwise specified, are conventional laboratory instruments and equipment; the test materials used in the following embodiments, unless otherwise specified, can be prepared or purchased according to the methods reported in existing literature.

[0026] Example 1: Synthesis of cyclic bis(3'-5')-(1'-azidophenylacetylene-2'-O-hydroxy-D-ribose-furanosyl)-phosphate TM-a

[0027] (1) Synthesis of cyclic bis(3'-5')-(1'-azidophenylacetylene-2'-O-acetyl-D-ribosyl-furanosyl)-2-chlorophenyl phosphate Synthesis of 3a

[0028]

[0029] Add a stirring magnet to a 25 mL reaction tube, add 0.1 mmol (1.0 eq) of cyclic bis (3'-5')-(1'-azido-2'-O-acetyl-D-ribose-furanosyl)-2-chlorophenyl phosphate and 2 mL of anhydrous tetrahydrofuran, replace nitrogen three times, add phenylacetylene 0.4 mmol (4.0 eq), triethylamine 0.2 mmol (2.0 eq), and cuprous iodide 0.04 mmol (0.4 eq) under nitrogen flow conditions. React at room temperature for 10 h. After the reaction is completed, dilute with dichloromethane, filter with diatomaceous earth, wash once with sodium chloride aqueous solution, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain a white solid 3a with a yield of 57%.

[0030] 1H NMR(600MHz,CDCl3):δ8.13(s,1H),7.89(d,J=7.2Hz,2H),7.84(s,1H),7.78(d,J=7.2Hz,2H),7.49(d,J=8.4Hz,1H),7.45-7.40(m,5H),7.37-7.33(m,4H),7.23(t,J=7.8Hz,1H),7.17-7.14(m,2H),7.04(t,J=7.8Hz,1H),6.30(d,J=2.4Hz,1H),6.05(s,1H),5.89(dd,J=2.4,5.4Hz,1H),5.89-5.84(m,1H),5.76(d,J=5.4Hz,1H),5.75-5.73(m,1H),4.73-4.68(m,2H),4.64-4.58(m,2H),4.57-4.56(m,1H),4.24-4.20(m,1H),2.14(s,3H),1.97(s,3H);

[0031] 13 C NMR(150MHz,CDCl3):δ169.41,169.07,146.10,146.06,145.83,145.78,131.19,130.89,130.04,129.88,129.04,129.02,128.74,128.59,128.40,128.25,126.94,126.82,126.09,126.03,125.42,125.37,125.36,125.31,121.45,121.44,121.28,121.27,119.38,118.63,90.44,89.92,80.23-80.12(m,1C),79.90-79.79(m,1C),75.75(d,J=4.05Hz),74.64(d,J=2.55Hz),74.41(d,J=3.60Hz),74.11(d,J=7.80Hz),66.93(d,J=4.95Hz),65.77(d,J=6.60Hz),20.61,20.44;

[0032] 31 PNMR(243MHz,CDCl3):δ-8.54,-9.94;

[0033] MALDI-TOF-HRMS calcd for C 42 H 38 Cl2N6O 14P2:982.1298; found [M+Na] + 1005.1192.

[0034] (2) Synthesis of cyclic bis(3'-5')-(1'-azidophenylacetylene-2'-O-hydroxy-D-ribose-furanosyl)-phosphate TM-a

[0035]

[0036] A stirring magnet was added to a 10 mL pressure tube, and 0.02 mmol (1.0 eq) of cyclic bis (3'-5')-(1'-azidophenylacetylene-2'-O-acetyl-D-ribose-furanosyl)-2-chlorophenyl phosphate and 2 mL of pyridine were added. 0.8 mmol (40.0 eq) of cis-pyridine-2-carbaldehyde oxime and 0.7 mmol (35.0 eq) of N,N,N,N-tetramethylguanidine were added, and the reaction was carried out at room temperature for 16 hours. Subsequently, 2 mL of ammonia water was added, and the reaction was carried out in an oil bath at 50 degrees Celsius for 2 days. After the reaction, 5 mL of dichloromethane was added, and H2O was used for extraction 3 times, 5 mL each time. The aqueous phase was combined and purified by reverse preparation column to obtain a white solid TM-a with a yield of 40%.

