CN107556355B - Nucleoside diphosphite amide and preparation method thereof - Google Patents
Nucleoside diphosphite amide and preparation method thereof Download PDFInfo
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Abstract
The invention relates to nucleoside diphosphite amide and a preparation method thereof. The method is a preparation method of nucleoside diphosphite amide based on 4-methoxy-4' -acetoxy trityl chloride. The nucleoside diphosphite amide designed and prepared by the invention can be used for impurity calibration and analysis in oligonucleotide synthesis, thereby being beneficial to large-scale synthesis, purification and quality control of oligonucleotide drugs.
Description
Technical Field
The invention belongs to the field of synthesis of nucleoside compounds, and particularly relates to a preparation method of novel nucleoside diphosphite amide.
Background
Antisense Technology (Antisense Technology) is a Technology for inhibiting or blocking gene expression by using a specific DNA or RNA fragment synthesized artificially or by organisms according to the principle of base complementarity, and is a novel drug development method. The medicine developed by using antisense technology becomes antisense nucleic acid medicine (antisense medicine for short), including antisense oligonucleotide (including antisense DNA and reaction RNA), ribozyme, antisense RNA and RNA interference. According to the nucleic acid hybridization principle, antisense drugs can hybridize with specific genes, interfering with the production process of pathogenic proteins at the gene level. Proteins are major substances of the structure and function of the organism, and play a very important role in human metabolism, and almost all human diseases are caused by the abnormality of proteins, whether host diseases (tumors and the like) or infectious diseases (hepatitis and the like). The traditional medicine mainly acts on the pathogenic protein directly, and the antisense medicine acts on the gene for coding the pathogenic protein, so that the traditional medicine can be applied to the treatment of various diseases. With the decoding of human genome maps and the approval of the first antisense nucleic acid, Fomivirsen, by the Food and Drug Administration, FDA, studies based on antisense nucleic acids have entered a new climax.
The research on antisense oligonucleotides includes first-generation antisense nucleic acids represented by thio modification, second-generation antisense nucleic acids represented by modification of the 2' -hydroxyl group of ribose, and third-generation antisense nucleic acids represented by modification of the phosphate backbone and ribose. In recent years, the synthesis and research of antisense oligonucleotides have advanced into a new stage, and are expected to become new drugs in the aspects of resisting viruses, tumors and inflammatory diseases, and are useful tools for researching gene functions.
In the synthesis and study of antisense oligonucleotides, the classical approach is often to first introduce a 4,4 '-bismethoxytrityl (DMT) protecting group at the 5' hydroxyl group of the nucleoside using 4,4 '-bismethoxytrityl chloride (DMTCl), thereby ensuring mono-selectivity for the 3' hydroxyl group of the nucleoside in subsequent monomer couplings. However, 4,4 '-bis-methoxytrityl chloride (DMTCl) is often accompanied by a small amount of degradation product 4-methoxy-4' -hydroxytrityl chloride during preparation and storage, as shown in formula 3. In the synthesis of oligonucleotide monomers, the impurity can cause the formation of another impurity nucleoside diphosphite amide monomer, as shown in formula (I), thereby affecting the synthesis efficiency, purification and quality control of oligonucleotide monomers.
Therefore, designing, preparing and applying the nucleoside diphosphonite amide to impurity calibration and analysis in oligonucleotide synthesis and research has positive significance for large-scale synthesis, purification and quality control of oligonucleotide drugs.
Disclosure of Invention
The invention aims to provide a preparation method of a novel nucleoside diphosphite amide.
In a first aspect of the present invention, there is provided a compound (nucleoside bisphosphinite) having a structure represented by the general formula (I):
in the formula (I), BPSelected from: n-protected adenine or a derivative thereof, guanine or a derivative thereof, cytosine or a derivative thereof, uracil or a derivative thereof, thymine or a derivative thereof;
R1selected from: hydrogen, halogen, alkoxy, alkyl.
In a preferred embodiment, the alkoxy group is a C1-C6 alkoxy group or an alkyl group, preferably a C1-C4 alkoxy group, more preferably a methoxy group; or the alkyl is C1-C6 alkyl, preferably C1-C4 alkyl.
In another preferred embodiment, the halogen is selected from: fluorine, chlorine, bromine, iodine; preferably, the halogen is fluorine.
