CN117551155B - Synthesis method of 5'-O-DMT-2' -O-propynyl-uridine - Google Patents
Synthesis method of 5'-O-DMT-2' -O-propynyl-uridine Download PDFInfo
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- CN117551155B CN117551155B CN202410023830.XA CN202410023830A CN117551155B CN 117551155 B CN117551155 B CN 117551155B CN 202410023830 A CN202410023830 A CN 202410023830A CN 117551155 B CN117551155 B CN 117551155B
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- uridine
- propynyl
- dmt
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- 229940045145 uridine Drugs 0.000 title claims abstract description 51
- 238000001308 synthesis method Methods 0.000 title description 4
- 238000000034 method Methods 0.000 claims abstract description 35
- -1 2' -O-propynyl-uridine Chemical compound 0.000 claims abstract description 31
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 26
- 230000002829 reductive effect Effects 0.000 claims abstract description 23
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 20
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000000746 purification Methods 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims description 101
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical class O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 claims description 45
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 21
- 239000000047 product Substances 0.000 claims description 19
- 238000003756 stirring Methods 0.000 claims description 18
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 claims description 16
- 238000004440 column chromatography Methods 0.000 claims description 16
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 claims description 15
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 claims description 15
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 14
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- UUGITDASWNOAGG-CCXZUQQUSA-N cyclouridine Chemical compound O=C1C=CN2[C@@H]3O[C@H](CO)[C@@H](O)[C@@H]3OC2=N1 UUGITDASWNOAGG-CCXZUQQUSA-N 0.000 claims description 10
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- QCPXVCYHAUXYFF-UHFFFAOYSA-N trimethyl(prop-1-ynoxy)silane Chemical compound CC#CO[Si](C)(C)C QCPXVCYHAUXYFF-UHFFFAOYSA-N 0.000 claims description 9
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a method for synthesizing 5' -O-DMT-2' -O-propynyl-uridine, which mainly adopts 2' -O-propynyl-uridine as a substrate, 4' -dimethoxy trityl chloride and pyridine are added, and the 2' -O-propynyl-uridine is converted into the 5' -O-DMT-2' -O-propynyl-uridine by a designed specific process. In the synthetic technical route of the application, isomers cannot be generated, the purification time is shortened, the cost is reduced, and the control reagent is not involved in the synthetic process, so that the method is beneficial to industrialized production.
Description
Technical Field
The invention belongs to the technical field of nucleotide synthesis, and particularly relates to a synthesis method of 5'-O-DMT-2' -O-propynyl-uridine.
Background
Oligonucleotides have broad application prospects in cancer treatment and genetics, such as: an oligonucleotide is a short single stranded DNA or RNA, typically consisting of several to tens of nucleotides. They can bind to complementary DNA or RNA sequences to form stable double stranded structures, and in particular, oligonucleotides can cross-link to internal or terminal positions of DNA or RNA within a cell, which cross-link can prevent replication of the cell. Thus, the oligonucleotide can be used as a specific drug that binds to DNA or RNA of cancer cells, preventing the replication of cancer cells, and thus killing cancer cells. This method of treatment is called gene-targeted therapy because it is directed to specific gene mutations, not to all healthy or diseased cells.
In genetics, oligonucleotides are used as probes for studying the structure and function of DNA and RNA. They can also be used for gene therapy, i.e. for the treatment of genetic diseases by modification of DNA or RNA sequences. In this process, the oligonucleotides may be designed to bind to abnormal gene sequences, thereby preventing the expression of the deleterious genes.
RNAi is a common gene expression control mechanism in organisms that can inhibit the expression of a specific gene by degrading the target RNA. During RNAi, double-stranded RNA (dsRNA) is a key effector molecule that can be cleaved by the intracellular RNaseIII enzyme into small fragments, which are called siRNAs (small interfering RNAs). The siRNA can bind to the target RNA, resulting in degradation of the target RNA, thereby inhibiting expression of a particular gene.
The 2' position is an important modification site during the synthesis of siRNA. At this position, the physicochemical properties and functions of the siRNA can be altered by the addition of chemical groups (e.g., methyl, ethyl, etc.). These modifications can increase the stability and specificity of the siRNA, allowing it to bind more efficiently to the target RNA, thereby more effectively inhibiting expression of a particular gene. Wherein: sugar-modified oligonucleotides are very important substances that alter the physicochemical properties and functions of siRNA, such as: the sugar-modified oligonucleotide can activate a subsequence of the 2' -deoxy-erythro-pentofuranosyl nucleoside of RNaseH, thereby promoting degradation of the target nucleic acid.
