Synthetic method of RNA monomer intermediate 2 '-fluoro-nucleoside modifier

Abstract

The invention provides an RNA monomer intermediate 2 '-fluoro-nucleoside modifier synthesis method, which comprises: carrying out an oxidation reaction on a compound represented by a structural formula I to obtain a compound represented by a structural formula II; performing fluorine substitution reaction on the compound shown in the structural formula II to obtain a compound shown in a structural formula III; and carrying out reduction reaction on the compound as shown in the structural formula III to obtain a compound as shown in a structural formula IV, namely the RNA monomer intermediate 2 '-fluoro-nucleoside modifier. According to the synthetic method of the RNA monomer intermediate 2 '-fluoro-nucleoside modifier, the raw materials are easy to obtain, the cost is low, the route is simple, operation is convenient, and the synthetic method is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceutical intermediates, and in particular relates to a method for synthesizing a 2'-fluoro-nucleoside modified RNA monomer intermediate. Background Art

[0002] Small nucleoside drugs have the characteristics of wide drug targets, long-lasting efficacy, and short R&D cycle. They are considered to be the third wave of pharmaceutical manufacturing after small molecule drugs and recombinant protein / antibody drugs. Research on small nucleic acid drugs has attracted more attention. According to data from 2022, the global market size of 15 listed small nucleic acid drugs reached US$3.784 billion, a year-on-year increase of 14%. It is estimated that by 2025, global sales of small nucleic acid drugs will exceed US$10 billion, with a compound growth rate of 66%.

[0003] At present, there is a huge demand for nucleoside modified monomers, raw materials for small nucleoside drugs. As one of the four common nucleoside modified compounds, the existing synthesis methods of 2′-fluoro-5′-DMTr nucleoside modified compounds are too long and the key step purification process is complicated. There is an urgent need for a new synthesis process with easy-to-obtain raw materials and simplified process.

[0004] For example, the patent publication US2004 / 29274 reports the following synthetic route of 2′-fluoro-5′-DMTr nucleoside modification:

[0005]

[0006] The limitation of this method is that the steps are too long. The double silicon protective group in the first step is not cheap. The subsequent protection and deprotection of too many protective groups are involved, which is costly and difficult to scale up the process.

[0007] Publication CN115725676A reports the use of an enzymatic method to synthesize the key intermediate 2′-fluorodeoxyadenosine, the route is as follows:

[0008]

[0009] Although this method can obtain 2′-fluoro-5′-DMTr nucleoside modification by protecting the intermediate 2′-fluorodeoxyadenosine through amino protection and 5′-DMTr protection in two steps, since its key step is enzyme catalysis, this step of the reaction is currently still in the laboratory stage, and there are still many problems in the synthesis efficiency and post-processing purification. Summary of the invention

[0010] Based on the above technical problems, the present invention provides a method for synthesizing an RNA monomer intermediate 2′-fluoro-nucleoside modification. The synthetic method has not only readily available raw materials and low cost, but also a simple route and convenient operation, and is suitable for industrial production.

[0011] The present invention provides a method for synthesizing a 2′-fluoro-nucleoside modified RNA monomer intermediate, comprising the following steps:

[0012] S1, subjecting the compound represented by structural formula I to oxidation reaction to obtain the compound represented by structural formula II;

[0013] S2, subjecting the compound represented by structural formula II to fluorine substitution reaction to obtain a compound represented by structural formula III;

[0014] S3, subjecting the compound represented by structural formula III to a reduction reaction to obtain a compound represented by structural formula IV, which is the RNA monomer intermediate 2′-fluoro-nucleoside modification;

[0015]

[0016] Wherein, P is a hydroxyl protecting group on the sugar group, and B is a naturally occurring or modified nucleoside base.

