Process for the preparation of desipramine
By using resin as a solid carrier for the direct condensation of large fragments and optimizing the lysis solution in the preparation of ground-free forests, the problems of low purity and industrial production in the existing technology have been solved, and high-purity and high-efficiency industrial production has been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHENGDU SINTANOVO BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-05-18
- Publication Date
- 2026-06-30
AI Technical Summary
Existing non-terrestrial forest preparation processes suffer from cumbersome condensation steps, long reaction cycles, and high risks of side reactions, resulting in low purity of crude products and making it difficult to meet the requirements of industrial production for product quality and batch consistency.
Using resin as a solid-phase support, the Boc-D-Phe-D-Phe-D-Leu-OH tripeptide fragment was directly coupled to Boc-Pip(Fmoc)-resin through a large fragment direct condensation mechanism. The lysis buffer and sedimentation process were optimized to reduce side reactions and improve purity.
It significantly improves the purity of crude forest products to over 95%, reduces the number of impurities and the probability of side reactions, is suitable for large-scale industrial production, simplifies the operation process, and reduces production costs.
Smart Images
Figure CN122301980A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of polypeptide preparation technology, specifically a preparation process for terrefinery. Background Technology
[0002] Difelikefalin, chemically known as 4-amino-1-(D-phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)piperidine-4-carboxylic acid acetate, is a selective κ-opioid receptor agonist primarily used clinically to treat chronic kidney disease-related pruritus (CKD). (aP) can significantly improve severe itching symptoms in patients with end-stage renal disease or non-dialysis-dependent chronic kidney disease, and has important clinical application value.
[0003] Currently, the preparation of terfenadine mainly employs solid-phase peptide synthesis. Traditional processes primarily utilize stepwise condensation of single protected amino acids, starting with a primary amino acid and sequentially coupling single amino acid units such as lysine, leucine, and phenylalanine. The target product is then obtained through deprotection, condensation, cleavage, and purification. However, this method has significant drawbacks in industrial production: the stepwise condensation steps are cumbersome, the reaction cycle is long, and the risk of side reactions and impurity formation is high, resulting in low purity of the crude terfenadine. This not only increases the difficulty and cost of subsequent purification but also makes it difficult to consistently meet the quality and batch-to-batch consistency requirements of large-scale production.
[0004] Although existing technologies have optimized the synthesis process to some extent, they still rely on the sequential coupling of single amino acids as the core route, failing to fundamentally solve problems such as multiple condensation steps, easy accumulation of impurities, and insufficient purity of crude products. While some improved schemes have improved yield or reaction conditions, they still have significant shortcomings in simplifying the condensation process, improving the purity of crude products, and adapting to industrial mass production, and cannot simultaneously meet the needs of high purity, high stability, and large-scale production. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a process for preparing non-terrestrial forests. This process effectively improves product purity while ensuring large-scale industrial production, and features simple steps and a short reaction cycle.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A process for preparing a type of illegal forest includes the following steps: (1) Using resin as a solid support, Boc-Pip(Fmoc)-OH is loaded and reacted with resin to obtain Boc-Pip(Fmoc)-resin. (2) Under the action of a condensing agent, Fmoc-D-Lys(Boc)-OH and Boc-D-Phe-D-Phe-D-Leu-OH are sequentially coupled onto the Boc-Pip(Fmoc)-resin to obtain the terfenadine resin. (3) The lysine peptide resin was lysed using a lysis buffer, and then concentrated, settled, and dried to obtain crude lysine.
[0007] Preferably, step (1) specifically involves: adding the resin to a solvent for swelling, adding Boc-Pip(Fmoc)-OH to the solvent for dissolution, adding an activator, mixing the activated Boc-Pip(Fmoc)-OH with the swollen resin for a first reaction, adding a resin end-capping agent for a second reaction, and using a deprotecting agent to deprotect the resin after the reaction is complete, thereby obtaining Boc-Pip(Fmoc)-resin.
[0008] Preferably, the resin is selected from one or more of the following: chloromethyl resin (Merrified Resin), 4-hydroxymethylphenoxymethyl resin (Wang Resin), 2-chlorotriphenylmethyl resin (2-CTC Resin), 4-hydroxymethylphenylacetyl resin (HMPA Resin), p-benzyloxybenzylamine resin (BHA Resin), and 9-Fmoc-aminoxanthine-3-yloxypolystyrene resin (Sieber Resin). More preferably, it is 2-chlorotriphenylmethyl resin (2-CTC Resin).
[0009] Preferably, the degree of substitution of the resin is 1.0~1.5 mmol / g.
[0010] Preferably, in step (1), the molar ratio of Boc-Pip(Fmoc)-OH to resin is (1~2):1. Exemplarily, in step (1), the molar ratio of Boc-Pip(Fmoc)-OH to resin is any one of 1:1, 1.5:1, or 2:1, or a value between two of these. More preferably, in step (1), the molar ratio of Boc-Pip(Fmoc)-OH to resin is 1.5:1.