Thus, it is important to synthesize sugar-modified oligonucleotides, 2' -O-propynyl being an organic chemical group, typically bound to the sugar moiety of the nucleotide, the presence of which can alter the chemical nature and biological activity of the nucleotide. The synthesis of 5' -O-DMT-2' -O-propynyl-uridine is a process for synthesizing 2' -O-propynyl-containing pharmaceutical intermediates, and is also an intermediate for synthesizing sugar-modified oligonucleotides.
Disclosure of Invention
In order to solve the problems, the application provides a new idea for synthesizing a medical intermediate 5'-O-DMT-2' -O-propynyl-uridine.
To achieve the above object, the present application is achieved by the following scheme:
the application provides a synthesis method of 5'-O-DMT-2' -O-propynyl-uridine, which comprises the following steps:
s1, adding pyridine into a first reaction container at the temperature of-5 ℃ to 5 ℃, continuously introducing inert gas into the first reaction container, sequentially adding 2 '-O-propynyl-uridine and 4,4' -dimethoxy trityl chloride into the first reaction container under the atmosphere of the inert gas, fully stirring at the temperature of-5 ℃ to 5 ℃ for complete reaction, washing an organic phase, concentrating the organic phase under reduced pressure, and purifying by column chromatography to obtain the target product of 5'-O-DMT-2' -O-propynyl-uridine.
As a further improvement of the present application, in step S1, the time for the complete stirring reaction is a first preset period of time, which may be set according to the addition amount of 2 '-O-propynyl-uridine, 4' -dimethoxytrityl chloride and pyridine, and may be, but not limited to, 1h to 10h, specifically, but not limited to, 1h, 2h, 3h, 4h, 5h, 6h, 7h, etc.
As a further improvement of the application, in the step S1, the volume mole ratio of the 2' -O-propynyl-uridine and the pyridine is 0.2mol/L to 0.6mol/L.
As a further improvement of the present application, in step S1, the volume molar ratio of the 4,4 '-dimethoxytrityl chloride and the 2' -O-propynyl-uridine is 0.8 to 1.5.
As a further improvement of the present application, in step S1, the progress of the stirring reaction is controlled by HPLC.
As a further improvement of the application, in the step S1, the eluent applied to the column chromatography purification is a mixed solution of dichloromethane and methanol, and the mixed solution also contains triethylamine with the volume percentage of 5%.
As a further improvement of the present application, step S2 and step S3 are further included before step S1:
s2, adding uridine and N, N-dimethylformamide into a second reaction vessel, fully dissolving, adding diphenyl carbonate and sodium bicarbonate, heating to 115-125 ℃, fully reacting, reducing the temperature to 20-30 ℃, separating out solids, filtering to obtain a solid substance filter cake, and washing the solid substance filter cake by adopting a first organic solvent to obtain 2,2' -dehydrated uridine;
s3, adding the 2,2 '-dehydrated uridine obtained in the step S2 into a third reaction container at the temperature of minus 5 ℃ to 5 ℃, continuously introducing inert gas into the third reaction container, sequentially adding a second organic solvent and propynyloxy silane into the third reaction container under the atmosphere of the inert gas, fully stirring for reaction, slowly dropwise adding boron trifluoride diethyl ether into the third reaction container at the temperature of minus 5 ℃ to 5 ℃, fully stirring at the temperature of minus 5 ℃ to 5 ℃ after dropwise adding, heating the third reaction container to 115 ℃ to 125 ℃, fully stirring for reaction at the temperature of 115 ℃ to 125 ℃, and sequentially carrying out reduced pressure concentration, column chromatography purification and reduced pressure concentration after the reaction is completed, thereby obtaining the 2' -O-propynyl-uridine.
As a further improvement of the present application, in step S2, the molar ratio of the sodium bicarbonate to the uridine is 1.5:1 to 3:1.
As a further improvement of the application, in the step S2, the molar ratio of the diphenyl carbonate to the uridine is 1.1:1-2:1.
As a further improvement of the application, in the step S2, the volume mole ratio of the uridine to the N, N-dimethylformamide is 0.5mol/L to 1.5mol/L.
As a further improvement of the present application, in step S2, the first organic solvent is at least one of ethanol, methanol, isopropanol, acetone, and ethyl acetate. Preferably, when the first organic solvent is methanol, the washing step is: mixing methanol and the filter cake in the step S2 together at 20-30 ℃, fully stirring, and filtering again to remove impurities and ions in the filter cake, thereby obtaining pure 2,2' -dehydrated uridine. Preferably, in step S2, the progress of the reaction is controlled by HPLC.
As a further improvement of the present application, in step S2, the time for the completion of the sufficient reaction is a second preset time period, which may be set according to the addition amount of uridine, diphenyl carbonate and sodium bicarbonate, and may be, but not limited to, 5h to 15h, specifically, but not limited to, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, etc.
