Environment-friendly and efficient Difelikefalin preparation method

Abstract

The invention relates to the technical field of biological medicine manufacturing, in particular to an environment-friendly and efficient Difelikefalin preparation method. According to the method, a solid-phase peptide synthesis technology is adopted, and the core is that a green mixed solvent system composed of 2-methyltetrahydrofuran and cyclopentyl methyl ether is used for comprehensively replacing traditional toxic solvents such as DMF and DCM. The method comprises the steps of resin swelling, Fmoc deprotection, amino acid coupling, peptide chain cutting, purification and freeze drying, a piperidine / 2-MeTHF solution is adopted in the deprotection, an optimized HATU / DIPEA activation system is adopted in the coupling reaction, a TFA / 2-MeTHF / water mixed solution is adopted in the cutting, and an ethanol-water chromatographic system is adopted in the purification. According to the invention, closed-loop circulation of the solvent is also realized, and 2-MeTHF and CPME are purified through rotary evaporation and reused in the synthesis process. According to the method, high purity and high yield of the product are guaranteed, production toxicity and environmental hazards are remarkably reduced, and the method has excellent industrial application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceutical manufacturing, and particularly relates to an environmentally friendly and efficient preparation method of Difelikefalin. BACKGROUND

[0002] The Chinese name of Difelikefalin is 4-amino-1-(p-phenylalanine-D-phenylalanine-D-leucine-D-lysine-piperidine-4-carboxylic acid, the English name is 4-amino-1-(D-phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)piperidine-4-carboxylic acid, the molecular formula is C 36 H 53 N7O6, and the molecular weight is 679.4 g / mol. Difelikefalin is a long-acting kappa opioid receptor agonist polypeptide that relieves itching by selectively targeting peripheral kappa opioid receptors.

[0003] The current mainstream method is solid-phase peptide synthesis, and the technical route mainly includes the following steps: fixing the C-terminal amino acid on a solid-phase resin, then sequentially performing the cycle reactions of deprotection and coupling on the resin, connecting the protected amino acid monomers one by one, finally cutting the complete peptide chain from the resin through strong acid (such as trifluoroacetic acid, TFA) and simultaneously removing the side chain protection group to obtain the crude peptide. The crude product is purified by preparative high-performance liquid chromatography, and is salified with acetic acid to finally obtain high-purity Difelikefalin raw material medicine.

[0004] Although the solid-phase peptide synthesis technology is mature and reliable, in the existing production practice of Difelikefalin, especially in the use of solvents, there are the following several significant problems, which are contrary to the current green and sustainable development concept: (1) Large amount of harmful organic solvents are used: a large amount of high-purity organic solvents such as N,N-dimethylformamide (DMF) and dichloromethane (DCM) are consumed in each step (washing, coupling, deprotection) of the synthesis process. These solvents are generally toxic and volatile, and most of them are classified as reproductive toxicants or persistent pollutants, which pose potential risks to the health of operators and the ecological environment; (2) The treatment cost of three wastes is high: a large amount of waste liquid containing residual amino acid derivatives, activators and the above harmful solvents is generated, and high cost needs to be invested for waste solvent recovery or harmless treatment.

[0005] In view of the above related art, there is an urgent need in the art to develop an environmentally friendly and efficient Difelikefalin preparation method, to construct a new and environmentally friendly solvent system, to reduce the use and emission of harmful substances in the production process under the premise of ensuring the quality and efficiency of Difelikefalin synthesis, and to meet the green and low-carbon development trend of the pharmaceutical industry. SUMMARY

[0006] In order to overcome the defects of the prior art, the present application provides an environmentally friendly and efficient Difelikefalin preparation method, which uses a mixed solvent system composed of 2-methyltetrahydrofuran (2-MeTHF) and cyclopentyl methyl ether (CPME) to replace traditional DMF and DCM for washing, swelling, coupling and deprotection steps in solid phase peptide synthesis, to obtain a synthesis process that can be implemented industrially and the solvent can be efficiently recycled, significantly reducing the emission of three wastes and production cost.

