Tripeptide compound and preparation method thereof

Abstract

The invention discloses a tripeptide compound and a preparation method thereof, the synthesis route is as follows: the process steps are brief, the used raw materials are cheap and easily available commercially available commodities, the reaction condition is mild, the post-treatment is simple, and the process is expected to effectively reduce the synthesis cost and is beneficial to popularization in the field of polypeptide synthesis.

Description

Technical Field

[0001] This invention belongs to the field of peptide synthesis technology, specifically, it relates to a tripeptide compound with neuroprotective effects and its preparation method. Background Technology

[0002] Pinealon is a tripeptide consisting of a short peptide chain of three amino acids linked by peptide bonds, with the amino acid sequence Glu-Asp-Arg. Current conventional tripeptide synthesis methods, such as chemical synthesis, often employ solid-phase or liquid-phase synthesis techniques, constructing the peptide chain through stepwise coupling of amino acids. Protecting and deprotecting the amino and carboxyl groups of the amino acids is necessary to avoid side reactions. For example, using Fmoc or Boc protecting groups and specific condensation reagents (such as HATU, EDCI, etc.) to achieve peptide bond formation. This method is suitable for the synthesis of well-defined tripeptides, but the steps are cumbersome, the yield is low, and there are many byproducts, limiting its application to small-scale preparations. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the purpose of this invention is to provide an efficient method for preparing tripeptide compounds.

[0004] This invention first discloses a method for preparing a tripeptide compound, the synthetic route of which is as follows:

[0005]

[0006] The specific preparation steps are as follows:

[0007] Preparation of Compound 2 (S1): In a three-necked flask, tert-butyloxycarbonyl-L-glutamic acid-5-tert-butyl ester (compound 1) was dissolved in N,N-dimethylformamide. Stirring was started until complete dissolution. The reaction was cooled to 0-10°C, more preferably 0-5°C, and N,N-diisopropylethylamine was added. Then, a condensing agent was added in batches. The reaction solution was stirred at 0-10°C for 2-3 hours. Then, L-aspartic acid-4-tert-butyl ester was added in batches. The reaction solution was stirred at 0-10°C for 12-15 hours. The consumption of the raw materials was monitored by TLC. The reaction solution was concentrated under reduced pressure using an oil pump to remove most of the solvent. Ethyl acetate was added, and then the solution was washed with water. The organic phase was dried and filtered. The filtrate was concentrated under reduced pressure to obtain a white solid, compound 2, which was directly used in the next step.

[0008] Preferably, tert-butyloxycarbonyl-L-glutamic acid-5-tert-butyl ester undergoes a condensation reaction directly with the amino group and the unprotected L-aspartic acid-4-tert-butyl ester of the amino group and the amino-ortho-carboxylic acid group.

[0009] Preferably, ethyl acetate of twice the volume of the starting material is added to dissolve the concentrate, then the concentrate is washed three times with water of twice the volume of the starting material, washed once with saturated brine, the organic phase is dried, and concentrated under reduced pressure to obtain white solid compound 2.

[0010] Preparation of compound 3 (S2): Compound 2 was dissolved in N,N-dimethylformamide in a three-necked flask and stirred until completely dissolved. The reaction was cooled to 0-10℃ (preferably 0-5℃), and N,N-diisopropylethylamine was added. Then, a condensing agent was added in batches. The reaction solution was stirred at 0-10℃ for 2-3 hours. Then, L-arginine was added in batches, and the reaction solution was stirred at 0-10℃ for 12-15 hours. The consumption of raw materials was monitored by TLC. The reaction solution was concentrated under reduced pressure using an oil pump to remove most of the solvent. Ethyl acetate was added, and the solution was washed with water. The organic phase was dried and filtered. The filtrate was concentrated under reduced pressure to obtain a light white solid compound 3, which was directly used in the next step.

[0011] Preferably, it undergoes a condensation reaction directly with unprotected L-arginine.