As a further improvement of the present application, in step S3, the second organic solvent is N, N-dimethylacetamide.
As a further improvement of the present application, in step S3, the propynyloxy silane is t-butyldimethyl (2-propynyloxy) silane or propynyloxy trimethylsilane. Preferably, the mol ratio of the tert-butyldimethyl (2-propynyloxy) silane to the 2,2 '-anhydrouridine is 1:4-1:6, or the mol ratio of the propynyloxy trimethylsilane to the 2,2' -anhydrouridine is 1:4-1:6.
As a further improvement of the application, in the step S3, the volume molar ratio of the boron trifluoride diethyl etherate to the 2,2' -dehydrated uridine is 1:2-1:3.
As a further improvement of the application, in the step S3, the reaction progress under the temperature condition of 115-125 ℃ is controlled by HPLC.
As a further improvement of the application, in the step S3, the eluent applied to the column chromatography purification is a mixed solution of dichloromethane and methanol, and the mixed solution also contains triethylamine with the volume percentage of 5%.
As a further improvement of the present application, in step S3: the time of the fully stirred reaction at the temperature of 5 ℃ to 5 ℃ is a third preset time period, and the third preset time period can be set according to the addition amount of the 2,2' -dehydrated uridine, the propynyloxy silane and the boron trifluoride diethyl etherate, and can be, but not limited to, 5min to 1h, and can be, but not limited to, 10min, 20min, 30min, 50min, 1h and the like; the time for fully stirring the mixture at the temperature of 115-125 ℃ is a fourth preset time period, and the fourth preset time period can be set according to the addition amount of 2,2' -anhydrouridine, propynyloxy silane and boron trifluoride diethyl ether, and can be 1-10 h, specifically but not limited to 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h and the like.
As a further improvement of the present application, in step S1, the inert gas may be, but is not limited to, nitrogen, argon, helium, neon, etc.; in step S3, the inert gas may be, but is not limited to, nitrogen, argon, helium, neon, or the like.
The method has the beneficial effects that the technical route for synthesizing the 5'-O-DMT-2' -O-propynyl-uridine has the following advantages:
1) Avoiding the generation of isomers and improving the purity and quality of the product.
2) The purification steps are reduced, the production cost is reduced, and the production efficiency is improved.
3) The whole process is reasonable in design, suitable for large-scale production, and high in practicality and economy.
4) Reduces the energy consumption and the environmental pollution, and meets the requirement of sustainable development.
In conclusion, the synthetic technology route of the application has excellent economic benefit and industrial application prospect, and has positive promotion effect on the development of the related fields.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of 2' -O-propynyl-uridine prepared in example 2;
FIG. 2 is a HPLC chart of 2' -O-propynyl-uridine prepared in example 2;
FIG. 3 is a nuclear magnetic resonance spectrum of 5'-O-DMT-2' -O-propynyl-uridine prepared in example 2;
FIG. 4 is a HPLC chart of 5'-O-DMT-2' -O-propynyl-uridine prepared in example 2.
Detailed Description
For the purposes, technical solutions and advantages of the present application, the technical solutions of the present application will be clearly and completely described below with reference to specific embodiments and drawings of the present application. It should be apparent that the described embodiments are only some, but not all, of the embodiments of the present application and are not intended to limit the scope of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
In order to solve the technical problems, the application provides a novel thinking for synthesizing a medical intermediate 5'-O-DMT-2' -O-propynyl-uridine, which has the following technical scheme:
the specific synthesis steps of 5'-O-DMT-2' -O-propynyl-uridine are as follows:
step one: 1 to 1.1L of pyridine is added into a first reaction vessel at the temperature of between minus 5 and 5 ℃ under the protection of nitrogen, 354.29 to 357.83mmol (100 to 101 g) of 2 '-O-propynyl-uridine (compound 10) and 425.15 to 428.1mmol (144 to 145 g) of 4,4' -dimethoxytrityl chloride (compound 2) are sequentially added into the first reaction vessel under the protection of nitrogen, and then the reaction is carried out for 3 to 5 hours under the stirring at the temperature of between minus 5 and 5 ℃. The progress of the reaction in the above experiment was controlled by HPLC, after completion of the reaction, the organic phase was washed three times with 1L of water, concentrated under reduced pressure, and purified by column chromatography to give 347.23mmol (203 g) of the objective product 5'-O-DMT-2' -O-propynyl-uridine in 98% yield. Wherein: in the process of column chromatography purification, the eluent is a mixed solution of dichloromethane and methanol, the mixed solution also contains 5% of triethylamine, the eluent can be added at one time or can be added for a plurality of times for washing in the process of elution, and the volume ratio of the dichloromethane to the methanol is reduced in gradient according to the addition sequence, for example: the volume ratio of dichloromethane to methanol can be reduced in a gradient way from 20:1 to 10:1, and the specific steps are as follows: if three eluents are adopted, the volume ratio of the methylene dichloride to the methanol is 20:1 for the first time, the volume ratio of the methylene dichloride to the methanol is 15:1 for the second time, and the volume ratio of the methylene dichloride to the methanol is 10:1 for the third time.