[0007] The present application provides an environmentally friendly and efficient Difelikefalin preparation method, which uses the following technical scheme: S1 resin swelling and pretreatment: add CTC resin to the reaction column, swell with mixed solvent for 2-3h, discharge and wash the resin with mixed solvent; S2 deprotection reaction: add deprotection solution to the reactor to treat the resin, then wash the resin with mixed solvent for 2-4 times until the filtrate is colorless; S3 coupling reaction: dissolve Fmoc-protected amino acid, coupling agent HATU and base DIPEA in step S2 system to form an activation solution, transfer the prepared activation solution to the solid phase synthesis reactor within 1-3min, and couple with the free amino group on the resin. After the reaction is completed, wash the resin with mixed solvent 3 times; repeat S2 and S3 operations, and connect the amino acids in the sequence in the order of C-terminal to N-terminal to obtain a resin polypeptide; S4 cleavage and preparation of crude peptide: use a mixed solution of trifluoroacetic acid, 2-MeTHF and water as the cleavage solution, add the pre-cooled cleavage solution to the resin polypeptide to 0-5℃, and cleave for 2-3h; collect the cleavage solution and add it dropwise to methyl tert-butyl ether at 0-5℃, continue stirring for 30min, then stand for 1h; precipitate the crude peptide, collect the precipitate by filtration to obtain Difelikefalin crude peptide; S5 purification: the Difelikefalin crude peptide of step S4 is purified by preparative high performance liquid chromatography using an ethanol-water system as the mobile phase; freeze-dry the collected pure solution to obtain high-purity white fluffy powder of Difelikefalin acetate.

[0008] By adopting the above scheme, the mixed solvent system composed of 2-MeTHF and CPME is adopted, 2-MeTHF has the characteristics of bio-based, low toxicity and biodegradability, CPME has the characteristics of low toxicity and good chemical stability, and the safety and environmental risk brought by the high-risk solvent is eliminated from the source; through the integrated S6 solvent recovery step, the waste solvent is efficiently rectified and recovered, the recovery rate is > 85%, the purity is > 99.8%, and it can be directly reused for production. This greatly reduces the purchase amount of fresh solvent and the discharge amount of hazardous waste liquid, realizes the in-plant circulation of the solvent, and minimizes the three wastes. The technical scheme changes the preparation process of Difelikefalin from the traditional high-pollution and high-cost mode to an environmentally friendly, economical, quality controllable and easy-to-industrialize green advanced manufacturing mode.

[0009] Preferably, the mixed solvent of step S1 is composed of 2-MeTHF and CPME in a volume ratio of (3-7):(7-3).

[0010] Preferably, the mixed solvent of step S1 is composed of 2-MeTHF and CPME in a volume ratio of 5:5.

[0011] By adopting the above scheme, 2-MeTHF has good swelling ability for the resin, and CPME has high chemical stability and moderate boiling point. Within this proportion range, the two complement each other, which can not only ensure that the solid-phase resin is fully swollen to facilitate the internal reaction, but also provide a suitable polar environment for the dissolution of amino acids, coupling agents and other reagents, and ensure efficient mass transfer of reactants inside and outside the resin.

[0012] Preferably, the deprotection solution of step S2 is a 2-MeTHF solution containing piperidine, and the volume percentage concentration of piperidine is 20-30%.

[0013] By adopting the above scheme, the 20%-30% piperidine concentration is obtained through optimization, which can provide sufficient alkaline environment to ensure that the Fmoc protecting group is quickly and completely removed, thereby exposing the highly reactive free amino group and preparing for subsequent coupling. At the same time, this concentration range effectively inhibits the occurrence of side reactions, and low concentration may lead to incomplete deprotection and produce impurities of missing sequences; while high concentration may increase the risk of racemization of amino acids and may cause unnecessary damage to the resin structure; using 2-MeTHF as the solvent instead of the traditional DMF may further reduce the risk of these side reactions due to different solvation effects.

[0014] Preferably, in step S2, the deprotection reaction is carried out twice, the deprotection solution is added for 10-15 min in the first time, and the deprotection solution is added for 10-15 min in the second time.

[0015] Preferably, the coupling reaction in step S3 is carried out at a temperature of 20-25°C with gentle stirring for 40-90 minutes.

