CN117866029A - Pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite and synthesis method thereof - Google Patents
Pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite and synthesis method thereof Download PDFInfo
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- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 6
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
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Landscapes
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Abstract
The invention relates to a novel pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite and a synthesis method thereof, wherein the pharmaceutical intermediate can be used for modifying nucleotide, can endow the nucleotide with brand-new chemical property and biological activity, and is beneficial to popularization of gene therapy means. The synthesis method of the medical intermediate provided by the application has the advantages of easily-selected raw materials, low price, simplicity in operation and high yield, and is favorable for industrialized popularization.
Description
Technical Field
The invention belongs to the technical field of nucleotide synthesis, and particularly relates to a pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite and a synthesis method thereof.
Background
Oligonucleotides, an important tool in our hands, have shown broad application prospects in cancer treatment and genetics. In complex biological mechanisms, these short single-stranded DNA or RNA, by virtue of their ability to stably bind to complementary sequences, open new approaches to cancer treatment. In particular, to the operational level, the oligonucleotides can be precisely positioned at the internal or terminal positions of the DNA or RNA in the cell, cross-linked therewith, and thus block the replication process of the cell. In gene-targeted therapies, this strategy of precise striking is commissioned, specifically for those cancer cells with specific gene mutations, while for normal cells autumn-disadvantaged.
In the genetic search, oligonucleotides are now the probes in our hands, helping us to interpret the structure and function of DNA and RNA. In the arena of gene therapy, they offer new possibilities for the treatment of genetic diseases, in particular by modifying the DNA or RNA sequences. At the same time, sugar-modified oligonucleotides have become an important regulatory tool. By altering the physicochemical properties and functions of the oligonucleotides, we can more precisely regulate gene expression in organisms. Wherein, the 2' -O-propynyl is taken as a key organic chemical group, and can endow the nucleotide with brand-new chemical property and biological activity after being combined with the sugar part of the nucleotide.
The research of novel pharmaceutical intermediates containing 2' -O-propynyl has very important influence on improving the preparation efficiency of sugar-modified oligonucleotides and improving the quality and effect of final products.
Disclosure of Invention
To solve the above problems, the present application provides a novel pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite and a synthesis method thereof.
To achieve the above object, the present application is achieved by the following scheme:
the application provides a pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite, which has the following structural formula:
in order to achieve the above purpose, the present application also provides a method for synthesizing the pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite, comprising the following steps:
adding a first organic solvent into a reaction container, adding 5' -O-DMT-2' -O-propynyl-guanosine into the reaction container at the temperature of minus 5 ℃ to 5 ℃, continuously introducing inert gas into the reaction container, adding diisopropylammonium tetrazole into the reaction container under the atmosphere of the inert gas, adding bis (diisopropylamino) (2-cyanoethoxy) phosphine into the reaction container, controlling the temperature of the reaction container at the temperature of minus 5 ℃ to 5 ℃, stirring for a first preset time period, controlling the temperature of the reaction container at the temperature of 20 ℃ to 30 ℃, stirring for a second preset time period, washing, concentrating under reduced pressure, purifying by column chromatography, concentrating under reduced pressure, and obtaining a medical intermediate named DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite.
As a further improvement of the application, the molar ratio of the 5'-O-DMT-2' -O-propynyl-guanosine to the diisopropylammonium salt tetrazole is 1:2-1:4.
As a further improvement of the present application, the molar ratio of the 5'-O-DMT-2' -O-propynyl-guanosine to the bis (diisopropylamino) (2-cyanoethoxy) phosphine is in the range of 1:1 to 1:3.
As a further improvement of the present application, the first organic solvent is methylene chloride.
As a further improvement of the application, the molar volume ratio of the 5'-O-DMT-2' -O-propynyl-guanosine and the dichloromethane is 0.1mol/L to 0.2mol/L.
As a further improvement of the present application, the inert gas is any one of nitrogen, argon, neon and helium.
As a further improvement of the present application, the specific steps of washing include washing with water followed by washing with saturated sodium chloride solution.
As a further improvement of the application, the washing temperature is 20-30 ℃, and the washing temperature of the saturated sodium chloride solution is 20-30 ℃.
As a further improvement of the present application, the temperature of the first reduced pressure concentration is 20 ℃ to 30 ℃, and the temperature of the second reduced pressure concentration is 20 ℃ to 30 ℃.
