CN118027100A - Synthesis method of N2-iBu-guanine- (S) -GNA phosphoramidite monomer - Google Patents
Synthesis method of N2-iBu-guanine- (S) -GNA phosphoramidite monomer Download PDFInfo
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- CN118027100A CN118027100A CN202410259411.6A CN202410259411A CN118027100A CN 118027100 A CN118027100 A CN 118027100A CN 202410259411 A CN202410259411 A CN 202410259411A CN 118027100 A CN118027100 A CN 118027100A
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- 239000000178 monomer Substances 0.000 title claims abstract description 17
- 238000001308 synthesis method Methods 0.000 title claims abstract description 11
- 239000002904 solvent Substances 0.000 claims abstract description 50
- 239000002994 raw material Substances 0.000 claims abstract description 44
- 238000006467 substitution reaction Methods 0.000 claims abstract description 38
- -1 2-isobutyryl-6-chloropurine Chemical compound 0.000 claims abstract description 33
- 239000002253 acid Substances 0.000 claims abstract description 15
- 238000007142 ring opening reaction Methods 0.000 claims abstract description 14
- 238000005859 coupling reaction Methods 0.000 claims abstract description 13
- JBWYRBLDOOOJEU-UHFFFAOYSA-N 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene Chemical compound C1=CC(OC)=CC=C1C(Cl)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 JBWYRBLDOOOJEU-UHFFFAOYSA-N 0.000 claims abstract description 12
- CIXHNHBHWVDBGQ-UHFFFAOYSA-N n-propan-2-ylpropan-2-amine;2h-tetrazole Chemical compound C1=NN=NN1.CC(C)NC(C)C CIXHNHBHWVDBGQ-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 6
- 239000003513 alkali Substances 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 34
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 15
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 12
- 230000035484 reaction time Effects 0.000 claims description 12
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
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- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 7
- GQHTUMJGOHRCHB-UHFFFAOYSA-N 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine Chemical compound C1CCCCN2CCCN=C21 GQHTUMJGOHRCHB-UHFFFAOYSA-N 0.000 claims description 6
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- 230000002194 synthesizing effect Effects 0.000 claims description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 6
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 claims description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 5
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- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 claims description 3
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 3
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 claims description 3
- 229910000024 caesium carbonate Inorganic materials 0.000 claims description 3
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- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
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- 238000010189 synthetic method Methods 0.000 claims description 2
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
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- ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 2,3-dimethylbutane Chemical group CC(C)C(C)C ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 0.000 description 1
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- BWGRDBSNKQABCB-UHFFFAOYSA-N 4,4-difluoro-N-[3-[3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-thiophen-2-ylpropyl]cyclohexane-1-carboxamide Chemical compound CC(C)C1=NN=C(C)N1C1CC2CCC(C1)N2CCC(NC(=O)C1CCC(F)(F)CC1)C1=CC=CS1 BWGRDBSNKQABCB-UHFFFAOYSA-N 0.000 description 1
- RYYIULNRIVUMTQ-UHFFFAOYSA-N 6-chloroguanine Chemical group NC1=NC(Cl)=C2N=CNC2=N1 RYYIULNRIVUMTQ-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a synthesis method of an N2-iBu-guanine- (S) -GNA phosphoramidite monomer, which comprises the following steps: step S1, in a first solvent, enabling 2-isobutyryl-6-chloropurine to undergo a first substitution reaction with a first raw material and alkali to obtain a first product system containing a first intermediate; step S2, in a second solvent, carrying out ring-opening hydrolysis reaction on the first intermediate in the presence of acid to obtain a second product system containing the second intermediate; s3, in a third solvent, enabling a second intermediate and 4,4' -dimethoxy triphenylchloromethane to undergo a second substitution reaction to obtain a third product system containing the third intermediate; and S4, in a fourth solvent, performing a coupling reaction on the third intermediate, the second raw material and the diisopropyl ammonium salt tetrazole to obtain a fourth product system, and separating and purifying to obtain the N2-iBu-guanine- (S) -GNA phosphoramidite monomer.
Description
Technical Field
The invention relates to the technical field of organic synthesis, in particular to a method for synthesizing an N2-iBu-guanine- (S) -GNA phosphoramidite monomer.
Background
RNA interference (RNA INTERFERENCE, RNAI) is a gene silencing phenomenon taking exogenous and endogenous RNA as degradation targets, and after exogenous genes are introduced or viruses invade, double-stranded RNA (dsRNA) artificially synthesized in cells or having the same sequence as the target genes in vivo degrades the target mRNA into small fragments of 21-22 nt, so that gene expression is specifically inhibited.
