A preparation method of polypeptide Epitalone acetate

Abstract

The present invention relates to the field of pharmaceutical technology, and in particular to a method for preparing a polypeptide Epitalone acetate. The specific technical scheme is as follows: the steps are as follows: (1) using HATU as a condensing agent, N-Boc-L-aspartic acid-4-benzyl ester and glycine benzyl hydrochloride are subjected to amidation reaction to obtain an intermediate compound 4; (2) removing the Boc protecting group of compound 4 with trifluoroacetic acid, and reacting with Boc-L-glutamic acid-O-benzyl 5 to obtain an intermediate compound 6; (3) after removing the Boc protecting group of compound 6 with trifluoroacetic acid, a condensation reaction is carried out with N-Cbz-L-alanine 7 to obtain an intermediate compound 8; (4) removing Cbz and three benzyl protecting groups of compound 8 by a palladium carbon hydrogenation method under acidic conditions to obtain the target compound Epitalone acetate. The synthetic route disclosed in the present invention is easy to operate, has good stereoselectivity, has no racemization problem in acid amine condensation, has mild reaction conditions, is simple to separate and purify, has a high overall yield, and can be prepared in an amplified manner.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a method for preparing polypeptide Epitalone acetate. Background Art

[0002] Epitalon is a synthetic polypeptide composed of four amino acid chains. It has three chiral centers. Its chemical name is Ala-Glu-Asp-Gly and its chemical structure is as follows.

[0003]

[0004] Epitalon is a cutting-edge biotechnology product that was originally discovered by Russian scientist Professor Vladimir Khavinson. Since then, it has attracted widespread attention and research in academia and medicine. It is not only a highly effective anti-aging agent, but also has been proven to be a telomerase activator with significant effects. This feature makes it show great potential in fighting against body aging and promoting cell health. Telomerase is a key factor in maintaining chromosome stability and cell lifespan. Epitalon can effectively extend the cell division cycle by activating telomerase, thereby slowing down the cell aging process and providing the body with more lasting vitality.

[0005] Epitalon has also demonstrated extraordinary capabilities in anti-tumor research. Experimental data show that it has a significant inhibitory effect on the development of spontaneous tumors in mice. This discovery has opened up new ideas for cancer treatment, especially in preventing tumor occurrence and delaying tumor progression. Epitalon may become a new type of treatment and bring good news to patients. Its geroprotective effect is not only reflected in its inhibitory effect on tumors, but also in its ability to comprehensively improve the body's physiological functions and enhance immunity; in addition, the scope of application of Epitalon goes far beyond this. Epitalon is also used as an auxiliary in cancer treatment, can significantly reduce the side effects of chemotherapy and radiotherapy, and improve patients' survival rate and quality of life.

[0006] In the field of anti-aging, Epitalon is regarded as a revolutionary product. Endogenous antioxidants such as Epitalon are polypeptides secreted by the pineal gland, which can significantly delay aging and keep tissue cells young by activating cell telomerase. Epitalon can improve the reproductive function of aged rats by improving the testosterone level in the blood of male rats and restoring the estrus cycle of female rats. At the same time, Epitalon can also protect the functional integrity of the retina of Campbell rats, improve the visual function of patients with pigmentary retinal degeneration, and show good prospects in the treatment of pigmentary retinitis, bringing new hope to this disease, which currently has no effective treatment.

[0007] In summary, Epitalon, as a multifunctional bioactive substance, has broad application prospects in anti-aging, anti-tumor and treatment of retinitis pigmentosa. With the continuous deepening of research and the continuous maturity of technology, it is believed that it will make greater contributions to human health in the future.