[0011] Preferably, the activator is selected from any one or more of N,N-diisopropylethylamine (DIEA), 4-dimethylaminopyridine (DMAP), triethylamine (TEA), and N-methylmorpholine (NMM).
[0012] Preferably, in step (1), the molar ratio of Boc-Pip(Fmoc)-OH to the activator is (1~2):3. For example, in step (1), the molar ratio of Boc-Pip(Fmoc)-OH to the activator is any one of 1:3, 1.5:3, 2:3, or a value between two of them.
[0013] Preferably, the reaction temperature of the first reaction in step (1) is 15~25℃ and the reaction time is 2~2.5h.
[0014] Preferably, the reaction temperature of the secondary reaction in step (1) is 15~25℃ and the reaction time is 0.5~1.5h.
[0015] Preferably, the resin end-capping agent in step (1) is a mixture of alcohol and alkali, wherein the alcohol is selected from any one of methanol (MeOH), ethanol (EtOH), and isopropanol (IPOH), and the alkali is selected from any one of N,N-diisopropylethylamine (DIEA), 4-dimethylaminopyridine (DMAP), triethylamine (TEA), and N-methylmorpholine (NMM).
[0016] Preferably, the removing agent in step (1) is selected from any one or more of piperidine (Pip), piperazine (Pipz), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBU), N-methylmorpholine (NMM), and pyrrolidine (Pyrr).
[0017] Preferably, the condensing agent in step (2) is selected from one or more of carbodiimides, benzotriazole salts, pyridinebenzotriazole salts, and phosphate esters. More preferably, it is one or more of N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), and N,N,N',N'-tetramethyl-O-(7-azabenzotriazole-1-yl)hexafluorophosphate (HATU). More preferably, it is N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in a molar ratio of 1:1.
[0018] Preferably, the molar ratio of the resin to the condensing agent is 1:(1~5). More preferably, it is 1:2.
[0019] Preferably, the molar ratio of the resin to Fmoc-D-Lys(Boc)-OH is 1:(1~5). More preferably, it is 1:2.
[0020] Preferably, the molar ratio of the resin to Boc-D-Phe-D-Phe-D-Leu-OH is 1:(1~5). More preferably, it is 1:2.
[0021] Preferably, the reaction conditions for step (2) are a reaction temperature of 15~25℃ and a reaction time of 2.0~2.5h.
[0022] Preferably, the lysis solution in step (3) comprises trifluoroacetic acid (TFA), triisopropylsilane (Tis), and H2O in a volume ratio of 9:0.5:0.5.
[0023] Preferably, in step (3), the ratio of lysis buffer to difolin peptide resin is (5~11):1 ml / g. Exemplarily, the ratio is any one or a combination of 5:1 ml / g, 5.4:1 ml / g, 9:1 ml / g, 9.5:1 ml / g, 10:1 ml / g, and 11:1 ml / g.
[0024] Preferably, the settling agent used in step (3) during the settling process is petroleum ether, methyl tert-butyl ether, ethyl tert-butyl ether, diethyl ether, or a mixture of any two of these ether reagents or a mixture of all three reagents. Methyl tert-butyl ether is preferred.
[0025] Preferably, the amount of settling agent used in step (3) is 1 to 20 times the volume ratio. Preferably, it is 5 times the volume ratio.
[0026] Preferably, the steps further include: purifying and converting the crude terrestrial forest product to salt.
[0027] The beneficial effects of this invention are: Compared to existing single-amino acid stepwise coupling processes, the non-Fmoc preparation process described in this application employs a large-fragment direct condensation mechanism to directly couple the Boc-D-Phe-D-Phe-D-Leu-OH tripeptide fragment to Boc-Pip(Fmoc)-resin. This significantly reduces the number of coupling steps, lowers the probability of side reactions such as single amino acid deletion and racemization, and solves the difficulties of impurity dispersion and control in existing technologies. Simultaneously, by optimizing process design parameters, side reactions are effectively reduced, resulting in a crude product purity of over 95%, a significant reduction in the total number of impurities, and effective control of high-content impurities. Furthermore, this process is stably adaptable to scale-up production from small-scale trials to 500 mmol industrial production, maintaining stable yield and purity. It enables mass production without complex operations, reducing subsequent purification pressure while improving production efficiency, thus balancing product quality and industrial production requirements. Attached Figure Description
[0028] Figure 1This is a flowchart of the process for preparing illegal forests provided by the present invention.
[0029] Figure 2 The image shows the HPLC analysis of the crude terrestrial forest product prepared in Example 4.
[0030] Figure 3 This is an LCMS image of the crude terrestrial forest product prepared in Example 4.
[0031] Figure 4 The image shows the HPLC analysis of the crude terrestrial forest product prepared in Comparative Example 1.
[0032] Figure 5 The image shows the HPLC analysis of the crude terrestrial forest product prepared in Example 1. Detailed Implementation
[0033] To enable those skilled in the art to better understand the technical solution of the invention, the invention will be further described in detail below with reference to specific embodiments.
