Novel synthesis method of 2 '-O-alkyl guanosine

Abstract

The invention discloses a new synthesis method of 2 '-O-alkyl guanosine, which provides a new thought for regioselective functionalization of hydroxyl on a ribose skeleton of a nucleoside compound and provides great help for development of drug synthesis. According to the method, nucleoside is taken as a raw material, a four-step strategy of acetalation and protection of 2 'and 3' hydroxyl groups at the same time, high-regioselectivity incomplete hydrogenation reduction of acetal protecting groups, Williamson etherification reaction and hydrodebenzylation reaction is adopted, the 2 '-O-alkyl guanosine is finally synthesized, and the method has the advantages of high selectivity, mild reaction conditions, green and environment-friendly reagents, short synthesis steps and the like. Main synthesis steps as follows: # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of drug and intermediate synthesis, and specifically relates to a new method for synthesizing 2'-O-alkylguanosine. Background Art

[0002] In recent years, with the in-depth study of the second-generation antisense drugs, it has been found that the incorporation of 2'-O-alkyl nucleosides, especially short-chain alkyl and methoxyethyl groups, into nucleotides can enhance biological activity, improve RNA stability, enhance immune escape, provide structural flexibility and increase biocompatibility, which makes it have important application value in the fields of drug research, RNA therapy and gene chips. 2'-O-alkyl nucleosides are also widely used in the development of new drugs or drug intermediates, such as key intermediates for nucleoside antiviral drugs (Remdesivir, Sofosbuvir, etc.). Its synthesis research began in the 1950s, but there is no relatively economical and efficient method so far. There are problems such as low selectivity, complex extraction steps, difficult separation and purification, and high cost, which limit its widespread use in the medical field.

[0003] So far, there are mainly the following synthetic routes: one is to use ribose as raw material, which has a long route, poor selectivity, difficult separation and low overall yield (Helv. Chim. Acta, 1996, 79, 1930; Tetrahedron, 1994, 50, 5361.); the second method is direct synthesis, that is, using nucleoside as raw material for direct alkylation. The ribose part has three hydroxyl groups that can be alkylated, which has poor regioselectivity and is difficult to separate 2'-O-alkyl nucleoside from 3'-O-alkyl nucleoside. Or the alkylating agent used, such as diazoalkanes, has the disadvantages of being difficult to obtain, highly toxic and explosive, which is not conducive to industrial large-scale production (J. Org. Chem., 2002, 67, 357; Eur. Org. Chem., 2009, 3265; J. Org. Chem., 2013, 78, 8545; Chem. Commun., 2017, 53, 541; CN 114634541,2022.); The third method is to protect first and then alkylate. Also using nucleosides as raw materials, use dichlorosilane TIPDSCl2 or MDPSCl2 to protect the two hydroxyl groups at the 3'- and 5'-positions, and then alkylate the 2'-hydroxyl group. This method was first used for the methylation reaction of adenosine in 1979, but it also has three disadvantages: First, under room temperature conditions, TIPDSCl2 or MDPSCl2 is very easy to fall off in the alkaline environment of the alkylation reaction, resulting in low yield and difficulty in separation. To ensure its stability, the alkylation reaction needs to be carried out at extremely low temperatures, which makes the alkylation reaction slow and increases the requirements for reaction equipment and reaction operation; second, if there is a strong acid in the post-treatment, it is easy to cause the nucleoside bond to break; third, the protective agent TIPDSCl2 or MDPSCl2 is not easy to obtain and is expensive, and the deprotecting agent tetrabutylammonium fluoride is also expensive, the atom economy is not high, and it is not conducive to industrial large-scale preparation (Nucleic Acids Res.,1989,18,41,; Tetrahedron,2000,56,1047; J.Org.Chem.,2002,67,7887; Bioorg.Med.Chem.Lett.,2003,13,1631 ; CN100355769, 2005; J. Org. Chem., 2014, 79, 4423; Angew. Chem. Int. Ed., 2015, 54, 4195; J. Org. Chem., 2015, 67, 9375). There are also Chinese patent reports that all three hydroxyl groups in adenosine can be protected with acetyl groups first, and then the acetyl group at the 2'-position is selectively deprotected with hydroxylamine acetate, followed by O-methylation, and finally the acetyl groups at the 3'- and 5'-positions are removed under alkaline conditions to obtain the target product (CN10331955A, 2013), but it has not been widely used.