[0008] At present, there are very few documents on the synthesis of Epitalone. Only a patent report by Vladimir Khavinson (WO2002090380) published its synthesis route, but the patent does not have any relevant nuclear magnetic characterization data (NMR). This route uses amino acids to react with NHS active esters to form amide bonds to obtain intermediates A and B; then the condensation reagent DCC is used to undergo a condensation reaction to form an amide bond to obtain intermediate C; finally, the Cbz and benzyl protecting groups are removed by palladium carbon hydrogenation to obtain the target compound Epitalone. This synthetic route has a high total yield and is simple to operate, but there are also some thorny problems in pilot-scale amplification: 1) A large amount of DMF must be used as a solvent in the reaction, which is troublesome to post-process; 2) Intermediates A and B are both acids with very high polarity, which are difficult to purify and characterize; 3) The purity of the target crude product may not be very high, and the literature reports that HPLC preparation and purification are required. Therefore, how to find a simple, fast, and large-scale synthetic route is of great significance for further biological research on Epitalone and its application in clinical practice as soon as possible. Summary of the invention

[0009] In view of the deficiencies in the prior art, the present invention provides a method for preparing polypeptide Epitalone acetate.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0011] The present invention discloses a method for preparing polypeptide Epitalone acetate, and the steps are as follows:

[0012] (1) using HATU as a condensation agent, N-Boc-L-aspartic acid-4-benzyl ester and glycine benzyl ester hydrochloride were subjected to an amidation reaction to obtain an intermediate compound 4;

[0013] (2) removing the Boc protecting group of compound 4 with trifluoroacetic acid, and reacting with Boc-L-glutamic acid-O-benzyl 5 to obtain the intermediate compound 6;

[0014] (3) After removing the Boc protecting group of compound 6 with trifluoroacetic acid, the compound 6 undergoes a condensation reaction with N-Cbz-L-alanine 7 to obtain the intermediate compound 8;

[0015] (4) The Cbz and three benzyl protecting groups of compound 8 were removed by palladium-carbon hydrogenation under acidic conditions to obtain the target compound epitalone acetate.

[0016] Preferably, the specific process of step (1) is: dissolving N-Boc-L-aspartic acid-4-benzyl ester and glycine benzyl ester hydrochloride in anhydrous dichloromethane, adding HATU under nitrogen protection, cooling to 0° C. in an ice-water bath, adding DIPEA dropwise, and heating to room temperature after the addition is completed, stirring and reacting to obtain compound 4.

[0017] Preferably, the molar ratio of N-Boc-L-aspartic acid-4-benzyl ester, glycine benzyl ester hydrochloride, HATU and DIPEA is 1:1.2-1.5:1.2-3.0:3.0-5.0, and the reaction time is 12-16 hours.

[0018] Preferably, the specific process of step (2) is: (a) dissolving compound 4 in anhydrous dichloromethane, adding trifluoroacetic acid, stirring and reacting at room temperature, and concentrating under reduced pressure to obtain trifluoroacetate; (b) dissolving trifluoroacetate and Boc-L-glutamic acid-O-benzyl in anhydrous dichloromethane, adding HATU under nitrogen protection, cooling to 0°C in an ice-water bath, adding DIPEA dropwise, and heating to room temperature after the addition to stir and react to obtain compound 6.

[0019] Preferably, the molar ratio of compound 4, trifluoroacetic acid, Boc-L-glutamic acid-O-benzyl, HATU and DIPEA is 1:10-50:0.9-1.0:1.2-3.0:3.0-5.0; the reaction time in step (a) is 2-5 hours, and the reaction time in step (b) is 12-16 hours.

[0020] Preferably, the specific process of step (3) is: (a) dissolving compound 6 in anhydrous dichloromethane, adding trifluoroacetic acid, stirring and reacting at room temperature, and concentrating under reduced pressure to obtain trifluoroacetate; (b) dissolving trifluoroacetate and N-Cbz-L-alanine in anhydrous dichloromethane, adding HATU under nitrogen protection, cooling to 0°C in an ice-water bath, adding DIPEA dropwise, and heating to room temperature after the addition to react and stirring to obtain compound 8.