In 1989, matzke et al reported for The first time that promoter-mediated cotransfection of homologous genes into tobacco caused silencing of transgene expression (M.A. Matzke, M. Primig, J. Trnovsky, A.J.M. Matzke, the EMBO Journal, 1989, 8:643-649.). In 1990, the Jorgensen group reported that exogenously transferred gene encoding chalcone synthase could inhibit the expression of endogenous chalcone synthase gene in flowers (C. Napoli, C. Lemieux, R. Jorgensen, PLANT CELL, 1990, 2 (4): 279-289). In 1998, fire et al found that double-stranded RNA added as a control showed a stronger inhibitory effect than sense or antisense RNA when subjected to an antisense RNA inhibition experiment in caenorhabditis elegans (C.elegans), and this phenomenon was designated as RNA interference (A.fire, S.Q. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello, 1998, 391:806-811.). The first global drug PATISIRAN based on RNAi technology (trade name: onpattro) was approved by the United states FDA for marketing in month 10 of 2018 for the treatment of hereditary thyroxine-mediated amyloidosis polyneuropathy, the first small interfering RNA (SMALL INTERFERING RNA, SIRNA) drug. 4 siRNA drugs, which were obtained by Alnylam pharmaceutical companies, were approved by FDA, such as Givlaari (trade name: givosiran, for treating acute hepatoporphyria in adults), oxlumo (trade name: lumasiran, for treating primary homooxaluria type 1), amvuttra (trade name: vutrisiran, for treating hereditary transthyretin amyloidosis with polyneuropathy in adults) and INCLISIRAN (trade name: leqvio, for treating hyperlipidemia) for norubiquitination (Novartis), respectively. With the continuous rise of research and development, up to 2023 and 3 months, the total of all siRNA drugs under research is 225, the total of all varieties under research in clinical stage is 58, wherein 8 varieties under clinical III, 23 varieties under clinical II, 26 varieties under clinical I and 1 variety under clinical application. At present, RNAi drugs are considered to be one of the most promising and rapidly developing fields in biology and drug development today.
In order to realize the efficient gene silencing of siRNA drugs, the drugs resist the degradation of nuclease through the chemical modification of the structural layer in design, so that the drugs can smoothly reach the inside of human cells, and the toxic and side effects of the siRNA drugs are reduced. Off-target effect is an important factor in the toxic side effect of nucleic acid drugs. By introducing S-glyceroglycerin (S-glycol nucleic acid, S-GNA) into the seed region of the antisense strand of siRNA, a right-handed helix structure (R-GNA cannot be formed) is formed in the A/T enrichment region of RNA, so that the stability of base pairing between nucleotides can be reduced, and the off-target effect can be relieved, thus being an important way for siRNA modification at present.
With the development of siRNA drugs, N2-iBu-guanine- (S) -GNA phosphoramidite monomer (G- (S) -GNA) is used as an important intermediate for S-GNA synthesis, and the structure is shown as follows:
。
In 1996, oscar reported for the first time the synthesis of G- (S) -GNA, which was prepared by ring opening substitution of purine with R-glycidol followed by substitution with DMTrCl, 2-cyanoethyl-N, N-diisopropylchlorophosphamide. In 1998 Hotoda et al reported a 2-amino-6-chloropurine substituted by acetonylidene protected glycerol sulfonate, followed by hydrolytic ring opening, iBu protection, and finally by coupling with bis (diisopropylamino) (2-cyanoethoxy) phosphine. In 2017, schlegel et al reported that G- (S) -GNA was obtained by DMTr-glycidol ring opening substitution of iBu-guanine followed by substitution with 2-cyanoethyl-N, N-diisopropylchlorophosphamide.
At present, G- (S) -GNA is synthesized by respectively introducing protective groups (isobutyryl ibU, benzoyl Bz and the like) at amino groups of purine and introducing a propylene glycol structure at an N9 position, so as to prepare a key second intermediate. The method for introducing propylene glycol structure at purine N9 position is that: glycidol ring opening substitution, mitsunobu reaction, and ring opening after propylidene protected glycidol substitution. The glycidol ring-opening substitution can simultaneously generate an isomer with the N7 position introduced into a propylene glycol structure, and the byproduct at the N7 position accounts for 40 to 60 percent of the total product, so that the yield of the target product is only about 30 percent, the subsequent separation and purification process is complex, and the method is only suitable for small-scale preparation. The Mitsunobu reaction needs to use triphenyl phosphorus, diisopropyl azodicarbonate and other reagents with high cost, yi Zhimin reagent, and triphenyl phosphorus oxide and other reagents generated by the reaction are difficult to remove, so that the Mitsunobu reaction has less application in industrial scale-up production. The glycidol substitution protected by acetonylidene is currently more frequently performed by methylsulfonate (Ms), but the production of the N7 isomer cannot be effectively controlled when the-OMs substrate is substituted due to the smaller steric hindrance. The above routes for synthesizing G- (S) -GNA have the problem of N7 and N9 selectivity in the alkylation step, and the obtained mixture is a mixture of isomers with a certain proportion, so that the method is only suitable for small-scale production in laboratories.