[0034] Unless otherwise specified, all reagents and instruments used in the following examples are commercially available products. Resins were purchased from Xi'an Lanxiao New Material Technology Co., Ltd., amino acid derivatives from Jier Biochemical (Shanghai) Co., Ltd., and other chemical reagents and solvents were of analytical or chemically pure grade, which can be purchased from reagent platforms of the appropriate grade.
[0035] The abbreviations and their English meanings in this invention are as follows:
[0036] Example 1: Preparation of crude products from illegal forests The preparation process of crude products from illegal forests is described in [link to documentation]. Figure 1 The specific steps are as follows: ① Preparation of terfenadine resin Weigh 5.14 g (1 eq) of 2-CTC Resin (1.05 mmol / g) and add it to a synthesis tube. Add 50 ml of DCM to swell the resin and drain the liquid. Add 4.67 g (2 eq) of Boc-Pip(Fmoc)-OH to an activation flask, dissolve it in 50 ml of DCM, add 2.5 ml (3 eq) of DIEA, activate in an ice bath for 5 min, and then add it to the synthesis tube. React at 25 °C for 2.0 h. After the reaction is complete, add 2.5 ml (3 eq) of DIEA and 5 ml of MeOH to the synthesis tube, react at 25 °C for 0.5 h, deprotect with 20% Pip / DMF solution for 30 min, wash with DMF 6 times to obtain Boc-Pip(Fmoc)-CTC. Resin; under the action of the HOBT / DIC condensation system (molar ratio of 1:1), Fmoc-D-Lys(Boc)-OH and Boc-D-Phe-D-Phe-D-Leu-OH were sequentially coupled. The protected amino acid monomer, the protected peptide fragment, and the HOBT / DIC condensation system were all added at a molar equivalent of 2-CTC Resin. DMF was added for dissolution, the reaction temperature was controlled at 25℃, the reaction time was 2.0 h, and the reaction progress was monitored by Kaiser colorimetric method. After the reaction was completed, the resin was washed 6 times with DMF. After the coupling was completed, the resin was shrunk with methanol and dried under vacuum at 30℃ to obtain 9.19 g of des Falin peptide resin.
[0037] ② Preparation of crude terfenadine At room temperature, 9.19 g of difenacin peptide resin was weighed and added to a round-bottom flask. A lysis buffer of TFA:Tis:H2O = 9:0.5:0.5 (V:V) was prepared in 100 ml (peptide resin reaction concentration: 10 ml / g). The buffer was added to the round-bottom flask, stirred at low temperature for 30 min, and then stirred at 30°C for 150 min. The mixture was filtered, and the filtrate was concentrated to obtain a concentrate. 5 V MTBE was added for precipitation. The precipitate was centrifuged to obtain a white solid, which was dried under vacuum at 30°C to obtain 3.62 g of crude difenacin, with a yield of 98.64% and a purity of 91.946%.
[0038] Example 2: Investigation of different lysis buffer formulations in the preparation process of crude forest products ① Preparation of terfenadine resin Weigh 38.31 g (1 eq) of 2-CTC Resin (1.05 mmol / g) and add it to a synthesis tube. Add 400 ml of DCM to swell the resin and drain the liquid. Add 28.17 g (1.5 eq) of Boc-Pip(Fmoc)-OH to an activation flask, dissolve it in 400 ml of DCM, add 21 ml (3 eq) of DIEA, activate in an ice bath for 5 min, and then add it to the synthesis tube and react at 25 °C for 2.0 h. After the reaction is complete, add 21 ml (3 eq) of DIEA and 40 ml of MeOH to the synthesis tube, react at 25 °C for 0.5 h, deprotect with 20% Pip / DMF solution for 30 min, wash with DMF 6 times to obtain Boc-Pip(Fmoc)-CTC. Resin; under the action of the HOBT / DIC condensation system (molar ratio of 1:1), Fmoc-D-Lys(Boc)-OH and Boc-D-Phe-D-Phe-D-Leu-OH were sequentially coupled. The protected amino acid monomer, the protected peptide fragment, and the HOBT / DIC condensation system were all added at a molar equivalent of 2-CTC Resin. DMF was added for dissolution, the reaction temperature was controlled at 25℃, the reaction time was 2.0 h, and the reaction progress was monitored by Kaiser colorimetric method. After the reaction was completed, the resin was washed 6 times with DMF. After the coupling was completed, the resin was shrunk with methanol and dried under vacuum at 30℃ to obtain 74.13 g of des Falin peptide resin.
[0039] The crude difalinin peptide was prepared from the difalinin peptide resin obtained by the above method using different lysis buffers, as follows: ② Preparation of crude terfenadine At room temperature, 5.05 g of difenacin peptide resin was weighed and added to a round-bottom flask. A lysis buffer of TFA:Tis:H2O = 9:0.5:0.5 (V:V) was prepared in 50 ml (peptide resin reaction concentration: 10 ml / g). The buffer was added to the round-bottom flask, stirred at low temperature for 30 min, and then stirred at 30℃ for 150 min. The mixture was filtered, and the filtrate was concentrated to obtain a concentrate. 5 V MTBE was added for precipitation. The precipitate was centrifuged to obtain a white solid. The solid was dried under vacuum at 30℃ to obtain 1.76 g of crude difenacin, with a yield of 95.05% and a purity of 97.86%.