[0016] Preferably, the activation solution in step S3 is formed by mixing Fmoc-protected amino acids, coupling agent HATU and base DIPEA in a molar ratio of 1:(0.9-1.1):(1.9-2.1) and dissolving them in the mixed solvent system. The concentration of Fmoc-protected amino acids in the activation solution is controlled at 0.1-0.3 mol / L.

[0017] By adopting the above scheme, HATU is approximately 1 equivalent, ensuring that each amino acid molecule can be efficiently activated, with a slight margin to ensure complete reaction; DIPEA is approximately 2 equivalents, one equivalent of which is used to neutralize the acid generated during activation, and the other equivalent ensures that the reaction system maintains the necessary alkaline environment to promote coupling; this ratio achieves the best balance between maximizing the reaction driving force and reducing the waste of expensive reagents, reflecting the economy of the process.

[0018] Preferably, in step S3, the following amino acids are linked one by one in order from C-terminus to N-terminus, in the order of D-lysine → D-leucine → D-phenylalanine → D-phenylalanine, and the chiral purity of each amino acid is >99.5%.

[0019] Preferably, the cutting fluid in step S4 is composed of TFA, 2-MeTHF and water in a volume ratio of (90-95):(2.5-5):(2.5-5).

[0020] Preferably, the purification conditions for the preparative high-performance liquid chromatography in step S5 are as follows: the chromatographic column is a C18 reversed-phase column, the mobile phase A is an aqueous solution containing 0.1% trifluoroacetic acid, the mobile phase B is ethanol, gradient elution is used, and the detection wavelength is 220 nm.

[0021] Preferably, step S5 involves freeze-drying the collected pure solution, which includes the following steps: S51 Pre-freezing: The pure solution is completely frozen at -45°C to -50°C; S52 single drying: Under a vacuum of 10-30 Pa, raise the temperature to -20℃ to -10℃ and maintain it for 20-40 hours; S53 Secondary drying: Raise the temperature to 20℃ to 30℃ and maintain it for 6-12 hours to obtain white, fluffy powdered Difelikefalin acetate with a moisture content ≤5.0%.

[0022] Preferably, the environmentally friendly and efficient method for preparing Difelikefalin further includes step S6, solvent recovery: the waste solvent containing 2-MeTHF and CPME generated in steps S1-S4 is purified by rotary evaporation, and the separated 2-MeTHF and CPME can be directly reused in the synthesis process to achieve a closed-loop cycle.

[0023] Preferably, in step S6, solvent recovery involves recovering the waste solvent generated in steps S1 to S4 using a distillation apparatus. The recovered 2-methyltetrahydrofuran and cyclopentyl methyl ether have a purity greater than 99.8% and are reused in steps S1 to S4.

[0024] Preferably, the rotary evaporation purification process includes: under a vacuum of 130-180 Pa, controlling the water bath temperature of the rotary evaporator at 45-55°C to collect a 2-methyltetrahydrofuran fraction with a purity ≥99.0%, and controlling the water bath temperature at 65-75°C to collect a cyclopentyl methyl ether fraction with a purity ≥98.5%.

[0025] By adopting the above scheme and achieving separation through precise control of the water bath temperature under vacuum, the purity fully meets or even exceeds the quality requirements of re-entering the core synthesis steps (S1 resin swelling, S3 coupling reaction), ensuring that the recycling process does not reduce product quality.

[0026] In summary, this application has the following beneficial effects: 1. This application discloses an environmentally friendly and efficient method for preparing Difelikefalin. By using a green mixed solvent system of 2-methyltetrahydrofuran / cyclopentylmethyl ether to replace traditional toxic solvents and integrating closed-loop recovery and ethanol-water purification processes, an environmentally friendly, efficient, and cost-controllable method for preparing Difelikefalin has been successfully constructed. While significantly reducing environmental toxicity and production costs, it ensures the high purity and excellent properties of the product, achieving synergistic optimization of environmental protection and industrial production.