The main reaction mechanism of the compound 10 to the target product in the first step is shown as the following reaction mechanism formula I:
a method for preparing the compound of formula I,
under the action of pyridine, 4 '-dimethoxy trityl chloride (compound 2) is decomposed to generate 4,4' -dimethoxy trityl carbonium ion, and hydroxyl oxygen at the 5 'position of 2' -O-propynyl-uridine (compound 10) attacks carbonium ion due to steric hindrance effect to obtain a target product. In the reaction of the third step: pyridine is used as a solvent, alkaline environment is provided, and the decomposition of 4,4' -dimethoxy trityl chloride is promoted; 4,4' -dimethoxy trityl chloride is an important organic compound, is mainly used as a hydroxyl protecting group and a deprotection group for synthesizing a nucleoside, and 4,4' -dimethoxy trityl chloride is used as a hydroxyl protecting group, can selectively protect hydroxyl in the nucleoside from being damaged by a reaction reagent in subsequent reactions, and can be used as a deprotection group when the protecting group needs to be removed, and the 4,4' -dimethoxy trityl chloride can be removed through specific reaction conditions so as to obtain a target product; 2' -O-propynyl-uridine was used as substrate, providing the structure of the uridine moiety. The feeding process is easy to instantaneously release heat, so that the reaction system becomes mixed, and the temperature of-5 ℃ to 5 ℃ is set to ensure the stability of the reaction system and control the occurrence of side reactions.
The technical route from the compound 10 to the target product is shown as a formula II:
II, the step of setting the position of the base plate,
in an embodiment, the method further includes the following steps before the first step:
step two: 410-414.1 mmol (100-101 g) of uridine (compound 1) and 400-450 mL of N, N-dimethylformamide are added into a second reaction vessel, 447.2-451.9 mmol (96-97 g) of diphenyl carbonate (compound 6) and 7.82-8.42 mmol (0.65-0.7 g) of sodium bicarbonate are added after full dissolution, the reaction is heated to 115-125 ℃ for 8-10 hours, the reaction progress of the experiment is controlled by HPLC, after the reaction is completed, the temperature of the reaction is reduced to 20-30 ℃, and solids are separated out and filtered, thus obtaining a solid filter cake. 400-500 mL of methanol and the solid filter cake are mixed together at 20-30 ℃, stirred for 2-3 hours, and filtered again so as to completely remove impurities and ions in the filter cake, and 88.2g of white powdery product (compound 7) is obtained, and the yield is 95.2%.
The main reaction mechanism of compounds 1 to 7 in step two is the following reaction mechanism formula iii:
III the number of the components to be processed,
after the basic sodium bicarbonate neutralizes the hydrogen ions on the nitrogen in compound 1, the electrons of compound 1 migrate to the carbonyl oxygen, then the oxygen with the negative nucleus attacks the carbon at the 2' position, then the electrons migrate to the hydroxyl oxygen at the 2' position, dehydroxylation becomes 2,2' -anhydrouridine (compound 7), and the dehydroxylated hydroxyl attacks the carbonyl of diphenyl carbonate (compound 6), which breaks down into phenol and monophenyl carbonate anions. In the reaction of the first step: uridine serves as a substrate for the reaction, providing the structure of the uridine moiety; sodium bicarbonate is used as an acid binding agent to help maintain a proper pH value so as to promote the reaction; diphenyl carbonate is used as a dehydrating agent and can react with the removed hydroxyl, so that the reaction is promoted; n, N-dimethylformamide is used as a solvent to help maintain the substrate and catalyst in a dissolved state to promote the reaction. The temperature of 115-125 ℃ is set to promote the reaction, break the initial bond of uridine, diphenyl carbonate and the like, and a certain amount of energy is required, and the energy can be provided at a high temperature. In addition, the high temperature may also help to increase the fluidity of the system, enabling better mixing and contacting of the substrate and reagents, thereby facilitating the progress of the reaction.
