A method for synthesizing a reverse deoxyribose phosphoramidite monomer and its intermediate compound
Through a simplified synthetic route and cheap raw materials, the problems of expensive raw materials and low yield in the synthesis of reverse deoxyribose residues were solved, and the efficient preparation of reverse deoxyribose phosphoramidite monomers was achieved.
Patent Information
- Application Number
- CN202310592339.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-24
- Publication Date
- 2025-09-30
- Estimated Expiration
- 2043-05-24
AI Technical Summary
The existing reverse deoxyribose residue synthesis method has expensive raw materials, a long synthesis route and low yield.
A simplified synthetic route was adopted, using inexpensive starting materials and specific protecting groups, and the reverse deoxyribose phosphoramidite monomer was synthesized through a series of steps, including the reaction of compound c with hexamethyldisilazane, followed by treatment with palladium on carbon and sodium hydroxide, and finally reaction with cyanoethyl N,N-diisopropylchlorophosphoramidite and dicyanoimidazole.
The method simplifies the synthetic route, reduces the cost of raw materials, improves the product yield, and provides an efficient method for preparing reverse deoxyribose phosphoramidite monomers.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of organic compound synthesis, and in particular to a method for synthesizing a reverse deoxyribose phosphite monomer and an intermediate compound thereof. Background Art
[0002] Nucleic acid drugs refer to nucleic acids themselves or closely related compounds that can be used to treat diseases. These include natural nucleotides and chemically modified nucleotides. They exert their effects by specifically recognizing endogenous nucleic acid sequences through a mechanism of complementary base pairing. In addition to gene therapy, therapeutic nucleic acids can also inhibit the expression of abnormal disease-associated proteins by inhibiting DNA or RNA expression without affecting the expression of other proteins. Compared to antibody drugs, nucleic acid drugs exhibit superior efficacy and safety, and their relatively small molecular weight facilitates mass production by pharmaceutical companies. These characteristics make nucleic acid drugs promising for previously difficult-to-treat cancers and genetic diseases, as well as diseases caused by viral infections such as influenza.
[0003] Inverted abasic residues (invAbs) are commonly added to the 3' or 5' ends of nucleic acid chains to enhance the activity of RNAi reagents. Inverted deoxyribose residues can be prepared by reacting inverted deoxyribose phosphate monomers with the 3' or 5' ends of RNAi. Currently, few methods for synthesizing inverted deoxyribose residues have been reported. Existing methods use expensive raw materials, long synthetic routes, and low yields (NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, Vol. 22, Nos. 5–8, pp. 1305–1307, 2003). Therefore, the development of new synthetic methods for preparing inverted deoxyribose residues is necessary.
[0004] In order to overcome the shortcomings of the prior art, the present invention provides a method for synthesizing a reverse deoxyribose phosphite monomer. The method has a short synthesis route, low raw material prices and good product yield. Summary of the Invention
[0005] The present invention provides an intermediate compound for synthesizing a reverse deoxyribose phosphoramidite monomer, or a stereoisomer thereof, having a structure shown in Formula I:
[0006]
[0007] in,
[0008] PG 1 is selected from silylhydroxy protecting groups, acyl protecting groups, and alkoxyalkyl protecting groups;
[0009] PG 2 is selected from triphenylmethyl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl.
[0010] Furthermore, the compound represented by formula I is represented by formula II,
[0011]
[0012] in,
[0013] PG 1 Selected from silyl hydroxy protecting groups; preferably, PG 1 is selected from the group consisting of tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, diethylisopropylsilyl, dimethylisopropylsilyl, di-tert-butylmethylsilyl, diphenylmethylsilyl, trimethylsilylethoxymethyl and trimethylsilylethyl; more preferably, PG 1 Selected from tert-butyldimethylsilyl;
[0014] PG 2 Selected from triphenylmethyl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl; preferably, PG 2 Selected from 4,4'-dimethoxytriphenyl group.