[0037] 1 H NMR (600MHz, D2O): δ8.66(s,2H),7.39(d,J=7.2Hz,4H),7.35(t,J=7.2Hz,2H),7.28(t,J=7.8Hz,4H),6.37(s,2H) ,4.75-4.72(m,2H),4.55(br,1H),4.54(d,J=3.6Hz,3H),4.50(s,1H),4.48(s,1H),4.17(dd,J=3.6,12.0Hz,2H);

[0038] 13 C NMR (150MHz, D2O): δ147.15,128.70,128.64,128.28,124.81,117.85,93.1 2,79.26(t,J=10.80Hz),74.91,70.02(d,J=4.65Hz),61.77(d,J=4.80Hz);

[0039] 31 PNMR (243MHz, D2O): δ-1.99;

[0040] MALDITOF-HRMS calculations for C 26 H 28 N6O 12P2:678.1240; found [MH] - 677.1163.

[0041] Example 2: Synthesis of cyclic bis(3'-5')-(1'-azido-2-fluorophenylacetylene-2'-O-hydroxy-D-ribose-furanosyl)-phosphate TM-b

[0042]

[0043] Add a stirring magnet to a 25 mL reaction tube, add 0.1 mmol (1.0 eq) of cyclic bis (3'-5')-(1'-azido-2'-O-acetyl-D-ribose-furanosyl)-2-chlorophenyl phosphate and 2 mL of anhydrous tetrahydrofuran, replace nitrogen three times, add 0.4 mmol (4.0 eq) of 1-ethynyl-2-fluorobenzene, 0.2 mmol (2.0 eq) of triethylamine, and 0.04 mmol (0.4 eq) of cuprous iodide under nitrogen flow conditions. React at room temperature for 10 h. After the reaction is completed, dilute with dichloromethane, filter with diatomaceous earth, wash once with sodium chloride aqueous solution, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain a white solid 3b with a yield of 71%.

[0044] 1 H NMR (600MHz, CDCl3): δ8.30(t,J=7.2Hz,1H),8.26(t,J=7.2Hz,1H),8.16(s,1H),8.00(d,J=3.0Hz,1H),7.48(d,J=8.4 Hz,1H),7.43(d,J=7.8Hz,1H),7.37(t,J=7.2Hz,2H),7.33-7.30(m,2H),7.25-7.22(m,3H),7.17-7.14(m,3H),7.13-7 .10(m,1H),7.05(t,J=7.8Hz,1H),6.24(d,J=2.4Hz,1H),6.07(s,1H),6.02-6.01(m,1H),5.88-5.85(m,1H),5.83-5.7 9(m,2H),4.72(br,1H),4.67-4.59(m,3H),4.56-4.52(m,1H),4.26(dd,J=8.4,17.4Hz,1H),2.12(s,3H),1.97(s,3H);

[0045] 13C NMR (150MHz, CDCl3): δ169.38, 169.22, 160.26 (d, J = 13.80Hz), 158.61 (d, J = 13. 35Hz),146.17,146.13,145.94,145.89,131.13,130.87,129.87(d,J=8.55Hz),1 29.79(d,J=8.40Hz),128.34,128.16,128.12(d,J=3.00Hz),128.01(d,J=3.00H z), 126.80, 126.74, 125.56 (d, J = 7.20Hz), 125.38 (d, J = 7.50Hz), 124.79 (d, J = 2. 55Hz), 124.72 (d, J = 2.40Hz), 121.50 (d, J = 1.65Hz), 121.24 (d, J = 1.65Hz), 118. 15-117.94(m,2C),115.94-115.73(m,2C),90.11,89.81,80.66-80.55(m,1C),80 .17-80.07(m,1C),75.68(d,J=4.20Hz),74.98(d,J=7.95Hz),74.58(d,J=2.85Hz ),74.27(d,J=3.60Hz),66.84(d,J=5.25Hz),66.01(d,J=6.15Hz),20.59,20.43;

[0046] 31 PNMR (243MHz, CDCl3): δ-8.30,-10.03;

[0047] MALDI-TOF-HRMS calculation for C 42 H 36 Cl2F2N6O 14 P2:1018.1110; found [M+Na] + 1041.1004.

[0048] The compound TM-b was prepared by deprotection according to the deprotection method described in Example 1.