Therefore, there is a need to study and develop a method for synthesizing N2-iBu-guanine- (S) -GNA phosphoramidite monomers.
Disclosure of Invention
The invention mainly aims to provide a synthesis method of N2-iBu-guanine- (S) -GNA phosphoramidite monomer, which aims to solve the problems of more byproducts, poor selectivity and low yield in the synthesis process of G- (S) -GNA in the prior art.
In order to achieve the above object, the present invention provides a method for synthesizing an N2-iBu-guanine- (S) -GNA phosphoramidite monomer, comprising: step S1, in a first solvent, enabling 2-isobutyryl-6-chloropurine to undergo a first substitution reaction with a first raw material and alkali to obtain a first product system containing a first intermediate;
Wherein the first raw material has the following structure: ；
The first intermediate has the following structure: ；
Step S2, in a second solvent, carrying out ring-opening hydrolysis reaction on the first intermediate in the presence of acid to obtain a second product system containing the second intermediate; the second intermediate has the following structure:
；
S3, in a third solvent, enabling a second intermediate and 4,4' -dimethoxy triphenylchloromethane to undergo a second substitution reaction to obtain a third product system containing the third intermediate; the third intermediate has the following structure:
；
S4, in a fourth solvent, performing a coupling reaction on the third intermediate, the second raw material and the diisopropyl ammonium salt tetrazole to obtain a fourth product system, and separating and purifying to obtain an N2-iBu-guanine- (S) -GNA phosphoramidite monomer; the second raw material has the following structure:
；
The N2-iBu-guanine- (S) -GNA phosphoramidite monomer has the following structure:
。
further, in the step S1, the molar ratio of the 2-isobutyryl-6-chloropurine to the first raw material is 1 (0.67-1.3).
Further, in the step S1, the molar ratio of the 2-isobutyryl-6-chloropurine to the base is 1 (1-3).
Further, in the step S1, the weight ratio of the 2-isobutyryl-6-chloropurine to the first solvent is 1 (5-50).
Further, the first solvent is selected from one or more of the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, toluene, acetonitrile, N-dimethylformamide, N-dimethylacetamide and dimethylsulfoxide.
Further, the base is selected from one or more of the group consisting of triethylamine, DBU, sodium bicarbonate, sodium carbonate, potassium carbonate and cesium carbonate.
Further, in the step S1, the reaction temperature of the first substitution reaction is 20-150 ℃ and the reaction time is 1-48 h.
Further, step S2 includes: mixing acid with a second solvent to obtain a first mixed system, and mixing the first mixed system with a first intermediate to obtain a second mixed system; and heating the second mixed system to 30-80 ℃, reacting for 1-24 h, and then regulating the pH to 7-8 by adopting alkaline resin to obtain a second product system.
Further, the concentration of the acid is 1-4 mol/L.
Further, the acid is selected from one or more of the group consisting of hydrochloric acid, p-toluene sulfonic acid, trifluoroacetic acid and acetic acid.
Further, in step S2, the weight ratio of the first intermediate to the second solvent is 1 (2-10).
Further, the second solvent is selected from dioxane and/or water mixtures.
Further, in the step S3, the molar ratio of the second intermediate to the 4,4' -dimethoxy triphenylchloromethane is 1 (0.6-1.3).
Further, in step S3, the weight ratio of the second intermediate to the third solvent is 1 (1-20).
Further, the third solvent is selected from pyridine.
Further, in the step S3, the reaction temperature of the second substitution reaction is 0-5 ℃ and the reaction time is 4-5 h.
Further, in the step S4, the molar ratio of the third intermediate to the diisopropylammonium tetrazole is 1 (0.7-2).
Further, in the step S4, the molar ratio of the third intermediate to the second raw material is 1 (0.8-2).
Further, in step S4, the weight ratio of the third intermediate to the fourth solvent is 1 (2-30).
Further, the fourth solvent is selected from one or more of the group consisting of dichloromethane, tetrahydrofuran and acetonitrile.
Further, in the step S4, the reaction temperature of the coupling reaction is 20-30 ℃ and the reaction time is 12-24 hours.
By applying the technical scheme of the application, the step S1 adopts the compound containing the p-toluenesulfonyl Ts as the first raw material, and the action site is the N-9 position in the 2-isobutyryl-6-chloropurine when the first substitution reaction is carried out, compared with the ring-opening substitution of-OMs and glycidol, the N-9 position with smaller steric hindrance is more prone to the substrate when the substrate is substituted due to higher steric hindrance of-OTs in the first raw material, and compared with the traditional synthetic route, the method can obviously inhibit the generation of N-7 position isomer, thereby improving the yield and purity of the first intermediate with the specific structure, and further improving the yield and purity of the target product G- (S) -GNA prepared later. In addition, the synthetic route does not need to use reagents with high cost and difficult removal such as triphenylphosphine, diisopropyl azodicarboxylate and the like, thereby reducing the synthetic cost and the difficulty of separation and purification.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 shows nuclear magnetic hydrogen spectra 1 H-NMR of the target product G- (S) -GNA obtained in example 1 of the present application;
FIG. 2 shows nuclear magnetic resonance spectrum 31 PNMR of the target product G- (S) -GNA prepared in example 1 of the present application.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to examples.