[0004] 2'-O-alkylguanosine has a wide range of application value in drug synthesis, and provides an important chemical intermediate for the development of new drugs, research on RNA modification and RNA technology, etc. As people further deepen their clinical research on antisense oligonucleotides, more and more oligonucleosides and their derivatives will be needed, and the demand for 2'-O-alkylguanosine and its modifications will also increase day by day. The market prospects are optimistic, so it is of great significance to find an economical and efficient synthesis method. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the existing synthesis technology such as unavailability of reaction reagents, low selectivity, difficulty in separation and purification, low synthesis efficiency, high cost and disadvantageous for industrial scale production, and to provide a new method for synthesizing 2'-O-alkylguanosine.

[0006] The technical solution to achieve the purpose of the present invention is:

[0007] A 2'-O-alkylguanosine, the structure of which is as follows:

[0008]

[0009] The preparation method of the above compound comprises the following steps:

[0010] (1) Under acid catalysis, the two hydroxyl groups of guanosine (I) at 2'- and 3'-positions are protected with benzyl groups using para-substituted benzaldehyde to prepare compound II;

[0011]

[0012] (2) Under the conditions of an acid catalyst and a reducing agent, the five-membered cyclic acetal in compound II is subjected to regioselective reduction ring-opening to prepare 3'-O-benzylguanosine (III);

[0013]

[0014] (3) Under alkaline conditions, 3'-O-benzylguanosine (III) is reacted with an alkylating agent to undergo a Williamson etherification reaction to prepare 3'-O-benzyl-2'-O-alkylguanosine (IV);

[0015]

[0016] (4) A step of removing the benzyl group on the 3'-hydroxyl group in compound IV in the presence of Pd / C and hydrogen to prepare 2'-O-alkylguanosine (V).

[0017]

[0018] Furthermore, in step (1), the acetalization reaction is carried out in an organic solvent such as benzene, toluene, xylene, etc. that can form an azeotrope with water; the acid catalyst can be a Lewis acid such as anhydrous aluminum chloride, anhydrous ferric chloride, anhydrous cobalt chloride, anhydrous copper chloride, anhydrous zinc chloride, copper trifluoromethanesulfonate, boron trifluoride ether complex, etc., can be a protonic acid such as hydrochloric acid, sulfuric acid, glacial acetic acid, p-toluenesulfonic acid, camphorsulfonic acid, etc., or can be other solid acids such as Amberlyst 15H + , acid salts such as potassium hydrogen sulfate, etc.; the acetalization protective agent can be an aromatic aldehyde, including benzaldehyde, p-fluorobenzaldehyde, p-chlorobenzaldehyde, p-bromobenzaldehyde, p-trifluoromethylbenzaldehyde, p-methoxybenzaldehyde, p-methylbenzaldehyde, p-ethylbenzaldehyde, p-isopropylbenzaldehyde, p-tert-butylbenzaldehyde, etc. The molar ratio of guanosine to aromatic aldehyde is 1:1.1-2; the molar ratio of guanosine to acid catalyst is 1:0.1-1; the acetalization reaction time is 12-24h.