[0049] Example 3: Synthesis of cyclic bis(3'-5')-(1'-azido-1-ethynyl-3-fluorobenzene-2'-O-hydroxy-D-ribose-furanosyl)-phosphate TM-c

[0050]

[0051] Add a stirring magnet to a 25 mL reaction tube, add 0.1 mmol (1.0 eq) of cyclic bis (3'-5')-(1'-azido-2'-O-acetyl-D-ribose-furanosyl)-2-chlorophenyl phosphate and 2 mL of anhydrous tetrahydrofuran, replace nitrogen three times, add 0.4 mmol (4.0 eq) of 1-ethynyl-3-fluorobenzene, 0.2 mmol (2.0 eq) of triethylamine, and 0.04 mmol (0.4 eq) of cuprous iodide under nitrogen flow conditions. React at room temperature for 10 h. After the reaction is completed, dilute with dichloromethane, filter with diatomaceous earth, wash once with sodium chloride aqueous solution, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain a white solid 3c with a yield of 43%.

[0052] 1 H NMR (400MHz, CDCl3): δ8.15(s,1H),7.84(s,1H),7.65-7.61(m,2H),7.54(d,J=8.0Hz,1H),7.52-7.50(m,1H),7.49 -7.48(m,1H),7.44(dt,J=1.2,8.0Hz,1H),7.42-7.34(m,4H),7.23(d,J=8.0Hz,1H),7.18-7.14(m,2H),7.08-7.00( m,3H),6.29(d,J=2.0Hz,1H),6.05(d,J=2.0Hz,1H),5.89(dd,J=2.0,4.8Hz,1H),5.87-5.82(m,1H),5.75(dd,J=2.0 ,5.2Hz,1H),5.73-5.69(m,1H),4.75-4.67(m,2H),4.65-4.54(m,3H),4.27-4.20(m,1H),2.15(s,3H),1.96(s,3H);

[0053] 13C NMR (150MHz, CDCl3): δ169.43, 169.07, 164.09 (d, J = 6.75Hz), 162.46 (d, J = 7.50 Hz),146.08,146.05,145.83,145.79,132.20(d,J=8.40Hz),132.00(d,J=8.40H z),131.20,130.91,130.69,130.64,128.42,128.27,126.97,126.86,125.40-1 25.31(m,2C),121.76(d,J=2.55Hz),121.63(d,J=2.70Hz),121.45(d,J=1.80Hz ),121.26,119.80,119.09,115.59(d,J=21.45Hz),115.43(d,J=21.00Hz),113. 06(d,J=6.45Hz),112.91(d,J=6.60Hz),90.51,89.96,80.24-80.14(m,1C),79. 95-79.84(m,1C),75.65(d,J=4.05Hz),74.67(d,J=2.40Hz),74.45(d,J=3.75Hz ),73.90(d,J=7.80Hz),66.89(d,J=5.10Hz),65.69(d,J=6.90Hz),20.61,20.42;

[0054] 31 PNMR (162MHz, CDCl3): δ-8.70,-9.95;

[0055] MALDI-TOF-HRMS calculation for C 42 H 36 Cl2F2N6O 14 P2:1018.1110; found [M+Na] + 1041.1007.

[0056] The compound TM-c was prepared by deprotection according to the deprotection method described in Example 1.

[0057] Example 4: Synthesis of cyclic bis(3'-5')-(1'-azido-1-ethynyl-4-fluorobenzene-2'-O-hydroxy-D-ribose-furanosyl)-phosphate TM-d

[0058]

[0059] Add a stirring magnet to a 25 mL reaction tube, add 0.1 mmol (1.0 eq) of cyclic bis (3'-5')-(1'-azido-2'-O-acetyl-D-ribose-furanosyl)-2-chlorophenyl phosphate and 2 mL of anhydrous tetrahydrofuran, replace nitrogen three times, add 0.4 mmol (4.0 eq) of 4-fluorophenylacetylene, 0.2 mmol (2.0 eq) of triethylamine, and 0.04 mmol (0.4 eq) of cuprous iodide under nitrogen flow conditions. React at room temperature for 10 h. After the reaction is completed, dilute with dichloromethane, filter with diatomaceous earth, wash once with sodium chloride aqueous solution, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain a white solid 3d with a yield of 76%.