[0017] Preferably, P is 4,4′-dimethoxytrityl, the structural formula of which is shown below:

[0018]

[0019] Preferably, B is a modified adenine base or guanine base, and its structural formula is as follows:

[0020]

[0021] Preferably, in step S1, the oxidation reaction is carried out under the reaction conditions of an oxidant, a catalyst, a phase transfer catalyst, a base and a solvent;

[0022] Preferably, the oxidant is at least one of sodium hypochlorite, calcium hypochlorite, trichloroisocyanuric acid, dichlorohydantoin or Dess-Martin reagent;

[0023] Preferably, the catalyst is at least one of 2,2,6,6-tetramethylpiperidinyl oxide, 4-amino-2,2,6,6-tetramethylpiperidinyl oxide or 4-hydroxy-2,2,6,6-tetramethylpiperidinyl oxide;

[0024] Preferably, the phase transfer catalyst is at least one of tetrabutylammonium bromide, tetramethylammonium bromide, tetramethylammonium chloride or tetraethylammonium chloride;

[0025] Preferably, the base is at least one of sodium bicarbonate, potassium bicarbonate, sodium phosphate, potassium phosphate, sodium acetate or potassium acetate;

[0026] Preferably, the solvent is at least one of toluene, dichloromethane, ethyl acetate or dichloroethane;

[0027] Preferably, the oxidation reaction temperature is 0-20°C and the time is 0.5-2h.

[0028] Preferably, the amount of the oxidant used is 1-1.5 times the molar amount of the compound shown in structural formula I; the amount of the catalyst used is 1-30% of the molar amount of the compound shown in structural formula I; the amount of the phase transfer catalyst used is 5-20% of the molar amount of the compound shown in structural formula I; the amount of the base used is 1-2 times the molar amount of the compound shown in structural formula I.

[0029] Preferably, in step S2, the fluorine substitution reaction is carried out under the reaction conditions of a fluorine substitution reagent, a chiral catalyst, a base and a solvent;

[0030] Preferably, the fluorine substitution reagent is at least one of N-fluorobisbenzenesulfonamide, 1-chloromethyl-4-fluoro-1,4-diazotized bicyclo-2.2.2-octane bis(tetrafluoroborate), N-fluoropyridine trifluoromethanesulfonate or 1-fluoro-4-hydroxy-1,4-diazotized bicyclo-2.2.2-octane bis(tetrafluoroborate);

[0031] Preferably, the chiral catalyst is a chiral amino acid ester or a chiral amino acid ester salt, preferably at least one of L-isoleucine methyl ester hydrochloride, L-valine methyl ester hydrochloride, L-phenylalanine tert-butyl ester hydrochloride, L-phenylalanine ethyl ester hydrochloride, L-phenylalanine methyl ester hydrochloride or L-leucine methyl ester hydrochloride;

[0032] Preferably, the base is at least one of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate or diammonium hydrogen phosphate;

[0033] Preferably, the solvent is at least one of tetrahydrofuran, 1,4-dioxane or acetonitrile;

[0034] Preferably, the temperature of the fluorine substitution reaction is 0-20°C and the time is 24-48h.

[0035] Preferably, the amount of the fluorine substitution agent is 1-1.5 times the molar amount of the compound shown in formula II; the amount of the chiral catalyst is 10-30% of the molar amount of the compound shown in formula II; the amount of the base is 1-3 times the molar amount of the compound shown in formula II.

[0036] Preferably, in step S3, the reduction reaction is carried out under the reaction conditions of a chiral catalyst, a reducing agent and a solvent;

[0037] Preferably, the chiral catalyst is (S)-2-methyl-CBS-oxazaborolidine;

[0038] Preferably, the reducing agent is at least one of borane tetrahydrofuran, sodium borohydride, sodium acetate borohydride or sodium cyanoborohydride;

[0039] Preferably, the solvent is at least one of methanol, ethanol, isopropanol or tetrahydrofuran;

[0040] Preferably, the reduction reaction is carried out at a temperature of 10-30°C and a time of 2-6 hours.

