Chemical synthesis method of beta-nicotinamide mononucleotide
Technical Field
The invention relates to the technical field of drug synthesis, in particular to a chemical synthesis method of beta-nicotinamide mononucleotide.
Background
Nicotinamide Mononucleotide (NMN) is Nicotinamide Adenine Dinucleotide (NAD)+) A synthetic intermediate of (1). It has been shown to function primarily as NAD in vivo+By conversion to exert its physiological function, e.g. activation of NAD+Substrate-dependent enzyme Sirt1 (histone deacetylase), RegulationNodal cell survival, death, and maintenance of redox status. Research has proved that the level of NMN is related to cardiovascular and cerebrovascular diseases, neurodegenerative diseases, diabetes and obesity.
Beta-nicotinamide mononucleotide is the cofactor NAD for the human longevity protein+The precursor of (1). NAD (nicotinamide adenine dinucleotide)+Is an important coenzyme of tricarboxylic acid cycle, promotes the metabolism of sugar, fat and amino acid, and participates in the synthesis of energy; NAD (nicotinamide adenine dinucleotide)+In the aspect of participating in human metabolism, NAD can be additionally supplemented+Can improve the function of human body.
The chemical structural formula of beta-Nicotinamide Mononucleotide (NMN) is as follows:
the production process of NMN mainly comprises an enzymatic method and a chemical synthesis method. The enzyme method has higher requirements on production conditions and large early investment, so the chemical synthesis method is still a hot spot in the current research.
One of the existing chemical synthesis methods is as follows: an effective chemical synthesis of nicotinamide riboside (NAR) and analogues report the process of synthesizing NMN by using tetraacetyl ribose and nicotinamide as starting materials, wherein the reaction yield of nicotinamide riboside salt is 58%, and the defects of rapid reaction, difficult control, more product impurities and low total yield exist.
The second existing chemical synthesis method: benzoyl ribofuranose or acetyl ribofuranose is used as a raw material, brominated or chlorinated, and then subjected to condensation reaction with nicotinamide to generate nucleoside, and then a protecting group is removed and phosphorylation is carried out to prepare a target product, wherein the yield of halogenation reaction is low and the environment is not friendly.
The third existing chemical synthesis method: the method takes acetylribofuranose and nicotinamide ethyl ester as raw materials, and prepares a finished product through condensation, deprotection, phosphorylation, ammonolysis and purification.
New synthetic techniques have also emerged, such as continuous reactions in microchannel reactors, in an attempt to achieve the highest yields with the shortest reaction times. However, the micro-channel and other related technologies have the problem of poor stepwise heat conduction effect, so that more impurities are generated, and the difficulty of later separation and purification is increased.
In view of this, the invention is particularly proposed.
Disclosure of Invention
Therefore, the invention provides a chemical synthesis method of beta-nicotinamide mononucleotide. The method adopts 1,2,3, 5-tetraphenyloyloxy-2-C-methyl-beta-D-ribofuranose and nicotinamide as starting materials, has mild reaction conditions, low impurity content and high total yield, and also has the advantages of short synthesis steps, suitability for operation and environmental friendliness.
A method for the chemical synthesis of β -nicotinamide mononucleotide, comprising: 1,2,3, 5-tetraphenyl formyloxy-2-C-methyl-beta-D-ribofuranose and nicotinamide are taken as initial raw materials, and the beta-nicotinamide mononucleotide is prepared by condensation reaction, debenzoyl protecting group removal and phosphorylation reaction in sequence.
The invention unexpectedly discovers that the ribofuranose of which the hydroxyl group is protected by the benzoyl has larger steric hindrance, and the ribofuranose and the nicotinamide are subjected to condensation reaction, so that the reaction condition is relatively mild, the byproducts are less, and the yield is high.