Step three: adding 0.442-0.446 mol (100-101 g) of 2,2' -dehydrated uridine (compound 7) into a third reaction container at the temperature of minus 5 ℃ to 5 ℃ under the protection of nitrogen, sequentially adding 400-450 ml of N, N-dimethylacetamide, 2.20-2.23 mol (375-380 g) of tert-butyldimethyl (2-propynyloxy) silane or 2.20-2.23 mol (382-387 g) of propynyloxy trimethyl silane into the third reaction container under the protection of nitrogen, fully stirring for reaction, then sucking 1.102-1.11 mol (156.5-157.5 g) of boron trifluoride diethyl ether (compound 9) at the temperature of minus 5 ℃ by using a 50ml syringe, dropwise adding boron trifluoride diethyl ether which is a solution of boron trifluoride in diethyl ether into the third reaction container, wherein the mass percentage of boron trifluoride in diethyl ether is 47%, the dropwise adding time is 2 h-4 h, stirring for the reaction at the temperature of minus 5 ℃ to 15 ℃ for 125-115 h, and the temperature of the third reaction container is increased to 125 ℃ to 115 min. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, purified by column chromatography and concentrated under reduced pressure to give 0.36mol (101.7 g) of 2 '-O-propynyl-uridine (compound 10) as a white solid product in a yield of 81.5% starting from t-butyldimethyl (2-propynyloxy) silane or 0.33mol (93.8 g) of 2' -O-propynyl-uridine (compound 10) as a white solid product in a yield of 75.2% starting from propynyloxy trimethylsilane. Wherein: in the process of column chromatography purification, the eluent is a mixed solution of dichloromethane and methanol, the mixed solution also contains 5% of triethylamine, the eluent can be added at one time or can be added for a plurality of times for washing in the process of elution, and the volume ratio of the dichloromethane to the methanol is reduced in gradient according to the addition sequence, for example: the volume ratio of dichloromethane to methanol can be reduced in a gradient way from 20:1 to 10:1, and the specific steps are as follows: if three eluents are adopted, the volume ratio of the methylene dichloride to the methanol is 20:1 for the first time, the volume ratio of the methylene dichloride to the methanol is 15:1 for the second time, and the volume ratio of the methylene dichloride to the methanol is 10:1 for the third time.
The main reaction mechanism of compounds 7 to 10 in step three is the following reaction mechanism formula vi:
a step VI of, in which,
compound 7, under the action of Lewis acid BF3, causes the carbon at the 2' position to be in an electron-deficient state, thereby causing S to occur with the oxygen atom of the alkyl (2-propynyloxy) silane N2 The reaction is favorable for generating a space structure which is in cis-position with the hydroxyl at the 3' position due to the steric hindrance effect. The reaction was then quenched with methanol to give compound 10. In the reaction of the third step: 2,2' -anhydrouridine is used as a source for providing uridine moieties; the boron trifluoride part in the boron trifluoride diethyl ether is Lewis acid as a catalyst, and the diethyl ether has the function of complexing the lone pair electron of ether linkage oxygen with electron-deficient boron of the boron trifluoride, so that the boron trifluoride is more stable and is favorable for canning and transportation as a commodity; n, N-dimethylacetamide provides a suitable solvent environment that allows the substrate and catalyst to interact effectively; the oxygen of propynyloxy has a pair of lone electrons, has higher reactivity, and reacts with SN2 of carbon atom in electron-deficient state at the 2' position in substrate molecule, thereby forming new chemical bond. The feeding process is easy to instantaneously release heat, so that the reaction system becomes mixed, and the temperature of-5 ℃ to 5 ℃ is set in the feeding and uniformly mixing process, so that the stability of the reaction system is maintained, and the occurrence of side reactions is controlled. The temperature of 115-125 ℃ is set to promote the reaction, break the initial combination of 2,2' -anhydrouridine and propynyloxy, and require a certain amount of energy which can be supplied by high temperature to further make them more susceptible to reaction with other molecules or groups. In addition, the high temperature may also help to increase the fluidity of the system, enabling better mixing and contacting of the substrate and reagents, thereby facilitating the progress of the reaction.
The technical route for synthesizing the 5'-O-DMT-2' -O-propynyl-uridine by the technical scheme is shown as a formula V:
and (V) a step of setting the position of the first element,
example 1
The specific synthesis steps of 5'-O-DMT-2' -O-propynyl-uridine are as follows:
step one: 1L of pyridine was charged into the first reaction vessel at 0℃under nitrogen protection, and 354.29mmol (100 g) of 2 '-O-propynyl-uridine (compound 10), 425.15mmol (144.05 g) of 4,4' -dimethoxytrityl chloride (compound 2) were sequentially added into the first reaction vessel under nitrogen protection, followed by stirring at 0℃for 4 hours. The progress of the reaction in the above experiment was controlled by HPLC, after completion of the reaction, the organic phase was washed three times with 1L of water, concentrated under reduced pressure, and purified by column chromatography to give 347.23mmol (203 g) of the objective product 5'-O-DMT-2' -O-propynyl-uridine in 98% yield. Wherein: in the process of column chromatography purification, the eluent is a mixed solution of dichloromethane and methanol, the mixed solution also contains 5% of triethylamine, the elution process adopts a mode of three eluents, the volume ratio of the first dichloromethane and the methanol is 20:1, the volume ratio of the second dichloromethane and the methanol is 15:1, and the volume ratio of the third dichloromethane and the methanol is 10:1.
