A method for preparing a locked nucleic acid intermediate

Abstract

The application discloses a synthesis method of a locked nucleic acid intermediate, and discloses an intermediate with a specific structure of formula I, wherein the definitions of the substitutions are as defined in the specification. The application adopts a step-by-step synthesis method, overcomes the defects of many by-products and high production cost in the prior art, has milder reaction conditions, higher reaction yield, and is more suitable for batch production.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry and fine chemical intermediate synthesis technology, specifically to a method for synthesizing oxoheterocyclic compounds. More specifically, it relates to a method for efficiently preparing locked nucleic acid intermediates through stepwise, mild base-promoted ring-cloning and silicon reagent-mediated demethylation reactions. Background Technology

[0002] The core efficacy of antisense oligonucleotides (such as ASO) lies in their specific and high-affinity hybridization with target RNA sequences. While first- and second-generation chemical modifications (such as the PS backbone and 2'-O-MOE) have improved pharmacokinetics, their affinity enhancement is limited. The discovery of locked nucleic acids (LNA) and their derivatives (LNA / cEt) is a breakthrough. Their key feature is the fixation of the 2'-O and 4'-C of furanose through a bridging structure, forming a rigid N-type conformation. This pre-organizes the oligonucleotide backbone, allowing it to bind to RNA without entropy compensation, resulting in a significant leap in binding affinity (Tm value).

[0003] However, this universal affinity enhancement is not uniform across different base sequence backgrounds. Studies have found that guanine residues play a particularly crucial and complex role in LNA / cEt-modified oligonucleotides, with an impact far exceeding that of other bases (A, C, U / T), directly affecting drug efficacy, specificity, and safety. A deep understanding and optimization of G-containing LNA / cEt units is a core technological step in developing efficient and safe nucleic acid drugs.

[0004] Compounds of general formula I are key intermediates in the synthesis of LNA / cEt.

[0005]

[0006] I,

[0007] Wherein, R1 and R2 represent protecting groups of hydroxyl groups, and R3 represents H, CN, C1-C6 alkyl or substituted C1-C6 alkyl.

[0008] Currently, the typical synthesis method for Formula I is the "one-pot method," with the specific reaction process as follows: Under anhydrous and oxygen-free conditions, sodium hydride is added to tetrahydrofuran, followed by the addition of 3-hydroxypropionitrile in an ice bath. The mixture is stirred at room temperature for 1 hour, and the tetrahydrofuran solution of the starting material, added in an ice bath, is then brought to room temperature and stirred for another 16 hours. This process utilizes the extremely strong alkalinity of sodium hydride to simultaneously complete two key transformations: (i) the deprotonation of hydroxypropionitrile to form an oxonium, which then performs a nucleophilic attack on the intramolecular potentiophilic site to achieve ring closure; (ii) the oxonium substitution of the chloride atom on guanine, followed by a β-elimination step under the action of a strong base to obtain the target product.

[0009]

[0010] The existing technical approach has the following drawbacks:

[0011] 1. Extremely high safety risks: Sodium hydride (NaH) is a hazardous material that reacts violently with water and releases hydrogen gas, posing a risk of combustion and explosion. During large-scale production, significant safety hazards exist in its feeding, reaction, quenching, and post-processing stages, placing extremely high demands on equipment, operating procedures, and plant fire safety.

[0012] 2. Stringent reaction conditions: The reaction must be carried out under strict anhydrous and inert gas (such as argon or nitrogen) protection. The dryness requirements for raw materials, solvents and equipment are extremely strict. Any trace amount of moisture will cause reagent failure and cause danger, which increases the complexity and cost of the process.

[0013] 3. Poor chemoselectivity: The strong basicity and high reactivity of NaH often lead to the formation of byproduct 1. These side reactions reduce the yield and selectivity of the main reaction, resulting in decreased product purity and difficulties in subsequent purification.

[0014] 4. Limited functional group tolerance: This strong base system has poor compatibility with many common functional groups (such as ester groups, amides, certain halogens, active hydrogen, etc.), which leads to the formation of byproduct 2. This severely limits its application in the synthesis of complex molecules or substrates with sensitive groups, and the method lacks universality.

[0015] The structures of byproducts 1 and 2 are as follows:

[0016] .

[0017] 5. Difficulty in process control and optimization: Because cyclization and demethylation occur continuously in a single batch, it is difficult to independently monitor and optimize the reaction progress and intermediate quality of each step. If a problem occurs, the entire batch of material may be scrapped, resulting in poor process robustness.