[0021] Preferably, the molar ratio of compound 6, trifluoroacetic acid, N-Cbz-L-alanine, HATU and DIPEA is 1:10-50:0.9-1.0:1.2-3.0:3.0-5.0; the reaction time in step (a) is 2-5 hours, and the reaction time in step (b) is 12-16 hours.

[0022] Preferably, the specific process of step (4) is: dissolving compound 8 in a mixed solvent of ethyl acetate, methanol and water, adding acetic acid and 10% palladium carbon catalyst, evacuating the air three times, and rapidly stirring the reaction at room temperature under a hydrogen pressure of 1 atm to obtain the target compound.

[0023] Preferably, the molar ratio of the compound 8 to acetic acid is 1:4.0-10.0; and the reaction time is 14-20 hours.

[0024] The present invention has the following beneficial effects:

[0025] 1. The synthetic route disclosed in the present invention meets the standards of green chemistry: such as utilizing renewable resources, being recyclable, using reagents with low toxicity, and having little environmental pollution after the reaction is processed; in addition, the overall yield is high, the stereoselectivity is good, the experimental operation is convenient, the reaction conditions are mild, the separation and purification are simple, and it can be used for large-scale preparation.

[0026] 2. The route disclosed by the present invention is easy to operate, has good stereoselectivity, has no racemization problem in acid-amine condensation, has mild reaction conditions, is simple to separate and purify, has a high total yield, and can be prepared in large quantities. The raw materials and reagents used are non-toxic, cheap and easily available, the production process is pollution-free and environmentally friendly, and creates good conditions for industrial-scale production and commercialization of the product. DETAILED DESCRIPTION

[0027] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0029] The present invention discloses a method for preparing polypeptide Epitalone acetate, and the synthetic route is as follows:

[0030]

[0031] The specific steps are as follows:

[0032] (1) Synthesis of Compound 4

[0033]

[0034] N-Boc-L-aspartic acid-4-benzyl ester and glycine benzyl ester hydrochloride were dissolved in anhydrous dichloromethane (DCM), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added under nitrogen protection. After cooling to 0°C in an ice-water bath, diisopropylethylamine (DIPEA) was added dropwise. After 30 minutes of addition, the temperature was raised to room temperature and stirred to react for 12 to 16 hours, preferably 14 hours. After the reaction was completed, water was added to quench the reaction, and dichloromethane was extracted. The organic phase was washed with 1M dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine in sequence. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. Rapid column chromatography using petroleum ether:ethyl acetate = 2:1 as the eluent gave a colorless oil, namely compound 4.

[0035] The molar ratio of N-Boc-L-aspartic acid-4-benzyl ester, glycine benzyl hydrochloride, HATU and DIPEA is 1:1.2-1.5:1.2-3.0:3.0-5.0.

[0036] Further preferably, the molar ratio of N-Boc-L-aspartic acid-4-benzyl ester, glycine benzyl hydrochloride, HATU and DIPEA is 1:1.2:1.5:3.0.

[0037] (2) Synthesis of Compound 6

[0038]

[0039] (2.1) Compound 4 was dissolved in anhydrous dichloromethane (DCM), trifluoroacetic acid (TFA) was added, and the mixture was stirred at room temperature for 2 to 5 h, preferably 3 h, and concentrated under reduced pressure to obtain the intermediate trifluoroacetate;

[0040] (2.2) The above intermediate trifluoroacetate and Boc-L-glutamic acid-O-benzyl (Compound 5) were dissolved in anhydrous dichloromethane (DCM), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added under nitrogen protection. After cooling to 0°C in an ice-water bath, diisopropylethylamine (DIPEA) was added dropwise. After 30 minutes of addition, the temperature was raised to room temperature and stirred for reaction for 12 to 16 hours (preferably 14 hours). After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phase was washed with 1M dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine in sequence. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was subjected to rapid column chromatography using petroleum ether:ethyl acetate = 1:1 as the eluent to obtain a white solid, i.e., Compound 6.