In another preferred embodiment, B isPSelected from: n-protected adenine, guanine, cytosine, uracil or thymine.
In another preferred embodiment, the compound has the structure shown in compound 2:
in another preferred embodiment, the compound has a structure selected from the group consisting of compounds 2a, 2b, 2c, 2d and 2 e:
in another aspect of the invention there is provided the use of said compounds for impurity calibration and/or analysis in oligonucleotide synthesis and research; or for the synthesis of antisense nucleotides.
In another aspect of the present invention, there is provided a process for preparing said compound, which process comprises:
(1) 4, 4' -bis (methoxy) trityl chloride is used as a raw material, and methyl on one benzene ring of the raw material is selectively removed and hydrolyzed;
(2) performing Ac protection on hydroxyl on a benzene ring of the product obtained in the step (1), and hydrolyzing;
(3) chlorinating the product obtained in step (2) to obtain a compound of formula 1;
(4) condensing the compound of formula 1 obtained in step (3) with a nucleoside to obtain a compound of formula C;
(5) removing Ac protection on the benzene ring of the compound of the formula C obtained in the step (4);
(6) carrying out double phosphorylation reaction on two hydroxyl groups in the product obtained in the step (5) to obtain a compound shown in a formula (I);
in the formula, BPSelected from: n-protected adenine or a derivative thereof, guanine or a derivative thereof, cytosine or a derivative thereof, uracil or a derivative thereof or thymine or a derivative thereof;
R1selected from: hydrogen, halogen, alkoxy, alkyl;
in a preferred embodiment, in the step (1), the methyl group on the benzene ring is removed by using boron tribromide; preferably, step (1) comprises: dissolving 4, 4' -bis (methoxy) trityl chloride, and adding BBr dropwise at 0 deg.C3And (3) reacting the dichloromethane solution until the raw materials are not converted any more, concentrating to dryness, extracting an organic phase, drying and concentrating to obtain a deprotection and hydrolysis product.
In another aspect of the present invention, there is provided the step (2) of reacting the deprotected and hydrolyzed product obtained in the step (1) with acetyl chloride to perform Ac protection; preferably, step (2) comprises: dissolving the product obtained in the step (1), reacting the product with acetyl chloride under the heating condition, and concentrating the reaction solution to be dry; dissolving in acetonitrile and water, and concentrating to dryness; then extracting an organic phase, drying and concentrating the organic phase to dryness, and carrying out silica gel column chromatography to obtain a product of which the hydroxyl on a benzene ring is protected by Ac.
In another aspect of the present invention, there is provided the step (3) of reacting the product obtained in the step (2) with acetyl chloride to perform chlorination; preferably, step (3) comprises: dissolving the product obtained in the step (2), reacting the product with acetyl chloride under the heating condition, and concentrating the reaction solution to be dry; dissolving, crystallizing, filtering, washing and drying to obtain the compound shown in the formula 1.
In another aspect of the present invention, the providing step (4) includes: mixing 2' -halogenated nucleoside with the compound shown in the formula 1, fully reacting, quenching, concentrating to remove the solvent to obtain a light yellow solid, diluting, washing, separating an organic phase, and drying to obtain a compound shown in the formula C; preferably, the 2' -halonucleoside is selected from the group consisting of: 2 ' -halogenated thymidine, 2 ' -halogenated uridine, 2 ' -halogenated adenosine, 2 ' -halogenated cytidine, 2 ' -halogenated guanosine.
In another aspect of the present invention, the providing step (5) comprises: dissolving the compound of the formula C obtained in the step (4) in methanol solution saturated by ammonia gas, stirring, and reacting until the compound of the formula C disappears; concentrating to dryness, and performing silica gel column chromatography.
In another aspect of the present invention, the providing step (6) includes: suspending the compound obtained in the step (5) and tetrazole in acetonitrile, concentrating under reduced pressure to remove water in the system, adding bis (diisopropylamino) (2-cyanoethoxy) phosphine, and reacting; washing; drying; concentrating; and (4) performing silica gel column chromatography to obtain the compound shown in the formula (I).
