Background technology
Cytidine(C (Cytidine), other the name citicoline, CDP-Cholion, diphosphate choline, Citicoline, cytidine diphosphate, Buddhist nun Kelin, the Buddhist nun can spirit or cytidine.It is made up of cytosine(Cyt) (base portion) and ribose (sugar moieties).Its structural formula is following:
The method of at present existing multiple synthesizing cytimidine nucleosides (cytidine), specific as follows:
(1) method that proposed in 1964 of people such as Nishimara is utilized the N of silicon etherificate protection
4-ethanoyl cytosine(Cyt) and 1-chloro triphenyl formyl radical ribose react under the reflux condition and generate the mix-configuration cytidine, utilize recrystallization and column chromatography with two kinds of isomer separation then.Its synthetic route is as shown in Figure 1.
Wherein, Bz is the phenyl formyl radical;
The shortcoming of this method is that flow process is complicated, yield is low, and its raw material 1-chloro triphenyl formyl radical ribose is difficult to obtain, and is α, two kinds of configuration mixtinites of β, and it participates in reaction back resultant also is two kinds of isomer, the aftertreatment difficulty.This method can't be accomplished scale production.
(2) people such as Helmut utilizes uridine to be raw material, through hexamethyldisilazane (HMDS) silicon etherificate obtain 4,2 ', 3 ', 5 '-four silicon etherificate uridines, a step high pressure ammonia is separated and is obtained cytidine in the exsiccant container then.Uridine expensive raw materials in this method, and the deprotection reaction condition is harsh, low suitability for industrialized production (the Adventures in Silicon-Organic Chemistry that is difficult to of yield; Acc.Chem.Res.1995,28 (12), 509-520).Its synthetic route is following:
(3) Zemlicka etc. is a raw material with the uridine; 150 ℃ of reflux in the presence of hexamethyldisilazane (HMDS) and trimethylchlorosilane; Make uridine silicon etherificate; High temperature reflux can obtain the cyclobutyl cytidine in the presence of tetramethyleneimine then, it is handled can obtain cytidine (Dephosp horylation of ribonucleoside 2 ' (3 ')-phosphates. in ammonia methyl alcohol; Tetrahedron Letters, 1969,9,715-718.).Its synthetic route is following:
This method and method (2) are identical substantially, when obtain 4,2 ', 3 ', behind 5 '-four silicon etherificate uridines, when making 4 to change amino into, used tetramethyleneimine, and then salify is converted into amino 4 nucleophilic substitution, this reaction yield is lower, operation is more complicated.
(4) people such as Sugiura Y. is raw material with the cytosine(Cyt), obtains N-isobutyryl cytosine(Cyt) with the isobutyric anhydride acylations, separates preparation cytidine (Syntheses of N with furanose through condensation, ammonia again
4, 2 ', 3 ', 5 '-Tetraacylcytidines from N
4-Acylcytosines, via Condensation with Tetraacylribose and Transribosylation with Acylated Purine Nucleosides; Chem.Pharm.Bull.36 (9), 1988,3253-3256).Its synthetic route is following:
Wherein, Py is a pyridine, and Leis acid is a lewis acid;
Raw materials used isobutyric anhydride of this method and 1-acetyl-2,3,5-tri-benzoyl ribofuranose price is higher and yield is lower, unsuitable suitability for industrialized production.
(5) people such as Vorbruggen H has reported the pyrimidine base of a part TMS protection and method (the A General Synthesis of N-Glycosides.I. of the shielded sugared condensation of a part
1Synthesis of Pyrimidine Nucleosides; J.Org.Chem.Vol.39, No.25,1974,3654-3660), and with reference to cytosine(Cyt) and the 1-acetyl-2,3 of this method with the total silicon baseization, the condensation of 5-tri-benzoyl ribofuranose, the synthetic route that the deprotection base prepares cytidine is following:
In this method, the cytosine(Cyt) of trimethyl silicon based etherificate is prone to deliquescence in air, cause the cytidine(C yield lower, and raw material 1-acetyl-2,3, and 5-tri-benzoyl ribofuranose price is higher, can't suitability for industrialized production.