[0041] Wherein, the molar ratio of the compound 4, trifluoroacetic acid, Boc-L-glutamic acid-O-benzyl, HATU and DIPEA is 1:10-50:0.9-1.0:1.2-3.0:3.0-5.0.

[0042] Further preferably, the molar ratio of compound 4, trifluoroacetic acid, Boc-L-glutamic acid-O-benzyl, HATU and DIPEA is 1:20:0.95:1.5:3.0.

[0043] (3) Synthesis of Compound 8

[0044]

[0045] (3.1) Compound 6 was dissolved in anhydrous dichloromethane (DCM), trifluoroacetic acid (TFA) was added, and the mixture was stirred at room temperature for 2 to 5 h, preferably 3 h, and concentrated under reduced pressure to obtain the intermediate trifluoroacetate;

[0046] (3.2) The above intermediate trifluoroacetate and N-Cbz-L-alanine (Compound 7) were dissolved in anhydrous dichloromethane (DCM), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added under nitrogen protection. After cooling to 0°C in an ice-water bath, diisopropylethylamine (DIPEA) was added dropwise. After 30 minutes of addition, the temperature was raised to room temperature and stirred for reaction for 12 to 16 hours (preferably 14 hours). After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phase was washed with 1M dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine in sequence. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was subjected to rapid column chromatography using petroleum ether:ethyl acetate = 2:3 as the eluent to obtain a white solid, i.e., Compound 8.

[0047] Wherein, the molar ratio of the compound 6, trifluoroacetic acid, N-Cbz-L-alanine, HATU and DIPEA is 1:10-50:0.9-1.0:1.2-3.0:3.0-5.0.

[0048] Further preferably, the molar ratio of compound 6, trifluoroacetic acid, N-Cbz-L-alanine, HATU and DIPEA is 1:20:0.95:1.5:3.0.

[0049] (4) Synthesis of Compound 1

[0050]

[0051] Compound 8 was dissolved in a mixed solvent of ethyl acetate, methanol and water (volume ratio of 2:2:1), acetic acid and 10% palladium-carbon catalyst were added, the air was evacuated three times, and the reaction was rapidly stirred at room temperature under a hydrogen pressure of 1 atm for 14 to 20 hours, preferably 16 hours. After the reaction, the hydrogen balloon was removed, and the mixture was directly filtered through diatomaceous earth. The filter residue was washed with water, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was slurried with ethyl acetate and then filtered to obtain a white solid, i.e., compound 1.

[0052] Wherein, the molar ratio of the compound 8 to acetic acid is 1:4.0-10.0, preferably 1:5.0.

[0053] The present invention will be further described below in conjunction with specific embodiments.

[0054] Example 1 Synthesis of Compound 4

[0055] N-Boc-L-aspartic acid-4-benzyl ester (Compound 2) (15.0 g, 46.4 mmol) and glycine benzyl ester hydrochloride (Compound 3) (11.2 g, 55.7 mmol) were dissolved in anhydrous dichloromethane (300 mL), HATU (26.5 g, 69.6 mmol) was added under nitrogen protection, and DIPEA (24.2 mL, 139.2 mmol) was added dropwise after cooling to 0°C in an ice-water bath. The temperature was raised to 40°C 30 minutes after the addition. The reaction was stirred at room temperature for 14 h, and water (200 mL) was added to quench the reaction. Dichloromethane (800 mL) was added for extraction, and the combined organic phase was washed with 1 M dilute hydrochloric acid (200 mL), saturated sodium bicarbonate solution (200 mL), and saturated brine (200 mL) in sequence. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was separated by rapid column chromatography using petroleum ether: ethyl acetate = 2:1 as the eluent to obtain compound 4, 20.7 g of an oily substance, with a yield of 95%.