The synthetic route of the invention is as follows:
the specific synthetic process is as follows: 1,2,3, 5-tetraphenyl formyloxy-2-C-methyl-beta-D-ribofuranose (compound A) containing active hydrogen is subjected to Claisen condensation reaction under the action of a catalyst to obtain nicotinamide tribenzoyl nucleoside (compound B), the benzoyloxy of the nicotinamide tribenzoyl nucleoside (compound B) is subjected to alkaline hydrolysis to obtain nicotinamide nucleoside salt (compound C), and the hydroxyl of the nicotinamide nucleoside salt (compound C) is subjected to phosphorylation to obtain beta-nicotinamide mononucleotide (compound D).
In some embodiments of the invention, the condensation reaction is at a temperature of 40-50 ℃.
In some embodiments of the present invention, the condensation reaction is performed in the presence of a first solvent and a catalyst, the first solvent is selected from at least one of dichloromethane, tetrahydrofuran, N-dimethylformamide, chloroform, dimethyl sulfoxide, acetone, preferably dichloromethane and/or tetrahydrofuran; the catalyst is selected from trifluoromethanesulfonic acid, toluenesulfonic acid or trimethylsilyl trifluoromethanesulfonate, preferably trifluoromethanesulfonic acid;
or, the mole ratio of the 1,2,3, 5-tetraphenyl formyloxy-2-C-methyl-beta-D-ribofuranose, nicotinamide and the catalyst is 1: (1.5-1.6): (2.0-2.1);
or the dosage (ml) of the first solvent is 5-6 times of the mass (g) of the 1,2,3, 5-tetraphenyl formyloxy-2-C-methyl-beta-D-ribofuranose.
In some embodiments of the invention, the temperature of the debenzoyl protecting group is 35-40 ℃.
In some embodiments of the invention, the debenzoyl protecting group is performed in a second solvent selected from at least one of ethanol, methanol, isopropanol, preferably ethanol, and an organic base; the organic base is selected from triethylamine, sodium methoxide, sodium ethoxide or tert-butyl potassium alkoxide, preferably triethylamine;
or the molar ratio of the organic base to the 1,2,3, 5-tetraphenyloloxy-2-C-methyl-beta-D-ribofuranose is (2.5-3): 1;
or dissolving the product of condensation reaction in a second solvent, adding organic base, and controlling the feeding speed to ensure that the temperature of the system does not exceed-5 ℃.
In some embodiments of the invention, the temperature of the phosphorylation reaction is 20-25 ℃.
In some embodiments of the present invention, the phosphorylation reagents used in the phosphorylation reaction are trimethyl phosphate and phosphorus oxychloride.
In some embodiments of the invention, the method further comprises anion exchange resin desalting purification of the product of the phosphorylation reaction. Preferably, the anion exchange resin is DD2 ion resin produced by shanghai huasha resin factory.
In some embodiments of the invention, the wash is first washed with water, the water wash is collected, and then the 1-10% CF is added3COOH eluting, collecting eluent, combining water washing liquid and eluent, and freeze-drying to obtain high-purity beta-nicotinamide mononucleotide.
In some embodiments of the invention, the method for synthesizing β -nicotinamide mononucleotide comprises the following steps:
(1) condensation reaction: dissolving 1,2,3, 5-tetraphenyl formyloxy-2-C-methyl-beta-D-ribofuranose, nicotinamide and a catalyst in a first solvent, reacting for 1-3h at 40-50 ℃, cooling to room temperature, adding methanol to quench the reaction, and removing the solvent under reduced pressure to obtain nicotinamide tribenzoyl nucleoside;
(2) debenzoyl protecting group: dissolving nicotinamide tribenzoyl nucleoside obtained in the step (1) in a second solvent, adding organic base at-10 to-5 ℃, controlling the feeding speed to ensure that the temperature of the system does not exceed-5 ℃, then heating to 35-40 ℃ for reaction for 2-3h, dropwise adding dilute hydrochloric acid to ensure that the system is neutral, adding methyl tert-butyl ether, precipitating solid at 0 ℃, and filtering to obtain nicotinamide nucleoside salt;
(3) phosphorylation reaction: dropwise adding a mixed solution of phosphorus oxychloride and trimethyl phosphate into the nicotinamide riboside salt obtained in the step (2) at the temperature of-5-0 ℃, keeping the temperature for reacting for 1-2 hours, turning to room temperature after the reaction is finished, adding ice water for quenching reaction, extracting an organic phase with ethyl acetate, and concentrating to obtain a crude product of beta-nicotinamide mononucleotide;
(4) desalting and purifying: dissolving the crude product of the beta-nicotinamide mononucleotide obtained in the step (3) in water, adsorbing the crude product by DD2 ionic resin, firstly washing the crude product by the water, collecting water washing liquid, and then using 5% CF3COOH eluting, collecting eluent, combining water washing liquid and eluent, and freeze-drying to obtain beta-nicotinamide mononucleotide.