[0018] Therefore, there is an urgent need in this field to develop a new method that is safer to operate, more moderate in conditions, more selective, and more suitable for industrial production. Summary of the Invention

[0019] To address the various shortcomings of existing technologies, this invention provides a method for preparing a compound of formula I, comprising the following steps:

[0020] Step 1) Compound II undergoes a cyclization reaction in the presence of a base to give compound III;

[0021] Step 2) Compound III is reacted in the presence of iodide and alkyl halosilane to prepare compound I;

[0022] Its synthetic route is as follows:

[0023] ,

[0024] in,

[0025] R1 and R2 are independently protecting groups for hydroxyl groups; R3 represents H, CN, C1-C6 alkyl, or substituted C1-C6 alkyl; R4 is a leaving group; and R5 is an acetoxy group (OAc).

[0026] Beneficial effects

[0027] 1. This invention completely eliminates the high-risk sodium hydride, replacing it with stable, inexpensive, and non-flammable potassium carbonate. The trimethylchlorosilane / sodium iodide system is also a conventional reagent with no special hazards. The entire process can be safely carried out in conventional glass or enamel-lined reactors, greatly reducing the production safety threshold and insurance costs.

[0028] 2. The ring-closing step of this invention does not require a strictly anhydrous and oxygen-free environment, significantly reducing the requirements for drying raw materials and solvents; it can even be operated in air. The demethylation step can usually be completed at room temperature or with mild heating. This simplifies the operation and reduces energy consumption and equipment requirements.

[0029] 3. Mild cyclization conditions minimize side reactions from sensitive groups such as nitrile groups. Stepwise reactions allow for independent optimization of each step (e.g., temperature, time, reagent equivalence), resulting in higher cyclization yields and intermediate purity. The trimethylchlorosilane / sodium iodide demethylation system is highly specific and has almost no impact on other functional groups in the molecule. Final product purity (HPLC) can typically be improved by 5-15%, reducing the purification burden.

[0030] 4. This method exhibits good compatibility with common functional groups in substrate molecules, such as esters, amides, halogens, alkenes, and acetals. This makes the method applicable not only to this compound but also to the synthesis of a range of derivatives with similar structures, providing a powerful tool for constructing compound libraries in drug discovery.

[0031] 5. Cost Reduction: The cost of the alkali used, such as potassium carbonate, is far lower than that of sodium hydride; reagent post-processing is simple, reducing waste. Strong Process Controllability: Intermediates can be separated, characterized, and quality controlled, facilitating process analysis (PAT) monitoring and ensuring batch-to-batch process consistency. Simple Operation: Conventional unit operations such as filtration, extraction, and washing can be completed, making it easy to scale up to kilogram-scale or even larger production. Improved Overall Yield: Although divided into two steps, the high efficiency and selectivity of each step often result in an overall yield that is superior to or at least equal to that of the original one-step method, with better product quality. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated components or steps, without excluding other substances or steps.

[0033] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments.

[0034] Those skilled in the art will understand that the present invention can be practiced even without certain specific details. In some embodiments, materials, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0035] In this invention, "halogenated group" refers to "halogen", such as fluorine, chlorine, bromine and iodine.

[0036] As used herein, the term "alkyl" (and in other groups containing alkyl, such as the alkyl portion of a haloalkyl group) in each case means a straight-chain or branched alkyl group generally having 1-20 carbon atoms, often 1-10 carbon atoms, preferably 1-6 carbon atoms, and especially 1-4 carbon atoms. Examples of C1-C4 alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), and 1,1-dimethylethyl (tert-butyl). Examples of C1-C6 alkyl groups, in addition to those mentioned for C1-C4 alkyl groups, include n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, or 1-ethyl-2-methylpropyl.

[0037] In this invention, the halogen is typically fluorine, chlorine, bromine, or iodine, preferably fluorine, bromine, or chlorine. Correspondingly, this also applies to halogens combined with other structures, such as alkyl halogens. Alkyl halogens preferably have a chain length of 1 to 6 carbon atoms, more preferably a chain length of 1 to 4 carbon atoms. Examples of alkyl halogens include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2-fluoroethyl, 2-chloroethyl, pentafluoroethyl, 1,1-difluoro-2,2,2-trichloroethyl, 2,2,3,3-tetrafluoroethyl, and 2,2,2-trichloroethyl; preferably fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, difluorochloromethyl, and dichlorofluoromethyl.

[0038] In this invention, substituted alkyl refers to alkyl groups that are replaced by halogens, hydroxyl groups, cyano groups, C1-C6 alkoxy groups, amino groups, ester groups, aryl groups, heteroaryl groups, etc., but are not limited thereto.