[0012] Preferably, ethyl acetate of twice the volume of the starting material is added to dissolve the concentrate, then the concentrate is washed three times with water of twice the volume of the starting material, washed once with saturated brine, the organic phase is dried and filtered, and the filtrate is concentrated under reduced pressure to obtain a light white solid compound 3, which can be directly used in the next step.

[0013] Preparation of compound 4 (S3): Compound 3 was dissolved in dichloromethane at 20-25℃, and then trifluoroacetic acid was added dropwise. The reaction solution was stirred at 20-25℃ for 2-3 hours. After the raw materials were consumed by TLC monitoring, methyl tert-butyl ether was added to the reaction solution, and stirring was continued until fine white solid particles were formed in the reaction solution. The filter cake was collected by filtration to obtain compound 4.

[0014] Preferably, the amount of trifluoroacetic acid used is half the volume of dichloromethane; the amount of methyl tert-butyl ether used is twice the volume of dichloromethane; and the solid is a powder or granular substance.

[0015] Preparation of S4 compound 5: Compound 4 was dissolved in deionized water. Anion exchange resin was weighed, and the aqueous solution of compound 4 was added to the activated anion exchange resin and diluted with deionized water. The mixture was stirred for 3-4 hours and then filtered under reduced pressure. The filtrate was freeze-dried to obtain a white powder of compound 5.

[0016] Preferably, compound 4 is dissolved in 2 times its volume of deionized water, the anion exchange resin is 5 times the weight of compound 4, and the mass of deionized water is 5 times the mass of compound 4.

[0017] Preferably, in step S1, the molar ratio of tert-butyloxycarbonyl-L-glutamic acid-5-tert-butyl ester (compound 1), L-aspartic acid-4-tert-butyl ester, condensing agent and N,N-diisopropylethylamine is 1:(1.0~1.05):(1.08~1.3):(1.1~1.5).

[0018] Preferably, in step S2, the molar ratio of compound 2, L-arginine, condensing agent and N,N-diisopropylethylamine is 1:(1.0-1.5):(1.08-1.8):(1.1-2.5).

[0019] Preferably, in step S1, the reaction temperature is 0-10℃.

[0020] Preferably, in step S2, the reaction temperature is 0-10℃.

[0021] Preferably, in step S3, the reaction temperature is 20-25℃.

[0022] Preferably, in step S1 or step S2, the condensing agent used is one or more of HATU, DCC, EDCI / HOBt, and TBTU. "One or more" in this invention means a combination of one, two, or more.

[0023] Preferably, in step S4, the anion exchange resin activation step is as follows: the anion exchange resin is activated with a 5% sodium hydroxide aqueous solution for 30-40 minutes, and then washed multiple times with deionized water until the wash water is neutral.

[0024] Preferably, in step S4, the filtrate is lyophilized under reduced pressure.

[0025] The present invention also provides a tripeptide compound, which is prepared using the above-described method for preparing tripeptide compounds; its optical purity is greater than 98%.

[0026] The present invention has the following beneficial technical effects:

[0027] ① The synthetic route is simplified. In the peptide bond construction process, this route prioritizes the use of unprotected amino acids. In the first two condensation reactions, the starting material, the carboxylic acid intermediate, directly condenses with amino acids whose amino and ortho-amino carboxylic acids are unprotected, effectively avoiding the selective deprotection step. ② It uses readily available and inexpensive raw materials. This route selects unprotected amino acids from amino and ortho-amino carboxylic acids as reaction intermediates, reducing the use of protecting groups and making the raw materials cheaper and more readily available. ③ The post-processing is simple. In the first two condensation reactions, the amount of solvent and condensing agent used is low, the reaction conditions are mild, and the reaction system is clean. The reaction solution is washed three times with water to effectively remove water-soluble condensing agent impurities, allowing the crude product obtained from the post-processing to be directly used in the next reaction. ④ The entire feeding and reaction process of the condensation reaction is controlled at a low temperature of 0-10 degrees Celsius, which effectively prevents the compound from racemic, and the optical purity of the product is greater than 98%. Attached Figure Description