As described in the background art, the existing G- (S) -GNA synthesis process has the problems of more byproducts, poor selectivity and low yield of target products. In order to solve the technical problems, the application provides a synthesis method of an N2-iBu-guanine- (S) -GNA phosphoramidite monomer, which comprises the following steps:
Step S1, in a first solvent, enabling 2-isobutyryl-6-chloropurine to undergo a first substitution reaction with a first raw material and alkali to obtain a first product system containing a first intermediate;
Wherein the first raw material has the following structure: ；
The first intermediate has the following structure: ；
Step S2, in a second solvent, carrying out ring-opening hydrolysis reaction on the first intermediate in the presence of acid to obtain a second product system containing the second intermediate; the second intermediate has the following structure:
；
S3, in a third solvent, enabling a second intermediate and 4,4' -dimethoxy triphenylchloromethane to undergo a second substitution reaction to obtain a third product system containing the third intermediate; the third intermediate has the following structure:
；
S4, in a fourth solvent, performing a coupling reaction on the third intermediate, the second raw material and the diisopropyl ammonium salt tetrazole to obtain a fourth product system, and separating and purifying to obtain an N2-iBu-guanine- (S) -GNA phosphoramidite monomer;
The second raw material has the following structure:
；
The N2-iBu-guanine- (S) -GNA phosphoramidite monomer has the following structure:
。
In the step S1, the compound containing the p-toluenesulfonyl Ts is used as a first raw material, and the action site is the N-9 position in 2-isobutyryl-6-chloropurine when the first substitution reaction is carried out, compared with ring-opening substitution of-OMs and glycidol, the N-9 position with smaller steric hindrance is more prone to being carried out on a substrate when the substrate is substituted due to higher steric hindrance of-OTs in the first raw material, and compared with the traditional synthetic route, the generation of N-7 position isomer can be obviously inhibited, so that the yield and purity of a first intermediate with the specific structure are improved, and the yield and purity of a target product G- (S) -GNA prepared later can be improved. In addition, the synthetic route does not need to use reagents with high cost and difficult removal such as triphenylphosphine, diisopropyl azodicarboxylate and the like, thereby reducing the synthetic cost and the difficulty of separation and purification.
The synthetic route of the target product is as follows:
。
wherein, in step S3, since the primary hydroxyl group is more active than the secondary hydroxyl group in the second intermediate, 4' -dimethoxytriphenylchloromethane (DMTrCl) preferentially substitutes the primary hydroxyl group, thereby forming a third intermediate having the above-described specific structure.
In a preferred embodiment, in step S1, the molar ratio of 2-isobutyryl-6-chloropurine to the first starting material is 1 (0.67 to 1.3), preferably 1 (0.9 to 1.1). The weight ratio of the 2-isobutyryl-6-chloropurine to the first raw material includes but is not limited to the above range, and the limitation of the weight ratio in the above range is beneficial to improving the utilization rate of the raw material, so that the yield and purity of the first intermediate are improved by fully reacting the two raw materials, the generation of the N7 substituted product is inhibited, and the yield of the target product is further improved.
In a preferred embodiment, the molar ratio of 2-isobutyryl-6-chloropurine to base is 1 (1-3). The weight ratio of 2-isobutyryl-6-chloropurine to base includes, but is not limited to, the above ranges, and limiting it to the above ranges is advantageous to obtain higher product conversions.
In a preferred embodiment, the weight ratio of 2-isobutyryl-6-chloropurine to first solvent is 1 (5-50). The weight ratio of the 2-isobutyryl-6-chloropurine to the first solvent includes, but is not limited to, the above range, and the limitation of the weight ratio is favorable for improving the solubility and the dispersibility of the 2-isobutyryl-6-chloropurine and the first raw material in the first solvent, and is favorable for improving the first substitution reaction efficiency, thereby being favorable for improving the yield of the first intermediate.
In order to enhance the solubility and dispersibility of the 2-isobutyryl-6-chloropurine and the first starting material, thereby enhancing the first substitution reaction efficiency, it is preferable that the first solvent include one or more of the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, toluene, acetonitrile, N-dimethylformamide, N-dimethylacetamide and dimethylsulfoxide.
In a preferred embodiment, the base includes, but is not limited to, one or more of the group consisting of triethylamine, DBU, sodium bicarbonate, sodium carbonate, potassium carbonate, and cesium carbonate. Compared with other kinds, the use of the above-mentioned kind of base is advantageous in improving the first substitution reaction efficiency and in suppressing the generation of N7 isomer.