[0039] This invention first provides a method for preparing a compound of formula I, which includes the following steps:

[0040] Step 1) Compound II undergoes a cyclization reaction in the presence of a base to give compound III;

[0041] Step 2) Compound III is reacted in the presence of iodide and alkyl halosilane to prepare compound I;

[0042] Its synthetic route is as follows:

[0043] ,

[0044] in,

[0045] R1 and R2 are each independently a protecting group for hydroxyl groups. Preferably, R1 is tert-butyldiphenylsilyl and R2 is naphthylmethyl; R3 represents H, CN, C1-C6 alkyl or substituted C1-C6 alkyl; R4 is a leaving group; and R5 is OAc.

[0046] In the above preparation method, the protecting group of the hydroxyl group is one of benzyl, benzoyl, 2,6-dichlorobenzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, methanesulfonic acid, toluenesulfonic acid, dimethoxytriphenylmethyl (DMT), 9-phenylxanthine-9-yl (Pixyl), 9-(p-methoxyphenyl)xanthine-9-yl (MOX), acetyl, naphthylmethyl, p-methoxybenzyl, methoxymethyl, trimethylsilyl, and benzyloxycarbonyl.

[0047] In the above preparation method, the leaving group is one of 4-toluenesulfonyloxy, methanesulfonyloxy, chloromethanesulfonyloxy, trifluoromethanesulfonyloxy, or a halogenated group, preferably methanesulfonyloxy.

[0048] In the above preparation method, the alkali is selected from one or more of potassium hydroxide, sodium hydroxide, potassium carbonate, sodium carbonate, potassium bicarbonate, and sodium bicarbonate, with potassium carbonate being preferred.

[0049] Preferably, step one of the above preparation method is carried out in a solvent, which is acetonitrile, tetrahydrofuran, acetone or methanol, preferably methanol.

[0050] In the above preparation method, the iodide is potassium iodide or sodium iodide.

[0051] In the above preparation method, the alkyl halosilane is trimethylsilyl trifluoromethanesulfonate, trimethylsilyl cyanide, trimethylchlorosilane or trimethylbromosilane, preferably trimethylchlorosilane.

[0052] Under preferred conditions, the method of the present invention achieves even better technical results.

[0053] I. Preparation Examples

[0054] In this specification, if there are any differences between chemical names and chemical structures, the structure is preferred. Generally, the compounds of this invention can be prepared by the methods described herein, unless further specified. The raw materials, reagents, etc., used to prepare the compounds of this invention are commercially available or can be prepared by methods conventional in the art.

[0055] Example 1: Synthesis of intermediate 1

[0056]

[0057] In a 2L three-necked round-bottom flask equipped with a mechanical stirrer, thermometer, and nitrogen inlet, compound A (100.0 g, 0.12 mol), anhydrous potassium carbonate (49.7 g, 0.36 mol, 3 equiv), and anhydrous methanol (800 mL) were added sequentially. The reaction was carried out under a nitrogen atmosphere at 25°C with stirring for 16 hours (HPLC monitoring showed no residual starting material and no detection of byproducts 1 and 2). After the reaction was complete, water (500 mL) and ethyl acetate (500 mL) were added to the reaction system. After stirring for 30 minutes, the mixture was allowed to stand and separated. The organic phase was washed once with saturated brine (500 mL), and 40 mL of the organic phase was separated. o Concentrate under reduced pressure below °C to obtain crude intermediate 1, a pale yellow foamy solid, which can be directly used in the next step.

[0058] Example 2: Synthesis of Compound 1

[0059] Acetonitrile (600 mL), water (30 mL), and sodium iodide (27.0 g, 0.18 mol, 1.5 equiv) were added to the crude intermediate 1 obtained in the previous step (corresponding to 0.12 mol). The mixture was cooled to 0–5°C in an ice-water bath. Trimethylchlorosilane (19.6 g, 0.18 mol, 1.5 equiv) was slowly added dropwise through a constant-pressure dropping funnel with vigorous stirring, controlling the internal temperature not to exceed 10°C. After the addition was complete, the ice bath was removed, the reaction solution was heated to 65°C, and stirring was continued for 12 hours (HPLC monitoring showed no remaining starting material and no detection of byproduct 1 or byproduct 2). After the reaction was complete, the reaction solution was cooled to room temperature, and saturated sodium thiosulfate (Na2S2O3) aqueous solution (500 mL) and ethyl acetate (500 mL) were slowly added. After stirring for 30 minutes, the mixture was allowed to stand and separated. The organic phase was washed once with saturated brine (500 mL), and 40 mL of the organic phase was separated. o Concentrate under reduced pressure below °C. The crude product was purified by silica gel column chromatography (eluent: gradient from dichloromethane / methanol = 20:1 to 10:1) to give a white solid (75.2 g, overall yield of 93% for both steps, HPLC purity >99.0%).