[0015] In some embodiments of the present invention, the compound represented by Formula I or Formula II is specifically:
[0016]
[0017] The present invention also provides a method for synthesizing compound d-1, comprising step 1:
[0018]
[0019] Step 1: Compound c is mixed with hexamethyldisilazane, and ammonium sulfate is added to react to obtain compound d-1;
[0020] Among them, R 1 Selected from
[0021] The present invention also provides a method for synthesizing a reverse deoxyribose phosphoramidite monomer using compound d-1, comprising steps 2 to 4:
[0022]
[0023] Step 2: Dissolve compound d-1 in ethyl acetate and ethanol, add palladium carbon, and stir the mixture at room temperature to obtain compound e-1;
[0024] Step 3: Dissolve compound e-1 in ethanol and water, add sodium hydroxide, and stir at 60-100°C for 5-24 hours to obtain compound f-1;
[0025] Step 4: Dissolve compound f-1 in dichloromethane, add 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and dicyanoimidazole, and stir at room temperature for 0.5 to 6 hours to obtain a reverse deoxyribose phosphoramidite monomer.
[0026] The present invention also provides a method for synthesizing a reverse deoxyribose phosphoramidite monomer, comprising step A, step B, and steps 2 to 4:
[0027]
[0028] Step A: Dissolve compound a in pyridine, add tert-butyldimethylsilyl chloride, and stir at room temperature for 5 to 24 hours to obtain compound b;
[0029] Step B: Dissolve compound b in pyridine, add DMTrCl, and react with stirring at 30-80°C for 8-24 hours to obtain compound c;
[0030] Step 1: Compound c is mixed with hexamethyldisilazane, and ammonium sulfate is added to react to obtain compound d-1;
[0031] Step 2: Dissolve compound d-1 in ethyl acetate and ethanol, add palladium carbon, and stir the mixture at room temperature to obtain compound e-1;
[0032] Step 3: Dissolve compound e-1 in ethanol and water, add sodium hydroxide, and stir at 60-100°C for 5-24 hours to obtain compound f-1;
[0033] Step 4: Dissolve compound f-1 in dichloromethane, add 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and dicyanoimidazole, and stir at room temperature for 0.5 to 6 hours to obtain a reverse deoxyribose phosphoramidite monomer;
[0034] Among them, R 1 Selected from
[0035] Furthermore, the reaction temperature in step 1 is 60-150°C; preferably, the reaction temperature in step 1 is 80-140°C; more preferably, the reaction temperature in step 1 is 80°C, 90°C, 100°C, 110°C, 120°C, 130°C, or 140°C.
[0036] Furthermore, the molar equivalent ratio of compound c to ammonium sulfate in step 1 is 1:1 to 3; preferably, the molar equivalent ratio of compound c to ammonium sulfate in step 1 is 1:1 to 2; more preferably, the molar equivalent ratio of compound c to ammonium sulfate in step 1 is 1:2.
[0037] Furthermore, the reaction time in step 1 is 1 to 5 hours; preferably, the reaction time in step 1 is 1 hour, 1.5 hours, 2 hours, or 2.5 hours.
[0038] Furthermore, the palladium carbon in step 2 is 10% w / w palladium carbon.
[0039] Furthermore, in step 2, the mass ratio of compound d-1 to palladium carbon is 1:0.1 to 0.2; preferably, in step 2, the mass ratio of compound d-1 to palladium carbon is 1:0.1.
[0040] Furthermore, the reaction time in step 2 is 5 to 24 hours; preferably, the reaction time in step 2 is 8 to 18 hours; more preferably, the reaction time is 10 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours.
[0041] Furthermore, in step 3, the molar equivalent ratio of e-1 to sodium hydroxide is 1:3 to 8; preferably, in step 3, the molar equivalent ratio of compound e-1 to sodium hydroxide is 1:5 to 7.