Disclose among the one Chinese patent application CN86101400 and a kind ofly used acyl group cytosine(Cyt) and the shielded monose of hydroxyl or prepared a kind of cytidine(C with method that the shielded nucleosides of hydroxyl (its base portion is not the nucleosides of cytosine(Cyt)) reacts; In case of necessity, comprise the reaction of removing hydroxyl-protection base.This method starting raw material is the acyl group cytosine(Cyt), and it is more expensive to compare the cytosine(Cyt) price, unsuitable suitability for industrialized production.
Summary of the invention
The invention provides a kind of preparation method of cytidine(C, operational path is simple, and cost is lower, can also reduce environmental pollution, produces safer.
A kind of preparation method of cytidine(C may further comprise the steps:
1. silylanization: cytosine(Cyt) and TERT-BUTYL DIMETHYL CHLORO SILANE are got N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE through Silanization reaction, N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE is dissolved the chloroform soln that obtains N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE in trichloromethane;
2. glucosides is synthetic: tetrem acyl ribose is dissolved the back add titanium tetrachloride solution in trichloromethane; Under 18 ℃~30 ℃ envrionment temperature, drip the chloroform soln of N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE, through glucosides react cytosine(Cyt) acetyl ribose;
3. ammonia is separated: be the methanol solution (being methanol ammonia solution) of 10% ammonia in the mass percentage concentration that in cytosine(Cyt) acetyl ribose, adds ammonia under 18 ℃~30 ℃ the envrionment temperature, get the cytidine(C bullion through ammonolysis reaction;
4. refining: the cytidine(C bullion is added in the ethanol, add water after the reflux dissolving under agitation, be cooled to 0 ℃ ± 2 ℃ crystallizatioies and isolate solid behind the 5h at least, drying obtains cytidine(C.
The concrete synthetic route of the inventive method is following:
Wherein, the formula I is a cytosine(Cyt), and the formula II is a TERT-BUTYL DIMETHYL CHLORO SILANE, and the formula III is N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE, and the formula IV is a tetrem acyl ribose, and the formula V is a cytosine(Cyt) acetyl ribose, and the formula VI is a cytidine(C.
The present invention finds to adopt the silica-based protection base as cytosine(Cyt) of tertiary butyl dimethyl-; Not only can effectively protect group; Can also effectively avoid the deliquescence of product N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE; For subsequent reactions provides strong assurance, finally significantly improve the yield of cytidine(C.
Step 1. in, described Silanization reaction preferably carries out in nitrogen or protection of inert gas, can further reduce the generation of by product, improves reaction yield and selectivity.Described rare gas element can be selected this area rare gas element commonly used for use, a kind of as in argon gas, the helium etc.
In order to improve speed of reaction, described Silanization reaction can adopt catalyst, and described catalyzer is selected ammonium sulfate for use.The add-on of catalyzer is preferably 1%~2% of cytosine(Cyt) quality.
The temperature of described Silanization reaction is preferably 110 ℃~130 ℃, can guarantee the carrying out of Silanization reaction better, further is preferably 120 ℃.
Step 2. in, the reaction of described glucosides is preferably carried out under whipped state, is more conducive to the carrying out of glucosides reaction.
Described titanium tetrachloride solution plays katalysis in the glucosides reaction, its consumption is preferably 0.9%~1.2% of tetrem acyl ribose quality.
Step 3. in, described ammonolysis reaction preferably carries out under whipped state, is more conducive to the carrying out of ammonolysis reaction.
Described ammonolysis reaction directly adopts commercially available methanol ammonia solution to separate solvent as ammonia, not only is beneficial to the carrying out of ammonolysis reaction but also draw materials conveniently, and cost is low.
Consumption to each raw material in each step reaction of the present invention does not have strict restriction, press generally that chemical reaction equation metering mol ratio or part material are suitable excessive all can.Consider from the equal angles that economizes in raw materials, step 1. in, the mol ratio of cytosine(Cyt) and TERT-BUTYL DIMETHYL CHLORO SILANE is preferably 1: 2~3; Step 2. in, the mol ratio of N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE and tetrem acyl ribose is preferably 1: 1~1.5; Step 3. in, the mol ratio of cytosine(Cyt) acetyl ribose and ammonia is preferably 1: 3~4.
Step 1., 2. step adopt existing method for judging reaction end such as tlc (TLC), liquid chromatography or gc etc. to carry out trace analysis with the method for judging reaction end of step in 3.; All react when wherein a certain raw material or plurality of raw materials and to finish, the terminal point that is regarded as reacting gets final product.Through overtesting, to carry out fully in order to make reaction, the reaction times of the Silanization reaction of step described in 1. is generally 6h to 10h; The reaction times of the glucosides reaction of step described in 2. is generally 2h to 5h; The reaction times of the ammonolysis reaction of step described in 3. is generally 1.5h to 2.5h.
