Novel small nucleic acid drug intermediate and preparation method thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and relates to a novel small nucleic acid drug intermediate and a preparation method thereof, the intermediate is a 2 '-FANA-dU phosphoramidite monomer, the preparation method comprises the following steps: 1, reacting a compound a with a 33% hydrogen bromide-acetic acid solution in dichloromethane, and performing post-treatment to obtain a compound b; 2, reacting uracil and the like to generate a white solid, and reacting the white solid with the compound b to obtain a compound c; 3, reacting the compound c in methanol and ammonia water, crystallizing and drying to obtain a compound d; 4, the compound d reacts with 4, 4 '-dimethoxytriphenylchloromethane, and a compound e is obtained through treatment; 5, the compound e reacts with anhydrous diisopropyl imidazole and a 2-cyanoethyl-N, N, N ', N'-tetraisopropyl phosphoramidite reagent, and the 2 '-FANA-dU phosphoramidite monomer is obtained.The 2'-FANA-dU phosphoramidite monomer prepared through the method can modify siRNA, the biological activity of the siRNA can be reserved or even enhanced, and the biological stability of the siRNA can be remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology and relates to a novel small nucleic acid drug intermediate and its preparation method. Background Technology

[0002] In recent years, nucleic acid therapy has achieved numerous breakthroughs, becoming a new global investment hotspot and a battleground for biopharmaceutical giants, propelling it into a period of rapid development. Nucleic acid drugs are expected to become the third largest class of drugs after small molecule chemical drugs and antibody drugs. Small nucleic acid drugs utilize the translational or regulatory functions of small nucleic acid molecules as interventions for diseases. Conventional treatments typically induce transient therapeutic effects because they target proteins rather than the root cause. In contrast, small nucleic acid drugs can achieve durable and fundamental therapeutic effects through gene inhibition, addition, substitution, or editing.

[0003] Small nucleic acid drugs can be mainly classified into antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), and RNA aptamers. The core of small nucleic acid drug design lies in the precise selection of sequences and strategies for chemical modification. Sequence design is crucial for ensuring drug specificity; it determines whether the drug molecule can effectively and specifically pair with the target mRNA, thereby achieving precise regulation of specific gene expression. Correct sequence selection can maximize drug efficacy while minimizing non-specific binding and potential off-target effects.

[0004] Small nucleic acid drugs, such as siRNA and ASO, are gradually becoming powerful tools for treating a variety of diseases. Through precise gene silencing or expression regulation mechanisms, they provide new therapeutic strategies for modern medicine. Nusinersen and Inclisiran represent the application of ASO and siRNA technologies, respectively. The design and modification strategies of these drugs, such as 2'-O-methylation and phosphate thioester bonds, aim to improve stability, prolong half-life, and reduce immunogenicity, thereby optimizing their therapeutic efficacy and safety.

[0005] Numerous chemical modification methods for RNA have been reported in the literature, mainly categorized into terminal and chemical group modifications of ribonucleic acid, backbone modifications or 2', 4'-position modifications of nucleotides, base modifications, and combinatorial modifications. Phosphothiophosphate modification is a first-generation nucleic acid modification method, producing products with strong resistance to nuclease digestion, but exhibiting significant non-specific toxicity. Alkyl modification of the 2-hydroxyl group of ribose is a second-generation nucleic acid modification method, with important examples being 2'-O-methyl (OMe) and 2'-O-methoxy-ethyl (MOE) RNA. While these products show some performance improvement, non-specific toxicity still exists. Based on this, many different modification methods have been developed, including Peptide nucleic acids (PNAs), N3,-P5,phosphoroamidates (NPs), 2-Deoxy-2,-fluoro-bD-arabinon nucleic acid (FANA), Locked nucleic acid (LNA), Morpholinooligonucleotides (MF), Cyclohexene nucleic acids (CeNA), Tricyclo-DNA (tcDNA), etc., which are called the third generation of nucleic acid modification methods (Xiaolan Chen, et al. Drug discovery today targets, 2005, 10(8):587-593).

[0006] FANA (2,-Deoxy-2,-fluoro-bD-arabinonucleic acid, 2,-deoxy-2'-fluoro-bD-arabinonucleotide) modified RNA refers to RNA analogs formed by replacing ribose with FANA to form the oligonucleotide backbone. Recent literature has confirmed that FANA modification is one of the very few methods that can enhance both the serum half-life and activity of siRNA (Chiu YL, et al. RNA, 2003, 9(9):1034-1048). However, there are currently no reports of siRNA modified by this method being used in tumor treatment. Appropriate modification of siRNA using FANA can preserve or even enhance its biological activity and significantly increase its biological stability, making it a promising siRNA modification method.