In a preferred embodiment, in step S1, the reaction temperature of the first substitution reaction is 20 to 150℃and the reaction time is 1 to 48 hours. The reaction temperature and the reaction time of the first substitution reaction include, but are not limited to, the above ranges, and limiting the above ranges is advantageous in suppressing the occurrence of side reactions, thereby being advantageous in improving the yield and purity of the first intermediate, and further in improving the yield of the target product.
In a preferred embodiment, step S2 comprises: mixing acid with a second solvent to obtain a first mixed system, and mixing the first mixed system with a first intermediate to obtain a second mixed system; and heating the second mixed system to 30-80 ℃, reacting for 1-24 h, and then regulating the pH to 7-8 by adopting alkaline resin to obtain a second product system. The adoption of the step S2 is beneficial to improving the reaction efficiency and inhibiting the occurrence of side reactions, thereby being beneficial to improving the yield and purity of the second intermediate.
In a preferred embodiment, the concentration of the acid is 1 to 4mol/L. The concentration of the acid includes, but is not limited to, the above-mentioned range, and limiting it to the above-mentioned range is advantageous in improving the reaction efficiency, thereby being advantageous in improving the yield of the second intermediate.
In a preferred embodiment, the acid includes, but is not limited to, one or more of the group consisting of hydrochloric acid, p-toluenesulfonic acid, trifluoroacetic acid and acetic acid. The acid raw materials of the type adopted by the application are easy to obtain and have proper cost.
In a preferred embodiment, the weight ratio of the first intermediate to the second solvent is 1 (2-10). The weight ratio of the first intermediate to the second solvent includes, but is not limited to, the above range, and limiting it to the above range is advantageous in improving the reaction efficiency, thereby being advantageous in improving the yield of the second intermediate.
In order to increase the solubility and dispersibility of the first intermediate, thereby increasing the reaction efficiency and the yield of the second intermediate, preferably, the second solvent includes, but is not limited to, a mixture of dioxane and water. The use of the second solvent of the above kind is advantageous in improving the solubility and dispersibility of the first intermediate, as compared with other kinds, and thus is advantageous in improving the reaction efficiency and improving the yield of the second intermediate.
In a preferred embodiment, in step S3, the molar ratio of the second intermediate to 4,4' -dimethoxytriphenylchloromethane is 1 (0.6-1.3). The weight ratio of the second intermediate to 4,4' -dimethoxy triphenylchloromethane includes but is not limited to the above range, and the limitation of the weight ratio in the above range is beneficial to improving the utilization rate of raw materials, thereby being beneficial to improving the efficiency of the second substitution reaction and further beneficial to improving the yield of the third intermediate.
In a preferred embodiment, the weight ratio of the second intermediate to the third solvent is 1 (1-20). The weight ratio of the second intermediate to the third solvent includes, but is not limited to, the above range, and limiting it to the above range is advantageous for improving the solubility and dispersibility of the second intermediate, thereby being advantageous for improving the second substitution reaction efficiency, and thus for improving the yield of the third intermediate.
In order to increase the solubility and dispersibility of the second intermediate and DMTrCl, thereby increasing the second substitution reaction efficiency and thus advantageously increasing the yield of the third intermediate, preferably the third solvent includes, but is not limited to, pyridine.
In a preferred embodiment, in step S3, the reaction temperature of the second substitution reaction is 0 to 5℃and the reaction time is 4 to 5 hours. The reaction temperature and the reaction time of the second substitution reaction include, but are not limited to, the above ranges, and limiting them to the above ranges is advantageous in improving the efficiency of the second substitution reaction while suppressing the occurrence of side reactions, thereby being advantageous in improving the yield and purity of the third intermediate.
In a preferred embodiment, in step S4, the molar ratio of the third intermediate to the diisopropylammonium tetrazolium salt is 1 (0.7-2). The weight ratio of the third intermediate to the diisopropylammonium tetrazole comprises but is not limited to the above range, and the weight ratio is limited to the above range, so that the conversion rate of the reaction raw materials is improved, and the hydrolysis of the target product in the post-treatment process is reduced.
In a preferred embodiment, the molar ratio of the third intermediate to the second starting material is 1 (0.8-2). The weight ratio of the third intermediate to the second raw material includes, but is not limited to, the above range, and limiting the weight ratio to the above range is advantageous for improving the raw material utilization ratio of the third intermediate and the second raw material, and for improving the coupling reaction efficiency, thereby being advantageous for improving the yield of the target product.
In a preferred embodiment, the weight ratio of the third intermediate to the fourth solvent is 1 (2-30). The weight ratio of the third intermediate to the fourth solvent includes, but is not limited to, the above range, and the limitation of the weight ratio is favorable for improving the solubility and the dispersibility of the third intermediate and the diisopropylammonium tetrazole, and is favorable for improving the coupling reaction efficiency, thereby being favorable for improving the yield of the target product.