The application has the beneficial effects that the application provides a novel pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite, and the pharmaceutical intermediate can be used for modifying nucleotide, can endow the nucleotide with brand-new chemical property and biological activity, and is beneficial to popularization of gene therapy means. The synthesis method of the medical intermediate provided by the application has the advantages of easily-selected raw materials, low price, simplicity in operation and high yield, and is favorable for industrialized popularization.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite, a pharmaceutical intermediate prepared in example 1;
FIG. 2 is a nuclear magnetic resonance spectrum of DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite, a pharmaceutical intermediate prepared in example 1;
FIG. 3 is a HPLC purity analysis chart of pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite prepared in example 1.
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 pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite and a synthesis method thereof, and the specific technical scheme is as follows:
adding 1L of dichloromethane into a reaction container, continuously introducing nitrogen into the reaction container, placing the reaction container at the temperature of minus 5 ℃ to 5 ℃ under the protection of nitrogen atmosphere, adding 150.2mmol (100 g) of 5'-O-DMT-2' -O-propynyl-guanosine (compound 1) into the reaction container, adding 449.6mmol (77 g to 78 g) of diisopropylammonium tetrazole (compound 3) into the reaction container, adding 298.6mmol (90 g to 91 g) of bis (diisopropylamino) (2-cyanoethoxy) phosphine (compound 2) into the reaction container, stabilizing the reaction container at the temperature of minus 5 ℃ to 5 ℃, stirring for 30 minutes to 60 minutes, adjusting the reaction container to the temperature of 20 ℃ to 30 ℃, stirring, controlling the reaction progress in HPLC (high performance liquid chromatography), and completely after the reaction. Adding 1L of water into the reaction container, controlling the temperature of the reaction container to be 20-30 ℃, carrying out catalytic washing for three times, adding 1L of saturated sodium chloride aqueous solution into the reaction container, and controlling the temperature of the reaction container to be 20-30 ℃ and carrying out catalytic washing for one time. Concentrating under reduced pressure at 20-30 ℃, purifying by column chromatography, eluting with dichloromethane/ethyl acetate=3/1-2/1, concentrating under reduced pressure at 20-30 ℃ to obtain 129.6mmol (112.2 g) of compound 4, and obtaining 86.3% yield.
In the application, the technical route for synthesizing the medical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite is shown as a formula I,
in the application, the synthesis mechanism of the pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite is shown as a formula II,
diisopropylammonium salt tetrazole (compound 3) with lone pair of electrons nitrogen attacks bis (diisopropylamino) (2-cyanoethoxy) phosphine (compound 2), followed by stripping off a part of diisopropylamine to form an intermediate, followed by 5'-O-DMT-2' -O-propynyl-guanosine (compound 1) with 3 '-hydroxy oxygen, and stripping off tetrazole to give DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite (compound 4).
In this application: 5'-O-DMT-2' -O-propynyl-guanosine is an oligonucleotide starting material, which is one of the main reactants in this application, bis (diisopropylamino) (2-cyanoethoxy) phosphine is another reaction starting material, which is used to introduce diisopropylamino groups, diisopropylammonium tetrazole is used as a catalyst to promote the reaction of 5'-O-DMT-2' -O-propynyl-guanosine and bis (diisopropylamino) (2-cyanoethoxy) phosphine, and methylene chloride is used as an organic solvent to dissolve and dilute the reactants, allowing them to be well mixed and react.
In order to verify that the technical solution of the present application has excellent effects, the following examples are also provided.
Example 1
The specific synthetic procedure for DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite is as follows:
1L of methylene dichloride is added into a reaction vessel, nitrogen is continuously introduced into the reaction vessel, the reaction vessel is placed under the protection of nitrogen atmosphere, 150.2mmol (100 g) of 5'-O-DMT-2' -O-propynyl-guanosine (compound 1) is added into the reaction vessel, 449.6mmol (77 g-78 g) of diisopropylammonium tetrazole (compound 3) is added into the reaction vessel, 298.6mmol (90-91 g) of bis (diisopropylamino) (2-cyanoethoxy) phosphine (compound 2) is added into the reaction vessel, the reaction vessel is stabilized at 0 ℃ and stirred for 45 minutes, the reaction vessel is adjusted to 25 ℃ and stirred for 13 hours, the progress of the reaction is controlled in HPLC, and the reaction is completed. 1L of water is added into the reaction vessel, the reaction vessel is controlled at 25 ℃ to be washed for three times, and then 1L of saturated sodium chloride aqueous solution is added into the reaction vessel, the reaction vessel is controlled at 25 ℃ to be washed for one time. Concentrating under reduced pressure at 25 ℃, purifying by column chromatography, eluting with dichloromethane/ethyl acetate=3/1-2/1, concentrating under reduced pressure at 25 ℃ to obtain 129.6mmol (112.2 g) of compound 4 in 86.3% yield.