[0042] Furthermore, the reaction temperature in step 3 is 80-100°C; preferably, the reaction temperature is 80°C, 85°C, 90°C, 95°C, or 100°C.
[0043] Furthermore, the reaction time in step 3 is 8 to 18 hours. Preferably, the reaction time is 10 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours.
[0044] Furthermore, in step 4, the molar equivalent ratio of f-1 to 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and dicyanoimidazole is 1:1-1.5:0.5-1.
[0045] Furthermore, the reaction time in step 4 is 0.5 to 3 hours. Preferably, the reaction time is 0.5 hour, 1 hour, 1.5 hours, 2 hours, or 2.5 hours.
[0046] Furthermore, the molar equivalent ratio of compound a to tert-butyldimethylchlorosilane in step A is 1:1 to 2; preferably, the molar equivalent ratio of compound a to tert-butyldimethylchlorosilane in step A is 1:1 to 1.5.
[0047] Furthermore, the reaction time in step A is 8 to 18 hours; preferably, the reaction time is 10 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours.
[0048] Furthermore, the molar equivalent ratio of compound b to DMTrCl in step B is 1:1 to 2; preferably, the molar equivalent ratio of compound b to DMTrCl in step B is 1:1 to 1.5.
[0049] Furthermore, the reaction time in step B is 10 to 18 hours; preferably, the reaction time is 10 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours.
[0050] Furthermore, the reaction temperature in step B is 40-60°C; preferably, the reaction temperature is 40°C, 45°C, 50°C, 55°C, or 60°C.
[0051] In the present invention, the term "hydroxyl protecting group" means a group known to those skilled in the art that can prevent a hydroxyl group from undergoing chemical reactions, including a silyl protecting group (such as tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, diethylisopropylsilyl, dimethylisopropylsilyl, di-tert-butylmethylsilyl, diphenylmethylsilyl, trimethylsilylethoxymethyl and trimethylsilylethyl), an alkoxyalkyl protecting group (such as methoxymethyl, 2-methoxyethoxymethyl, 2,2,2-trichloroethoxymethyl, 1-ethoxyethyl, 1-methyl-1-methoxyethyl and tetrahydropyranyl), an acyl protecting group (such as ... protecting groups (e.g., formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, fluoroacetyl, difluoroacetyl, trifluoroacetyl, bromoacetyl, tribromoacetyl, methoxyacetyl, pivaloyl and benzoyl), alkoxycarbonyl-type protecting groups (e.g., methoxycarbonyl, 2,2,2-trichloroethoxycarbonyl and benzyloxycarbonyl), benzyl-type protecting groups (e.g., benzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,5-dimethoxybenzyl, 2,3,4-trimethoxybenzyl, 3,4,5-trimethoxybenzyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 2,6-dichlorobenzyl, 4-cyanobenzyl, diphenylmethyl, triphenylmethyl and 4,4'-dimethoxytrityl).
[0052] In the present invention, DMTr represents 4,4'-dimethoxytriphenyl group.
[0053] The term "inverted abasic residue" is also referred to as an inverted abasic site. The inverted abasic residue may be linked via phosphate, phosphorothioate or other internucleoside linkages.
[0054] The synthetic method of the reverse deoxyribose phosphoramidite monomer of the present invention has a short route, low raw material price and good product yield.
[0055] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.
[0056] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0057] Figure 1 is the H NMR spectrum of compound d-1;
[0058] Figure 2 is the H NMR spectrum of compound e-1;
[0059] Figure 3 is the H NMR spectrum of compound f-1;
[0060] Figure 4 is the H NMR spectrum of compound g-1. Specific implementation plan
[0061] In order to make the purpose, technical solutions and advantages of the present invention clearer, preferred embodiments of the present invention will be described in detail below.
[0062] The abbreviations used are as follows: DMTrCl: 4,4'-bismethoxytrityl chloride; HMDS: hexamethyldisilazane.
[0063] Room temperature refers to 20℃~30℃.