[0060] 1 H NMR (400MHz, CDCl3): δ8.17(s,1H),7.88(br,2H),7.83(s,1H),7.77-7.74(m,2H), 7.47(d,J=8.0Hz,1H),7.44(d,J=8.0Hz,1H),7.36-7.34(m,2H),7.25-7.21(m,1H), 7.17-7.03(m,7H),6.31(s,1H),6.05(s,1H),5.88-5.84(m,2H),5.76-5.69(m,2H), 4.76-4.67(m,2H),4.64-4.55(m,3H),4.26-4.19(m,1H),2.15(s,3H),1.96(s,3H);

[0061] 13C NMR (150MHz, CDCl3): δ169.43, 169.05, 163.84 (d, J = 5.40Hz), 162.20 (d, J = 5.25Hz), 146.08, 146.04, 145.82, 145.78, 131.18, 13 0.92,128.40,128.27,127.89,127.83,127.81,127.75,126.94,126.86,126.35(d,J=2.55Hz),126.12(d,J=2.10Hz),125.36,12 5.31,121.41,121.40,121.27,121.26,116.15,116.12,116.00,115.98,90.62,89.98,80.10-80.00(m,1C),79.86-79.75(m,1C) ,75.72(d,J=3.60Hz),74.72,74.47(d,J=3.60Hz),73.78(d,J=7.95Hz),66.93(d,J=5.10Hz),65.66(d,J=6.90Hz),20.62,20.44;

[0062] 31 PNMR (162MHz, CDCl3): δ-8.72,-9.92;

[0063] MALDI-TOF-HRMS calculation for C 42 H 36 Cl2F2N6O 14 P2:1018.1110; found [M+Na] + 1041.1009.

[0064] The compound TM-d was prepared by deprotection according to the deprotection method described in Example 1.

[0065] Example 5: Synthesis of cyclic bis(3'-5')-(1'-azido-1-ethynyl-3,5-difluorobenzene-2'-O-hydroxy-D-ribose-furanosyl)-phosphate TM-e

[0066]

[0067] Add a stirring magnet to a 25 mL reaction tube, add 0.1 mmol (1.0 eq) of cyclic bis (3'-5')-(1'-azido-2'-O-acetyl-D-ribose-furanosyl)-2-chlorophenyl phosphate and 2 mL of anhydrous tetrahydrofuran, replace nitrogen three times, add 0.4 mmol (4.0 eq) of 1-ethynyl-3,5-difluorobenzene, 0.2 mmol (2.0 eq) of triethylamine, and 0.04 mmol (0.4 eq) of cuprous iodide under nitrogen flow conditions. React at room temperature for 10 h. After the reaction is completed, dilute with dichloromethane, filter with diatomaceous earth, wash once with sodium chloride aqueous solution, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain a white solid 3e with a yield of 63%.

[0068] 1 H NMR (600MHz, CDCl3): δ8.17(s,1H),7.87(s,1H),7.49(d,J=8.4Hz,1H),7.44(d,J=7.8Hz,1H),7.42(d,J=6.0Hz,2 H),7.38-7.34(m,2H),7.29(d,J=6.0Hz,2H),7.24(d,J=7.8Hz,1H),7.16(t,J=7.8Hz,2H),7.08(t,J=7.8Hz,1H),6 .77(q,J=8.4Hz,2H),6.28(s,1H),6.05(s,1H),5.90(d,J=4.2Hz,1H),5.83-5.81(m,1H),5.74(d,J=4.8Hz,1H),5. 72-5.69(m,1H),4.75-4.73(m,1H),4.70(br,1H),4.61-4.56(m,3H),4.26-4.23(m,1H),2.15(s,3H),1.96(s,3H);

[0069] 13C NMR (150MHz, CDCl3): δ169.46, 169.10, 164.37 (d, J=3.00Hz), 164.28 (d, J= 2.55Hz),162.72(d,J=3.60Hz),162.64(d,J=3.45Hz),146.61,146.32,146 .03(d,J=5.85Hz),145.82(d,J=6.45Hz),133.24-133.10(m,1C),133.04-1 32.90(m,1C),131.19,130.93,128.45,128.27,126.98,126.92,125.36,12 5.31,121.48(d,J=1.65Hz),121.22(d,J=1.65Hz),120.23,119.59,109.06 -108.79(m,4C),104.14-103.64(m,2C),90.57,90.01,80.34-80.24(m,1C) ,80.04-79.92(m,1C),75.44(d,J=3.75Hz),74.62,74.47(d,J=3.75Hz),73 .77(d,J=7.65Hz),66.77(d,J=5.10Hz),65.65(d,J=7.05Hz),20.61,20.43;