In another aspect of the present invention, there is provided a method for preparing an antisense nucleotide using the compound, the method comprising:
(a) carrying out monomer coupling on the compound shown in the formula (I) and the compounds shown in the formulas 3 and 4, and then oxidizing trivalent phosphine and deprotecting TBDMS to obtain a reaction product;
(b) carrying out monomer coupling on the reaction product obtained in the previous step and the compounds shown in the formulas 3 and 4, and then oxidizing trivalent phosphine and carrying out deprotection on TBDMS to obtain a further reaction product;
(c) repeating the step (b) to obtain a polymer with the required length, and removing the base of the polymer and the protecting group on the phosphine to obtain the antisense nucleotide.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Detailed Description
The present inventors have made intensive studies to disclose a novel nucleoside bisphosphinite compound which can be used for the synthesis of antisense oligonucleotides. Meanwhile, the invention also discloses a preparation method of the nucleoside diphosphite amide based on AcO-MMT-Cl.
Compound (I)
The invention provides a nucleoside diphosphite amide with a structure shown in a chemical general formula (I), which can be used for synthesizing antisense oligonucleotide:
in the formula (I), BPSelected from: n-protected adenine or a derivative thereof, guanine or a derivative thereof, cytosine or a derivative thereof, uracil or a derivative thereof or thymine or a derivative thereof;
R1selected from: hydrogen, halogen, alkoxy;
R2selected from: phosphoramidate groups, or phosphate groups;
R3selected from: phosphoramidate groups, or phosphate groups.
The term "halogen" as used herein refers to F, Cl, Br or I, especially F, Cl or Br. Preferably, the halogen is fluorine.
The term "alkoxy" as used herein refers to oxygen-containing alkyl groups such as methoxy, ethoxy, n-propoxy, isopropoxy, and the like. Preferably it contains 1 to 6 carbon atoms; more preferably 1 to 4 carbon atoms; more preferably 1-3, such as 2 carbon atoms.
As used herein, "derivatives" of adenine, guanine, cytosine, uracil or thymine include, without limitation, nitrogen: adenine, guanine, cytosine, uracil or thymine isomers, compounds in which substitution of groups occurs at some sites, for example, the H atom may be substituted by a hydroxyl group, a C1-C4 alkyl group, a C1-C4 alkoxy group or a halogen group; for another example, methyl is substituted by ethyl or propyl.
In a preferred embodiment of the present invention, B isPSelected from: n-protected adenine, guanine, cytosine, uracil or thymine.
In a preferred embodiment of the present invention, the compound has a structure represented by compound 2:
in a preferred embodiment of the present invention, the compound has a structure selected from the group consisting of compounds 2a, 2b, 2c, 2d and 2 e:
preparation method
The invention provides a novel synthesis method of nucleoside diphosphite amide, which mainly comprises the step of introducing a synthesized novel compound 4-methoxy-4' -acetoxy trityl chloride (AcO-MMT-Cl, shown as a formula 1) to nucleoside so as to further introduce the diphosphite amide.
As a preferred mode of the invention, the reaction process of the method is as follows:
in short, the method for preparing nucleoside diphosphite amide takes 4, 4' -bismethoxytrityl chloride (DMTCl) as a raw material, and after a new compound AcO-MMT-Cl is synthesized by selective demethylation protection, hydrolysis, Ac protection, hydrolysis and chlorination in sequence, the target product is obtained by condensation, Ac protection removal and diphosphite acylation with nucleoside in sequence. More specifically, the above method may comprise the following steps in sequence:
(1) 4, 4' -bis (methoxy) trityl chloride (DMTCl) is used as a raw material, and is hydrolyzed after one methyl on a benzene ring is selectively removed for protection, so that a compound shown as a formula A is obtained;
(2) reacting the deprotection product obtained in the step (1) with acetyl chloride for Ac protection, and hydrolyzing to obtain a compound shown as a formula B;
(3) reacting the hydrolysate B subjected to Ac protection in the step (2) with acetyl chloride, and chlorinating to obtain a compound shown in a formula 1;
(4) condensing a compound represented by formula 1 with a nucleoside; the base B on the nucleoside may be adenine, guanine, cytosine, uracil or a corresponding derivative;
(5) deprotection is carried out on Ac on the benzene ring in the step (4);
(6) and (4) carrying out double phosphorylation reaction on two hydroxyl groups in the intermediate product obtained in the step (5) to obtain the required nucleoside diphosphite amide.