[0041] Preferably, the 2′-fluoro-nucleoside modification is a 2′-fluoro-5′-DMTr adenine nucleoside modification, and its synthesis route is as follows:

[0042]

[0043] Preferably, the 2′-fluoro-nucleoside modification is a 2′-fluoro-5′-DMTr guanine nucleoside modification, and its synthesis route is as follows:

[0044]

[0045] The present invention proposes a method for synthesizing a RNA monomer intermediate 2′-fluoro-nucleoside modification, which uses readily available non-fluorinated nucleoside modifications as starting materials, and efficiently synthesizes a RNA monomer intermediate—2′-fluoro-5′-DMTr nucleoside modification after oxidation, fluorine substitution, and reduction reactions. Compared with other synthesis methods in the prior art, the present invention can efficiently obtain 2′-fluoro-5′-DMTr nucleoside modification on the one hand; on the other hand, the entire synthesis steps are streamlined, the conditions are mild, the product yield is high, and it can be extended to industrial production. DETAILED DESCRIPTION

[0046] Hereinafter, the present invention will describe the technical solution in detail through specific embodiments, but it should be clearly stated that these embodiments are for illustration only and are not to be construed as limiting the scope of the present invention.

[0047] Example 1

[0048] This example provides a method for synthesizing an RNA monomer intermediate 2′-fluoro-5′-DMTr adenine nucleoside modification, and its synthesis route and specific synthesis method are as follows:

[0049]

[0050] (1) Compound A1 (30 g, 1 eq) and DCM (150 mL, 5 v) were added to a 500 mL three-necked flask, followed by water (100 mL), sodium bicarbonate (5.8 g, 1.5 eq), 2,2,6,6-tetramethylpiperidinoxide (360 mg, 5%) and tetrabutylammonium bromide (1.5 g, 0.1 eq), and the mixture was cooled to 0°C under stirring. A 10 wt% sodium hypochlorite solution (40 g, 1.2 eq) was then added dropwise. After the addition was completed for 1 h, the mixture was stirred at 0°C for 0.5 h, and the mixture was allowed to stand for separation. The organic phase was separated and concentrated under reduced pressure. Acetonitrile (150 mL, 5 v) was added, and the mixture was stirred at 0°C for 1 h. The mixture was filtered, and the filter cake was washed with acetonitrile and dried to obtain a white solid (27.1 g), which was Compound A2 (yield 90.3%, purity 98.6%).

[0051] (2) Compound A2 (30 g, 1 eq) was added to a 500 mL three-necked flask, followed by L-isoleucine methyl ester hydrochloride (1.7 g, 0.2 eq), tetrahydrofuran (150 mL, 5 v), water (8.2 g, 10 eq) and sodium bicarbonate (7.6 g, 2 eq). The mixture was cooled to 10°C under stirring, and N-fluorobisbenzenesulfonamide (17.3 g, 1.2 eq) was slowly added in batches. After 150 min of dropwise addition, the mixture was stirred at 10°C for 24 h, water (150 mL) was added, the mixture was concentrated under reduced pressure, filtered, and the filter cake was washed with water. The obtained solid was added to acetonitrile (200 mL), stirred at 0°C for 1 h, filtered, and the filter cake was washed with acetonitrile. After drying, a light yellow solid (26.5 g) was obtained, which was Compound A3 (yield 86.4%, purity 98.3%).

[0052] (3) Compound A3 (30 g, 1 eq) was added to a 500 mL three-necked flask, followed by tetrahydrofuran (150 mL, 5 v) and (S)-2-methyl-CBS-oxazaborolidine (1.2 g, 0.1 eq), and the mixture was stirred and dispersed at 15°C. Borane tetrahydrofuran (4.5 g, 1.2 eq) was added in batches. After 1 h, the mixture was stirred and reacted at 15°C for 2 h. Water (20 mL) was added and stirred for 2 h. Saturated sodium chloride solution (80 mL) was added for separation. The organic phase was concentrated under reduced pressure. Ethanol (150 mL) was added to the obtained solid, and water (75 mL) was slowly added dropwise. The mixture was stirred at room temperature for 2 h, filtered, and dried to obtain Compound A4 (27.7 g, yield 92.4%, purity 99.3%).