The invention has the following technical effects:
1. the invention takes 1,2,3, 5-tetraphenyl formyloxy-2-C-methyl-beta-D-ribofuranose and nicotinamide as initial raw materials to carry out condensation reaction, the reaction condition is mild, the by-products are less, after quenching reaction, the nicotinamide tribenzoyl nucleoside with higher purity can be obtained by removing the solvent under reduced pressure, and the nicotinamide tribenzoyl nucleoside can be directly used for next reaction without purification.
2. The method can obtain the high-purity beta-nicotinamide mononucleotide by three steps of reactions (each step of reaction does not need purification) and one step of desalting purification, and has the advantages of easily obtained raw materials, short reaction route, simple post-treatment, environmental protection, high reaction total yield and suitability for industrial production.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
Nicotinamide and 1,2,3, 5-tetraphenyloyloxy-2-C-methyl-beta-D-ribofuranose (CAS number: 15397-15-6) used in the following examples are all technical grade, and the purity is more than 97%.
Example 1
This example provides a method for synthesizing β -nicotinamide mononucleotide, comprising the following steps:
(1) condensation reaction
58 g of 1,2,3, 5-tetraphenyloyloxy-2-C-methyl-beta-D-ribofuranose and 18.3 g of nicotinamide were weighed out and dissolved in 300ml of tetrahydrofuran, and the resulting solution was transferred to a 1000ml three-necked flask with mechanical stirring for further use. Transferring 30 g of trifluoromethanesulfonic acid into a dropping funnel, slowly dropping into the system for 40 minutes, controlling the temperature not to exceed 50 ℃ in the dropping process, keeping the temperature at 50 ℃ after dropping, refluxing for 2 hours, and cooling to room temperature after HPLC detection reaction is completed. Adding 100ml of water to quench and react; the product was extracted by adding 200ml of dichloromethane with stirring, the organic phase was washed three times with saturated brine, once with purified water, and concentrated to dryness under reduced pressure to give nicotinamide tribenzoyl nucleoside in a yield of 72.0% and a quantitative purity of 87.5% by HPLC.
(2) Debenzoyl protecting group
Dissolving nicotinamide tribenzoyl nucleoside obtained by the reaction in 300ml of absolute ethanol, cooling to-5 ℃, dropwise adding triethylamine 40ml while stirring, controlling the dropwise adding speed, controlling the temperature to be not higher than-5 ℃ because the temperature is increased in the reaction process, completing dropwise adding within about 1.5 hours, stirring for 30 minutes, heating to 35 ℃, and reacting for 2.5 hours; after the HPLC detection reaction is finished, a certain amount of dilute hydrochloric acid is dripped until the reaction system is neutral. 200ml of methyl tert-ether were added and the organic phase was obtained with stirring. Cooling to 0 ℃, separating out the product, separating, decompressing and drying to obtain nicotinamide riboside salt, wherein the yield is 95.6%, and the HPLC quantitative purity is 93.8%.