[0027] 2. This application presents an environmentally friendly and efficient method for preparing Difelikefalin. Through optimized deprotection procedures, precise coupling feed ratios, and mild coupling temperatures, it systematically suppresses the generation of key impurities such as missing sequences and racemization. The parameters in this method have been optimized and given clear ranges. The process flow is clear, and it has excellent scalability and batch-to-batch reproducibility, providing a solid and reliable technical foundation for large-scale industrial production. Attached Figure Description

[0028] Figure 1 This is a high-performance liquid chromatogram of the crude Difelikefalin peptide obtained in Example 1; Figure 2The image shows a high-performance liquid chromatogram of the purified Difelikefalin product obtained in Example 1. Detailed Implementation

[0029] The technical solution of this application is further illustrated by specific embodiments below. These specific embodiments do not represent a limitation on the scope of protection of this application. Any non-essential modifications and adjustments made by others based on the concept of this application still fall within the scope of protection of this application.

[0030] Unless otherwise specified, the experimental methods shown in the following examples are conventional methods.

[0031] CTC resin, Merck AG, Germany, brand: Millipore, product name: Cysteamine 2-chlorotrityl resin Novabio chem®, product number: 856000.

[0032] Solvents: 2-Methyltetrahydrofuran (2-MeTHF, anhydrous grade, >99.5%), cyclopentyl methyl ether (CPME, anhydrous grade, >99.5%), methyl tert-butyl ether (MTBE, industrial grade), ethanol (anhydrous grade, >99.8%), piperidine (>99%); HATU (>98%), HOAt (>98%), N,N-diisopropylethylamine (DIPEA, >99.5%), trifluoroacetic acid (TFA, >99.5%); all reagents and materials shown are commercially available products.

[0033] Solid-phase synthesis reactor: CS Bio, USA; Model: CS936; HPLC: Thermo Fisher Scientific, Equipment model: UltiMate 3000 HPLC; C18 preparation column, Shimadzu Corporation, Japan, Shim-pack GIS C18, 10µm, 10x250mm; The present application will be further described in detail below with reference to embodiments and comparative examples.

[0034] Example

[0035] Example 1: An environmentally friendly and efficient method for preparing Difelikefalin employs the following technical solution: S1 resin swelling and pretreatment: 1 kg of CTC resin (loading 0.5 mmol / g) was added to the reaction column, and the resin was fully swollen with 6 L of mixed solvent for 2-3 hours and then discharged. The resin was washed with 3 L of mixed solvent. S2 Deprotection Reaction: Add 5L of deprotection solution to the reactor to treat the resin. The first addition of the deprotection solution is followed by a 10-minute reaction, and the second addition of 5L of the deprotection solution is followed by a 15-minute reaction. Then, wash the resin quickly with 3L of mixed solvent 2-4 times until the filtrate is colorless. S3 Coupling Reaction: The prepared activation solution was transferred to a solid-phase synthesis reactor within 2 min and coupled with the free amino groups on the resin from step S2. The mixture was stirred gently at 25°C for 65 min. After the reaction, the resin was washed three times with 3 L of mixed solvent. S2 and S3 were repeated, and the following amino acids were linked one by one in order from C-terminus to N-terminus, in the order of D-lysine → D-leucine → D-phenylalanine → D-phenylalanine, to obtain the resin peptide. S4 cleavage and preparation of crude peptide: 8L of cleavage fluid pre-cooled to 0-5℃ was added to the resin peptide and cleaved for 2h; the cleavage fluid was collected and added dropwise to 80L of methyl tert-butyl ether at 0-5℃, and stirred for 30min, then allowed to stand for 1h; crude peptide was precipitated, filtered and collected to obtain crude Difelikefalin peptide. S5 Purification: The crude Difelikefalin peptide from step S4 was purified by preparative high-performance liquid chromatography; the collected purified solution was then freeze-dried, including the following steps: S51 Pre-freezing: The pure solution is completely frozen at -45°C; S52 single drying: Under a vacuum of 20 Pa, the temperature is raised to -15℃ and maintained for 30 hours; S53 Secondary drying: The temperature is raised to 25℃ and maintained for 9 hours to obtain white, fluffy powdered Difelikefalin acetate with a moisture content of ≤5.0%.

[0036] The specific addition amounts of the mixed solvent, 2-MeTHF and CPME, are 10L of 2-MeTHF and 10L of CPME.