In a preferred embodiment, the fourth solvent includes, but is not limited to, one or more of the group consisting of dichloromethane, tetrahydrofuran, and acetonitrile. Compared with other types, the fourth solvent of the type is favorable for improving the solubility and the dispersibility of the third intermediate and the diisopropyl ammonium salt tetrazole, so that the coupling reaction efficiency is improved, and meanwhile, the subsequent separation and purification are facilitated, so that the yield of a target product is improved.
In a preferred embodiment, in step S4, the reaction temperature of the coupling reaction is 20 to 30℃and the reaction time is 12 to 24 hours. The reaction temperature and the reaction time of the coupling reaction include, but are not limited to, the above ranges, and limiting the same to the above ranges is advantageous in improving the coupling reaction efficiency and in suppressing the occurrence of side reactions, thereby being advantageous in improving the yield and purity of the target product.
The application is described in further detail below in connection with specific examples which are not to be construed as limiting the scope of the application as claimed.
Example 1
A synthetic method of G- (S) -GNA comprises the following synthetic route:
。
the synthesis steps comprise:
(1) The synthesis of compound 2 was performed using 2-isobutyryl-6-chloropurine (compound 1) as starting material:
10.00g, 41.93mmol of 2-isobutyryl-6-chloropurine (compound 1) (see STEFAN PITSCH ET al HELVETICA CHIMICA ACTA, 2003, 86 (12): 4270-4363. Prepared) was dissolved in 142g, 150mL of N, N-Dimethylformamide (DMF) and stirred to clarify at room temperature, 13.14g, 46.90mmol of starting material A (see Iwona Dams et al Tetrahedron, 2013, 69 (5): 1634-1648) and 17.30g, 125.18mmol of K 2CO3 were then added to give a white suspension, which was warmed to 80℃to form a brown clarified liquid, stirred at this temperature for 12h with heat preservation, after the reaction was completed the first product system containing compound 2 was poured into 1000mL of water, extracted with ethyl acetate, the organic phase was dried by shrinkage, 11.5g of white solid was obtained by beating with ethyl acetate, which was verified to be compound 2 in a single configuration, yield was 81.1%;
the nuclear magnetic hydrogen spectrum data for compound 2 is as follows:
1HNMR（400MHz,DMSO-d6） δ 9.80（s,1H）,7.98（s,1H）,4.48（m,1H）,4.26-4.11（m,2H）,4.00（dd,J=8.7,6.5Hz,1H）,3.82（dd,J=8.7,5.3Hz,1H）,2.97（d,J=7.1Hz,1H）,1.29（s,3H）,1.24（s,3H）,1.08（s,3H）,1.07（s,3H）;
Wherein, the raw material A has the following chemical structure: ；
(2) Synthesis of Compound 3:
Adding 10g and 28.26mmol of compound 2 into 200mL of mixed solvent of water and dioxane, wherein the volume ratio of water to dioxane is 1:1, stirring to be clear, adding 50mL of 4mol/L HCl solution into the system, heating to 50 ℃, keeping the temperature and stirring for 1h, regulating the pH of the reaction system to 7 by using alkaline resin (Mitsubishi, dianion wa30 ion exchange resin) after the reaction is finished, filtering, eluting a filter cake by using ethyl acetate, extracting the filtrate by using ethyl acetate, merging and drying the organic phases, purifying by using a silica gel pad, and obtaining 6.9g of yellow oily substance, namely compound 3, wherein the yield is 82.6%;
the nuclear magnetic hydrogen spectrum data for compound 3 is as follows:
1HNMR（400MHz,DMSO-d6） δ 7.85（s,1H）,4.16（dd,1H）,3.90（dd,J=13.8,8.5Hz,1H）,3.76（m,1H）,3.43-3.20（m,2H）,2.74（m,1H）,2.28（m,1H）,1.07（d,J=6.8Hz,3H）,0.98（d,J=6.9Hz,3H）;
(3) Synthesis of Compound 4:
13.0g and 44.05mmol of compound 3 are dissolved by 196g and 200mL of pyridine at room temperature, the mixture is cooled to 0 ℃, 16.78g and 48.45mmol of DMTrCl are dissolved by 60mL of pyridine and then slowly added into the mixture in a dropwise manner, and a third product system containing the compound 4 is obtained after the reaction for 12 hours; directly shrinking the third product system to dryness, purifying a silica gel pad, and shrinking the fraction to dryness to obtain 17.4g of white solid, namely compound 4, with the yield of 66.1%;
The nuclear magnetic hydrogen spectrum data for compound 4 is as follows:
1HNMR（400MHz,Chloroform-d） δ 7.56（s,1H）,7.51-7.45（m,2H）,7.42-7.33（m,4H）,7.30（s,1H）,7.26-7.18（m,1H）,6.84（d,J=8.6Hz,4H）,4.47（d,J=7.3Hz,1H）,4.31（d,J=13.7Hz,1H）,4.02（m,1H）,3.80（s,6H）,3.33（dd,J=9.5,4.5Hz,1H）,3.21（dd,J=9.4,6.1Hz,1H）,2.71（m,3H）,1.27（t,6H）;
(4) Synthesizing a target product:
7.5g, 25.10mmol of starting material B (CAS: 102691-36-1) and 3.4g of diisopropylammonium tetrazole are added to 100mL of Dichloromethane (DCM) at 20℃and stirred for 5min, 10g, 16.73mmol of compound 4 are added and reacted at room temperature for 12h, and the consumption of the starting material is followed by sampling TLC. After the reaction is finished, filtering, condensing the filtrate, separating and purifying by a silica gel pad to obtain 11.2G of white solid, namely a target product G- (S) -GNA, wherein the yield is 83.8%;