In a preferred embodiment of the present invention, in step (1), a methyl group on a benzene ring is removed by boron tribromide; preferably, step (1) comprises: dissolving 4, 4' -bis (methoxy) trityl chloride, and adding BBr dropwise at 0 deg.C3And (3) reacting the dichloromethane solution until the raw materials are not converted any more, concentrating to dryness, extracting an organic phase, drying and concentrating to obtain a deprotection and hydrolysis product. The 4, 4' -bis (methoxy) trityl chloride can be dissolved in an organic solvent, and the organic solvent is dichloromethane. The reaction until no conversion of the starting material is carried out means that the starting material is obtained in the amount of the product<HPLC can be used to monitor for the presence of starting material at 7%.
In a preferred embodiment of the present invention, in step (2), Ac protection is performed by reacting the deprotected and hydrolyzed product obtained in step (1) with acetyl chloride; preferably, step (2) comprises: dissolving the product obtained in step (1), preferably in an organic solvent, more preferably toluene; reacting with acetyl chloride under heating (preferably 80-90 ℃), and concentrating the reaction solution to dryness; dissolving in acetonitrile and water (preferably acetonitrile and water at a volume ratio of 1:1), and concentrating to dryness; then extracting an organic phase, drying and concentrating the organic phase to dryness, and carrying out silica gel column chromatography to obtain a product of which the hydroxyl on a benzene ring is protected by Ac.
In a preferred embodiment of the present invention, in step (3), the product obtained in step (2) is reacted with acetyl chloride to carry out chlorination; preferably, step (3) comprises: dissolving the product obtained in step (2), preferably in an organic solvent, more preferably toluene; reacting with acetyl chloride under heating (preferably 80-90 ℃), and concentrating the reaction solution to dryness; dissolving, preferably in an organic solvent, more preferably toluene; crystallizing; carrying out suction filtration; washing; drying to obtain the compound of formula 1.
As a preferred embodiment of the present invention, the step (4) includes: mixing the 2' -halonucleoside with the compound of formula 1, preferably, dissolving in dry pyridine with stirring; fully reacting; quenching, preferably with methanol; concentrating to remove the solvent to obtain a light yellow solid; diluting, preferably with dichloromethane; washing; separating out an organic phase; drying to obtain the compound of formula C; preferably, the 2' -halonucleoside is selected from the group consisting of: 2 ' -halogenated thymidine, 2 ' -halogenated uridine, 2 ' -halogenated adenosine, 2 ' -halogenated cytidine, 2 ' -halogenated guanosine.
As a preferred embodiment of the present invention, the step (5) includes: dissolving the compound of the formula C obtained in the step (4) in methanol solution saturated by ammonia gas, stirring, and reacting until the compound of the formula C disappears; concentrating to dryness, and performing silica gel column chromatography. The disappearance includes substantial disappearance, such as over 90% disappearance.
As a preferred embodiment of the present invention, the step (6) includes: carrying out water addition on the compound obtained in the step (5) and tetrazole in acetonitrile for a plurality of times, such as 3-10 times, preferably 4-8 times; adding bis (diisopropylamino) (2-cyanoethoxy) phosphine, and reacting; washing, preferably washing with a mixture of an aqueous sodium bicarbonate solution and an aqueous saturated sodium chloride solution; drying; concentrating; the compound is obtained by silica gel column chromatography.
In the specific examples of the present invention, 2' -fluoro-thymidine is taken as an example to describe in further detail the method of synthesizing nucleoside bisphosphoramide monomers of the present invention: 4, 4' -bis (methoxy) trityl chloride (DMTCl) reacts with boron tribromide in dichloromethane to selectively remove methyl group and protect the product, and then the product is hydrolyzed to obtain the compound shown in the formula A. And reacting the hydrolyzed crude product with acetyl chloride to protect the hydroxyl on a benzene ring, and hydrolyzing in acetonitrile and water to obtain the compound shown in the formula B. The overall yield of these two steps was 53.9% (mol). The compound shown in the formula 1 is condensed with 2 ' -fluoro-thymidine in pyridine to obtain a crude product of 5 ' - (4-methoxy-4 ' -acetoxy trityl) -2 ' -fluoro-thymidine, and the crude product is further subjected to Ac deprotection in ammonia methanol solution to obtain the compound of 5 ' - (4-methoxy-4 ' -hydroxytrityl) -2 ' -fluoro-thymidine, wherein the yield of the two steps is 81% (mol). Reaction of 5 ' - (4-methoxy-4 ' -hydroxytrityl) -2 ' -fluoro-thymidine with bis (diisopropylamino) (2-cyanoethoxy) phosphine under standard conditions gave the nucleoside bisphosphinite amide of formula (II) in 70-85% yield.