[0019] Furthermore, in step (2), the regioselective reduction ring-opening reaction is carried out in an organic solvent such as acetonitrile, tetrahydrofuran, dichloroethane, 1,4-dioxane, benzene, toluene, etc.; the acid catalyst can be a halide salt such as anhydrous aluminum chloride, anhydrous ferric chloride, anhydrous cobalt chloride, anhydrous copper chloride, anhydrous zinc chloride, zinc iodide, etc., can be a protonic acid such as sulfuric acid, glacial acetic acid, p-toluenesulfonic acid, etc., can be a triflate such as zinc triflate, scandium triflate, copper triflate , silver trifluoromethanesulfonate, praseodymium trifluoromethanesulfonate, europium trifluoromethanesulfonate, samarium trifluoromethanesulfonate, indium trifluoromethanesulfonate, lanthanum trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, gadolinium trifluoromethanesulfonate, neodymium trifluoromethanesulfonate, or other Lewis acids such as trimethylsilyl trifluoromethanesulfonate, boron trifluoride ethyl ether, etc.; the reducing agent can be 9-borabicyclo[3.3.1]nonane, sodium cyanoboride, borane dimethyl sulfide complex, borane trimethylamine complex, lithium aluminum hydride, diisobutylaluminum hydride, etc. The molar ratio of acetal protected guanosine to acid catalyst is 1:0.05-0.5; the molar ratio of acetal protected guanosine to reducing agent is 1:2-10; the temperature of hydrogenation incomplete reduction ring opening reaction is 30℃-60℃; the reaction time is 4-24h.

[0020] Furthermore, in step (3), the alkylation reaction is carried out in the presence of an organic solvent such as acetonitrile, acetone, dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, etc. The base can be sodium hydride, alkali metal hydroxide or alkaline earth metal, and can be an alkali metal amide salt such as lithium diisopropylamide, lithium hexamethyldisilazide, sodium hexamethyldisilazide, potassium hexamethyldisilazide, etc. The alkylating agent can be a halogenated alkane or a sulfonate, such as bromoalkane, iodoalkane, dialkyl sulfate, alkyl p-toluenesulfonate, 2-bromoethyl methyl ether, 2-methoxyethyl methanesulfonate, etc., and the alkyl group is mainly methyl, ethyl, etc. The molar ratio of 3'-O-benzylguanosine to the base is 1:1.0-1.5; the molar ratio of 3'-O-benzylguanosine to the alkylating agent is 1:1.0-1.5; the alkylation reaction temperature is 35°C-60°C; and the alkylation reaction time is 6-24h.

[0021] Furthermore, in step (4), the hydrodebenzylation reaction is carried out in the presence of an organic solvent such as methanol, ethanol, or tetrahydrofuran, using hydrogen as a hydrogen source and palladium carbon (5% to 10%) as a catalyst; the molar ratio of the palladium carbon catalyst to 3'-O-benzyl-2'-O-alkylguanosine is 1:5 to 10; the reaction temperature is 25° C. to 60° C.; the reaction time is 2 to 18 h; and the pressure is 0.1 to 4 MPa.

[0022] Compared with the prior art, the present invention has the following advantages: (1) the raw materials of the present invention are cheap and readily available, green and environmentally friendly, the synthesis steps are short and simple, easy to operate, and easy to scale up for preparation; (2) the selectivity of each step of the reaction in the present invention is high, and the product is easy to separate. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below with reference to the embodiments.

[0024] For ease of understanding, a representative overall synthesis route of one of the target compounds 2'-O-methoxyethylguanosine described in the present invention is described, and its synthesis route is as follows:

[0025]

[0026] The synthetic method of the present invention comprises the following specific steps:

[0027] (1) first dissolving guanosine in toluene, then adding benzaldehyde and p-toluenesulfonic acid in sequence, heating to reflux for acetalization reaction and separating the generated water, and continuing the reflux reaction for half an hour after no obvious water is separated, and obtaining 2',3'-benzylideneguanosine through post-treatment and separation and purification;

[0028] (2) Dissolve 2',3'-benzylidene guanosine in an organic solvent such as dichloromethane, add a reducing agent, and stir at room temperature to 60°C for 20 minutes under nitrogen protection. Then add an acid catalyst and stir at 30°C to 60°C for 4 to 24 hours under nitrogen protection to produce a reduction ring-opening reaction to synthesize 3'-O-benzyl guanosine;