[0040] ③ Preparation of crude terfenadine At room temperature, 5.05 g of difenacin peptide resin was weighed and added to a round-bottom flask. 50 ml of lysis buffer (TFA:H2O = 9:1 (V:V)) was prepared (peptide resin reaction concentration: 10 ml / g). The solution was added to the round-bottom flask, stirred at low temperature for 30 min, then stirred at 30°C for 150 min. The mixture was filtered, and the filtrate was concentrated to obtain a concentrate. 5 V MTBE was added for precipitation. The precipitate was centrifuged to obtain a white solid. The solid was dried under vacuum at 30°C to obtain 1.02 g of crude difenacin, with a yield of 55.09% and a purity of 86.64%.
[0041] ④ Preparation of crude terfenadine At room temperature, 5.05 g of difenacin peptide resin was weighed and added to a round-bottom flask. A lysis buffer of TFA:H2O:PhSMe = 9:0.5:0.5 (V:V) was prepared in 50 ml (peptide resin reaction concentration: 10 ml / g). The buffer was added to the round-bottom flask, stirred at low temperature for 30 min, and then stirred at 30°C for 150 min. The mixture was filtered, and the filtrate was concentrated to obtain a concentrate. 5 V MTBE was added for precipitation. The precipitate was centrifuged to obtain a white solid. The solid was dried under vacuum at 30°C to obtain 0.95 g of crude difenacin, with a yield of 51.31% and a purity of 89.90%.
[0042] This embodiment uses the controlled variable method, with terafolin peptide resin prepared by the same process as raw material, to compare the effects of three different lysis buffer systems. The results show that the lysis buffer combination of TFA:Tis:H2O=9:0.5:0.5 (V:V) is the optimal scheme, with a crude product yield of 95.05% and a purity of 97.86%, significantly better than the other two groups. This is because, during the peptide resin lysis process, the resin linkage bonds break, and the removal of the Boc side chain protecting group generates a large number of active intermediates such as carbocations and alkyl cations. In group ③, only water is used as a cation scavenger, which cannot fully quench the large number of carbocations in the system. These carbocations are prone to covalent modification side reactions with the peptide chain, which not only reduces the product yield, but also makes it easy for the tert-butyl group generated by Boc removal to recouple with the peptide chain, further reducing the product purity. In group ④, phenyl methyl sulfide (PhSMe) is added. This reagent mainly has a good stabilizing effect on benzyl cations, but its tert-butyl cation scavenging effect is poor, and it cannot effectively quench the tert-butyl cation. Effective removal of reactive cations is achieved, but 2-CTC resin is highly sensitive to acidic environments. Prolonged exposure to a strongly acidic pyrolysis system can lead to excessive pyrolysis, generating a large number of peptide chain fragments, reducing the proportion of the target product, and ultimately causing a simultaneous decrease in yield and purity. In contrast, group ② simultaneously adds water and triisopropylsilane (Tis). Water can quench carbocations through nucleophilic interaction to generate neutral alcohol molecules, while Tis, as a hydrogen donor, can quench carbocations through reduction to generate alkane molecules. The resulting small molecule impurities can be effectively dissolved and removed during the precipitation of methyl tert-butyl ether, thereby significantly improving the yield and purity of the target product.
[0043] Example 3: Investigation of different starting material input amounts in the preparation process of crude products from illegal forests Boc-Pip(Fmoc)-OH is a non-natural amino acid, resulting in high raw material procurement costs. If the feed equivalent is too low, incomplete reaction at the resin sites can easily occur, leading to the formation of missing peptide impurities and reducing product purity. Conversely, blindly increasing the feed equivalent does not significantly improve product purity but instead greatly increases raw material consumption and industrial production costs. Therefore, considering both the synthesis reaction efficiency and industrial production economics, this embodiment systematically investigates and screens the optimal feed equivalent and reasonable upper limit of Boc-Pip(Fmoc)-OH.