[0053] Example 2-15

[0054] The preparation method of reference example 1 is different in that, in Example 2-15, in step (2), the same equivalent of different chiral catalysts, bases and solvents are used to prepare compound A3, and the yield of the obtained compound A3 is calculated. The results are shown in Table 1 below:

[0055] Table 1

[0056]

[0057]

[0058] As can be seen from Table 1 above, referring to Example 5, when the traditional L-phenylalanine methyl ester hydrochloride is used as a chiral catalyst for the fluorine substitution reaction of compound A2, the yield of the obtained compound A3 is poor. This may be because the fluorine substitution site here is the 2-position on the sugar group, and L-isoleucine methyl ester hydrochloride and the like increase the selectivity of steric hindrance. For example, when the chiral amino acid ester salts described in Examples 1-3 are used as chiral catalysts for the fluorine substitution reaction of compound A2, the yield of the obtained compound A3 is better. When the chiral amino acid ester salts described in other Examples 4-6 are used instead of Example 1 as the chiral catalyst, the yield of compound A3 will be reduced to varying degrees; in addition, referring to Examples 7-11, it can be seen that different bases will have some effect on the yield of compound A3, but the effect is not significant; referring to Examples 12-15, it can be seen that when the solvents of 1,4-dioxane, acetonitrile, dichloromethane, and methanol are used instead of tetrahydrofuran, the yield of compound A3 will be reduced to varying degrees.

[0059] Example 16

[0060] This example provides a method for synthesizing an RNA monomer intermediate 2′-fluoro-5′-DMTr guanine nucleoside modification, and its synthesis route and specific synthesis method are as follows:

[0061]

[0062] (1) Compound G1 (30 g, 1 eq) and DCM (150 mL, 5 v) were added to a 500 mL three-necked flask, followed by water (100 mL), sodium bicarbonate (5.9 g, 1.5 eq), 2,2,6,6-tetramethylpiperidinoxide (370 mg, 5%) and tetrabutylammonium bromide (1.5 g, 0.1 eq), and the mixture was cooled to 10° C. under stirring, and a 10 wt % sodium hypochlorite solution (41 g, 1.2 eq) was added dropwise. After the addition was completed for 1 h, the mixture was stirred at 10° C. for 0.5 h, and the mixture was allowed to stand for separation. The organic phase was separated and concentrated under reduced pressure, acetonitrile (150 mL, 5 v) was added, and the mixture was stirred at 10° C. for 1 h. The mixture was filtered, and the filter cake was washed with acetonitrile and dried to obtain a white solid (25.9 g), which was compound G2 (yield 86.2%, purity 98.1%).

[0063] (2) Compound G2 (30 g, 1 eq) was added to a 500 mL three-necked flask, followed by L-isoleucine methyl ester hydrochloride (1.7 g, 0.2 eq), tetrahydrofuran (150 mL, 5 v), water (8.4 g, 10 eq), and diammonium hydrogen phosphate (12.4 g, 2 eq). The mixture was cooled to 15° C. under stirring, and N-fluorobisbenzenesulfonamide (17.3 g, 1.2 eq) was slowly added in batches. After the addition was completed for 150 min, the mixture was stirred at 15° C. for 24 h, water (150 mL) was added, the mixture was concentrated under reduced pressure, filtered, and the filter cake was washed with water. The obtained solid was added to acetonitrile (200 mL), stirred at 5° C. for 1 h, filtered, and the filter cake was washed with acetonitrile. After drying, a light yellow solid (26.9 g) was obtained, which was compound G3 (yield 87.3%, purity 97.9%).