[0037] The deprotection solution is a 2-MeTHF solution containing piperidine, with a volume percentage concentration of 25%. Specifically, 1.25 L of piperidine is measured and diluted to 5 L with 2-MeTHF solution.

[0038] The activation solution is formed by dissolving a mixture of Fmoc-protected amino acids, coupling agent HATU, and base DIPEA in a 1:1:2 molar ratio in a mixed solvent, resulting in a final total volume of 4 L. The concentration of Fmoc-protected amino acids in the activation solution is controlled at 0.25 mol / L. The specific addition amounts are shown in Table 1. Table 1. Dosage of each amino acid activation solution component in Example 1 Amino acid Molecular weight about (g / mol) Fmoc-amino acid mass (g) HATU mass (g) DIPEA mass (g) Fmoc-D-Lys(Boc)-OH 468.5 468.5 380.2 258.5 Fmoc-D-Leu-OH 353.4 353.4 380.2 258.5 Fmoc-D-Phe-OH 387.4 387.4 380.2 258.5 Fmoc-D-Phe-OH 387.4 387.4 380.2 258.5 The cutting fluid is composed of TFA, 2-MeTHF and water in a volume ratio of 92.5:5:2.5; specifically, it is a mixture of 92.5L TFA, 5L 2-MeTHF and 2.5L water.

[0039] The crude Difelikefalin peptide was purified by preparative high-performance liquid chromatography (HPLC) using a C18 reversed-phase column, a detection wavelength of 220 nm, a flow rate of 150 mL / min, mobile phase A being an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B being ethanol. The gradient elution conditions were: 0-5 min (20% B); 5-35 min (20% → 50% B); 36-41 min (5% B); 42-50 min (20% B).

[0040] Example 2: An environmentally friendly and efficient method for preparing Difelikefalin employs the following technical solution: The difference from Example 1 is that: S1 resin swelling and pretreatment: Swelling with a mixed solvent for 2 hours; the mixed solvent consisted of 2-MeTHF and CPME in a volume ratio of 3:7. S2 deprotection reaction: The deprotection solution contains 20% (v / v) piperidine in 2-MeTHF solution; after deprotection, the resin is washed twice rapidly with a mixed solvent; S3 Coupling Reaction: The molar ratio of the activated solution to amino acids:HATU:DIPEA was 1:0.9:1.9, and the amino acid concentration was 0.1 mol / L; the coupling reaction was carried out at 20℃ with gentle stirring for 40 min. S4 cleavage and preparation of crude peptide: The cleavage solution was prepared at a volume ratio of TFA:2-MeTHF:water = 90:5:5 and cleaved for 2 hours. The cleavage solution was collected and added dropwise to methyl tert-butyl ether (MTBE) at 0℃ to precipitate crude peptide. The precipitate was collected by filtration to obtain crude Difelikefalin peptide. S5 purification: Freeze-drying includes the following steps: S51 Pre-freezing: The pure solution is completely frozen at -45°C; S52 single drying: Under a vacuum of 10 Pa, the temperature is raised to -20℃ and maintained for 20 hours; S53 Secondary drying: Raise the temperature to 20℃ and maintain it for 6 hours.

[0041] The mixed solvent is added in the form of 6L of 2-MeTHF and 14L of CPME.

[0042] The deprotection solution is specifically prepared by measuring 1 L of piperidine and diluting it to 5 L with 2-MeTHF solution.

[0043] The activation solution is formed by mixing Fmoc-protected amino acids, coupling agent HATU, and base DIPEA in a molar ratio of 1:0.9:1.9 and dissolving them in the mixed solvent system. The concentration of Fmoc-protected amino acids in the activation solution is controlled at 0.1 mol / L. Specific addition amounts are shown in Table 2. Table 2. Dosage of each amino acid activation solution component in Example 2 The cutting fluid is a mixture of 90L TFA, 5L 2-MeTHF, and 5L water.