(3) Phosphorylation reactions
Dissolving nicotinamide riboside salt obtained by the reaction in 200ml of acetonitrile, dissolving 23 g of phosphorus oxychloride in 50ml of trimethyl phosphate for later use, slowly dripping the mixed solution of the trimethyl phosphate and the phosphorus oxychloride into the solution at the temperature of-5-0 ℃, keeping the constant speed, slowly dripping for 1.5 hours, detecting the reaction progress by HPLC, and finishing the reaction for about 3 hours. Adding about 200ml of water to quench the reaction; adding 150ml ethyl acetate to extract twice under stirring, combining organic phases, concentrating to dryness to obtain a crude product of the beta-nicotinamide mononucleotide, wherein the yield is 88.0 percent, and the quantitative purity of HPLC is 91.5 percent.
(4) Desalting and purifying
Dissolving the crude product of beta-nicotinamide mononucleotide obtained in the above step in water, adsorbing with DD2 ion resin, washing with water, and then using 5% CF by volume percentage3COOH elution, collecting chromatographic liquid, and freeze drying to obtain beta-nicotinamide mononucleotide with yield of 75.6% and HPLC quantitative purity of 98.32%.
Example 2
This example provides a method for synthesizing β -nicotinamide mononucleotide, comprising the following steps:
(1) condensation reaction
58 g of 1,2,3, 5-tetraphenyloyloxy-2-C-methyl-beta-D-ribofuranose and 18.3 g of nicotinamide were weighed out and dissolved in 300ml of dichloromethane, and the solution was transferred to a 1000ml three-necked flask with mechanical stirring for use after stirring. Transferring 48 g of trimethylsilyl trifluoromethanesulfonate into a dropping funnel, slowly dropping into the system for 40 minutes, controlling the temperature not to exceed 50 ℃ in the dropping process, keeping the temperature at 50 ℃ after dropping, refluxing for 3 hours, and cooling to room temperature after HPLC detection reaction is completed. Adding 200ml of water to quench and react; the product was extracted with 200ml of dichloromethane and the organic phase was washed three times with saturated brine, once with purified water and concentrated to dryness under reduced pressure to give nicotinamide tribenzoyl nucleoside in 71.5% yield and 86.8% quantitative purity by HPLC.
(2) Debenzoyl protecting group
Dissolving 46 g of sodium methoxide in 50ml of ethanol for later use; dissolving nicotinamide tribenzoyl nucleoside obtained by the reaction in 300ml of absolute ethanol, cooling to-5 ℃, dropwise adding an ethanol solution of sodium methoxide while stirring, controlling the dropwise adding speed, controlling the temperature to be not higher than-5 ℃ because the temperature is increased in the reaction process, completing dropwise adding within about 2 hours, stirring for 30 minutes, heating to 35 ℃, and reacting for 4 hours; after the HPLC detection reaction is finished, a certain amount of dilute hydrochloric acid is dripped until the reaction system is neutral. 200ml of methyl tert-ether were added and the organic phase was obtained with stirring. Cooling to 0 ℃, separating out the product, separating, decompressing and drying to obtain nicotinamide riboside salt, wherein the yield is 95.0%, and the HPLC quantitative purity is 93.2%.
(3) Phosphorylation reactions
Dissolving nicotinamide riboside salt obtained by the reaction in 200ml of acetonitrile, dissolving 23 g of phosphorus oxychloride in 50ml of trimethyl phosphate for later use, slowly dripping the mixed solution of the trimethyl phosphate and the phosphorus oxychloride into the solution at the temperature of-5-0 ℃, keeping the constant speed, slowly dripping for 1.5 hours, detecting the reaction progress by HPLC, and finishing the reaction for about 3 hours. Adding about 200ml of water to quench the reaction; adding 150ml ethyl acetate to extract twice under stirring, combining organic phases, concentrating to dryness to obtain a crude product of the beta-nicotinamide mononucleotide, wherein the yield is 87.5 percent, and the quantitative purity of HPLC is 90.5 percent.
(4) Desalting and purifying
Dissolving the crude product of beta-nicotinamide mononucleotide obtained in the above step in water, adsorbing with DD2 ion resin, washing with water, and then using 5% CF by volume percentage3COOH eluting, collecting chromatographic solution, and freeze-drying to obtain beta-nicotinamideMononucleotide, yield 72.3% and quantitative purity of 97.50% by HPLC.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.