Raw material that the present invention is used and reagent all can adopt the commercially available prod, and raw material is easy to get, and the price of used raw material and reagent is lower, greatly reduces production cost.
The method of calculation of yield are among the present invention: Theoretical Mass * 100% of the actual mass ÷ product of yield=product, total recovery=respectively the go on foot product of reaction yield.
The detection method of cytidine(C purity: HPLC among the present invention detects wavelength 242nm, moving phase acetonitrile: water=88: 12 (volume ratio), flow velocity 0.5ml/min, C18 chromatographic column, 30 ℃ of column temperatures, sample size 1 μ L.
Compared with prior art, the present invention has following advantage:
With prior art relatively, the present invention through the protection of tertiary butyl dimethylsilane, C-N key glucosides is synthetic, ammonia is separated and the preparation of make with extra care the completion cytidine(C, this process step is simple; The inexpensive amount of the solvent of selecting for use is few; The raw material that uses be easy to get and price lower, preparation of industrialization yield Gao Keda is more than 92%, purity Gao Keda is more than 99.5%; Can effectively be controlled to the product cost, make production cost lower.Operational path of the present invention is simple, and cost is low, and environmental pollution is little, and production safety is a route that is suitable for suitability for industrialized production, is with a wide range of applications.
Description of drawings
Fig. 1 is the synthetic route chart of the method that proposed in 1964 of people such as Nishimara, and wherein, Bz is the phenyl formyl radical.
Embodiment
Embodiment 1
In the 250ml there-necked flask, add cytosine(Cyt) 10g (0.09mol), ammonium sulfate 150mg keeps dry, and in there-necked flask, feeds nitrogen protection, adds TERT-BUTYL DIMETHYL CHLORO SILANE 30g (0.20mol) then, stirs at a slow speed.Finish, be warming up to 120 ℃ of insulation reaction 8h, reaction is finished.Obtain product N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE.Product is dissolved with the 100ml trichloromethane.
In the 250ml there-necked flask; Add tetrem acyl ribose 35g (0.11mol) and 10ml trichloromethane stirring and dissolving; Toward wherein dripping titanium tetrachloride 32g; And under 20 ℃ envrionment temperature, drip the chloroform soln of N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE, and drip and finish, stir 2h.Reaction is finished, and adds 15ml water, stirs the 2h cancellation, filters, and it is dried with revolving behind the anhydrous sodium sulfate drying to filtrate, and obtains cytosine(Cyt) acetyl ribose.
In the 250ml there-necked flask, add cytosine(Cyt) acetyl ribose, the mass percentage concentration that adds ammonia is 10% methanol ammonia solution 60g, stirs 2h under 20 ℃ the envrionment temperature.Reaction is finished, and is evaporated to dried cytidine(C bullion 21.5g, the yield 98.2% of obtaining.
In the 250ml there-necked flask, add cytidine(C bullion 21.5g, add the 80ml absolute ethyl alcohol, reflux dissolving 2h adds entry 10ml, stirs down, is cooled to 0 ℃ of crystallization, the 5h after-filtration, oven dry obtains cytidine(C elaboration 21.0g.Yield 95.9%, detecting HPLC purity is 99.8%.
The cytidine(C elaboration:
1H-NMR (DMSO-d
6, δ): 3.52 ~ 3.64 (m, 2H, 5 '-H), 3.77 ~ 3. 87 (m, 1H, 4 '-H), 3.93 ~ 4.05 (m, 2H, 2 '-H and 3 '-H); 4.97 (s, 1H, OH), 5.08 (s, 1H, OH), 5.26 (s, 1H, OH); 5.72 (d, 1H, 5-H), 5.77 (d, 1H, 1 '-H), 7.11 (s, 2H, NH
2), 7.8 (d, 1H, 6-H).MS(ESI,m/z):(M+H)
+244。
Embodiment 2
In the 250ml there-necked flask, add cytosine(Cyt) 10g (0.09mol), ammonium sulfate 200mg keeps dry, and in there-necked flask, feeds the helium protection, adds TERT-BUTYL DIMETHYL CHLORO SILANE 27g (0.18mol) then, stirs at a slow speed.Finish, be warming up to 110 ℃ of insulation reaction 10h, reaction is finished.Obtain product N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE.Product is dissolved with the 100ml trichloromethane.