wherein, the raw material B has the following structure: 。
The structure of the target product G- (S) -GNA is confirmed by nuclear magnetic hydrogen spectrum (shown in figure 1) and nuclear magnetic phosphorus spectrum (shown in figure 2), and the characterization result is as follows:
1H-NMR（400MHz,Chloroform-d） δ 11.97(s,1H),8.76(d,1H),7.54(d,1H),7.44(s,2H),7.35-7.26(m,7H),6.87-6.75(m,4H),4.33(t,2H),3.79(s,7H),3.54(s,3H),3.18(d,2H),2.65(s,1H),2.61-2.53(m,1H),2.47(s,1H),1.36-1.19(m,17H),1.19-1.08(m,12H);
31PNMR(162MHz，Chloroform-d) δ 148.59，148.48。
Example 2
The difference from example 1 is that: the molar ratio of the 2-isobutyryl-6-chloropurine to the raw material A is 1:0.67, and the specific steps are as follows:
10.00g, 41.93mmol of 2-isobutyryl-6-chloropurine (compound 1) are dissolved in 150mL of N, N-Dimethylformamide (DMF) and stirred for clarification at room temperature, then 8g, 27.96mmol of raw material A and 17.30g, 125.18mmol of K 2CO3 are added to obtain a white suspension, the white suspension is heated to 80 ℃ to form brown clarified liquid, the first product system containing compound 2 is poured into 1000mL of water after the reaction is completed and stirred for 12h at the temperature, extracted with ethyl acetate, the organic phase is dried by shrinking, and the obtained white solid is 9.1g, which is verified to be the compound 2 with a single configuration, and the yield is 92.0 percent.
Example 3
The difference from example 1 is that: the molar ratio of the 2-isobutyryl-6-chloropurine to the raw material A is 1:1.3, and the specific steps are as follows:
10.00g, 41.93mmol of 2-isobutyryl-6-chloropurine (compound 1) is dissolved in 150mL of N, N-Dimethylformamide (DMF) and stirred for clarification at room temperature, then 15.5g, 54.24mmol of raw material A and 17.30g, 125.18mmol of K 2CO3 are added to obtain a white suspension, the white suspension is heated to 80 ℃ to form brown clarified liquid, the temperature is kept at the temperature and stirred for 12h, after the reaction is completed, the first product system containing compound 2 is poured into 1000mL of water and extracted with ethyl acetate, the organic phase is dried by condensation, and the mixture is pulped and purified by ethyl acetate to obtain 11.0g of white solid, the yield of the compound 2 is 74.5%, and a small amount of N7 substituted product can be detected after the reaction.
Example 4
The difference from example 1 is that: the molar ratio of the 2-isobutyryl-6-chloropurine to the raw material A is 1:1.7.
After the reaction, the N7-substituted impurity of the product is obviously increased to 20%, the subsequent purification is difficult, and the yield is reduced to 50.0%.
Comparative example 1
The difference from example 1 is that: substituting a raw material C for the raw material A in the step (1), wherein the raw material C contains methylsulfonyl and has the following chemical structure:
. The yield of compound 2 was only 50.0%. /(I)
The inventors also performed the following amplification experiments:
Example 5
The difference from example 1 is that: the reaction in the step (1) was amplified to 1.0kg, and after completion of the reaction, 1.2kg of compound 2 was obtained, which had a yield of 81.2% and an HPLC purity of 96%, and was used for the next ring-opening hydrolysis reaction.
Example 6
The difference from example 1 is that: the reaction of step (2) was scaled up to 1.0kg, and after completion of the reaction, 0.7kg of compound 3 was obtained in a yield of 82.0% and an HPLC purity of 95% and was used for the next second substitution reaction.
From the above description, it can be seen that the above embodiments of the present application achieve the following technical effects: in the step S1, the compound containing the p-toluenesulfonyl Ts is used as a first raw material, and the action site is the N-9 position in 2-isobutyryl-6-chloropurine when the first substitution reaction is carried out, compared with ring-opening substitution of-OMs and glycidol, the N-9 position with smaller steric hindrance is more prone to being carried out on a substrate when the substrate is substituted due to higher steric hindrance of-OTs in the first raw material, and compared with the traditional synthetic route, the generation of N-7 position isomer can be obviously inhibited, so that the yield and purity of a first intermediate with the specific structure are improved, and the yield and purity of a target product G- (S) -GNA prepared later can be improved. In addition, the synthetic route does not need to use reagents with high cost and difficult removal such as triphenylphosphine, diisopropyl azodicarboxylate and the like, thereby reducing the synthetic cost and the difficulty of separation and purification.