[0064] (3) Compound G3 (30 g, 1 eq) was added to a 500 mL three-necked flask, followed by tetrahydrofuran (150 mL, 5 v) and (S)-2-methyl-CBS-oxazaborolidine (1.2 g, 0.1 eq), and the mixture was stirred and dispersed at 10°C. Borane tetrahydrofuran (4.6 g, 1.2 eq) was added in batches. After 1 h of addition, the mixture was stirred and reacted at 10°C for 2 h. Water (20 mL) was added and stirred for 2 h. Saturated sodium chloride solution (80 mL) was added for separation. The organic phase was concentrated under reduced pressure. Ethanol (150 mL) was added to the obtained solid, and water (75 mL) was slowly added dropwise. The mixture was stirred at room temperature for 2 h, filtered, and dried to obtain compound G4 (26.9 g, yield 89.7%, purity 99.0%).

[0065] Examples 17-30

[0066] The preparation method of reference example 16 is different in that, in Example 17-30, in step (2), the same equivalent of different chiral catalysts, bases and solvents are used to prepare compound G3, and the yield of the obtained compound G3 is calculated. The results are shown in Table 2 below:

[0067] Table 2

[0068]

[0069]

[0070] As can be seen from Table 2 above, referring to Example 20, when the traditional L-phenylalanine methyl ester hydrochloride is used as a chiral catalyst for the fluorine substitution reaction of compound G2, the yield of the obtained compound G3 is poor. This may be because the fluorine substitution site here is the 2-position on the sugar group, and L-isoleucine methyl ester hydrochloride and the like increase the selectivity of steric hindrance. For example, when the chiral amino acid ester salts described in Examples 16-18 are used as chiral catalysts for the fluorine substitution reaction of compound G2, the yield of the obtained compound G3 is better. When the chiral amino acid ester salts described in other Examples 19-21 are used instead of Example 1 as the chiral catalyst, the yield of compound G3 will be reduced to varying degrees; in addition, referring to Examples 22-26, it can be seen that different bases will have some effect on the yield of compound G3, but the effect is not significant; referring to Examples 27-30, it can be seen that when 1,4-dioxane, acetonitrile, dichloromethane, and methanol are used instead of tetrahydrofuran as solvents, the yield of compound G3 will be reduced to varying degrees.

[0071] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for synthesizing a 2'-fluoro-nucleoside modified RNA monomer intermediate, characterized in that: The steps include: S1, subjecting the compound represented by structural formula I to oxidation reaction to obtain the compound represented by structural formula II; S2, subjecting the compound represented by structural formula II to fluorine substitution reaction to obtain a compound represented by structural formula III; S3, subjecting the compound represented by structural formula III to a reduction reaction to obtain a compound represented by structural formula IV, which is the RNA monomer intermediate 2'-fluoro-nucleoside modification; Wherein, P is a hydroxyl protecting group on the sugar group, and B is a natural or modified nucleoside base.

2. The method for synthesizing the RNA monomer intermediate 2'-fluoro-nucleoside modification according to claim 1, characterized in that: P is 4,4'-dimethoxytrityl, and its structural formula is shown below:

3. The method for synthesizing the RNA monomer intermediate 2'-fluoro-nucleoside modification according to claim 1 or 2, characterized in that: B is a modified adenine base or guanine base, and its structural formula is shown below:

4. The method for synthesizing the RNA monomer intermediate 2'-fluoro-nucleoside modification according to any one of claims 1 to 3, characterized in that: In step S1, the oxidation reaction is carried out under the reaction conditions of an oxidant, a catalyst, a phase transfer catalyst, a base and a solvent; Preferably, the oxidant is at least one of sodium hypochlorite, calcium hypochlorite, trichloroisocyanuric acid, dichlorohydantoin or Dess-Martin reagent; Preferably, the catalyst is at least one of 2,2,6,6-tetramethylpiperidinyl oxide, 4-amino-2,2,6,6-tetramethylpiperidinyl oxide or 4-hydroxy-2,2,6,6-tetramethylpiperidinyl oxide; Preferably, the phase transfer catalyst is at least one of tetrabutylammonium bromide, tetramethylammonium bromide, tetramethylammonium chloride or tetraethylammonium chloride; Preferably, the base is at least one of sodium bicarbonate, potassium bicarbonate, sodium phosphate, potassium phosphate, sodium acetate or potassium acetate; Preferably, the solvent is at least one of toluene, dichloromethane, ethyl acetate or dichloroethane; Preferably, the oxidation reaction temperature is 0-20°C and the time is 0.5-2h.