[0060] Example 3-5 Synthesis of intermediate compound 1

[0061] The preparation method is the same as in Example 1, except that only the type of reaction solvent is changed.

[0062] Table 1. Reaction results of Examples 3-5

[0063]

[0064] Example 6: Synthesis of Compound 1

[0065]

[0066] Intermediate 1 (1.0 mmol) was added to tetrahydrofuran (5 mL), methanol (5 mL), and hydrochloric acid (2 M, 5 mL), and stirred at 60°C for 24 hours. HPLC showed that a small amount of starting material remained. The reaction was stopped, and after processing, HPLC calculation showed that 3% of the starting material remained. Byproduct 1 was not detected, and byproduct 2 accounted for 10%.

[0067] Example 7: Synthesis of Compound 1

[0068]

[0069] Intermediate 1 (1.0 mmol) was added to dichloromethane (10 mL) and boron tribromide (1.5 mmol), and stirred at 25°C for 16 hours. HPLC showed that a small amount of starting material remained. The reaction was stopped, and after processing, HPLC calculation showed that 5% of the starting material remained. Byproduct 1 was not detected, and byproduct 2 accounted for 15%.

[0070] Examples 8-10 Synthesis of Compound 1

[0071] The preparation method is the same as in Example 2, except that only the type of alkylsilane is changed.

[0072] Table 2 Reaction results of Examples 8-10

[0073]

[0074] Comparative Example 1: Synthesis of Compound 1

[0075] Sodium hydride (28.8 g, 0.72 mol, 6 equiv) dispersed in 60% mineral oil was added to a reaction flask filled with high-purity nitrogen. Under continuous nitrogen protection, anhydrous tetrahydrofuran (THF, 300 mL) was added to the flask. Then, 3-hydroxypropionitrile (34.2 g, 0.48 mol, 4 equiv) was slowly added dropwise through a constant-pressure dropping funnel in an ice-water bath (0°C), controlling the dropping rate to maintain the reaction solution temperature below 5°C. During this process, a large number of hydrogen bubbles were generated violently. After the addition was complete, the ice bath was removed, the temperature was raised to 25°C, and the mixture was stirred for 1 hour. Then, under an ice-water bath (0°C), an anhydrous tetrahydrofuran (THF, 700 mL) solution of compound A (100.0 g, 0.12 mol) was slowly added dropwise through a constant-pressure dropping funnel. After the addition was complete, the reaction mixture was heated to 25°C and stirred vigorously at this temperature for 18 hours. The reaction process was monitored by HPLC. The remaining raw material was 10%, byproduct 1 accounted for 5%, and byproduct 2 accounted for 11%.

[0076] After the reaction was complete, the reaction system was cooled to 0°C. Under vigorous mechanical stirring and external ice bath cooling, 500 mL of pre-cooled saturated ammonium chloride aqueous solution (approximately 2 mL / min) was added dropwise via a syringe pump at a very slow rate (approximately 2 mL / min) to quench excess sodium hydride. After quenching, 500 mL of ethyl acetate was added. After stirring for 30 minutes, the mixture was allowed to stand and separated. The organic phase was washed once with saturated brine (500 mL), and 40 mL of the organic phase was separated. oConcentrate under reduced pressure below °C. The crude product was purified by silica gel column chromatography (eluent: gradient from dichloromethane / methanol = 20:1 to 10:1) to give a pale yellow solid (40.6 g, overall yield of 51% for both steps, HPLC purity 91.8%).

[0077] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing a compound of formula I, characterized in that, Includes the following steps: Step 1) Compound II undergoes a cyclization reaction in the presence of a base to give compound III; Step 2) Compound III is reacted in the presence of iodide and alkyl halosilane to prepare compound I; Its synthetic route is as follows: ， in, R1 is tert-butyldiphenylsilyl, R2 is naphthylmethyl; R3 is H; R4 is Ms; R5 is acetoxy; Step 1) The reaction is carried out in a solvent, namely methanol; The iodide mentioned is potassium iodide or sodium iodide; The alkyl halosilane is trimethylchlorosilane, trimethylbromosilane, or trimethylsilyltrifluoromethanesulfonate or trimethylsilyl cyanide, which replaces the alkyl halosilane.

2. The preparation method according to claim 1, characterized in that, The alkali is selected from one or more of potassium hydroxide, sodium hydroxide, potassium carbonate, sodium carbonate, potassium bicarbonate, and sodium bicarbonate.

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