[0070] 31 PNMR (243MHz, CDCl3): δ-8.82,-9.95;

[0071] MALDI-TOF-HRMS calculation for C 42 H 34 Cl2F4N6O 14 P2:1054.0921; found [M+Na] + 1077.0817.

[0072] The compound TM-e was prepared by deprotection according to the deprotection method described in Example 1.

[0073] Example 6: Synthesis of cyclic bis(3'-5')-(1'-azido-4-ethynyl-1,2-difluorobenzene-2'-O-hydroxy-D-ribose-furanosyl)-phosphate TM-f

[0074]

[0075] Add a stirring magnet to a 25 mL reaction tube, add 0.1 mmol (1.0 eq) of cyclic bis (3'-5')-(1'-azido-2'-O-acetyl-D-ribose-furanosyl)-2-chlorophenyl phosphate and 2 mL of anhydrous tetrahydrofuran, replace nitrogen three times, add 0.4 mmol (4.0 eq) of 4-ethynyl-1,2-difluorobenzene, 0.2 mmol (2.0 eq) of triethylamine, and 0.04 mmol (0.4 eq) of cuprous iodide under nitrogen flow conditions. React at room temperature for 10 h. After the reaction is completed, dilute with dichloromethane, filter with diatomaceous earth, wash once with sodium chloride aqueous solution, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain a white solid 3f with a yield of 60%.

[0076] 1 H NMR (600MHz, CDCl3): δ8.16(s,1H),7.81(s,1H),7.76(dd,J=8.4,10.2Hz,1H),7.63-7.60(m,2H),7.48(d,J=7.8Hz,2H), 7.45(d,J=8.4Hz,1H),7.37(t,J=7.2Hz,2H),7.24(d,J=7.2Hz,1H),7.22-7.19(m,2H),7.17(t,J=7.8Hz,2H),7.08(t,J= 7.2Hz,1H),6.30(s,1H),6.04(s,1H),5.88(d,J=4.8Hz,1H),5.86-5.83(m,1H),5.73(d,J=4.2Hz,1H),5.69(dd,J=8,4,1 2.0Hz,1H),4.75-4.74(m,1H),4.68(br,1H),4.63-4.56(m,3H),4.23(dd,J=7.2,13.8Hz,1H),2.16(s,3H),1.96(s,3H);

[0077] 13C NMR (150MHz, CDCl3): δ169.48,169.07,151.64-151.34(m,2C),150.11-149.74(m,2C),146.03,145.99,145.79,145.75,131.20,13 0.94,128.44,128.30,127.00,126.93,125.31(t,J=6.00Hz,2C),122.31-122.26(m,1C),122.16-122.10(m,1C),121.42,121.24(d, J=1.65Hz),118.10,118.08,117.98,117.96,115.26,115.19,115.13,115.07,90.66,90.00,80.09-80.00(m,1C),79.88-79.77(m,1 C),75.60(d,J=3.90Hz),74.72,74.51(d,J=3.45Hz),73.52(d,J=7.65Hz),66.89(d,J=5.25Hz),65.58(d,J=6.75Hz),20.66,20.47;

[0078] 31 PNMR (243MHz, CDCl3): δ-8.92,-9.93;

[0079] MALDI-TOF-HRMS calculation for C 42 H 34 Cl2F4N6O 14 P2:1054.0921; found [M+Na] + 1077.0820.

[0080] The compound TM-f was prepared by deprotection according to the deprotection method described in Example 1.