The nuclear magnetic hydrogen spectrum of the pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite prepared in the embodiment is shown in figure 1, the nuclear magnetic phosphorus spectrum is shown in figure 2, and the HPLC purity spectrum is shown in figure 3.
For DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite prepared in this example 1 H NMR， 31 P NMR and HPLC purity profile analysis is as follows:
1 H NMR(400MHz,DMSO-d6):12.07(s,1H),11.63(s,1H),8.10(s,2H),7.34-7.38(m,4H),7.21-7.30(m,14H),6.82-6.87(m,8H),5.91-5.96(m,2H),4.76-4.78(m,1H),4.50-4.51(m,1H),4.16-4.34(m,4H),3.78-3.81(m,1H),3.72-3.73(m,2H),3.73(s,6H),3.72(s,6H),3.54-3.66(m,6H),3.44-3.46(m,2H),3.24-3.30(m,5H),2.77-2.80(m,1H),2.59-2.62(m,2H),2.18(s,6H),1.17-1.20(m,1H),1.12-1.15(m,21H),0.99-1.01(m,5H)。
31 P NMR(162MHz,DMSO):δ149.98,149.74
HPLC purity analysis for DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite prepared in this example is shown in FIG. 3, and HPLC purity is greater than 95%.
The sources of the raw materials used in this example are specifically as follows:
dichloromethane is a product with a lot number of 20230721 produced by tin-free crystal chemical industry Co., ltd, 5'-O-DMT-2' -O-propynyl-guanosine is a product with a lot number of NVC0033-55 produced by Noveukang Biotechnology Co., ltd, diisopropylammonium salt tetrazole is a product with a lot number of 230433 produced by Noveukang Biotechnology Co., ltd, and bis (diisopropylamino) (2-cyanoethoxy) phosphine is a product with a lot number of RV1361230412-RP172 produced by Runyu New Material Co., ltd; the sodium chloride adopts a product with a lot number of 20230208 produced by Jiangsu Qiangsheng functional chemistry Co., ltd; the ethyl acetate is a product with a lot number of 20230421 produced by Shanghai Lingfeng chemical reagent Co.
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 pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite characterized by the following structural formula:
2. a method for synthesizing a pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite according to claim 1, comprising the steps of:
adding a first organic solvent into a reaction container, adding 5'-O-DMT-2' -O-propynyl-guanosine into the reaction container at the temperature of minus 5 ℃ to 5 ℃, continuously introducing inert gas into the reaction container, adding diisopropylammonium tetrazole into the reaction container under the atmosphere of the inert gas, adding bis (diisopropylamino) (2-cyanoethoxy) phosphine into the reaction container, controlling the temperature of the reaction container at the temperature of minus 5 ℃ to 5 ℃, stirring for a first preset time period, controlling the temperature of the reaction container at the temperature of 20 ℃ to 30 ℃, stirring for a second preset time period, washing, concentrating under reduced pressure, purifying by column chromatography, and concentrating under reduced pressure to obtain a medical intermediate.
3. The method for synthesizing pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite according to claim 2, wherein the molar ratio of said 5' -O-DMT-2' -O-propynyl-guanosine to said diisopropylammonium tetrazole is 1:2-1:4.
4. The method for synthesizing pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite according to claim 2, wherein the molar ratio of said 5' -O-DMT-2' -O-propynyl-guanosine to said bis (diisopropylamino) (2-cyanoethoxy) phosphine is 1:1 to 1:3.
5. The method for synthesizing a pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite according to claim 2, wherein the first organic solvent is dichloromethane.
6. The method for synthesizing pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite according to claim 5, wherein the molar volume ratio of said 5' -O-DMT-2' -O-propynyl-guanosine to said dichloromethane is 0.1mol/L to 0.2mol/L.
7. The method for synthesizing pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite according to claim 2, wherein said inert gas is any one of nitrogen, argon, neon and helium.
8. The method for synthesizing pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite according to claim 2, wherein said washing comprises a specific step of washing with water followed by a washing with saturated sodium chloride solution.
9. The method for synthesizing pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite according to claim 8, wherein the washing temperature is 20-30 ℃, and the washing temperature of the saturated sodium chloride solution is 20-30 ℃.
10. The method for synthesizing pharmaceutical intermediate DMT-2' -O-propynyl-G (Ac) -CE-phosphoramidite according to claim 2, wherein the first reduced pressure concentration is at a temperature of 20 ℃ to 30 ℃ and the second reduced pressure concentration is at a temperature of 20 ℃ to 30 ℃.
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