Example 2
The difference between this embodiment and embodiment 1 is that the method further includes the following steps before the first step:
step two: 410mmol (100 g) of uridine (compound 1) and 400mL of N, N-dimethylformamide were added to a second reaction vessel, after complete dissolution, 450mmol (96.6 g) of diphenyl carbonate (compound 6) and 8.18mmol (0.68 g) of sodium hydrogencarbonate were added, the reaction was heated to 120℃and conducted for 9 hours, the progress of the reaction in the above-mentioned experiment was controlled by HPLC, after completion of the reaction, the temperature of the above-mentioned reaction was lowered to 25℃and solids were precipitated and filtered to obtain a solid cake. 450mL of methanol and the above solid cake were mixed together at 25℃and stirred for 2.5 hours, and filtered again to sufficiently remove impurities and ions in the cake, to give 88.2g of a white powdery product (Compound 7) in a yield of 95.2%.
Step three: 0.442mol (100 g) of 2,2' -dehydrated uridine (compound 7) was added to the third reaction vessel at 0℃under nitrogen protection, 400ml of N, N-dimethylacetamide, 2.21mol (377 g) of t-butyldimethyl (2-propynyloxy) silane or 2.21mol (384 g) of propynyloxy trimethyl silane were sequentially added to the third reaction vessel under nitrogen protection, after the reaction was sufficiently stirred, 1.105mol (156.9 g) of boron trifluoride diethyl ether (compound 9) was sucked by a 50ml syringe at 0℃and added dropwise to the third reaction vessel, wherein boron trifluoride diethyl ether means a solution of boron trifluoride in diethyl ether, and the mass percentage of boron trifluoride in diethyl ether was 47%, the dropwise addition time was 3 hours, after the completion of dropwise addition, stirring was carried out at 0℃for 25 minutes, and then the third reaction vessel was heated to 120℃and stirred at 120℃for 15 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, purified by column chromatography and concentrated under reduced pressure to give 0.36mol (101.7 g) of 2 '-O-propynyl-uridine (compound 10) as a white solid product in a yield of 81.5% starting from t-butyldimethyl (2-propynyloxy) silane or 0.33mol (93.8 g) of 2' -O-propynyl-uridine (compound 10) as a white solid product in a yield of 75.2% starting from propynyloxy trimethylsilane. Wherein: in the process of column chromatography purification, the eluent is a mixed solution of dichloromethane and methanol, the mixed solution also contains 5% of triethylamine, the elution process adopts a mode of three eluents, the volume ratio of the first dichloromethane and the methanol is 20:1, the volume ratio of the second dichloromethane and the methanol is 15:1, and the volume ratio of the third dichloromethane and the methanol is 10:1.
The spectrum of 1H NMR analysis for 2' -O-propynyl-uridine (compound 10) prepared in example 2 is shown in FIG. 1, and specifically comprises the following:
1 H NMR (400 MHz, DMSO-d6): 11.34 (s, 1H), 7.91-7.93 (d, 2H), 5.87-5.88 (m, 1H), 5.64-5.67 (m, 1H), 5.23-5.24 (d, 1H), 5.15-5.17 (t, 1H), 4.20-4.33 (m, 2H), 4.08-4.15 (m, 2H), 3.86-3.89 (q, 1H), 3.53-3.66 (m, 2H), 3.42-3.43 (t, 1H)。
HPLC purity analysis for 2' -O-propynyl-uridine prepared in the present example 2 is shown in FIG. 2, and HPLC purity is more than 95%.
The 1H NMR spectrum of 5'-O-DMT-2' -O-propynyl-uridine prepared in this example 2 is shown in FIG. 3, and is specifically as follows:
1 H NMR (400 MHz, DMSO-d6): 11.40 (s, 1H), 7.72-7.74 (d, 2H), 7.37-7.39 (m, 2H), 7.31-7.35 (m, 2H), 7.24-7.26 (m, 5H), 6.89-6.92 (d, 4H), 5.84-5.85 (m, 1H), 5.32-5.34 (d, 1H), 5.28-5.30 (d, 1H), 4.25-4.35 (m, 3H), 4.16-4.19 (m, 1H), 3.96-3.99 (m, 1H), 3.74 (s, 6H), 3.49-3.50 (t, 1H), 3.19-3.30 (m, 2H)。
HPLC purity analysis for 5'-O-DMT-2' -O-propynyl-uridine prepared in the present example 2 is shown in FIG. 4, and HPLC purity is more than 95%.