[0044] Example 3: An environmentally friendly and efficient method for preparing Difelikefalin employs the following technical solution: The difference from Example 1 is that: S1 resin swelling and pretreatment: Swelling with a mixed solvent for 3 hours; the mixed solvent consisted of 2-MeTHF and CPME in a volume ratio of 7:3. S2 deprotection reaction: The deprotection solution contains 30% (v / v) piperidine in 2-MeTHF solution; after deprotection, the resin is washed twice rapidly with a mixed solvent; S3 Coupling Reaction: The molar ratio of the activated solution to amino acids:HATU:DIPEA was 1:1.1:2.1, and the amino acid concentration was 0.3 mol / L; the coupling reaction was carried out at 25℃ with gentle stirring for 90 min. S4 cleavage and preparation of crude peptide: The cleavage solution was prepared at a volume ratio of TFA:2-MeTHF:water = 95:2.5:2.5 and cleaved for 3 hours. The cleavage solution was collected and added dropwise to methyl tert-butyl ether (MTBE) at 5°C to precipitate crude peptide. The precipitate was collected by filtration to obtain crude Difelikefalin peptide. S5 purification: Freeze-drying includes the following steps: S51 Pre-freezing: The pure solution is completely frozen at -50°C; S52 single drying: Under a vacuum of 30 Pa, the temperature is raised to -10℃ and maintained for 40 hours; S53 Secondary drying: Raise the temperature to 30℃ and maintain it for 12 hours.

[0045] The mixed solvent is added in the amount of 14L of 2-MeTHF and 6L of CPME.

[0046] The deprotection solution is specifically prepared by measuring 1.5 L of piperidine and diluting it to 5 L with 2-MeTHF solution.

[0047] The activation solution contains Fmoc-protected amino acids, HATU coupling agent, and DIPEA in a molar ratio of 1:1.1:2.1, with an amino acid concentration of 0.3 mol / L. Specific addition amounts are shown in Table 3. Table 3. Dosage of each amino acid activation solution component in Example 3 Molecular weight about (g / mol) Fmoc-amino acid mass (g) HATU mass (g) DIPEA mass (g) Fmoc-D-Lys(Boc)-OH 468.5 468.5 418.2 271.4 Fmoc-D-Leu-OH 353.4 353.4 418.2 271.4 Fmoc-D-Phe-OH 387.4 387.4 418.2 271.4 Fmoc-D-Phe-OH 387.4 387.4 418.2 271.4 The cutting fluid is a mixture of 95L TFA, 2.5L 2-MeTHF and 2.5L water.

[0048] Example 4: An environmentally friendly and efficient method for preparing Difelikefalin adopts the following technical solution: The difference from Example 1 is that it also includes step S6 solvent recovery: the waste solvent containing 2-MeTHF and CPME generated in steps S1-S4 is recovered by a distillation device, and the recovered 2-methyltetrahydrofuran and cyclopentylmethyl ether are reused in steps S1 to S4.

[0049] The rotary evaporation purification process includes: under a vacuum of 150 Pa, controlling the water bath temperature of the rotary evaporator at 50°C to collect the 2-methyltetrahydrofuran fraction with a purity ≥99.0%, and controlling the water bath temperature at 70°C to collect the cyclopentyl methyl ether fraction with a purity ≥98.5%. Comparative Example

[0050] Comparative Example 1: The difference from Example 1 is that conventional DMF (for deprotection and coupling) and DCM (for swelling and washing) are used as solvent systems instead of the 2-MeTHF / CPME mixed solvent system of the present invention. All other steps remain the same.

[0051] Comparative Example 2: The difference from Example 1 is that 100% 2-MeTHF is used as the solvent instead of the mixed solvent.

[0052] Comparative Example 3: The difference from Example 1 is that only one deprotection reaction is performed, and the reaction time is 20 minutes.

[0053] Comparative Example 4: The difference from Example 1 is that the coupling reaction temperature is increased to 35°C.