[0028] Figure 1This is the 1H-NMR spectrum of compound 4 obtained in Example 1;

[0029] Figure 2 This is the 1H-NMR spectrum of compound 5 obtained in Example 1;

[0030] Figure 3 This is the HPLC-SFC chromatogram of compound 5 obtained in Example 1. Detailed Implementation

[0031] The core idea of ​​this invention is as follows: tert-butyloxycarbonyl-L-glutamic acid-5-tert-butyl ester is directly condensed with L-aspartic acid-4-tert-butyl ester with unprotected amino and ortho-carboxyl groups to obtain compound 2. Then, compound 2 is directly condensed with L-arginine with unprotected amino and ortho-carboxyl groups to prepare compound 3. Compound 3 is the precursor of compound 5 with a protecting group. Finally, compound 3 is deprotected to obtain compound 4. Compound 4 is then prepared by anion exchange resin to exchange the TFA group (CF3COO-) of compound 4 with OH-, finally obtaining the salt-free free tripeptide compound 5.

[0032] The synthetic route is as follows:

[0033]

[0034] More preferred synthesis conditions are:

[0035]

[0036] The specific process for preparing the tripeptide compound of the present invention is as follows:

[0037] Step 1: Preparation of Compound 2 (Condensation Reaction)

[0038] tert-Butoxycarbonyl-L-glutamic acid-5-tert-butyl ester (compound 1) was dissolved in DMF. The reaction solution was cooled to 0-10°C, and then a condensing agent (one of the following ratios: HATU, DCC, EDCI / HOBt, TBTU) was added. DIEA was added dropwise, and the mixture was stirred at 0-10°C for 3 hours. Then, L-aspartic acid-4-tert-butyl ester was added in batches, and the reaction was carried out at 0-10°C for 12-15 hours. TLC (DCM:MeOH = 5:1) showed that the starting material was completely consumed. Most of the DMF was recovered by vacuum concentration using an oil pump. The concentrate was dissolved in ethyl acetate, washed three times with water, and washed once with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a white solid compound 2, which could be used directly in the next step without purification.

[0039] Step 2: Preparation of compound 3 (condensation reaction)

[0040] Compound 2 was dissolved in DMF, and the reaction solution was cooled to 0-10°C. Then, a condensing agent (one of the following ratios: HATU, DCC, EDCI / HOBt, TBTU) was added, followed by dropwise addition of DIEA. The mixture was stirred at 0-10°C for 3 hours, and then L-arginine was added in batches. The reaction was carried out at 0-10°C for 12-15 hours. TLC (DCM:MeOH = 5:1) showed that the starting material was completely consumed. Most of the DMF was recovered by vacuum concentration using an oil pump. The concentrate was dissolved in ethyl acetate, washed three times with water, and washed once with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a light white solid compound 3, which can be used directly in the next step without purification.

[0041] Step 3: Preparation of compound 4 (deprotection)

[0042] Compound 3 was dissolved in DCM. TFA was added dropwise to the above reaction solution at 20-25°C. The reaction solution was stirred at 20-25°C for 3 hours. TLC (DCM:MeOH = 5:1) showed that the starting material was consumed. MTBE was added to the reaction solution and stirred until white fine solid particles were formed. The mixture was filtered, and the filter cake was collected to obtain compound 4, which could be used directly in the next step without purification.

[0043] Step 4: Preparation of compound 5 (free)

[0044] Compound 4 was dissolved in deionized water. The activated anion exchange resin was weighed, and the aqueous solution of compound 4 was added to the anion exchange resin. Deionized water was then added to the mixture, and the mixture was stirred at 20-25°C for 2 hours. The filtrate was collected after filtration and lyophilized to obtain a white powder of free compound 5 with an HPLC purity greater than 98%. The TFA atom (CF3COO-) bound in compound 4 was exchanged for OH-, ultimately yielding a salt-free free tripeptide (compound 5).