Other nucleoside bisphosphinites can be synthesized by the same method as described above or by a similar method.
The nucleoside diphosphite amide designed and prepared by the invention can be applied to impurity calibration and analysis in oligonucleotide synthesis, thereby being beneficial to large-scale synthesis, purification and quality control of oligonucleotide drugs.
The present invention will be described in further detail with reference to specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention; those skilled in the art will appreciate that, on the basis of the present invention, suitable modifications or changes may be made without any inventive step, and all such modifications or changes are therefore intended to be included within the scope of the present invention. The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions.
The conditions for High Performance Liquid Chromatography (HPLC) in the following examples of the invention are as follows:
column: luna C18, 4.6 x 150 mm;
flow rate: 1.0ml/min, column temperature: 25 ℃;
detection wavelength: 260 nm;
mobile phase: the gradient conditions were as follows:
| Time | solution A | Liquid B |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 15 | 5 | 95 |
| 20 | 5 | 95 |
| 25 | 65 | 35 |
| 30 | 65 | 35 |
Solution A: TEAA buffer (0.1 mol/l aqueous acetic acid adjusted to PH 7.0 with triethylamine).
And B, liquid B: chromatographic grade acetonitrile.
During the analysis, the liquid A and the liquid B are degassed on line, and the helium flow rate is 50 ml/min.
Example 1 Synthesis of thymidine bisphosphoramide
1. Synthesis of Compound A
100g of DMTCl (295.8mmol) was weighed into a dry three-necked flask, and after dissolving in 400ml of Dichloromethane (DCM), the air in the system was purged by bubbling argon gas. After the temperature of the reaction solution was reduced to 0 ℃ by an ice water bath, 444ml of 1M boron tribromide (BBr) was gradually added dropwise to the above system3443.7mmol) of dichloromethane solution, the dropwise addition time being about 60 min. After the addition, the reaction was stirred at 20. + -. 5 ℃ for about 12 hours until the starting material was no longer converted. HPLC monitors the reaction if starting material<Stopping the reaction when the reaction rate is 7 percent; otherwise, the reaction is continued, and samples are taken every 50-60 minutes for HPLC analysis until the starting material<7 percent. After the reaction, the reaction solution was concentrated to dryness at 37 ℃ under reduced pressure. To the dry sample was added 500ml acetonitrile and 500ml water and stirred for 30 min. The reaction mixture was concentrated to dryness at 40 ℃ under reduced pressure, and 500ml of ethyl acetate and 500ml of water were added to dissolve and extract. The layers were then allowed to settle, the organic phase was separated and the aqueous phase was extracted twice more with ethyl acetate (200ml x 2). The organic phases are combined, dried over 40g of anhydrous sodium sulfate and concentrated to dryness to give about 100g of crude product. The crude product was used in the next reaction without purification.
2. Synthesis of Compound B
In three dry mouths, about 100g of crude compound A (295.8mmol) from the previous step was dissolved in 500ml of toluene, and the air in the system was purged by bubbling argon. 170ml of acetyl chloride (2.4mol) were added to the system. After the addition, the reaction is heated to 80-90 ℃ and refluxed for about 3 hours. TLC and HPLC detection reaction, TLC: developing agent n-heptane: climbing a board with ethyl acetate of 6: 1; HPLC can monitor the formation of the reaction product. After the reaction, the reaction solution was concentrated to dryness at 40 ℃ under reduced pressure. To the dry sample was added 500ml acetonitrile and 500ml water and stirred for 30 min. The reaction mixture was concentrated to dryness at 40 ℃ under reduced pressure, and 500ml of ethyl acetate and 500ml of water were added to dissolve and extract. The organic phase is dried over 40g of anhydrous sodium sulfate and concentrated to dryness. 55.5g of product is obtained by flash silica gel column chromatography, the total separation yield of two steps is 53.9% (mol), 55.5% (w) and the purity is 99.35%.