[0081] Example 7: Synthesis of cyclic bis(3'-5')-(1'-azido-1-bromo-4-ethynylbenzene-2'-O-hydroxy-D-ribose-furanosyl)-phosphate TM-g

[0082]

[0083] Add a stirring magnet to a 25mL reaction tube, add 0.1mmol (1.0eq) of cyclic bis(3'-5')-(1'-azido-2'-O-acetyl-D-ribose-furanosyl)-2-chlorophenyl phosphate and 2mL of anhydrous tetrahydrofuran, replace nitrogen three times, add 0.4mmol (4.0eq) of (4-bromophenyl)acetylene, 0.2mmol (2.0eq) of triethylamine, and 0.04mmol (0.4eq) of cuprous iodide under nitrogen flow conditions. React at room temperature for 10h. After the reaction is completed, dilute with dichloromethane, filter with diatomaceous earth, wash once with sodium chloride aqueous solution, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain 3g of white solid with a yield of 54%.

[0084] 1 H NMR (600MHz, CDCl3): δ8.21(br,1H),7.86(s,1H),7.78(d,J=7.2Hz,2H),7.66(d,J=7.8Hz,2H),7.55(dd,J=7 .2,12.6Hz,4H),7.47(d,J=8.4Hz,1H),7.44(d,J=8.4Hz,1H),7.35(d,J=7.8Hz,2H),7.24(t,J=7.2Hz,1H),7 .18-7.14(m,2H),7.05(t,J=7.8Hz,1H),6.31(s,1H),6.05(s,1H),5.88-5.85(m,2H),5.75(br,1H),5.72-5. 69(m,1H),4.75-4.73(m,1H),4.68(br,1H),4.63-4.56(m,3H),4.24-4.20(m,1H),2.15(s,3H),1.96(s,3H);

[0085] 13C NMR (150MHz, CDCl3): δ169.44,169.05,146.07,146.03,145.80,145.76,132.23,131.20,130.93,129.0 6,128.84,128.42,128.28,127.62,127.51,126.98,126.89,125.36,125.35,125.32,125.30,122.74,12 2.59,121.41,121.26,90.65,90.00,80.10-80.00(m,1C),79.85-79.74(m,1C),75.66(d,J=3.15Hz),74 .73,74.49(d,J=3.45Hz),73.70(d,J=8.25Hz),66.91(d,J=4.95Hz),65.64(d,J=6.60Hz),20.63,20.44;

[0086] 31 PNMR (243MHz, CDCl3): δ-8.81,-9.92;

[0087] MALDI-TOF-HRMS calculation for C 42 H 36 Br2Cl2N6O 14 P2:1139.9488; found [M+Na] + 1162.9377.

[0088] The compound TM-g was prepared by deprotection according to the deprotection method described in Example 1.

[0089] Example 8: Synthesis of cyclic bis(3'-5')-(1'-azido-4-ethynylbenzyloxy-2'-O-hydroxy-D-ribose-furanosyl)-phosphate TM-h

[0090]

[0091] Add a stirring magnet to a 25mL reaction tube, add 0.1mmol (1.0eq) of cyclic bis(3'-5')-(1'-azido-2'-O-acetyl-D-ribose-furanosyl)-2-chlorophenyl phosphate and 2mL of anhydrous tetrahydrofuran, replace nitrogen three times, add 0.4mmol (4.0eq) of 4-methoxyphenylacetylene, 0.2mmol (2.0eq) of triethylamine, and 0.04mmol (0.4eq) of cuprous iodide under nitrogen flow conditions. React at room temperature for 10h. After the reaction is completed, dilute with dichloromethane, filter with diatomaceous earth, wash once with sodium chloride aqueous solution, dry with anhydrous sodium sulfate, concentrate, and purify with column chromatography to obtain a white solid 3h with a yield of 77%.

[0092] 1 H NMR (400MHz, CDCl3): δ8.05(s,1H),7.84-7.83(m,1H),7.82-7.80(m,1H),7.75(s,1H),7.73-7.71(m,1H),7.70-7. 69(m,1H),7.48(d,J=8.0Hz,1H),7.43(d,J=8.0Hz,1H),7.37-7.34(m,2H),7.22(td,J=8.0Hz,1H),7.15(t,J=8.0H z,2H),7.04(t,J=7.6Hz,1H),6.99-6.92(m,4H),6.28(d,J=2.0Hz,1H),6.03(d,J=2.0Hz,1H),5.88-5.83(m,2H),5 .77-5.71(m,2H),4.72-4.65(m,2H),4.63-4.53(m,3H),4.24-4.18(m,1H),3.84(s,6H),2.14(s,3H),1.96(s,3H);