In the 250ml there-necked flask; Add tetrem acyl ribose 50g (0.135mol) and 20ml trichloromethane stirring and dissolving; Toward wherein dripping titanium tetrachloride 60g; And under 18 ℃ envrionment temperature, drip the chloroform soln of N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE, and drip and finish, stir 5h.Reaction is finished, and adds 15ml water, stirs the 2h cancellation, filters, and it is dried with revolving behind the anhydrous sodium sulfate drying to filtrate, and obtains cytosine(Cyt) acetyl ribose.
In the 250ml there-necked flask, add cytosine(Cyt) acetyl ribose, the mass percentage concentration that adds ammonia is 10% methanol ammonia solution 45.9g, stirs 2.5h under 18 ℃ the envrionment temperature.Reaction is finished, and is evaporated to dried cytidine(C bullion 21.0g, the yield 95.9% of obtaining.
In the 250ml there-necked flask, add cytidine(C bullion 21.0g, add the 80ml absolute ethyl alcohol, reflux dissolving 2h adds entry 10ml, stirs down, is cooled to-2 ℃ of crystallizations, the 5h after-filtration, oven dry obtains cytidine(C elaboration 20.3g.Yield 92.7%, detecting HPLC purity is 99.5%.
The cytidine(C elaboration:
1H-NMR (DMSO-d
6, δ): 3.52 ~ 3.64 (m, 2H, 5 '-H), 3.77 ~ 3.87 (m, 1H, 4 '-H), 3.93 ~ 4.05 (m, 2H, 2 '-H and 3 '-H); 4.97 (s, 1H, OH), 5.08 (s, 1H, OH), 5.26 (s, 1H, OH); 5.72 (d, 1H, 5-H), 5.77 (d, 1H, 1 '-H), 7.11 (s, 2H, NH
2), 7.8 (d, 1H, 6-H).MS(ESI,m/z):(M+H)
+244。
Embodiment 3
In the 250ml there-necked flask, add cytosine(Cyt) 10g (0.09mol), ammonium sulfate 100mg keeps dry, and in there-necked flask, feeds nitrogen protection, adds TERT-BUTYL DIMETHYL CHLORO SILANE 40.5g (0.27mol) then, stirs at a slow speed.Finish, be warming up to 130 ℃ of insulation reaction 6h, reaction is finished.Obtain product N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE.Product is dissolved with the 100ml trichloromethane.
In the 250ml there-necked flask; Add tetrem acyl ribose 28.6g (0.09mol) and 10ml trichloromethane stirring and dissolving; Toward wherein dripping titanium tetrachloride 28.6g; And under 30 ℃ envrionment temperature, drip the chloroform soln of N-(tertiary butyl dimethyl-is silica-based)-2-(tertiary butyl dimethyl Si base)-4-PYRIMITHAMINE, and drip and finish, stir 3.5h.Reaction is finished, and adds 15ml water, stirs the 2h cancellation, filters, and it is dried with revolving behind the anhydrous sodium sulfate drying to filtrate, and obtains cytosine(Cyt) acetyl ribose.
In the 250ml there-necked flask, add cytosine(Cyt) acetyl ribose, the mass percentage concentration that adds ammonia is 10% methanol ammonia solution 53.55g, stirs 1.5h under 30 ℃ the envrionment temperature.Reaction is finished, and is evaporated to dried cytidine(C bullion 20.8g, the yield 95.0% of obtaining.
In the 250ml there-necked flask, add cytidine(C bullion 20.8g, add the 80ml absolute ethyl alcohol, reflux dissolving 2h adds entry 10ml, stirs down, is cooled to 2 ℃ of crystallizations, the 6h after-filtration, oven dry obtains cytidine(C elaboration 20.2g.Yield 92.3%, detecting HPLC purity is 99.6%.