[0056] The synthesized compound 4 was 1 HNMR, 13 CNMR and HRMS tests show that the product is a pure compound, and its performance indicators or characterization data are as follows:

[0057] (c 0.65, EtOAc); 1 H NMR (600MHz, CDCl3) δ7.38–7.31(m,10H),7.03(s,1H),5.70(d,J=8.1Hz,1H),5.18–5.10(m,4H),4 .59(s,1H),4.05(s,2H),3.06(dd,J=17.1,3.5Hz,1H),2.74(dd,J=17.1,5.7Hz,1H),1.45(s,9H); 13C NMR (151MHz, CDCl3) δ171.62,170.94,169.23,155.48,135.32,135.10,128.60,128.55,1 28.49,128.33,128.24,80.57,67.14,66.82,50.46,41.43,38.56,36.01,28.23; HR-ESIMS m / z:calculated forC 25 H 30 N2O7Na + [M+Na] + :493.1945, found 493.1947.

[0058] Example 2 Synthesis of Compound 6

[0059] Compound 4 (18.0 g, 38.3 mmol) was dissolved in anhydrous DCM (200 mL), TFA (57 mL, 766 mmol) was added, and the mixture was stirred at room temperature for 3 h, and concentrated under reduced pressure to obtain a residue, which was then added with 200 mL of chlorobenzene and concentrated under reduced pressure to obtain the intermediate trifluoroacetate, which was directly used in the next step;

[0060] The above intermediate trifluoroacetate and Boc-L-glutamic acid-O-benzyl (Compound 5) (12.3 g, 36.4 mmol) were dissolved in anhydrous dichloromethane (300 mL), HATU (21.9 g, 57.5 mmol) was added under nitrogen protection, and DIPEA (20.0 mL, 115 mmol) was added dropwise after cooling to 0°C in an ice-water bath. After 30 minutes of addition, the temperature was raised to room temperature and stirred for 14 h. Water (200 mL) was added to quench the reaction, and dichloromethane (800 mL) was added to extract. The combined organic phase was washed with 1M dilute hydrochloric acid (200 mL), saturated sodium bicarbonate solution (200 mL), and saturated brine (200 mL) in sequence. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was separated by rapid column chromatography using petroleum ether: ethyl acetate = 1:1 as the eluent to obtain compound 6 as a white solid of 22.6 g with a yield of 90%.

[0061] The synthesized compound 6 was 1 HNMR, 13 CNMR and HRMS tests show that the product is a pure compound, and its performance indicators or characterization data are as follows:

[0062] (c 0.96, EtOAc); 1H NMR (600MHz, CDCl3) δ7.38–7.31(m,10H),7.03(s,1H),5.70(d,J=8.1Hz,1H),5.18–5.10(m,4H),4 .59(s,1H),4.05(s,2H),3.06(dd,J=17.1,3.5Hz,1H),2.74(dd,J=17.1,5.7Hz,1H),1.45(s,9H); 13 C NMR (151MHz, CDCl3) δ173.21,171.53,171.42,170.38,169.01,155.95,135.53,135.31,135.24,128.50,128.39,128.33 ,128.26,128.24,128.18,80.47,66.90,66.81,66.62,54.51,49.05,41.39,38.53,35.42,30.35,28.19,26.91; HR-ESIMS m / z:calculated for C 37 H 43 N3O 10 Na + [M+Na] + :712.2841, found 712.2843.

[0063] Example 3 Synthesis of Compound 8

[0064] Compound 6 (10.0 g, 14.5 mmol) was dissolved in anhydrous DCM (150 mL), TFA (22 mL, 290 mmol) was added, and the mixture was stirred at room temperature for 3 h, and concentrated under reduced pressure to obtain a residue, which was then added with 200 mL of chlorobenzene and concentrated under reduced pressure to obtain the intermediate trifluoroacetate, which was directly used in the next step;

[0065] The above intermediate trifluoroacetate and N-Cbz-L-alanine (compound 7) (3.1 g, 13.8 mmol) were dissolved in anhydrous dichloromethane (300 mL), HATU (8.3 g, 21.8 mmol) was added under nitrogen protection, and DIPEA (7.6 mL, 43.5 mmol) was added dropwise after cooling to 0°C in an ice-water bath. After 30 minutes of addition, the temperature was raised to room temperature and stirred for 14 h. Water (200 mL) was added to quench the reaction, and dichloromethane (800 mL) was added to extract. The combined organic phase was washed with 1 M dilute hydrochloric acid (200 mL), saturated sodium bicarbonate solution (200 mL), and saturated brine (200 mL) in sequence. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was separated by rapid column chromatography using petroleum ether: ethyl acetate = 2:3 as the eluent to obtain compound 8 as a white solid (9.3 g) with a yield of 85%.