3. Synthesis of Compound 1
55.5g of Compound B (0.136mol) was weighed into a dry three-necked flask, dissolved in 450ml of toluene, and then purged with argon by bubbling. 80.5ml of acetyl chloride (1.116mol) was added to the system. And after the addition is finished, heating the reaction to 80-90 ℃ and refluxing for 3 hours to finish the reaction. After the reaction is finished, the reaction solution is decompressed and concentrated to be dry at the temperature of 40 ℃ to obtain a white solid crude product. After dissolving in 50ml of toluene, 350ml of n-heptane were added and the crystals were crystallized by stirring at 0 ℃. The precipitated solid was suction-filtered under an argon atmosphere. The filter cake was washed twice (120ml x 2) with n-heptane/toluene (7/1, v/v) and dried in vacuo to give 43g of product with HPLC purity 99.42%, yield: 73.6% (mol), 77.5% (w).
1H NMR(600MHz,DMSO-d6,TMS):δ2.25(3H,s),3.72(3H,s),6.86(2H,d,J＝8.4Hz),7.04(2H,d,J＝8.4Hz),7.11(2H,d,J＝9.0Hz),7.22-7.23(5H,m),7.28-7.31(2H,m)。
4. Synthesis of Compound C-1
30g of 2-F-thymidine (115.4mmol) and 44g of compound 1(121.2mmol) are weighed out in a 250ml three-necked flask and dissolved with 180ml of dry pyridine with stirring. After stirring the reaction at room temperature for 5h, TLC (CH)2Cl2MeOH 95:5, v/v) and HPLC (DMT-30min) monitored the reaction until the starting material had substantially disappeared (conversion 90%), and the reaction was stopped. The system was quenched by addition of 6ml of methanol and concentrated to remove all solvents to give a crude pale yellow solid. The crude product is diluted with 600ml of dichloromethane, 600ml of water is washed, the organic phase separated off is dried over 10g of anhydrous sodium sulfate to obtain 80g of crude product. The crude product was used in the next reaction without purification.
Synthesis of Compound D-1
The crude compound C-1 was dissolved in 200ml of ammonia saturated methanol solution, stirred at room temperature for 12 hours, and then TLC (CH)2Cl2MeOH 95:5, v/v) andthe reaction was stopped by HPLC monitoring until the starting material disappeared. Vacuum concentrating to remove solvent, and performing flash silica gel column chromatography to obtain product 45.5g, with total separation yield of 72.0% (mol), 152% (w), and purity of 99.0%.
Synthesizing a compound 2a, which has a structure shown in a formula (II):
20g of the synthesized compound D-1(36.5mmol) and 2.0g of tetrazole (29.2mmol) are respectively suspended in 200ml of acetonitrile, and then the suspension is decompressed and concentrated for several times to remove the water in the system, and the water content is measured to be 36ppm after the suspension is dissolved in 200ml of DCM. Then, 12.1g of a reagent of bis (diisopropylamino) (2-cyanoethoxy) phosphine (40.1mmol) was added thereto, and the reaction was carried out at room temperature for 3 hours. After the reaction (3h), the reaction solution is washed 3 times by a mixed solution of 0.6mol/L sodium bicarbonate water solution and saturated sodium chloride water solution (the volume ratio is 1:1, 400ml is 6), an organic phase is dried by 30g of anhydrous sodium sulfate and then concentrated, and a crude product is subjected to flash silica gel column chromatography to obtain 28.7g of a product, wherein the separation yield is 83% (mol), 144% (w) and the purity is 99%. Mass spectrum: MS (ESI) M/z 949.5(M + H)+,100)。
Example 2 Synthesis of uridine diphosphonite
Compound 1 was prepared according to the procedure described in example 1.