[0044] ① Preparation of terfenadine resin At room temperature, 4.86 g, 4.87 g, and 4.88 g of 2-CTC Resin (1.05 mmol / g) were weighed and added to synthesis tubes, followed by 50 ml of DCM to swell the resin, and the liquid was drained. 2.35 g (molar ratio of Boc-Pip(Fmoc)-OH to 2-CTC Resin was 1:1), 3.57 g (molar ratio of Boc-Pip(Fmoc)-OH to 2-CTC Resin was 1.5:1), and 4.67 g (molar ratio of Boc-Pip(Fmoc)-OH to 2-CTC Resin was 2:1) were weighed and added to activation flasks, dissolved in 50 ml of DCM, and then 2.5 ml (3.0 eq) of DIEA was added. The mixture was activated in an ice bath for 5 min, then added to synthesis tubes and reacted at 25 °C for 2.0 h. After the reaction was complete, DIEA (3.0 eq) and MeOH were added to the synthesis tubes. 5 ml of the solution was reacted at 25°C for 0.5 h, followed by deprotection with 20% Pip / DMF solution for 30 min. The mixture was washed 6 times with DMF to obtain Boc-Pip(Fmoc)-CTC Resin. Fmoc-D-Lys(Boc)-OH and Boc-D-Phe-D-Phe-D-Leu-OH were sequentially coupled in a HOBT / DIC condensation system (molar ratio 1:1). The protected amino acid monomer, protected peptide fragment, and HOBT / DIC condensation system were all added at a molar equivalent of 2.0 times that of 2-CTC Resin. DMF was added for dissolution, and the reaction temperature was controlled at 25°C for 2.0 h. The reaction progress was monitored using the Kaiser method. After the reaction was complete, the mixture was washed 6 times with DMF. The resin was then shrunk with methanol and vacuum dried at 30°C to obtain 6.28 g, 9.07 g, and 8.70 g of des Falin peptide resin.
[0045] ② Preparation of crude terfenadine A lysis buffer of TFA:Tis:H2O = 9:0.5:0.5 (V:V) was prepared. 62 ml, 90 ml, and 87 ml of the above-mentioned amounts of terfenadine peptide resin (reaction concentration: 10 ml / g) were added at room temperature. The mixture was stirred at low temperature for 30 min, then stirred at 30℃ for 150 min. The mixture was filtered, and the filtrate was concentrated to obtain a concentrate. MTBE at 5V was added for precipitation. The precipitate was centrifuged to obtain a white solid. The solid was vacuum dried at 30℃ to obtain 1.95 g, 3.28 g, and 2.95 g of crude terfenadine, with yields of 57.40%, 96.56%, and 86.84%, and purities of 60.63%, 95.25%, and 95.92%, respectively.
[0046] As can be seen from the above, in liquid-phase condensation systems, reactants are uniformly dispersed and molecules collide freely, allowing for complete reaction with a 1:1 equivalent ratio. In contrast, solid-phase condensation systems are heterogeneous reactions, with the amino terminus immobilized on the resin surface, resulting in restricted molecular diffusion, significant local steric hindrance, and a significantly lower overall reaction rate. Therefore, appropriately increasing the concentration of the carboxyl component in the liquid phase is an effective technique for improving the efficiency of solid-phase coupling reactions. Simultaneously, industrial production must prioritize material cost control. Boc-Pip(Fmoc)-OH is a non-natural amino acid with high synthesis and procurement costs; therefore, clearly defining its appropriate upper limit for the equivalent ratio is of significant practical importance for cost reduction and efficiency improvement in industrial applications. Product data shows that when the feed amount is 1.0 equivalent, the reaction is incomplete, easily generating a large number of missing peptide impurities, resulting in low purity and yield of the crude product. Feeding amounts of 1.5 equivalent and 2.0 equivalent yield roughly the same purity of the crude product. Further increasing the amount of Boc-Pip (Fmoc)-OH will increase side reactions and complicate the system, thus reducing the condensation reaction efficiency and causing a decrease in the crude product yield. Considering both reaction performance and production costs, 1.5 equivalent is the optimal feed ratio.
[0047] To verify the stability, repeatability, and adaptability of the preparation process of this invention under different industrial production scales, the following examples were examined through scale-up experiments.
[0048] Example 4: Preparation of 50 mmol-level non-felting forest Weigh 46.785 g (1 eq) of 2-CTC Resin (1.07 mmol / g) and add it to a synthesis tube. Add 500 ml of DCM to swell the resin and drain the liquid. Add 35.021 g (1.5 eq) of Boc-Pip(Fmoc)-OH to an activation flask, dissolve it in 500 ml of DCM, add 38.935 g of DIEA, activate in an ice bath for 5 min, and add to the synthesis tube. React at 15-25°C for 2.0-2.5 h. After the reaction is complete, add 39.025 g of DIEA and 50 ml of MeOH to the synthesis tube and react at 15-25°C for 0.5-1.5 h. Deprotect the product by reacting with 20% Pip / DMF solution for 30 min, and wash 6 times with DMF to obtain Boc-Pip(Fmoc)-CTC. Resin; under the action of the HOBT / DIC condensation system (molar ratio of 1:1), Fmoc-D-Lys(Boc)-OH and Boc-D-Phe-D-Phe-D-Leu-OH were sequentially coupled. The protected amino acid monomer, protected peptide fragment, and HOBT / DIC condensation system were all added at a molar equivalent of 2-CTC Resin. DMF was added for dissolution, and the reaction temperature was controlled at 15–25 °C for 2.0–2.5 h. The reaction progress was monitored using the Kaiser colorimetric method. After the reaction was completed, the resin was washed 6 times with DMF. After coupling, the resin was shrunk with methanol and dried under vacuum at 30 °C to obtain 90.8 g of des-folin peptide resin.