It should be noted that the terms "first," "second," and the like in the description and in the claims are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments of the application described herein are, for example, capable of operation in sequences other than those described herein.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for synthesizing an N2-iBu-guanine- (S) -GNA phosphoramidite monomer, the method comprising:
Step S1, in a first solvent, enabling 2-isobutyryl-6-chloropurine to undergo a first substitution reaction with a first raw material and alkali to obtain a first product system containing a first intermediate;
wherein the first raw material has the following structure: ；
the first intermediate has the following structure: ；
step S2, in a second solvent, carrying out ring-opening hydrolysis reaction on the first intermediate in the presence of acid to obtain a second product system containing the second intermediate; the second intermediate has the following structure:
；
Step S3, in a third solvent, enabling the second intermediate and 4,4' -dimethoxy triphenylchloromethane to undergo a second substitution reaction to obtain a third product system containing the third intermediate; the third intermediate has the following structure:
；
S4, in a fourth solvent, carrying out a coupling reaction on the third intermediate, a second raw material and diisopropyl ammonium salt tetrazole to obtain a fourth product system, and separating and purifying to obtain the N2-iBu-guanine- (S) -GNA phosphoramidite monomer;
the second raw material has the following structure:
；
the N2-iBu-guanine- (S) -GNA phosphoramidite monomer has the following structure:
。
2. the synthesis method according to claim 1, wherein in the step S1, the molar ratio of the 2-isobutyryl-6-chloropurine to the first raw material is 1 (0.67 to 1.3); and/or the number of the groups of groups,
The molar ratio of the 2-isobutyryl-6-chloropurine to the alkali is 1 (1-3); and/or the number of the groups of groups,
The weight ratio of the 2-isobutyryl-6-chloropurine to the first solvent is 1 (5-50); and/or the number of the groups of groups,
The first solvent is selected from one or more of tetrahydrofuran, 2-methyltetrahydrofuran, toluene, acetonitrile, N-dimethylformamide, N-dimethylacetamide and dimethyl sulfoxide; and/or the number of the groups of groups,
The base is selected from one or more of the group consisting of triethylamine, DBU, sodium bicarbonate, sodium carbonate, potassium carbonate and cesium carbonate.
3. The synthesis method according to claim 1 or 2, wherein in the step S1, the reaction temperature of the first substitution reaction is 20 to 150 ℃ and the reaction time is 1 to 48 hours.
4. The synthesis method according to claim 1, wherein the step S2 comprises:
Mixing the acid with the second solvent to obtain a first mixed system, and mixing the first mixed system with the first intermediate to obtain a second mixed system; and heating the second mixed system to 30-80 ℃, and regulating the pH value to 7-8 by adopting alkaline resin after reacting for 1-24 hours to obtain the second product system.
5. The method according to claim 4, wherein the concentration of the acid is 1 to 4mol/L; and/or the number of the groups of groups,
The acid is selected from one or more of the group consisting of hydrochloric acid, p-toluene sulfonic acid, trifluoroacetic acid and acetic acid.
6. The synthesis method according to claim 1, 4 or 5, wherein in the step S2, the weight ratio of the first intermediate to the second solvent is 1 (2-10); and/or the number of the groups of groups,
The second solvent is selected from dioxane and/or water mixture.
7. The synthesis method according to claim 1, wherein in the step S3, a molar ratio of the second intermediate to the 4,4' -dimethoxytriphenylchloromethane is 1 (0.6 to 1.3); and/or the number of the groups of groups,
The weight ratio of the second intermediate to the third solvent is 1 (1-20); and/or the number of the groups of groups,
The third solvent is selected from pyridine.
8. The method according to claim 1 or 7, wherein in the step S3, the reaction temperature of the second substitution reaction is 0 to 5 ℃ and the reaction time is 4 to 5 hours.
9. The synthesis method according to claim 1, wherein in the step S4, the molar ratio of the third intermediate to the diisopropylammonium tetrazole is 1 (0.7-2); and/or the number of the groups of groups,
The mol ratio of the third intermediate to the second raw material is 1 (0.8-2); and/or the number of the groups of groups,
The weight ratio of the third intermediate to the fourth solvent is 1 (2-30); and/or the number of the groups of groups,
The fourth solvent is selected from one or more of the group consisting of dichloromethane, tetrahydrofuran and acetonitrile.
10. The synthetic method according to claim 1 or 9, wherein in the step S4, the reaction temperature of the coupling reaction is 20 to 30 ℃ and the reaction time is 12 to 24 hours.
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