[0045] The compound abbreviations in this invention specifically refer to the following compounds: DMF is N,N-dimethylformamide, HATU is 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate, EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, HOBt is 1-hydroxybenzotriazole, TBTU is O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, DCC is dicyclohexylcarbodiimide, DIEA is N,N-diisopropylethylamine, EA is ethyl acetate, TFA is trifluoroacetic acid, MTBE is methyl tert-butyl ether, TLC refers to thin-layer chromatography, DCM is dichloromethane, and MeOH is methanol.

[0046] The above describes the experimental procedure of the present invention. The following is a further description of specific embodiments of the present invention.

[0047] Example 1

[0048] Step 1: Preparation of compound 2 (condensation reaction), the process is as follows:

[0049] Compound 1 (50 g, 164.8 mmol) was dissolved in 250 mL of DMF. The reaction solution was cooled to 0-10 °C, and HATU (67.7 g, 178.0 mmol) was added sequentially, followed by dropwise addition of DIEA (27.7 g, 217.4 mmol). The mixture was stirred at 0-10 °C for 3 h, and then L-aspartic acid-4-tert-butyl ester (31.8 g, 168.1 mmol) was added in portions. The reaction was carried out at 0-10 °C for 12-15 h. TLC (DCM:MeOH = 5:1) showed that the starting material was completely consumed. Most of the DMF was recovered by vacuum concentration using an oil pump. The concentrate was dissolved in 500 mL of EA and washed three times with 400 mL of water and once with 400 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a white solid compound 2 (61 g), which could be used directly in the next step without purification.

[0050] Step 2: Preparation of compound 3 (condensation reaction), the process is as follows:

[0051] Compound 2 (61 g, 128.6 mmol) was dissolved in 300 mL of DMF. The reaction solution was cooled to 0-10 °C, and HATU (73.4 g, 192.9 mmol) was added sequentially, followed by dropwise addition of DIEA (29.9 g, 231.5 mmol). The mixture was stirred at 0-10 °C for 3 h, and then L-arginine (26.9 g, 154.4 mmol) was added in portions. The reaction was carried out at 0-10 °C for 12-15 h. TLC (DCM:MeOH = 5:1) showed that the starting material was completely consumed. Most of the DMF was recovered by vacuum concentration. The concentrate was dissolved in 400 mL of EA and washed three times with 400 mL of water and once with 400 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a light white solid compound 3 (70 g), which could be used directly in the next step without purification.

[0052] Step 3: Preparation of compound 4 (deprotection), the process is as follows:

[0053] Compound 3 (17 g, 27.1 mmol) was dissolved in 90 mL of DCM. TFA (45 mL) was added dropwise to the reaction mixture at 20-25 °C. The reaction mixture was stirred at 20-25 °C for 3 h. TLC (DCM:MeOH = 5:1) showed that the starting material was completely consumed. 180 mL of MTBE was added to the reaction mixture, and the mixture was stirred until fine white particles formed. The mixture was filtered, and the filter cake was collected to obtain compound 4 (14 g), which could be used directly in the next step without purification. Compound 4 was then analyzed to obtain…1 H-NMR spectrum as follows Figure 1 , 1 HNMR (400MHz, D2O), δ4.72 (m, 1H), 4.27 (q, 1H), 3.98 (q, 1H), 3.10 (m, 2H), 2.85-2.65 (m, 2H), 2.45 (m, 2H), 2.10 (m, 2H), 1.78 (m, 1H), 1.70 (m, 1H), 1.48 (m, 2H), MS (ESI) m / z: [M+1]=419.40.

[0054] Step 4: Preparation of compound 5 (free), the process is as follows:

[0055] 14g of compound 4 was dissolved in 20ml of deionized water. 160g of activated anion exchange resin was weighed out, and the aqueous solution of compound 4 was added to the anion exchange resin. Then, 100ml of deionized water was added to the mixture, and the mixture was stirred at 20-25℃ for 2 hours. After filtration, the filtrate was collected and lyophilized to obtain 6g of free compound 5 as a white powder with an HPLC purity of 99.5% and an optical purity greater than 98%. Compound 5 was then analyzed to obtain… 1 H-NMR spectrum as follows Figure 2 , 1 ¹H NMR (400 MHz, D₂O), δ 4.72 (m, 1H), 4.18 (t, 1H), 3.98 (q, 1H), 3.07 (t, 2H), 2.85–2.60 (m, 2H), 2.45 (q, 2H), 2.05 (m, 2H), 1.78 (m, 1H), 1.70 (m, 1H), 1.48 (m, 2H), MS (ESI) m / z: [M+1] = 419.40. Compound five was obtained after detection. Figure 3 The HPLC-SFC chromatogram showed that the main peak (RT = 5.551 min) was the target compound peak, accounting for 99.50% of the area, and the purity met the high purity sample standard; there was only one obvious impurity peak (RT = 8.164 min), accounting for 0.50% of the area, with no other impurity peaks interfering, and the sample purity was uniform.

[0056] Example 2

[0057] Step 1: Preparation of compound 2 (condensation reaction), the process is as follows:

[0058] Compound 1 (50 g, 164.8 mmol) was dissolved in 250 mL of DMF. The reaction solution was cooled to 0-10 °C, and TBTU (57.2 g, 178.0 mmol) was added sequentially, followed by dropwise addition of DIEA (27.7 g, 217.4 mmol). The mixture was stirred at 0-10 °C for 3 h, and then L-aspartic acid-4-tert-butyl ester (31.8 g, 168.1 mmol) was added in portions. The reaction was carried out at 0-10 °C for 12-15 h. TLC (DCM:MeOH = 5:1) showed that the starting material was completely consumed. Most of the DMF was recovered by vacuum concentration using an oil pump. The concentrate was dissolved in 500 mL of EA and washed three times with 400 mL of water and once with 400 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a white solid compound 2 (59 g), which could be used directly in the next step without purification.

[0059] Step 2: Preparation of compound 3 (condensation reaction), the process is as follows:

[0060] Compound 2 (59 g, 124.4 mmol) was dissolved in 300 mL of DMF. The reaction solution was cooled to 0-10 °C, and TBTU (59.9 g, 186.6 mmol) was added sequentially, followed by dropwise addition of DIEA (28.9 g, 223.9 mmol). The mixture was stirred at 0-10 °C for 3 h, and then L-arginine (25.9 g, 149.3 mmol) was added in portions. The reaction was carried out at 0-10 °C for 12-15 h. TLC (DCM:MeOH = 5:1) showed that the starting material was completely consumed. Most of the DMF was recovered by vacuum concentration using an oil pump. The concentrate was dissolved in 500 mL of EA and washed three times with 400 mL of water and once with 400 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a light white solid compound 3 (68 g), which could be used directly in the next step without purification.

[0061] Step 3: Preparation of compound 4 (deprotection), the process is as follows:

[0062] Compound 3 (12 g, 19.1 mmol) was dissolved in 60 ml of DCM. TFA (30 ml) was added dropwise to the above reaction solution at 20-25 °C. The reaction solution was stirred at 20-25 °C for 3 h. TLC (DCM:MeOH = 5:1) showed that the starting material was consumed. 120 ml of MTBE was added to the reaction solution and stirred until fine white solid particles were formed. The mixture was filtered, and the filter cake was collected to obtain compound 4 (8 g), which could be used directly in the next step without purification.

[0063] Step 4: Preparation of compound 5 (free), the process is as follows:

[0064] Dissolve 6g of compound 4 in 20ml of deionized water, add 10% sodium bicarbonate solution to bring the pH of the solution to 7.5 (measured with a pH meter), add the free compound 4 solution to a dialysis membrane (100-500 Da), clamp both ends with a rack, place 500ml of deionized water in a beaker, place the dialysis membrane containing the compound 5 solution into the beaker, and stir with a magnetic stirrer. Change the water for the first time after 4 hours, and then change the water every 6-8 hours thereafter, for a total of 4 deionized water changes. Then pour out the aqueous solution from the dialysis bag, freeze-dry to obtain 2.0g of free compound 5 white powder with HPLC purity greater than 98% and optical purity greater than 98%.