[0054] Performance testing The efficiency and cleanliness of the synthesis reaction were determined by HPLC analysis of the crude peptides and purified Difelikefalin in Examples 1-4 and Comparative Examples 1-4. The overall yield, calculated based on the initial resin loading, reflects the process efficiency. The moisture content of the purified Difelikefalin is used to verify the key indicator of product stability. The results of each performance test are shown in Table 4. Table 4 Performance Test Results Crude peptide purity (%) Fine peptide purity (%) Maximum single impurity (%) Total yield (%) Moisture content (%) Example 1 92.5 99.7 0.12 79.2 2.8 Example 2 92 99.6 0.15 78.5 3.1 Example 3 91.8 99.6 0.16 77.8 2.9 Example 4 91.7 99.7 0.15 78.2 2.9 Comparative Example 1 91 99.3 0.16 77.2 3 Comparative Example 2 89.1 99.1 0.25 75 3.3 Comparative Example 3 89.5 99.1 0.31 73.2 3.5 Comparative Example 4 90.8 99 0.45 76.1 3 The Difelikefalin samples prepared using the environmentally friendly and efficient preparation methods of Examples 1-4 and Comparative Examples 1-4 were subjected to the above performance tests, and the test results are shown in Table 4.

[0055] The HPLC peak chromatogram of the crude Difelikefalin peptide obtained by the preparation method in Example 1 is as follows: Figure 1 As shown, the elution time was 11.2657 min. The HPLC peak chromatogram of the purified Difelikefalin sample is as follows. Figure 2 As shown, the peak elution time was 11.2471 min; and the high purity (99.7%), low impurities (0.12%), and high yield (79.2%) demonstrate the high efficiency and reliability of the entire process; at the same time, the extremely low solvent cost and high recovery rate highlight its economic and environmental advantages. Examples 2-4 show indicators very close to E1, fully demonstrating the beneficial effects of the green mixed solvent, optimized process parameters, and closed-loop recovery of the present invention.

[0056] Comparative Example 1 uses DMF (for deprotection and coupling) and DCM (for swelling and washing) as solvent systems to replace the 2-MeTHF / CPME mixed solvent system of the present invention. Comparative Example 1 is not much different from Example 1 in terms of product purity and yield, which proves the effectiveness of the traditional method. However, its fatal drawbacks are environmental protection and economy: the three wastes are highly toxic, and the solvent cost is higher than that of the example due to the difficulty and low efficiency of DMF / DCM recovery. This also proves that the present technical solution achieves a dual breakthrough in environmental protection and cost without sacrificing quality.

[0057] Comparative Example 2 used 100% 2-MeTHF as the solvent instead of the mixed solvent; all quality indicators (purity, yield) were significantly worse than those in Example 1; indicating that 2-MeTHF alone is insufficient in terms of resin swelling, reagent dissolution, or mass transfer efficiency, proving that the combined use of 2-MeTHF and CPME produces the necessary synergistic effect, which is the key to ensuring high-quality synthesis.

[0058] Comparative Example 3 underwent only one deprotection reaction, with a reaction time of 20 min. The purity of the crude peptide obtained and the purity of the purified Difelikefalin sample were significantly reduced, while the maximum single impurity was increased. This demonstrates that the two-step deprotection method is crucial for the complete removal of the Fmoc group and the reduction of missing sequence impurities, and is a necessary step to ensure high purity.

[0059] Comparative Example 4 increased the coupling reaction temperature to 35℃; after purification, the purity of the Difelikefalin sample decreased, and the maximum single impurity increased significantly (0.45%), which is likely due to amino acid racemization caused by high temperature; this strongly demonstrates the key role of the present invention in controlling the mild coupling temperature to suppress racemization and ensure the chiral purity of the product.

[0060] The above experimental results show that the environmentally friendly and efficient Difelikefalin preparation method of this application, by using a 2-methyltetrahydrofuran / cyclopentylmethyl ether green mixed solvent system to replace traditional toxic solvents and integrating closed-loop recovery and ethanol-water purification processes, has successfully constructed an environmentally friendly, efficient, and cost-controllable Difelikefalin preparation method. While significantly reducing environmental toxicity and production costs, it ensures the high purity and excellent properties of the product, achieving synergistic optimization of environmental protection and industrial production.

[0061] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but as long as they are within the scope of the present invention, they are protected by patent law.