Synthesis of Compound C-2
28.4g of 2-F-uridine (115.4mmol) and 44g of compound 1(121.2mmol) were weighed out in a 250ml three-necked flask and dissolved with stirring in 180ml of dry pyridine. After stirring the reaction at room temperature for 5h, TLC (CH)2Cl2MeOH 95:5, v/v) and HPLC monitored the reaction until the starting material was substantially gone (90% conversion), and the reaction was stopped. The system was quenched by addition of 6ml of methanol and concentrated to remove all solvents to give a crude pale yellow solid. The crude product is diluted with 600ml of dichloromethane, washed with 600ml of water, the organic phase separated off is dried over 10g of anhydrous sodium sulfate to give 76g of crude product. The crude product was used in the next reaction without purification.
Synthesis of Compound D-2
The crude compound C-2 was dissolved in 200ml of ammonia saturated methanol solution, and stirred at room temperatureAfter stirring for 12h, TLC (CH)2Cl2MeOH 95:5, v/v) and HPLC monitored the reaction until the starting material disappeared and the reaction was stopped. Vacuum concentrating to remove solvent, and performing flash silica gel column chromatography to obtain product 43.2g with total separation yield of 70.1% (mol), 152% (w), and purity of 99.5%.
Synthesis of the Structure shown in Compound 2b
19.5g of the synthesized compound D-2(36.5mmol) and 2.0g of tetrazole (29.2mmol) were respectively suspended in 200ml of acetonitrile with water for 6 times, and then dissolved in 200ml of DCM to measure the water content to be 36 ppm. Then, 12.1g of a reagent of bis (diisopropylamino) (2-cyanoethoxy) phosphine (40.1mmol) was added thereto, and the reaction was carried out at room temperature for 3 hours. After the reaction (3h), the reaction solution was washed 3 times with a mixture of 0.6mol/L aqueous sodium bicarbonate and saturated aqueous sodium chloride (volume ratio 1:1, 400 ml. about.6), the organic phase was dried over 30g of anhydrous sodium sulfate and concentrated, and the crude product was subjected to flash silica gel column chromatography to obtain 26.9g of product with an isolated yield of 79% (mol), 138% (w) and a purity of 98.8%. Mass Spectrometry MS (ESI) M/z 935.7(M + H)+,100)。
Example 3 Synthesis of Cytidine bisphosphoryl imine
Compound 1 was prepared according to the procedure described in example 1.
Compound 1 was reacted with 4-acetyl-2' -fluoro-dC according to the procedure described in example 1 to obtain cytidine bisphosphoryl imine 2c having the structure shown. Mass Spectrometry MS (ESI) M/z 976.6(M + H)+,100)。
Example 4 Synthesis of adenosine bisphosphoryl imine
Compound 1 was prepared according to the procedure described in example 1.
The compound 1 was reacted with 6-benzoyl-2' -fluoro-dA according to the method described in example 1 to prepare adenosine bisphosphoryl imide 2 d. Mass spectrum MS (ESI) m/z 1062.8(M+H+,100)。
Example 5 Synthesis of guanosine bisphosphoryl imine
Compound 1 was prepared according to the procedure described in example 1.
Compound 1 was reacted with 2-benzoyl-2' -fluoro-dA according to the method described in example 1 to obtain guanosine bisphosphoryl imide 2 e. Mass Spectrometry MS (ESI) M/z 1078.6(M + H)+,100)。
Example 6 Synthesis of antisense nucleotides
Monomeric coupling of phosphoramidite monomers with protected nucleosides 3 and 4 using nucleoside bisphosphonates 2 a-2 e prepared as in examples 1-5 above, respectively, followed by oxidation of trivalent phosphine and deprotection of TBDMS gives 5.5 repeating the coupling-oxidation-deprotection step to obtain 6, and finally 6 removing the base and the protecting group on the phosphine to obtain the formal antisense nucleotide.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (18)
1. A process for preparing a compound having the structure of formula (I) wherein formula (I) is:
in the formula (I), BPSelected from: n-protected adenine, guanine, cytosine, uracil, thymine; r1Selected from: hydrogen, halogen, alkoxy, alkyl; the alkoxy is C1-C6 alkoxy, and the alkyl is C1-C6 alkyl;
the method comprises the following steps:
(1) using 4, 4' -bis (methoxy) trityl chloride as a raw material, selectively removing methyl on one benzene ring of the raw material by using boron tribromide and hydrolyzing; the method comprises the following steps: dissolving 4, 4' -bis (methoxy) trityl chloride, and adding BBr dropwise at 0 deg.C3Reacting a dichloromethane solution until the raw materials are not converted any more, concentrating to dryness, extracting an organic phase, drying and concentrating to obtain a deprotection and hydrolysis product;
(2) performing Ac protection on hydroxyl on a benzene ring of the product obtained in the step (1), and hydrolyzing;
(3) chlorinating the product obtained in step (2) to obtain a compound of formula 1;
(4) condensing the compound of formula 1 obtained in step (3) with a nucleoside to obtain a compound of formula C;
(5) removing Ac protection on the benzene ring of the compound of the formula C obtained in the step (4);
(6) carrying out double phosphorylation reaction on two hydroxyl groups in the product obtained in the step (5) to obtain the compound shown in the formula (I);
2. the method of claim 1, wherein in step (2), the deprotected and hydrolyzed product obtained in step (1) is reacted with acetyl chloride to effect Ac protection.