5. The method for synthesizing the RNA monomer intermediate 2'-fluoro-nucleoside modification according to claim 4, characterized in that: The amount of the oxidant is 1-1.5 times the molar amount of the compound of formula I; the amount of the catalyst is 1-30% of the molar amount of the compound of formula I; the amount of the phase transfer catalyst is 5-20% of the molar amount of the compound of formula I; The amount of the base used is 1-2 times the molar amount of the compound represented by structural formula I.

6. The method for synthesizing the RNA monomer intermediate 2'-fluoro-nucleoside modification according to any one of claims 1 to 5, characterized in that: In step S2, the fluorine substitution reaction is carried out under the reaction conditions of a fluorine substitution reagent, a chiral catalyst, a base and a solvent; Preferably, the fluorine substitution reagent is at least one of N-fluorobisbenzenesulfonamide, 1-chloromethyl-4-fluoro-1,4-diazotized bicyclo-2.2.2-octane bis(tetrafluoroborate), N-fluoropyridine trifluoromethanesulfonate or 1-fluoro-4-hydroxy-1,4-diazotized bicyclo-2.2.2-octane bis(tetrafluoroborate); Preferably, the chiral catalyst is a chiral amino acid ester or a chiral amino acid ester salt, preferably at least one of L-isoleucine methyl ester hydrochloride, L-valine methyl ester hydrochloride, L-phenylalanine tert-butyl ester hydrochloride, L-phenylalanine ethyl ester hydrochloride, L-phenylalanine methyl ester hydrochloride or L-leucine methyl ester hydrochloride; Preferably, the base is at least one of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate or diammonium hydrogen phosphate; Preferably, the solvent is at least one of tetrahydrofuran, 1,4-dioxane or acetonitrile; Preferably, the temperature of the fluorine substitution reaction is 0-20°C and the time is 24-48h.

7. The method for synthesizing the RNA monomer intermediate 2'-fluoro-nucleoside modification according to claim 6, characterized in that: The amount of the fluorine substitution agent is 1-1.5 times the molar amount of the compound shown in formula II; the amount of the chiral catalyst is 10-30% of the molar amount of the compound shown in formula II; the amount of the base is 1-3 times the molar amount of the compound shown in formula II.

8. The method for synthesizing the RNA monomer intermediate 2'-fluoro-nucleoside modification according to any one of claims 1 to 7, characterized in that: In step S3, the reduction reaction is carried out under the reaction conditions of a chiral catalyst, a reducing agent and a solvent; Preferably, the chiral catalyst is (S)-2-methyl-CBS-oxazaborolidine; Preferably, the reducing agent is at least one of borane tetrahydrofuran, sodium borohydride, sodium acetate borohydride or sodium cyanoborohydride; Preferably, the solvent is at least one of methanol, ethanol, isopropanol or tetrahydrofuran; Preferably, the reduction reaction is carried out at a temperature of 10-30°C and a time of 2-6 hours.

9. The method for synthesizing the RNA monomer intermediate 2'-fluoro-nucleoside modification according to any one of claims 1 to 8, characterized in that: The 2'-fluoro-nucleoside modification is a 2'-fluoro-5'-DMTr adenine nucleoside modification, and its synthesis route is as follows:

10. The method for synthesizing the RNA monomer intermediate 2'-fluoro-nucleoside modification according to any one of claims 1 to 8, characterized in that: The 2'-fluoro-nucleoside modification is a 2'-fluoro-5'-DMTr guanine nucleoside modification, and its synthesis route is as follows:

Images