[0049] Take 90.79g of TFA:Tis:H2O=90:5:5(v / v) and prepare 900ml of lysis buffer. Add the peptide resin to the lysis buffer and stir for 30min at 0-10℃. Then, raise the temperature to 15-30℃ and stir for 120-150min. Filter to separate the resin. Wash the peptide resin with 30ml of lysis buffer, filter, combine the filtrates, and concentrate by rotary evaporation at 30℃ to obtain 420ml of concentrate.
[0050] 2100 ml of MTBE was pre-cooled to below 0°C. 420 ml of the concentrate was slowly added to the MTBE, and the mixture was stirred until sedimentation, resulting in a white emulsion. The solid was separated using a refrigerated centrifuge. The solid was washed twice with 420 ml of MTBE each time, and then separated again using a refrigerated centrifuge. The solid was then vacuum-dried at 30°C to obtain 43.4 g of crude *Diplophora fargesii*, with a purity of 90.961% and a yield of 127.6% (theoretical yield 34.0 g). The HPLC chromatogram of the crude *Diplophora fargesii* is shown below. Figure 2 LCMS detection graph is shown below. Figure 3 .
[0051] 43.4 g of crude difenamarene was dissolved in 1.6 L of 0.1% AcOH / H2O. After dissolution, the solution was filtered and set aside. Purification was performed by high-performance liquid chromatography (HPLC). The chromatographic column was a UniSil 10-200 C8 10 μm DAC200. The mobile phases were: A: methanol; B: 0.1% acetic acid; gradient: 5% A to 30% A (80 min); flow rate: 800 mL / min. The target peak was collected, and the solution was concentrated by rotary evaporation to obtain the purified solution. The purified solution was then converted to salt. The mobile phases were: A: methanol; B: 50 mM ammonium acetate; gradient: 5% A to 5% A (30 min). The target peak was collected, concentrated, and lyophilized to obtain 19.8 g of difenamarene acetate with a purity of 99.90% and an overall yield of 58.22%.
[0052] Example 5: Preparation of 200 mmol-level non-felting forest Weigh 187.44 g (1 eq) of 2-CTC Resin (1.07 mmol / g) and add it to a synthesis tube. Add 2 L of DCM to swell the resin and drain the liquid. Add 140.67 g (1.5 eq) of Boc-Pip(Fmoc)-OH to an activation flask, add 2 L of DCM to dissolve it, add 155.30 g of DIEA, and activate in an ice bath for 5 min. Add this to the synthesis tube and react at 15-25°C for 2.0-2.5 h. After the reaction is complete, add 155.15 g of DIEA and 200 ml of MeOH to the synthesis tube and react at 15-25°C for 0.5-1.5 h. Deprotect the product by reacting with 20% Pip / DMF solution for 30 min, and wash 6 times with DMF to obtain Boc-Pip(Fmoc)-CTC. Resin; under the action of the HOBT / DIC condensation system (molar ratio of 1:1), Fmoc-D-Lys(Boc)-OH and Boc-D-Phe-D-Phe-D-Leu-OH were sequentially coupled. The protected amino acid monomer, the protected peptide fragment, and the HOBT / DIC condensation system were all added at a molar equivalent of 2-CTC Resin. DMF was added for dissolution, and the reaction temperature was controlled at 15~25℃. The reaction time was 2.0~2.5h. The reaction progress was monitored by Kaiser colorimetric method. After the reaction was completed, the resin was washed 6 times with DMF. After the coupling was completed, the resin was shrunk with methanol and dried under vacuum at 30℃ to obtain 331.44g of des-folin peptide resin.
[0053] Take 326.42g of TFA:Tis:H2O=90:5:5(v / v) and prepare 3.3L of lysis buffer. Add the peptide resin to the lysis buffer and stir for 30min at 0-10℃. Then raise the temperature to 15-30℃ and stir for 120-150min. Filter to separate the resin. Wash the peptide resin with 0.7L of lysis buffer, filter, combine the filtrates, and concentrate by rotary evaporation at 30℃ to obtain 1.2L of concentrate.
[0054] 6L of MTBE was pre-cooled to below 0℃. 1.2L of the concentrate was slowly added to the MTBE, and the mixture was stirred and allowed to settle to obtain a white emulsion. The solid was separated using a refrigerated centrifuge. The solid was washed twice with methyl tert-butyl ether (1.2L each time) and separated again using a refrigerated centrifuge. The solid was then dried under vacuum at 30℃ to obtain 177.28g of crude terfenadine with a purity of 94.864% and a yield of 130.5% (theoretical yield 135.88g).
[0055] 177.28 g of crude difoolin was dissolved in 6 L of 0.1% AcOH / H2O. After dissolution, the solution was filtered and set aside. Purification was performed by high-performance liquid chromatography (HPLC). The chromatographic column was a UniSil 10-200 C8 10 μm DAC200. The mobile phases were: A: methanol; B: 0.1% acetic acid; gradient: 5% A to 30% A (80 min); flow rate: 800 mL / min. The target peak was collected, and the solution was concentrated by rotary evaporation to obtain the purified solution. The purified solution was then converted to salt. The mobile phases were: A: methanol; B: 50 mM ammonium acetate; gradient: 5% A to 5% A (30 min). The target peak was collected, concentrated, and lyophilized to obtain 80.0 g of difoolin acetate with a purity of 99.32% and an overall yield of 58.66%.