[0007] Chinese invention patent CN115960147A [A method for preparing azvudine and its intermediates] discloses a method for constructing a parent nucleus using 1,3,5-tribenzoyl-2-fluoroarabinose as a raw material, through bromination and subsequent SN2 nucleophilic substitution reaction with pyrimidine bases. Chinese invention patent CN103570784A [A method for synthesizing 2'-FANA-dU fluoro oligonucleotides] discloses a method for obtaining 1-bromo-2-deoxy-2-fluoro-3,5-di-O-benzoyl-α-D-arabinose through fluorination and bromination reactions, followed by an SN2 nucleophilic substitution reaction with the corresponding base. N 2. Nucleophilic substitution reaction, followed by hydrolysis to prepare 2'-FANA-dU modified nucleosides. All of the above methods can prepare 2'-FANA-dU modified nucleosides, but neither Patent 1 nor Patent 2 involves a method for preparing the 2'-FANA-dU phosphorous amide monomer. Therefore, this invention combines the process characteristics of Patent 1 and Patent 2 to provide a method for preparing the 2'-FANA-dU phosphorous amide monomer with high reaction efficiency and high product quality. Summary of the Invention

[0008] The purpose of this invention is to provide a novel small nucleic acid drug intermediate and its preparation method, so as to solve the problem of the lack of a preparation method for 2'-FANA-dU phosphorous amide monomer in the prior art.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] An intermediate for a novel small nucleic acid drug, a 2'-FANA-dU phosphoridamide monomer, has the following structure:

[0011]

[0012] A method for preparing a novel small nucleic acid drug intermediate includes the following steps:

[0013] 1.1 Step 1: Compound a, 1-O-benzyl-2-fluoro-3,4-di-O-benzoyl-α-D-arabinose was added to dichloromethane, and a 33% hydrobromide-acetic acid solution was added dropwise at 0-10℃. The reaction was then carried out at 20-25℃ for 6 hours. After the reaction was completed, water was added to quench the reaction, and the phases were separated. The organic phase was extracted three times with 5% sodium bicarbonate solution and then concentrated to dryness under reduced pressure to obtain compound b, 1-bromo-2-fluoro-3,4-di-O-benzoyl-α-D-arabinose.

[0014] 1.2. Step 2: Uracil, hexamethyldisilazane and ammonium sulfate were added to the flask and heated to 125-130℃ for 10 h. The hexamethyldisilazane was distilled off under reduced pressure to obtain a white solid. Toluene was added to the reaction flask, and compound b was added with stirring. The reaction was kept at 55-60℃ for 10 h. After the reaction was completed, the organic phase was extracted twice with 5% sodium bicarbonate solution. The organic phase was concentrated to dryness under reduced pressure to obtain compound c, 1-uracil-2-fluoro-3,4-di-O-benzoyl-α-D-arabinose.

[0015] 1.3 Step 3: Add compound c to the flask, add methanol and ammonia, stir and react at 30-35℃ for 10h. After the reaction is completed, concentrate under reduced pressure until no condensate drips, add ethanol and dichloromethane, stir and crystallize at 20-25℃ for 2h, filter, wash with a small amount of anhydrous ethanol, and dry under vacuum at 50℃ to obtain compound d, 1-uracil-2-fluoro-3,4-di-hydroxy-α-D-arabinose;

[0016] 1.4 Step 4: Add dry pyridine to the flask, add compound d with stirring, add 4,4'-dimethoxytriphenylchloromethane at 2-8℃, keep the reaction at this temperature for 4 hours, after the reaction is complete, add methanol to quench the reaction, concentrate under reduced pressure until no condensate drips, add dichloromethane to dissolve, extract with 5% sodium bicarbonate and water respectively, concentrate under reduced pressure with dichloromethane until solid precipitates, add n-heptane, crystallize at 0-10℃ for 2 hours, filter, wash the filter cake with n-heptane, dry with forced air at 40℃ to obtain compound e, 1-uracil-2-fluoro-3-hydroxy-4-O-DMTr-α-D-arabinose;

[0017] 1.5 Step 5: Add dry dichloromethane to the flask, add compound e with stirring, add anhydrous diisopropylimidazolium, stir at 20-25℃ for 30 min, purge with nitrogen three times, add 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonamide reagent, react at 20-25℃ for 6 h, add water to quench the reaction, allow to stand and separate the layers, extract the organic layer twice with 5% sodium bicarbonate solution, concentrate to dryness under reduced pressure, purify by column chromatography, and dry under vacuum to obtain compound f, 2'-FANA-dU phosphorimamide monomer.