[0093] 13C NMR (150MHz, CDCl3): δ169.41,169.07,160.05,159.97,146.06,146.04,145.82,145.78,131.18,130.89,128.40 ,128.26,127.42,127.35,126.94,126.81,125.36(t,J=7.95Hz),122.77,122.57,121.43,121.42,121.29,121.2 8,114.47,114.43,90.45,89.89,80.14-80.04(m,1C),79.83-79.72(m,1C),75.77(d,J=4.05Hz),74.66(d,J=2.4 0Hz), 74.42 (d, J = 3.60Hz), 74.06 (d, J = 7.80Hz), 66.95 (d, J = 5.25Hz), 65.76 (d, J = 6.75Hz), 55.46, 20.61, 20.44;

[0094] 31 PNMR (162MHz, CDCl3): δ-8.59,-9.92;

[0095] MALDI-TOF-HRMS calculation for C 44 H 42 Cl2N6O 16 P2:1042.1510; found [M+Na] + 1065.1047.

[0096] The compound TM-h was prepared by deprotection according to the deprotection method described in Example 1.

[0097] Example 9: Synthesis of cyclic bis(3'-5')-(1'-azido-2-ethynylpyridine-2'-O-hydroxy-D-ribose-furanosyl)-phosphate TM-i

[0098]

[0099] Add a stirring magnet to a 25 mL reaction tube, add 0.1 mmol (1.0 eq) of cyclic bis (3'-5')-(1'-azido-2'-O-acetyl-D-ribose-furanosyl)-2-chlorophenyl phosphate and 2 mL of anhydrous tetrahydrofuran, replace nitrogen three times, add 0.4 mmol (4.0 eq) of 2-ethynylpyridine, 0.2 mmol (2.0 eq) of triethylamine, and 0.04 mmol (0.4 eq) of cuprous iodide under nitrogen flow conditions. React at room temperature for 10 h. After the reaction is completed, dilute with dichloromethane, filter with diatomaceous earth, wash once with sodium chloride aqueous solution, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain white solid 3i with a yield of 75%.

[0100] 1 H NMR (600MHz, CDCl3): δ8.59(s,1H),8.56(s,1H),8.51(s,1H),8.35(s,1H),8.21(d,J=7.8Hz,1H),8.16(d, J=7.8Hz,1H),7.84-7.80(m,2H),7.45(dd,J=8.4,16.2Hz,2H),7.37(t,J=6.6Hz,2H),7.30-7.24(m,3H),7 .16(t,J=7.2Hz,2H),7.05(t,J=7.2Hz,1H),6.24(s,1H),6.10(s,1H),6.03(s,1H),5.86-5.84(m,2H),5.8 1-5.80(m,1H),4.73(br,1H),4.66-4.60(m,3H),4.55-4.52(m,1H),4.29(m,1H),2.11(s,3H),1.97(s,3H);

[0101] 13C NMR (150MHz, CDCl3): δ169.26,169.20,149.38,148.42,146.14,146.10,145.97,145.93,137.57,137.40,131.0 8,130.89,128.34,128.17,126.77,125.56,125.51,125.43,125.38,123.38,122.17,121.52,121.51,121.22,1 21.21,120.87,120.67,90.09,89.88,80.80-80.70(m,1C),80.23-80.13(m,1C),75.43(d,J=3.75Hz),75.15(d, J=7.50Hz),74.45(d,J=2.25Hz),74.23(d,J=3.00Hz),66.64(d,J=5.10Hz),66.03(d,J=6.30Hz),20.57,20.41;

[0102] 31 PNMR (243MHz, CDCl3): δ-8.23,-10.12;

[0103] MALDI-TOF-HRMS calculation for C 40 H 36 Cl2N8O 14 P2:984.1203; found [M+Na] + 1007.1098.

[0104] The compound TM-i was prepared by deprotection according to the deprotection method described in Example 1.