[0056] Example 6: Preparation of 6500 mmol-level non-felting forest Weigh 467.71 g (1 eq) of 2-CTC Resin (1.07 mmol / g) and add it to a synthesis tube. Add 5 L of DCM to swell the resin and drain the liquid. Add 350.88 g (1.5 eq) of Boc-Pip(Fmoc)-OH to an activation flask, dissolve it in 5 L of DCM, add 387.43 g of DIEA, and activate in an ice bath for 5 min. Add this to the synthesis tube and react at 15-25°C for 2.0-2.5 h. After the reaction is complete, add 388.30 g of DIEA and 500 ml of MeOH to the synthesis tube and react at 15-25°C for 0.5-1.5 h. Deprotect the product by reacting with 20% Pip / DMF solution for 30 min, and wash 6 times with DMF to obtain Boc-Pip(Fmoc)-CTC. Resin; under the action of the HOBT / DIC condensation system (molar ratio of 1:1), Fmoc-D-Lys(Boc)-OH and Boc-D-Phe-D-Phe-D-Leu-OH were sequentially coupled. The protected amino acid monomer, the protected peptide fragment, and the HOBT / DIC condensation system were all added at a molar equivalent of 2-CTC Resin. DMF was added for dissolution, and the reaction temperature was controlled at 15~25℃. The reaction time was 2.0~2.5h. The reaction progress was monitored by Kaiser colorimetric method. After the reaction was completed, the resin was washed 6 times with DMF. After the coupling was completed, the resin was shrunk with methanol and dried under vacuum at 30℃ to obtain 799.60g of des-folin peptide resin.
[0057] Take 795.00 g of TFA:Tis:H2O = 90:5:5 (v / v) and prepare 8 L of lysis buffer. Add the peptide resin to the lysis buffer and stir for 30 min at 0-10℃. Then, raise the temperature to 15-30℃ and stir for 120-150 min. Filter to separate the resin, wash the peptide resin with 1.5 L of lysis buffer, filter again, combine the filtrates, and concentrate by rotary evaporation at 30℃ to obtain 1.8 L of concentrated solution.
[0058] 9 L of MTBE was pre-cooled to below 0°C. 1.8 L of the concentrated solution was slowly added to the MTBE, and the mixture was stirred and allowed to settle, resulting in a white emulsion. The solid was separated using a refrigerated centrifuge. The solid was washed twice with 1.8 L of MTBE each time, and then separated again using a refrigerated centrifuge. The solid was then vacuum dried at 30°C to obtain 398.96 g of crude difenamarene. The purity was 96.199%, and the yield was 117.4% (theoretical yield 339.71 g).
[0059] 398.96 g of crude difoolin was dissolved in 14 L of 0.1% AcOH / H2O. After dissolution, the solution was filtered and set aside. Purification was performed by high-performance liquid chromatography (HPLC). The chromatographic column was a UniSil 10-200 C8 10 μm DAC200. The mobile phases were: A: methanol; B: 0.1% acetic acid; gradient: 5% A to 30% A (80 min); flow rate: 800 mL / min. The target peak was collected, and the solution was concentrated by rotary evaporation to obtain the purified solution. The purified solution was then converted to salt. The mobile phases were: A: methanol; B: 50 mM ammonium acetate; gradient: 5% A to 5% A (30 min). The target peak was collected, concentrated, and lyophilized to obtain 217.60 g of difoolin acetate with a purity of 99.89% and an overall yield of 63.94%.
[0060] Comparative Example 1: Traditional Preparation Process ① Preparation of terfenadine resin Weigh 5.12 g (1 eq) of 2-CTC Resin (1.05 mmol / g) and add it to a synthesis tube. Add 50 ml of DCM to swell the resin and drain the liquid. Add 4.88 g (2 eq) of Boc-Pip(Fmoc)-OH to an activation flask, dissolve it in 50 ml of DCM, add 2.5 ml (3 eq) of DIEA, activate in an ice bath for 5 min, and then add it to the synthesis tube. React at 25 °C for 2.0 h. After the reaction is complete, add 2.5 ml (3 eq) of DIEA and 5 ml of MeOH to the synthesis tube, react at 25 °C for 0.5 h, deprotect with 20% Pip / DMF solution for 30 min, wash with DMF 6 times to obtain Boc-Pip(Fmoc)-CTC. Resin; under the action of the HOBT / DIC condensation system (molar ratio of 1:1), Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, and Fmoc-Phe-OH were sequentially coupled. The protected amino acid monomers and the HOBT / DIC condensation system were both added at a molar equivalent of 2-CTC Resin. DMF was added for dissolution, and the reaction temperature was controlled at 25℃ for 2.0 h. The reaction progress was monitored by Kaiser colorimetric method. After the reaction was completed, the resin was washed 6 times with DMF. After the coupling was completed, the resin was shrunk with methanol and dried under vacuum at 30℃ to obtain 8.58 g of desfarin peptide resin.