[0066] The synthesized compound 8 was 1 HNMR, 13 CNMR and HRMS tests show that the product is a pure compound, and its performance indicators or characterization data are as follows:

[0067] (c 0.46, EtOAc); 1 H NMR(600MHz, CDCl3)δ7.75(d,J=5.6Hz,1H),7.63(d,J=8.5Hz,1H),7.41–7.26(m,20H),5.4 6(d,J=5.9Hz,1H),5.14–5.05(m,6H),5.05–5.02(m,2H),4.95(d,J=6.3Hz,1H),4.37(dt,J =7.6,6.0Hz,1H),4.15(ddd,J=24.2,15.7,6.5Hz,2H),3.93(dd,J=18.0,5.2Hz,1H),2.96( ddd,J=21.6,16.8,6.0Hz,2H),2.58–2.42(m,2H),2.22–2.04(m,2H),1.36(d,J=7.1Hz,3H); 13 C NMR (151MHz, CDCl3) δ174.28,173.69,171.11,170.76,170.37,169.27,156.43,135.94,135.38,135.28,128.58,128.50,128.39,128.31 ,128.26,128.22,128.16,128.11,128.00,67.22,66.92,66.87,66.67,54.11,51.25,49.48,41.38,35.70,30.43,25.79,18.02; HR-ESIMS m / z:calculatedfor C 43 H 46 N4O 11 Na + [M+Na] + :817.3055, found 817.3058.

[0068] Example 4 Synthesis of Compound 1

[0069] Compound 8 (5.0 g, 6.3 mmol) was dissolved in a mixed solvent (250 mL) of ethyl acetate / methanol / water (2 / 2 / 1), acetic acid (1.8 ml, 31.5 mmol) and 10% palladium-carbon catalyst (3.0 g) were added, the mixture was evacuated and ventilated three times, and the mixture was rapidly stirred at room temperature under a hydrogen pressure of 1 atm for 16 h, the hydrogen balloon was removed, the mixture was directly filtered through diatomaceous earth, the residue was washed with water (200 mL), the filtrate was concentrated under reduced pressure to obtain a crude product, the crude product was then pulped with ethyl acetate (500 mL), and then filtered, and the residue was fully dried to obtain compound 1, a white solid of 2.55 g, with a yield of 90%. The purity of compound 1 measured by HPLC was 96.79%, and the total yield of the entire reaction route was 65.4%.

[0070] The synthesized compound 1 was 1 HNMR, 13 CNMR and HRMS tests show that the product is a pure compound, and its performance indicators or characterization data are as follows:

[0071] (c 0.33,H2O); 1 H NMR(600MHz,D2O)δ4.72(dd,J=7.7,5.4Hz,1H),4.38(dd,J=8.5,5.9Hz,1H),4.10(q,J=7.0Hz,1H),3.84(s,2H),2.87(dd,J=16.6,5. 2Hz,1H),2.76(dd,J=16.6,8.1Hz,1H),2.43(t,J=7.2Hz,2H),2.12(dd,J=13.9,6.6Hz,1H),2.02–1.96(m,1H),1.53(d,J=7.1Hz,3H); 13 C NMR (151MHz, D2O) δ178.03,175.38,175.03,172.86,172.27,170.88,53.36,50.56,48.93,42.62,36.56,30.72,26.21,16.39; HR-ESIMSm / z: calculated for C 14 H 23 N4O9 + [M+H] + :391.1460, found 391.1462.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing polypeptide Epitalone acetate, characterized in that: Here are the steps: (1) using HATU as a condensation agent, N-Boc-L-aspartic acid-4-benzyl ester and glycine benzyl ester hydrochloride were subjected to an amidation reaction to obtain an intermediate compound 4; (2) removing the Boc protecting group of compound 4 with trifluoroacetic acid, and reacting with Boc-L-glutamic acid-O-benzyl 5 to obtain the intermediate compound 6; (3) After removing the Boc protecting group of compound 6 with trifluoroacetic acid, the compound 6 undergoes a condensation reaction with N-Cbz-L-alanine 7 to obtain the intermediate compound 8; (4) The Cbz and three benzyl protecting groups of compound 8 were removed by palladium-carbon hydrogenation under acidic conditions to obtain the target compound epitalone acetate.