[0105] Example 10: Study on the biofilm inhibition activity of compounds

[0106] Three strains of bacteria were used: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus

[0107] Dilute the bacteria to 5 × 10 5Colony forming units / unit milliliter (CFU / mL), or OD595nm of about 0.4 to 0.6. Add 75 μL of the above-diluted bacterial dilution to each well of a 96-well plate, dissolve the compound in 25 μL of sterile saline, and add the compound to the wells according to the gradient concentration of 1.25mM, 625μM, 312μM, 156μM, 78μM, 39μM, 19μM, and 10μM. There are 3 replicates in each group. After incubation at 37°C overnight for 18h, aspirate the bacterial solution and wash 3 times with PBS. (Note: Be careful not to puncture the biofilm during the washing process) Turn the plate upside down on paper, dry for 30min, add 150μL of crystal violet and let it stand for 15min. Remove the crystal violet, wash with PBS to remove the residual crystal violet, and dry for 30min. Add 30% ethanol and incubate in a 37°C incubator for 30min to dissolve the biofilm. Take 100 μL and add it to a new 96-well plate, and measure the absorbance at 595 nm using an ELISA reader.

[0108] Table 1. Biofilm inhibition rate (%) of the compounds. Biofilm inhibition rate of the nine compounds at a concentration of 156 μM.

[0109]

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A cyclic dinucleotide analog containing a triazole structural unit, the structure of which is as follows: in, X is any one of carbon or nitrogen, and R1 is any one or more of unsubstituted, monosubstituted or polysubstituted alkyl, alkoxy and halogen.

2. The cyclic dinucleotide analog containing a triazole structural unit according to claim 1, characterized in that: R1 is any one of unsubstituted, 2-fluoro substituted, 3-fluoro substituted, 4-fluoro substituted, 3,5-difluoro substituted, 3,4-difluoro substituted, 4-bromo substituted, and 4-methoxy substituted; when X is nitrogen, R is unsubstituted.

3. A method for preparing a cyclic dinucleotide analog containing a triazole structural unit, characterized in that: The method uses a macrocyclic ribose phosphate skeleton 1 containing a diazide group as a substrate and an aromatic cycloalkynyl compound 2 as a raw material reagent, and reacts under alkali and metal ion catalysis conditions and nitrogen conditions to obtain a compound 3, and the compound 3 is deprotected to obtain a cyclic dinucleotide analog TM containing a triazole structural unit, wherein the reaction route is as follows: Wherein, X is any one of carbon or nitrogen, R1 is any one or more of unsubstituted, monosubstituted or polysubstituted alkyl, alkoxy, halogen, and the base is any one or more of triethylamine, N-methylimidazole, sodium carbonate, and dimethylamine.

4. The method for preparing a cyclic dinucleotide analog containing a triazole structural unit according to claim 3, characterized in that: The reaction solvent in the process of preparing compound 3 from compound 1 is tetrahydrofuran, the base is triethylamine, and the metal ion catalyst is cuprous iodide.

5. The method for preparing a cyclic dinucleotide analog containing a triazole structural unit according to claim 4, characterized in that: The reaction temperature in the process of preparing compound 3 from compound 1 is room temperature.

6. The method for preparing a cyclic dinucleotide analog containing a triazole structural unit according to claim 5, characterized in that: The material feed ratio in the process of preparing compound 3 from compound 1 is: 1.0 molar equivalent of the diazide macrocyclic ribose phosphate compound 1, 4.0 molar equivalents of the aromatic cycloalkynyl compound 2, 2.0 molar equivalents of the base, and 0.4 molar equivalents of the metal ion catalyst.

7. The method for preparing a cyclic dinucleotide analog containing a triazole structural unit according to claim 4, characterized in that: The reaction conditions for preparing TM from compound 3 are: first add pyridine, cis-pyridine-2-carbaldehyde oxime, and N,N,N,N-tetramethylguanidine to react at room temperature, and then add ammonia water to react at a higher temperature.

8. The method for preparing a cyclic dinucleotide analog containing a triazole structural unit according to claim 7, characterized in that: The reaction conditions for preparing TM from compound 3 are as follows: take 0.02 mmol of compound 3, add 2 mL of pyridine, add 0.8 mmol of cis-pyridine-2-carbaldehyde oxime, and 0.7 mmol of N,N,N,N-tetramethylguanidine, react at room temperature for 16 h, then add 2 mL of aqueous ammonia, and react in an oil bath at 50 degrees Celsius for 2 days.

9. Use of the cyclic dinucleotide analogue containing a triazole structural unit according to any one of claims 1 to 2 in the preparation of biofilm inhibitor drugs.

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