3. The method of claim 2, wherein step (2) comprises: dissolving the product obtained in the step (1), reacting the product with acetyl chloride under the heating condition, and concentrating the reaction solution to be dry; dissolving in acetonitrile and water, and concentrating to dryness; then extracting an organic phase, drying and concentrating the organic phase to dryness, and carrying out silica gel column chromatography to obtain a product of which the hydroxyl on a benzene ring is protected by Ac.
4. The method of claim 1, wherein in step (3), the product obtained in step (2) is reacted with acetyl chloride to carry out chlorination.
5. The method of claim 4, wherein step (3) comprises: dissolving the product obtained in the step (2), reacting the product with acetyl chloride under the heating condition, and concentrating the reaction solution to be dry; dissolving, crystallizing, filtering, washing and drying to obtain the compound shown in the formula 1.
6. The method of claim 1, wherein step (4) comprises: mixing the 2' -halogenated nucleoside with the compound shown in the formula 1, fully reacting, quenching, concentrating to remove the solvent to obtain a light yellow solid, diluting, washing, separating an organic phase, and drying to obtain the compound shown in the formula C.
7. The method of claim 6, wherein said 2' -halonucleoside is selected from the group consisting of: 2 ' -halogenated thymidine, 2 ' -halogenated uridine, 2 ' -halogenated adenosine, 2 ' -halogenated cytidine, 2 ' -halogenated guanosine.
8. The method of claim 1, wherein step (5) comprises: dissolving the compound of the formula C obtained in the step (4) in methanol solution saturated by ammonia gas, stirring, and reacting until the compound of the formula C disappears; concentrating to dryness, and performing silica gel column chromatography.
9. The method of claim 1, wherein step (6) comprises: suspending the compound obtained in the step (5) and tetrazole in acetonitrile, concentrating under reduced pressure to remove water in the system, adding bis (diisopropylamino) (2-cyanoethoxy) phosphine, and reacting; washing; drying; concentrating; and (4) performing silica gel column chromatography to obtain the compound shown in the formula (I).
10. The method of claim 1, wherein the alkoxy group is a C1 to C4 alkoxy group.
11. The method of claim 1, wherein the alkyl group is a C1 to C4 alkyl group.
12. The method of claim 10, wherein the alkoxy group is methoxy.
13. The method of claim 1, wherein the halogen is selected from the group consisting of: fluorine, chlorine, bromine, iodine.
14. The method of claim 13, wherein said halogen is fluorine.
15. The method of claim 1, wherein the compound has the structure shown in compound 2:
16. the method of claim 15, wherein the compound has a structure selected from the group consisting of compounds 2a, 2b, 2c, 2d and 2 e:
17. the method of claim 1, wherein the compound is used to synthesize an antisense nucleotide.
18. The method of claim 17, wherein the method of synthesizing antisense nucleotides using the compound comprises:
(a) monomeric coupling of a compound of claim 1 with a compound of formula 3 and formula 4, followed by oxidation of the trivalent phosphine and deprotection of TBDMS to obtain a reaction product;
(b) carrying out monomer coupling on the reaction product obtained in the previous step and the compounds shown in the formulas 3 and 4, and then oxidizing trivalent phosphine and carrying out deprotection on TBDMS to obtain a further reaction product;
(c) repeating the step (b) to obtain a polymer with a required length, and removing the base of the polymer and the protecting group on the phosphine to obtain the antisense nucleotide;
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