Claims

1. An environmentally friendly and efficient method for preparing Difelikefalin, characterized by, The technical scheme is as follows: S1 resin swelling and pretreatment: CTC resin is added to the reaction column, and mixed solvents are used for sufficient swelling for 2-3 hours, and the resin is washed with mixed solvents; S2 deprotection reaction: a deprotection solution is added to the reactor to treat the resin, and the resin is washed with mixed solvents for 2-4 times until the filtrate is colorless; S3 coupling reaction: Fmoc-protected amino acids, coupling agent HATU and base DIPEA are dissolved in the system of step S2 to form an activation solution, and the prepared activation solution is transferred to the solid-phase synthesis reactor within 1-3 minutes to couple with the free amino group on the resin. After the reaction is completed, the resin is washed with mixed solvents for 3 times; repeat S2 and S3 operations, and connect the amino acids in the sequence in the order from C terminal to N terminal to obtain a resin polypeptide; S4 cleavage and preparation of crude peptide: a mixed solution of trifluoroacetic acid, 2-MeTHF and water is used as a cleavage solution, and the resin polypeptide is added with the cleavage solution pre-cooled to 0-5°C, and cleaved for 2-3 hours; Collect the cleavage solution and add it dropwise to methyl tert-butyl ether at 0-5°C, continue to stir for 30 minutes, and then stand for 1 hour; precipitate the crude peptide, collect the precipitate by filtration, and obtain Difelikefalin crude peptide; S5 purification: the Difelikefalin crude peptide of step S4 is purified by preparative high performance liquid chromatography with an ethanol-water system as the mobile phase; the collected pure product solution is freeze-dried to obtain high-purity white fluffy powder of Difelikefalin acetate.

2. The environmentally friendly process for the preparation of Difelikefalin as claimed in claim 1, wherein, The mixed solvent of step S1 is composed of 2-MeTHF and CPME in a volume ratio of (3-7):(7-3).

3. The environmentally friendly process for the preparation of Difelikefalin as claimed in claim 1, wherein, The deprotection solution of step S2 is a 2-MeTHF solution containing piperidine, and the volume percentage concentration of piperidine is 20-30%.

4. The environmentally friendly process for the preparation of Difelikefalin as claimed in claim 1, wherein, In step S2, the deprotection reaction is carried out twice, the first time for 10-15 minutes and the second time for 10-15 minutes.

5. The environmentally friendly process for the preparation of Difelikefalin as claimed in claim 1, wherein, The activation solution of step S3 is prepared by mixing Fmoc-protected amino acids, coupling agent HATU and base DIPEA in a molar ratio of 1:(0.9-1.1):(1.9-2.1) to form an activation solution in the mixed solvent system. In the activation solution, the concentration of Fmoc-protected amino acids is controlled at 0.1-0.3 mol / L.

6. The environmentally friendly process for the preparation of Difelikefalin as claimed in claim 1, wherein, The cleavage solution of step S4 is composed of TFA, 2-MeTHF and water in a volume ratio of (90-95):(2.5-5):(2.5-5).

7. The environmentally friendly process for the preparation of Difelikefalin as claimed in claim 1, wherein, The purification conditions of preparative high performance liquid chromatography in step S5 are as follows: the chromatographic column is a C18 reversed-phase column, the mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, the mobile phase B is ethanol, gradient elution is adopted, and the detection wavelength is 220 nm.

8. The environmentally friendly process for the preparation of Difelikefalin as claimed in claim 1, wherein, It also includes step S6 solvent recovery: the waste solvents containing 2-MeTHF and CPME generated in steps S1-S4 are purified by rotary evaporation, and 2-MeTHF and CPME are separated and can be directly reused in the synthesis process to realize closed-loop circulation.

9. The environmentally friendly process for the preparation of Difelikefalin as claimed in claim 8, wherein, The rotary evaporation purification process comprises: under a vacuum of 130-180 Pa, controlling the water bath temperature of the rotary evaporator at 45-55 ℃ to collect a 2-methyltetrahydrofuran fraction with a purity ≥ 99.0%, and controlling the water bath temperature at 65-75 ℃ to collect a cyclopentyl methyl ether fraction with a purity ≥ 98.5%.

10. Difelikefalin, characterized in that: prepared by a method as claimed in any one of claims 1 to 9.

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