The cytidine(C elaboration:
1H-NMR (DMSO-d
6, δ): 3.52 ~ 3.64 (m, 2H, 5 '-H), 3.77 ~ 3.87 (m, 1H, 4 '-H), 3.93 ~ 4.05 (m, 2H, 2 '-H and 3 '-H); 4.97 (s, 1H, OH), 5.08 (s, 1H, OH), 5.26 (s, 1H, OH); 5.72 (d, 1H, 5-H), 5.77 (d, 1H, 1 '-H), 7.11 (s, 2H, NH
2), 7.8 (d, 1H, 6-H).MS(ESI,m/z):(M+H)
+244。
Comparative Examples 1
N
2Under the protection, 10g (0.09mol) cytosine(Cyt), triethylamine 150mg adds 21.8g (0.20mol) trimethylchlorosilane, is warming up to 120 ℃ of reaction 8h, and concentrating under reduced pressure gets the oily product, and product is dissolved with the 100ml trichloromethane.
In the 250ml there-necked flask, add tetrem acyl ribose 35g (0.11mol) and 10ml trichloromethane stirring and dissolving, toward wherein dripping titanium tetrachloride 32g, and under 20 ℃ envrionment temperature, drip the chloroform soln of above-mentioned oily matter, drip and finish, stirring 2h.Reaction is finished, and adds 15ml water, stirs 2h and filters, and it is dried with revolving behind the anhydrous sodium sulfate drying to filtrate, and obtains cytosine(Cyt) acetyl ribose.
In the 250ml there-necked flask, add cytosine(Cyt) acetyl ribose, the mass percentage concentration that adds ammonia is 10% methanol ammonia solution 60g, stirs 2h under 20 ℃ the envrionment temperature.Reaction is finished, and is evaporated to dried cytidine(C bullion 18.1g, the yield 82.68% of obtaining.
In the 250ml there-necked flask, add cytidine(C bullion 18.1g, add the 80ml absolute ethyl alcohol, reflux dissolving 2h adds entry 10ml, stirs down, is cooled to 0 ℃ of crystallization, the 5h after-filtration, oven dry obtains cytidine(C elaboration 15.8g.Yield 72.2%, detecting HPLC purity is 99.0%.
In this Comparative Examples, except substituting TERT-BUTYL DIMETHYL CHLORO SILANE with trimethylchlorosilane, substitute ammonium sulfate with triethylamine, all the other operation stepss are with embodiment 1.
Its synthetic route is following:
In this method, the cytosine(Cyt) of trimethyl silicon based silicon etherificate is prone to deliquescence in air, cause the cytidine(C yield lower.
Comparative Examples 2
N
2Protection down; 10g (0.09mol) cytosine(Cyt), hexamethyldisilazane 150mg and 21.8g (0.20mol) trimethylchlorosilane are warming up to 120 ℃ of reaction 8h, concentrating under reduced pressure in acetonitrile solution; Get the oily product, product is dissolved with the 100ml trichloromethane.
In the 250ml there-necked flask, add 1-acetyl-2,3,5-tri-benzoyl ribofuranose 0.11mol and 10ml ethylene dichloride stirring and dissolving are toward wherein dripping (CH
3)
3SiSO
2CF
332g, and under 20 ℃ envrionment temperature, drip the chloroform soln of above-mentioned oily matter, drip and finish, stir 2h.Reaction is finished, and adds 15ml water, stirs 2h and filters, and it is dried with revolving behind the anhydrous sodium sulfate drying to filtrate, and obtains cytosine(Cyt) acetyl ribose.
In the 250ml there-necked flask, add cytosine(Cyt) acetyl ribose, the mass percentage concentration that adds ammonia is 10% methanol ammonia solution 60g, stirs 2h under 20 ℃ the envrionment temperature.Reaction is finished, and is evaporated to dried cytidine(C bullion 18.5g, the yield 84.5% of obtaining.
In the 250ml there-necked flask, add cytidine(C bullion 18.5g, add the 80ml absolute ethyl alcohol, reflux dissolving 2h adds entry 10ml, stirs down, is cooled to 0 ℃ of crystallization, the 5h after-filtration, oven dry obtains cytidine(C elaboration 16.2g.Yield 74%, detecting HPLC purity is 99.1%.
In this Comparative Examples,, substitute ammonium sulfate with hexamethyldisilazane except substituting TERT-BUTYL DIMETHYL CHLORO SILANE with trimethylchlorosilane in the Silanization reaction, and with acetonitrile as reaction solvent; With 1-acetyl-2,3,5-tri-benzoyl ribofuranose substitutes tetrem acyl ribose, (CH in the glucosides reaction
3)
3SiSO
2CF
3Substitute titanium tetrachloride, all the other operation stepss are with embodiment 1.