[0064] Example 1
[0065] Synthesis of compound b-1:
[0066]
[0067] 2'-Deoxythymidine (a-1, 20 g, 82.6 mmol) was dissolved in pyridine (100 mL) under ice bath, tert-butyldimethylsilyl chloride (13.6 g, 90.9 mmol) was added, and the mixture was stirred at room temperature for 16 hours. Pyridine was evaporated under reduced pressure, and the mixture was cooled to room temperature. The solid was dissolved in ethyl acetate (200 mL), washed three times with water (200 mL), ethyl acetate was evaporated under reduced pressure, and the mixture was cooled to room temperature. Petroleum ether (400 mL) was added to the solid, and the mixture was stirred at room temperature for 4 hours. The mixture was filtered and the solid was dried to obtain compound b-1 (28 g, 78.7 mmol, yield 96%).
[0068] Synthesis of compound c-1:
[0069]
[0070] Compound b-1 (18 g, 50.6 mmol) was dissolved in pyridine (90 mL) at room temperature, and DMTrCl (20.5 g, 60.7 mmol) was added. The mixture was stirred at 50 ° C for 16 hours, and pyridine was evaporated under reduced pressure. The mixture was cooled to room temperature, and the solid was dissolved in dichloromethane (200 mL). The solid was washed three times with water (200 mL), and the dichloromethane was evaporated under reduced pressure. The product was purified by column chromatography (PE / EA = 5 / 1) to give compound c-1 (26.4 g, 40.1 mmol, yield 79%).
[0071] Synthesis of compound d-1:
[0072]
[0073] Compound c-1 (658 mg, 1 mmol, 79% yield) was mixed with HMDS (4 mL) at room temperature, and ammonium sulfate (264 mg, 2 mmol) was added. The mixture was stirred at 140 °C for 1.5 hours, cooled to room temperature, and the solvent was evaporated under reduced pressure. The mixture was purified by column chromatography to obtain compound d-1 (442 mg, 0.83 mmol, 83% yield).
[0074] Synthesis of compound e-1:
[0075]
[0076] Compound d-1 (13 g, 24.3 mmol) was dissolved in ethyl acetate (100 mL) and ethanol (100 mL) at room temperature, and 10% w / w Pd / C (1.3 g) was added. The mixture was stirred at room temperature for 16 hours, filtered, and the solvent was evaporated under reduced pressure to obtain compound e-1 (12.5 g, 23.4 mmol, yield 96%).
[0077] Synthesis of compound f-1:
[0078]
[0079] Compound e-1 was dissolved in ethanol (80 mL) and water (80 mL) at room temperature, sodium hydroxide (13.6 g, 160 mmol) was added, and the mixture was stirred at 90 ° C for 16 hours. The solvent was evaporated under reduced pressure and cooled to room temperature. The solid was dissolved in dichloromethane (100 mL), washed three times with water (200 mL), and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (PE / EA = 3 / 1) to obtain compound f-1 (7.3 g, 17.4 mmol, yield 74%).
[0080] Synthesis of compound g-1 (reverse deoxyribose phosphoramidite monomer):
[0081]
[0082] Compound f-1 was dissolved in dichloromethane (50 mL) at room temperature, and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (6.86 g, 22.8 mmol) and dicyanoimidazole (1.35 g, 11.4 mmol) were added. The mixture was stirred at room temperature for 1 hour, diluted with dichloromethane (50 mL), washed three times with water (100 mL), and the solvent was evaporated under reduced pressure. The mixture was cooled to room temperature to obtain an oily substance, which was dissolved in dimethyl sulfoxide (20 mL) and purified by reverse phase column (C18) chromatography (water / acetonitrile = 1 / 4) to obtain compound g-1 (4.6 g, 7.4 mmol, yield 64%).