[0061] ② Preparation of crude terfenadine At room temperature, 8.58 g of difaflint peptide resin was weighed and added to a round-bottom flask. 86 ml of a lysis buffer (TFA:Tis:H2O = 9:0.5:0.5 (V:V)) was prepared and added to the round-bottom flask. The mixture was stirred at low temperature for 30 min, then at 30°C for 150 min. The mixture was filtered, and the filtrate was concentrated to obtain a concentrate. 5V MTBE was added for precipitation. The precipitate was centrifuged to obtain a white solid, which was then vacuum dried at 30°C to obtain 3.12 g of crude difaflint, with a yield of 85.48% and a purity of 88.151%.
[0062] Example 1: Analysis of impurities in illegal forests The crude products of ground-fed forests obtained in Example 1 and Comparative Example 1 were analyzed by HPLC (see...). Figure 4 and Figure 5 The impurity levels were analyzed, and the results are shown in Table 1: Table 1
[0063] As shown in Table 1, the crude terrestris obtained by the single amino acid stepwise coupling process in Comparative Example 1 had a purity of only 88.151%, with a total of 30 impurities, of which as many as 15 were greater than 0.5%, indicating a significantly high impurity level. Furthermore, it contained multiple single-point amino acid deletion impurities, such as Des-D-Phe 1&2 Des-D-Phe 1 / 2 Impurities such as Des-D-Leu, Des-Pip, and Des-D-Lys present complex side reactions and varied impurity sources, resulting in poor process controllability. However, the crude product purity of the large-fragment condensation process in Example 1 of this application is improved to 91.946%, nearly 4 percentage points higher than the comparative example; the total number of impurities is reduced to 15, with only 5 impurities exceeding 0.5%, and all impurity contents are controlled below 3.0%. Impurity localization shows that Example 1 only contains Des-Pip, Des-D-Lys, and overall missing tripeptide fragments, with no multi-point dispersed missing impurities, indicating a single and controllable side reaction pattern. These data directly demonstrate that the process of this application can significantly reduce coupling steps, decrease side reactions and impurity accumulation, achieve a substantial increase in crude product purity, and significantly reduce the number and content of impurities, making it more suitable for the quality control and subsequent purification requirements of industrial production.
[0064] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A process for preparing illegal forest land, characterized in that, Includes the following steps: (1) Using resin as a solid support, Boc-Pip(Fmoc)-OH is loaded and reacted with resin to obtain Boc-Pip(Fmoc)-resin. (2) Under the action of a condensing agent, Fmoc-D-Lys(Boc)-OH and Boc-D-Phe-D-Phe-D-Leu-OH are sequentially coupled onto the Boc-Pip(Fmoc)-resin to obtain the terfenadine resin. (3) The lysine peptide resin was lysed using a lysis buffer, and then concentrated, settled, and dried to obtain crude lysine.
2. The preparation process according to claim 1, characterized in that, The specific steps (1) are as follows: the resin is added to the solvent for swelling, Boc-Pip(Fmoc)-OH is added to the solvent for dissolution and then an activator is added. The activated Boc-Pip(Fmoc)-OH is mixed with the swollen resin for a first reaction. After the reaction, a resin end-capping agent is added for a second reaction. After the reaction is completed, a deprotecting agent is used to deprotect the resin, thus obtaining Boc-Pip(Fmoc)-resin.
3. The preparation process according to claim 1, characterized in that, The resin is selected from any one or more of chloromethyl resin, 4-hydroxymethylphenoxymethyl resin, 2-chlorotriphenylmethyl resin, 4-hydroxymethylphenylacetyl resin, p-benzyloxybenzylamine resin, and 9-Fmoc-aminoxanthine-3-yloxypolystyrene resin.
4. The preparation process according to claim 1, characterized in that, In step (1), the molar ratio of Boc-Pip(Fmoc)-OH to resin is (1~2):
1.
5. The preparation process according to claim 1, characterized in that, The condensing agent in step (2) is selected from any one or more of carbodiimides, benzotriazole salts, pyridine benzotriazole salts, and phosphate esters.
6. The preparation process according to claim 1, characterized in that, The molar ratio of the resin to Fmoc-D-Lys(Boc)-OH is 1:(1~5).
7. The preparation process according to claim 1, characterized in that, The molar ratio of the resin to Boc-D-Phe-D-Phe-D-Leu-OH is 1:(1~5).
8. The preparation process according to claim 1, characterized in that, The reaction conditions for step (2) are a reaction temperature of 15~25℃ and a reaction time of 2.0~2.5h.
9. The preparation process according to claim 1, characterized in that, The pyrolysis solution in step (3) comprises trifluoroacetic acid, triisopropylsilane and H2O in a volume ratio of 9:0.5:0.
5.
10. The preparation process according to any one of claims 1 to 9, characterized in that, The steps also include: purifying and converting the crude arborescent material to salt.