In the above examples 1 and 2, the sources of the drugs used were as follows: pyridine is a product with lot number 20230209 produced by national pharmaceutical group chemical company, 2' -O-propynyl-uridine is a product with lot number NVC0033-27 produced by Norvigneaux Biotechnology Co., ltd. In Suzhou, 4' -dimethoxytrityl chloride is a product with lot number 22042805 produced by Undah Hua Ren technology Co., ltd., uridine is a product with lot number 20112006 produced by Undah Hua Ren technology Co., ltd., diphenyl carbonate is a product with lot number 90WQRM4U produced by Anhui Hirshini technology Co., ltd., sodium bicarbonate is a product with lot number 20230316 produced by Jiangsu-strong functional chemical Co., ltd., methanol is a product with lot number 20230318 produced by tin-free crystal chemical Co., ltd., 2,2' -anhydrouridine was a lot number MPC42-1569533-2 manufactured by Jiangsu Aikang Biochemical Co., ltd, N, N-dimethylacetamide was a lot number P2637035 manufactured by Shanghai Teitatan technologies Co., ltd, t-butyldimethyl (2-propynyloxy) silane was a lot number AZF23-1159477-1 manufactured by Jiangsu Aikang biological medicine Co., ltd, propynyloxy trimethylsilane was a lot number OWEE4RDR manufactured by Anhui Technophora Co., ltd, boron trifluoride diethyl ether was a lot number OFPQR4XE manufactured by Anhui Technophora Co., ltd, methylene chloride was a lot number 20230308 manufactured by Wuxi Chemicals Co., ltd, and triethylamine was a lot number 20230218 manufactured by Jiangsu Fusheng Fuchen chemical Co., ltd.
Comparative example 1
In order to prove that the technical scheme of the application has excellent technical effects, the technical scheme for preparing the 5'-O-DMT-2' -O-propynyl-uridine, which is adopted in the prior art, comprises the following steps:
step one: 410mmol (100 g) of uridine (compound 1) and 1L of pyridine were added to the reaction vessel at 20℃to 30℃and 512 mmol (173.4 g) of 4,4' -dimethoxytrityl chloride (compound 2) was added four times, followed by stirring at 20℃to 30℃for 1 to 2 hours. The reaction progress of the experiment is controlled by HPLC, after the reaction is completed, the reaction is concentrated under reduced pressure at 40-50 ℃, after 2L of dichloromethane is used for dissolving, the organic phase is washed three times by 2L of water, 100-200 g of anhydrous sodium sulfate is used for drying for 1 hour, the reaction is concentrated under reduced pressure at 40-50 ℃, the crude product is purified by column chromatography, the eluent is a mixed solution of methanol and dichloromethane, the volume percentage of the methanol in the mixed solution is 0-10%, and the white solid compound 3 (220 g,402.5mmol, yield 98%) is obtained by compression concentration.
Step two: 4.03mmol (2.2 g) of compound 3 and a solvent are placed in a reaction vessel, fully mixed, the solvent is a mixed solvent of 20ml of benzene and acetonitrile, the volume ratio of benzene and acetonitrile is 1:1, then 4.43mmol of dibutyltin oxide (compound 4), 8.06mmol of chlorpropyne (compound 5) and 2.01mmol of tetrabutylammonium iodide (TBAI) are sequentially added into the reaction vessel, after being fully stirred, the reaction is carried out for 4-6 hours at the temperature of 95-105 ℃, the reaction progress of the experiment is controlled by HPLC, after the reaction is completed, the pressure is reduced, the column chromatography is carried out, the eluent is a mixed solution of methanol and dichloromethane, the volume percentage of the methanol in the mixed solution is 0-5%, and the white solid target product (0.94 g,1.612mmol and 40% yield) is obtained through pressure concentration.
The two steps mentioned above are taken together, that is, the technical route for the synthesis of 5'-O-DMT-2' -O-propynyl-uridine of comparative example 1 is as follows:
and the formula VI.
In summary, the existing technical route for synthesizing 5'-O-DMT-2' -O-propynyl-uridine contains dibutyl tin oxide (compound 4), which is a highly toxic drug, belongs to a controlled reagent, is unfavorable for industrialization, and has lower synthesis yield because of the generation of 5'-O-DMT-3' -O-propynyl-uridine isomer in the synthesis process. Therefore, the application provides a new idea for synthesizing 5' -O-DMT-2' -O-propynyl-uridine, which mainly adopts 2' -O-propynyl-uridine as a substrate, and converts 2' -O-propynyl-uridine into 5' -O-DMT-2' -O-propynyl-uridine by adding 4,4' -dimethoxy trityl chloride and pyridine and by a designed specific process. Avoiding the generation of isomers, reducing the difficulty of the purification process and lowering the cost.