[0018] The beneficial effects of this invention are as follows:

[0019] The method for synthesizing 2'-FANA-dU phosphorus amide monomer provided by this invention combines the process features of existing patents, resulting in a highly efficient reaction and improved product quality. The 2'-FANA-dU phosphorus amide monomer prepared using this method can be used to prepare FANA-modified siRNA. This modification not only retains or even enhances the biological activity of siRNA but also significantly increases its biological stability, providing strong support for the application of siRNA in disease treatment, especially in tumor treatment, and possessing significant clinical and commercial value. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure of compound af, a novel small nucleic acid drug intermediate proposed in this invention. Detailed Implementation

[0021] The present invention will be further illustrated by specific embodiments below, but the scope of protection of the present invention is not limited to the following embodiments.

[0022] The 2'-FANA-dU phosphoridamide monomer was prepared by purchasing 2'-fluoroarabinose as raw material and reacting it with phosphorus reagent through bromination, SN2 nucleophilic substitution, deprotection and addition of a protecting group.

[0023] Example: Please refer to the appendix Figure 1 Synthetic route for 2'-FANA-dU provided by the present invention

[0024] Step 1: Synthesis of compound b

[0025] Under stirring conditions, 100g of compound a was added to 1000mL of dichloromethane. The temperature was controlled at 0-10℃, and 33% hydrogen bromide-acetic acid solution was added dropwise. After the addition was complete, the temperature was controlled at 20-25℃ for 6 hours. After the reaction was completed, 80mL of water was added to quench the reaction, and the layers were separated. The organic phase was extracted three times with 5% sodium bicarbonate solution and then concentrated to dryness under reduced pressure to obtain compound b (oil).

[0026] Step 2: Synthesis of compound c

[0027] Uracil (10.0 g, 89 mmol), hexamethyldisilazane (145.0 g, 900 mmol), and ammonium sulfate (1.17 g, 8.9 mmol) were added to a 500 ml flask. The mixture was heated to 125–130 °C and reacted for 10 h. The hexamethyldisilazane was then concentrated under reduced pressure to obtain a white solid. 200 g of toluene was added to the reaction flask, and compound b (38.1 g, 90 mmol) was added under stirring. The reaction temperature was maintained at 55–60 °C for 10 h. After the reaction was completed, the organic phase was extracted twice with 5% sodium bicarbonate solution. The organic phase was then concentrated to dryness under reduced pressure to obtain compound c.

[0028] Step 3: Synthesis of compound d

[0029] Compound c (8g, 17.6mmol) was added to a 250ml flask, followed by 80ml of methanol and 45ml of ammonia. The mixture was stirred at 30-35℃ for 10 hours. After the reaction was completed, the mixture was concentrated under reduced pressure until no condensate dripped. Then, 60ml of ethanol and 50ml of dichloromethane were added. The mixture was stirred at 20-25℃ for 2 hours to crystallize. The mixture was filtered, washed with a small amount of anhydrous ethanol, and dried under vacuum at 50℃ to obtain compound d.

[0030] Step 4: Synthesis of compound e

[0031] Add 90 ml of dry pyridine to a 500 ml flask, and add compound d (11 g, 44.6 mmol) with stirring. Add 4,4'-dimethoxytriphenylchloromethane (18.2 g, 53.6 mmol) while controlling the temperature at 2–8 °C. Keep the reaction at this temperature for 4 h. After the reaction is complete, add 8 ml of methanol to quench the reaction. Concentrate under reduced pressure until the condensate drips down. Add 120 ml of dichloromethane to dissolve the precipitate. Extract with 100 ml of 5% sodium bicarbonate and 100 ml of water. Concentrate under reduced pressure with dichloromethane until a solid precipitates. Add 150 ml of n-heptane and crystallize at 0–10 °C for 2 h. Filter the mixture, wash the filter cake with n-heptane, and dry it in a forced-air environment at 40 °C to obtain compound e.