[0083] The above-mentioned embodiments are only preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above-mentioned embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. A compound of formula I, or a stereoisomer thereof: in, PG 1 is selected from silylhydroxy protecting groups, acyl protecting groups, and alkoxyalkyl protecting groups; PG 2 Selected from 4,4'-dimethoxytriphenyl group.
2. The compound according to claim 1, characterized in that: The compound shown in formula I is shown in formula II, in, PG 1 Selected from tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, diethylisopropylsilyl, dimethylisopropylsilyl, di-tert-butylmethylsilyl, diphenylmethylsilyl, trimethylsilylethoxymethyl or trimethylsilylethyl; PG 2 Selected from 4,4'-dimethoxytriphenyl group.
3. The compound according to claim 2, characterized in that: The compound is specifically:
4. A method for synthesizing compound d-1, comprising step 1: Step 1: Compound c is mixed with hexamethyldisilazane, and ammonium sulfate is added to react to obtain compound d-1; in, R 1 Selected from 5. A method for synthesizing a reverse deoxyribose phosphoramidite monomer from compound d-1, comprising steps 2 to 4: Step 2: Dissolve compound d-1 in ethyl acetate and ethanol, add palladium carbon, and stir the mixture at room temperature to obtain compound e-1; Step 3: Dissolve compound e-1 in ethanol and water, add sodium hydroxide, and stir at 60-100°C for 5-24 hours to obtain compound f-1; Step 4: Dissolve compound f-1 in dichloromethane, add 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and dicyanoimidazole, and stir at room temperature for 0.5 to 6 hours to obtain a reverse deoxyribose phosphoramidite monomer.
6. A method for synthesizing a reverse deoxyribose phosphoramidite monomer, comprising step A, step B, and steps 2 to 4: Step A: Dissolve compound a in pyridine, add tert-butyldimethylsilyl chloride, and stir at room temperature for 5 to 24 hours to obtain compound b; Step B: Dissolve compound b in pyridine, add DMTrCl, and stir at 30-80°C for 8-24 hours to obtain compound c; Step 1: Compound c is mixed with hexamethyldisilazane, and ammonium sulfate is added to react to obtain compound d-1; Step 2: Dissolve compound d-1 in ethyl acetate and ethanol, add palladium carbon, and stir the mixture at room temperature to obtain compound e-1; Step 3: Dissolve compound e-1 in ethanol and water, add sodium hydroxide, and stir at 60-100°C for 5-24 hours to obtain compound f-1; Step 4: Dissolve compound f-1 in dichloromethane, add 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and dicyanoimidazole, and stir at room temperature for 0.5 to 6 hours to obtain a reverse deoxyribose phosphoramidite monomer; in, R 1 Selected from 7. The synthesis method according to any one of claims 4 to 6, characterized in that: The reaction temperature in step 1 is 60-150° C.; The molar equivalent ratio of compound c to ammonium sulfate in step 1 is 1:1 to 3; The reaction time in step 1 is 1 to 5 hours.
8. The synthesis method according to claim 5 or 6, characterized in that: In step 2, the mass ratio of compound d-1 to palladium carbon is 1:0.1-0.2; In step 3, the molar equivalent ratio of e-1 to sodium hydroxide is 1:3 to 8; In step 4, the molar equivalent ratio of f-1 to 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and dicyanoimidazole is 1:1-1.5:0.5-1.
9. The synthesis method according to claim 5 or 6, characterized in that: The palladium carbon in step 2 is 10% w / w palladium carbon; The reaction time in step 2 is 5 to 24 hours; The reaction temperature in step 3 is 80-100° C.; The reaction time in step 4 is 0.5 to 3 hours.
10. The synthesis method according to claim 6, characterized in that: In step A, the molar equivalent ratio of compound a to tert-butyldimethylchlorosilane is 1:1-2; The reaction time in step A is 8 to 18 hours; In step B, the molar equivalent ratio of compound b to DMTrCl is 1:1-2; The reaction time in step B is 10 to 18 hours; The reaction temperature in step B is 40-60°C.