Although the present disclosure describes embodiments, not every embodiment is described in terms of a single embodiment, and such description is for clarity only, and one skilled in the art will recognize that the embodiments may be combined in any suitable manner to form other embodiments that will be apparent to those skilled in the art.
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for synthesizing 5'-O-DMT-2' -O-propynyl-uridine, which is characterized by comprising the following steps:
s1, adding pyridine into a first reaction container at the temperature of minus 5 ℃ to 5 ℃, continuously introducing inert gas into the first reaction container, sequentially adding 2 '-O-propynyl-uridine and 4,4' -dimethoxy trityl chloride into the first reaction container under the atmosphere of the inert gas, fully stirring at the temperature of minus 5 ℃ to 5 ℃ for complete reaction, washing an organic phase, concentrating the organic phase under reduced pressure, and purifying by column chromatography to obtain a target product of 5'-O-DMT-2' -O-propynyl-uridine;
step S3 is further included before step S1:
s3, adding 2,2 '-dehydrated uridine into a third reaction container at the temperature of minus 5 ℃ to 5 ℃, continuously introducing inert gas into the third reaction container, sequentially adding a second organic solvent and propynyloxy silane into the third reaction container under the atmosphere of the inert gas, fully stirring for reaction, slowly dropwise adding boron trifluoride diethyl ether into the third reaction container at the temperature of minus 5 ℃ to 5 ℃, fully stirring at the temperature of minus 5 ℃ to 5 ℃ after dropwise adding, heating the third reaction container to 115 ℃ to 125 ℃, fully stirring for reaction at the temperature of 115 ℃ to 125 ℃, and sequentially carrying out reduced pressure concentration, column chromatography purification and reduced pressure concentration after the reaction is complete, thereby obtaining 2' -O-propynyl-uridine.
2. The method for synthesizing 5' -O-DMT-2' -O-propynyl-uridine according to claim 1, wherein in the step S1, the volume molar ratio of said 2' -O-propynyl-uridine to said pyridine is 0.2mol/L to 0.6mol/L.
3. The method for synthesizing 5'-O-DMT-2' -O-propynyl-uridine according to claim 1, wherein in the step S1, the molar ratio of the 4,4 '-dimethoxytrityl chloride to the 2' -O-propynyl-uridine is 0.8 to 1.5.
4. A method of synthesizing 5'-O-DMT-2' -O-propynyl-uridine according to any of claims 1-3, further comprising step S2, before step S3:
s2, adding uridine and N, N-dimethylformamide into a second reaction vessel, fully dissolving, adding diphenyl carbonate and sodium bicarbonate, heating to 115-125 ℃, fully reacting, reducing the temperature to 20-30 ℃, separating out solids, filtering to obtain a solid filter cake, and washing the solid filter cake by adopting a first organic solvent to obtain the 2,2' -dehydrated uridine.
5. The method for synthesizing 5'-O-DMT-2' -O-propynyl-uridine according to claim 4, wherein in the step S2, the molar ratio of the sodium bicarbonate to the uridine is 1.5:1 to 3:1.
6. The method for synthesizing 5'-O-DMT-2' -O-propynyl-uridine according to claim 4, wherein in the step S2, the molar ratio of diphenyl carbonate to uridine is 1.1:1 to 2:1.
7. The method for synthesizing 5'-O-DMT-2' -O-propynyl-uridine according to claim 4, wherein in the step S2, the volume molar ratio of said uridine to said N, N-dimethylformamide is 0.5mol/L to 1.5mol/L.
8. The method for synthesizing 5'-O-DMT-2' -O-propynyl-uridine according to claim 1, wherein in step S3, said propynyloxy silane is t-butyldimethyl (2-propynyloxy) silane or propynyloxy trimethyl silane.
9. The method for synthesizing 5'-O-DMT-2' -O-propynyl-uridine according to claim 8, wherein the molar ratio of said t-butyldimethyl (2-propynyloxy) silane to said 2,2 '-anhydrouridine is 1:4 to 1:6 or the molar ratio of said propynyloxy trimethylsilane to said 2,2' -anhydrouridine is 1:4 to 1:6.
10. The method for synthesizing 5' -O-DMT-2' -O-propynyl-uridine according to claim 1, wherein in the step S3, the molar ratio of boron trifluoride diethyl etherate to 2,2' -anhydrouridine is 1:2 to 1:3.
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