2. The preparation method according to claim 1, characterized in that: The specific process of step (1) is as follows: N-Boc-L-aspartic acid-4-benzyl ester and glycine benzyl ester hydrochloride are dissolved in anhydrous dichloromethane, HATU is added under nitrogen protection, the mixture is cooled to 0°C in an ice-water bath, DIPEA is added dropwise, and the mixture is heated to room temperature and stirred to react to obtain compound 4.

3. The preparation method according to claim 2, characterized in that: The molar ratio of N-Boc-L-aspartic acid-4-benzyl ester, glycine benzyl ester hydrochloride, HATU and DIPEA is 1:1.2-1.5:1.2-3.0:3.0-5.0, and the reaction time is 12-16 hours.

4. The preparation method according to claim 1, characterized in that: The specific process of step (2) is as follows: (a) dissolving compound 4 in anhydrous dichloromethane, adding trifluoroacetic acid, stirring the mixture at room temperature, and concentrating under reduced pressure to obtain trifluoroacetate; (b) dissolving trifluoroacetate and Boc-L-glutamic acid-O-benzyl in anhydrous dichloromethane, adding HATU under nitrogen protection, cooling the mixture to 0°C in an ice-water bath, adding DIPEA dropwise, and heating the mixture to room temperature and stirring the mixture to obtain compound 6.

5. The preparation method according to claim 4, characterized in that: The molar ratio of compound 4, trifluoroacetic acid, Boc-L-glutamic acid-O-benzyl, HATU and DIPEA is 1:10-50:0.9-1.0:1.2-3.0:3.0-5.0; the reaction time in step (a) is 2-5 hours, and the reaction time in step (b) is 12-16 hours.

6. The preparation method according to claim 1, characterized in that: The specific process of step (3) is as follows: (a) dissolving compound 6 in anhydrous dichloromethane, adding trifluoroacetic acid, stirring the mixture at room temperature, and concentrating under reduced pressure to obtain trifluoroacetate; (b) dissolving trifluoroacetate and N-Cbz-L-alanine in anhydrous dichloromethane, adding HATU under nitrogen protection, cooling the mixture to 0°C in an ice-water bath, adding DIPEA dropwise, and heating the mixture to room temperature and stirring the mixture to obtain compound 8.

7. The preparation method according to claim 6, characterized in that: The molar ratio of compound 6, trifluoroacetic acid, N-Cbz-L-alanine, HATU and DIPEA is 1:10-50:0.9-1.0:1.2-3.0:3.0-5.0; the reaction time in step (a) is 2-5 hours, and the reaction time in step (b) is 12-16 hours.

8. The preparation method according to claim 1, characterized in that: The specific process of step (4) is as follows: dissolving compound 8 in a mixed solvent of ethyl acetate, methanol and water, adding acetic acid and 10% palladium carbon catalyst, evacuating the air three times, and rapidly stirring the reaction at room temperature under a hydrogen pressure of 1 atm to obtain the target compound.

9. The preparation method according to claim 8, characterized in that: The molar ratio of the compound 8 to acetic acid is 1:4.0-10.0; and the reaction time is 14-20 hours.

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