Claims

1. A method for preparing a tripeptide compound, characterized in that, The synthetic route is as follows: The specific preparation steps are as follows: Preparation of compound 2 (S1): Compound 1 was dissolved in N,N-dimethylformamide in a three-necked flask and stirred until completely dissolved. The reaction was cooled, and N,N-diisopropylethylamine was added. Then, a condensing agent was added in batches, and the reaction solution was stirred for 2-3 hours. Then, L-aspartic acid-4-tert-butyl ester was added in batches, and the reaction solution was stirred for 12-15 hours. The consumption of the raw materials was monitored by TLC. The reaction solution was concentrated under reduced pressure using an oil pump to remove most of the solvent. Ethyl acetate was added, and then the solution was washed with water. The organic phase was dried, filtered, and concentrated under reduced pressure to obtain compound 2. Preparation of compound 3 (S2): Compound 2 was dissolved in N,N-dimethylformamide in a three-necked flask and stirred until completely dissolved. The reaction was cooled, and N,N-diisopropylethylamine was added. Then, a condensing agent was added in batches, and the reaction solution was stirred for 2-3 hours. L-arginine was then added in batches, and the reaction solution was stirred for 12-15 hours. The consumption of the raw materials was monitored by TLC. The reaction solution was concentrated under reduced pressure using an oil pump to remove most of the solvent. Ethyl acetate was added, and the solution was washed with water. The organic phase was dried, filtered, and concentrated under reduced pressure to obtain compound 3. Preparation of compound 4 of S3: Compound 3 was dissolved in dichloromethane in a three-necked flask, and then trifluoroacetic acid was added dropwise. After the addition was complete, the reaction solution was stirred for 2-3 hours. TLC was used to monitor the consumption of the raw materials. Methyl tert-butyl ether was added to the reaction solution, and the mixture was stirred until white fine solid particles were formed in the reaction solution. The filter cake was collected by filtration to obtain compound 4. Preparation of compound 5 (S4): Compound 4 was dissolved in deionized water in a beaker. Anion exchange resin was weighed, and the aqueous solution of compound 4 was added to the activated anion exchange resin. Deionized water was added to dilute the solution, and the mixture was stirred for 3-4 hours. The mixture was then filtered under reduced pressure, and the filtrate was freeze-dried to obtain compound 5.

2. The preparation process of compound 1 according to claim 1, characterized in that, In step S1, the molar ratio of compound 1, L-aspartic acid-4-tert-butyl ester, condensing agent and N,N-diisopropylethylamine is 1:(1.0~1.05):(1.08~1.3):(1.1~1.5).

3. The method for preparing the tripeptide compound according to claim 1, characterized in that, In step S2, the molar ratio of compound 2, L-arginine, condensing agent and N,N-diisopropylethylamine is 1:(1.0-1.5):(1.08-1.8):(1.1-2.5).

4. The method for preparing the tripeptide compound according to claim 1, characterized in that, In step S1, the reaction temperature is 0-10℃.

5. The method for preparing the tripeptide compound according to claim 1, characterized in that, In step S2, the reaction temperature is 0-10℃.

6. The method for preparing the tripeptide compound according to claim 1, characterized in that, In step S3, the reaction temperature is 20-25℃.

7. The method for preparing the tripeptide compound according to claim 1, characterized in that, In step S1 or step S2, the condensing agent used is one or more of HATU, DCC, EDCI / HOBt, and TBTU.

8. The method for preparing the tripeptide compound according to claim 1, characterized in that, In step S4, the anion exchange resin activation step is as follows: the anion exchange resin is activated with a 5% sodium hydroxide aqueous solution for 30-40 minutes, and then washed multiple times with deionized water until the wash water is neutral.

9. The method for preparing the tripeptide compound according to claim 1, characterized in that, In step S4, vacuum filtration is performed using a dialysis membrane.

10. A tripeptide compound, characterized in that, The tripeptide compound was prepared using the preparation method of any one of claims 1-9, and its optical purity was greater than 98%.

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