[0029] (3) dissolving 3'-O-benzylguanosine in an organic solvent, adding sodium hydride, stirring at 35°C to 60°C for 1 to 2 hours, then adding bromoethyl methyl ether to the reaction system, stirring at 35°C to 60°C for 6 to 16 hours under nitrogen protection, and performing Williamson etherification reaction to synthesize 3'-O-benzyl-2'-O-methoxyethylguanosine;

[0030] (4) 3'-O-benzyl-2'-O-methoxyethylguanosine is dissolved in an organic solvent, palladium carbon is added, air is replaced by nitrogen gas three times under normal pressure, hydrogen gas is introduced to 2 MPa, and the mixture is stirred at 25°C to 60°C for 2 to 18 hours to produce a reductive debenzylation reaction to synthesize 2'-O-methoxyethylguanosine.

[0031] (I) Preparation of 2',3'-benzylideneguanosine

[0032] Embodiment 1:

[0033]

[0034] Add 2.832g (10mmol) of guanosine to 60mL of toluene, then add 1.166g (11mmol) of benzaldehyde and 0.344 (2mmol) of p-toluenesulfonic acid in sequence, install a reflux water separator, heat to reflux, react for 10h without obvious water release, and continue to react for 0.5h. After the reaction is completed, add saturated sodium bicarbonate solution to adjust the pH to 7-8, and remove toluene from the organic phase under reduced pressure to obtain a yellow oily liquid. Neutral alumina column chromatography to obtain 2.971g of white solid, with a yield of 80%.

[0035] Embodiment 2~5:

[0036] The amount of acid catalyst was changed, and other operating conditions were the same as in Example 1 to explore the effect of the amount of acid catalyst on the reaction results. The results are shown as follows:

[0037]

[0038] Embodiments 6 to 9

[0039] The amount of benzaldehyde was changed, and other operating conditions were the same as in Example 1 to explore the effect of the amount of benzaldehyde on the reaction results. The results are shown as follows:

[0040]

[0041] (II) Preparation of 3'-O-benzylguanosine

[0042] Example 10

[0043]

[0044] Dissolve 1.857g (5mmol) of 2',3'-benzylidene guanosine in 20mL of acetonitrile, add 1.571g (25mmol) of sodium cyanoboride and 2.75mL (25mmol) of titanium tetrachloride at 0℃, and stir for 8h. After the reaction is completed, quench the reaction with saturated sodium bicarbonate solution, remove the solvent by vacuum distillation, dissolve and extract with ethyl acetate, wash with saturated bicarbonate solution, wash with saturated brine, and vacuum distill the organic phase to obtain a reddish brown solid. Neutral alumina column chromatography, 3'-O-benzylguanosine 0.84g, yield 45%; 2'-O-benzylguanosine 0.112g, yield 6%; guanosine 0.552g, yield 39%; recovered raw material 0.182g.

[0045] Example 11

[0046]

[0047] Dissolve 1.857g (5mmol) of 2',3'-benzylidene guanosine in 20mL of dichloromethane, add 1.824g (25mmol) of borane trimethylamine complex, protect with nitrogen, stir at 40℃ for 20min, then add 3.620g (10mmol) of copper trifluoromethanesulfonate, stir at 40℃ for 16h. Add 0.7mL of triethylamine and 9mL of methanol dropwise to quench the reaction, remove the solvent by vacuum distillation, dissolve and extract with ethyl acetate, wash with saturated bicarbonate solution, wash with saturated brine, and vacuum distill the organic phase to obtain a reddish brown solid. Neutral alumina column chromatography, obtain 1.325g of brown solid, yield 71%, and recover 0.464g of raw material.