[0032] Step 5: Synthesis of compound f

[0033] Add 80 ml of dry dichloromethane to a 250 ml flask, add compound e (9.2 g, 16.7 mmol) with stirring, add anhydrous diisopropylimidazolium (0.98 g, 8.9 mmol), stir for 30 min at 20–25 °C, purge with nitrogen three times, add 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonamide reagent (5.5 g, 18.3 mmol) to the reaction solution, react for 6 h at 20–25 °C, quench the reaction with 20 ml of water, allow to stand and separate the layers, extract the organic layer twice with 5% sodium bicarbonate solution, concentrate to dryness under reduced pressure to obtain an oily substance, purify by column chromatography, and dry under vacuum to obtain compound f.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An intermediate for a novel small nucleic acid drug, characterized in that, It is a 2'-FANA-dU phosphorus amide monomer, with the structure shown below:

2. A method for preparing a novel small nucleic acid drug intermediate, characterized in that, Includes the following steps: 1.1 Step 1: Compound a, 1-O-benzyl-2-fluoro-3,4-di-O-benzoyl-α-D-arabinose was added to dichloromethane, and a 33% hydrobromide-acetic acid solution was added dropwise at 0-10℃. The reaction was then carried out at 20-25℃ for 6 hours. After the reaction was completed, water was added to quench the reaction, and the phases were separated. The organic phase was extracted three times with 5% sodium bicarbonate solution and then concentrated to dryness under reduced pressure to obtain compound b, 1-bromo-2-fluoro-3,4-di-O-benzoyl-α-D-arabinose. 1.

2. Step 2: Uracil, hexamethyldisilazane and ammonium sulfate were added to the flask and heated to 125-130℃ for 10 h. The hexamethyldisilazane was distilled off under reduced pressure to obtain a white solid. Toluene was added to the reaction flask, and compound b was added with stirring. The reaction was kept at 55-60℃ for 10 h. After the reaction was completed, the organic phase was extracted twice with 5% sodium bicarbonate solution. The organic phase was concentrated to dryness under reduced pressure to obtain compound c, 1-uracil-2-fluoro-3,4-di-O-benzoyl-α-D-arabinose. 1.3 Step 3: Add compound c to the flask, add methanol and ammonia, stir and react at 30-35℃ for 10h. After the reaction is completed, concentrate under reduced pressure until no condensate drips, add ethanol and dichloromethane, stir and crystallize at 20-25℃ for 2h, filter, wash with a small amount of anhydrous ethanol, and dry under vacuum at 50℃ to obtain compound d, 1-uracil-2-fluoro-3,4-di-hydroxy-α-D-arabinose; 1.4 Step 4: Add dry pyridine to the flask, add compound d with stirring, add 4,4'-dimethoxytriphenylchloromethane at 2-8℃, keep the reaction at this temperature for 4 hours, after the reaction is complete, add methanol to quench the reaction, concentrate under reduced pressure until no condensate drips, add dichloromethane to dissolve, extract with 5% sodium bicarbonate and water respectively, concentrate under reduced pressure with dichloromethane until solid precipitates, add n-heptane, crystallize at 0-10℃ for 2 hours, filter, wash the filter cake with n-heptane, dry with forced air at 40℃ to obtain compound e, 1-uracil-2-fluoro-3-hydroxy-4-O-DMTr-α-D-arabinose; 1.5 Step 5: Add dry dichloromethane to the flask, add compound e with stirring, add anhydrous diisopropylimidazolium, stir at 20-25℃ for 30 min, purge with nitrogen three times, add 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonamide reagent, react at 20-25℃ for 6 h, add water to quench the reaction, allow to stand and separate the layers, extract the organic layer twice with 5% sodium bicarbonate solution, concentrate to dryness under reduced pressure, purify by column chromatography, and dry under vacuum to obtain compound f, 2'-FANA-dU phosphorimamide monomer.

3. The method for preparing the novel small nucleic acid drug intermediate according to claim 2, characterized in that, In step one, the mass-to-volume ratio of compound a to dichloromethane is 1 g: 10 mL.

4. The method for preparing the novel small nucleic acid drug intermediate according to claim 2, characterized in that, In step two, the ratio of uracil, hexamethyldisilazane and ammonium sulfate is 10.0g:145.0g:1.17g.

5. The method for preparing the novel small nucleic acid drug intermediate according to claim 2, characterized in that, In step three, the ratio of compound c, methanol, and ammonia is 8g:80ml:45ml.

6. The method for preparing the novel small nucleic acid drug intermediate according to claim 2, characterized in that, In step four, the molar ratio of compound d to 4,4'-dimethoxytriphenylchloromethane is 1:1.

2.

7. The method for preparing the novel small nucleic acid drug intermediate according to claim 2, characterized in that, In step five, the molar ratio of compound e, anhydrous diisopropylimidazolium, and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonamide reagent is 1:0.53:1.1.

Images