[0048] Example 12

[0049]

[0050] Dissolve 1.857g (5mmol) of 2',3'-benzylidene guanosine in a mixed solution of 20mL of dichloromethane and ether, then add 0.949g (25mmol) of lithium aluminum hydride, protect with nitrogen, stir at 45℃ for 20min, then add 3.333g (25mmol) of anhydrous aluminum chloride, continue to stir and react at reflux for 16h. After the reaction is completed, add deionized water to quench the reaction, remove the solvent by vacuum distillation, dissolve and extract with ethyl acetate, wash with saturated bicarbonate solution, wash with saturated brine, and vacuum distill the organic phase to obtain a reddish brown solid. Neutral alumina column chromatography, obtain 0.970mg of brown solid, yield 52%; separate and obtain 0.283g of deep reduction product guanosine, yield 20%; recover 0.464g of raw material.

[0051] (III) Preparation of 3'-O-benzyl-2'-O-methoxyethylguanosine

[0052] Example 13

[0053]

[0054] 1.865 g (5 mmol) of 3'-O-benzylguanosine was dissolved in 20 mL of DMF, and then 0.3 g (60%, 7.5 mmol) of sodium hydride was added, and the mixture was stirred at 35°C for 1 h under nitrogen protection, and then 1.043 g (7.5 mmol) of bromoethyl methyl ether was added, and the mixture was stirred at 35°C for 6 h. After the reaction was completed, the reaction mixture was poured into 30 mL of ice water to quench the reaction, and the solvent was removed by distillation under reduced pressure, dissolved and extracted with ethyl acetate, washed with saturated bicarbonate solution, washed with saturated brine, and the organic phase was distilled under reduced pressure, and chromatographed on a neutral alumina column to obtain 1.358 g of a yellow solid with a yield of 63%.

[0055] (IV) Preparation of 2'-O-methylguanosine

[0056] Embodiment 14

[0057]

[0058] 1.078 g (2.5 mmol) of 3'-O-benzyl-2'-O-methoxyethyl guanosine was added to 30 mL of ethanol solution, and 2.120 g (10%, 0.5 mmol) of palladium carbon was added. The atmosphere was replaced with nitrogen three times, and hydrogen was introduced to 2 MPa. The mixture was heated to 50°C and reacted for 2 h. The mixture was filtered through a sand core, washed with ethyl acetate, and the filtrate was distilled under reduced pressure to obtain 0.852 g of a white solid with a yield of 99%.

Claims

1. 2'-O-alkylguanosine, characterized in that Has the following structure:

2. A method for preparing 2'-O-alkylguanosine, characterized in that: The following steps are involved: (1) Under acid catalysis, the two hydroxyl groups of guanosine (I) at 2'- and 3'-positions are protected with benzyl groups using para-substituted benzaldehyde to prepare compound II; (2) Under the conditions of an acid catalyst and a reducing agent, the five-membered cyclic acetal in compound II is subjected to regioselective reduction and ring-opening to prepare 3'-O-benzylguanosine (III); (3) Under alkaline conditions, 3'-O-benzylguanosine (III) is reacted with an alkylating agent to undergo a Williamson etherification reaction to prepare 3'-O-benzyl-2'-O-alkylguanosine (IV); (4) a step of removing the benzyl group on the 3'-hydroxyl group in compound IV in the presence of Pd / C and hydrogen to prepare 2'-O-alkylguanosine (V); 3. The preparation method according to claim 2, characterized in that: In step (1), the acetalization reaction is carried out in an organic solvent such as benzene, toluene, xylene, etc. that can form an azeotrope with water; the acid catalyst can be a Lewis acid such as anhydrous aluminum chloride, anhydrous ferric chloride, anhydrous cobalt chloride, anhydrous copper chloride, anhydrous zinc chloride, copper trifluoromethanesulfonate, boron trifluoride ether complex, etc., can be a protonic acid such as hydrochloric acid, sulfuric acid, glacial acetic acid, p-toluenesulfonic acid, camphorsulfonic acid, etc., can also be other solid acids such as Amberlyst 15H + , acid salts such as potassium hydrogen sulfate, etc.; the acetalization protective agent can be an aromatic aldehyde, including benzaldehyde, p-fluorobenzaldehyde, p-chlorobenzaldehyde, p-bromobenzaldehyde, p-trifluoromethylbenzaldehyde, p-methoxybenzaldehyde, p-methylbenzaldehyde, p-ethylbenzaldehyde, p-isopropylbenzaldehyde, p-tert-butylbenzaldehyde, etc. The molar ratio of guanosine to aromatic aldehyde is 1:1.1-2; the molar ratio of guanosine to acid catalyst is 1:0.1-1; the acetalization reaction time is 12-24h.

4. The preparation method according to claim 2, characterized in that: In step (2), the regioselective reduction ring-opening reaction is carried out in an organic solvent such as acetonitrile, tetrahydrofuran, dichloroethane, 1,4-dioxane, benzene, toluene, etc.; the acid catalyst can be a halide such as anhydrous aluminum chloride, anhydrous ferric chloride, anhydrous cobalt chloride, anhydrous copper chloride, anhydrous zinc chloride, zinc iodide, etc., can be a protonic acid such as sulfuric acid, glacial acetic acid, p-toluenesulfonic acid, etc., can be a triflate such as zinc triflate, scandium triflate, copper triflate, trifluoromethanesulfonate Silver methanesulfonate, praseodymium trifluoromethanesulfonate, europium trifluoromethanesulfonate, samarium trifluoromethanesulfonate, indium trifluoromethanesulfonate, lanthanum trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, gadolinium trifluoromethanesulfonate, neodymium trifluoromethanesulfonate, or other Lewis acids such as trimethylsilyl trifluoromethanesulfonate, boron trifluoride ethyl ether, etc.; the reducing agent can be 9-borabicyclo[3.3.1]nonane, sodium cyanoboride, borane dimethyl sulfide complex, borane trimethylamine complex, lithium aluminum hydride, diisobutylaluminum hydride, etc. The molar ratio of acetal protected guanosine to acid catalyst is 1:0.05-0.5; the molar ratio of acetal protected guanosine to reducing agent is 1:2-10; the temperature of hydrogenation incomplete reduction ring opening reaction is 30℃-60℃; the reaction time is 4-24h.

5. The preparation method according to claim 2, characterized in that: In step (3), the alkylation reaction is carried out in the presence of an organic solvent such as acetonitrile, acetone, dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, etc. The base can be sodium hydride, alkali metal hydroxide or alkaline earth metal, and can be an alkali metal amide salt such as lithium diisopropylamide, lithium hexamethyldisilazide, sodium hexamethyldisilazide, potassium hexamethyldisilazide, etc. The alkylating agent can be a halogenated alkane or a sulfonate, such as bromoalkane, iodoalkane, dialkyl sulfate, alkyl p-toluenesulfonate, 2-bromoethyl methyl ether, 2-methoxyethyl methanesulfonate, etc., and the alkyl group is mainly methyl, ethyl, etc. The molar ratio of 3'-O-benzylguanosine to the base is 1:1.0-1.5; the molar ratio of 3'-O-benzylguanosine to the alkylating agent is 1:1.0-1.5; the alkylation reaction temperature is 35°C-60°C; and the alkylation reaction time is 6-24h.

6. The preparation method according to claim 2, characterized in that: In step (4), the hydrodebenzylation reaction is carried out in the presence of an organic solvent such as methanol, ethanol, or tetrahydrofuran, using hydrogen as a hydrogen source and palladium carbon (5% to 10%) as a catalyst; the molar ratio of the palladium carbon catalyst to 3'-O-benzyl-2'-O-alkylguanosine is 1:5 to 10; the reaction temperature is 25° C. to 60° C.; the reaction time is 2 to 18 h; and the pressure is 0.1 to 4 MPa.

Images