Synthesis method of N-methylated polypeptide

Abstract

The invention provides a method for synthesizing N-methylated polypeptide, which comprises the following steps: dissolving a carboxylic acid compound, N-methyl amino-acid ester hydrochloride or N-methyl amino-acid ester, an alkaline substance and pivaloic anhydride in an organic solvent, reacting at 15-80 DEG C for 3-10 hours, and post-treating the obtained reaction liquid to obtain an N-methyl dipeptide compound, the method comprises the following steps: removing a protecting group in an N-methyl dipeptide compound to obtain a free-state N-methyl dipeptide compound, and condensing the free-state N-methyl dipeptide compound and amino acid or N-methyl amino-acid ester protected by amino to obtain the N-methylated polypeptide, the mixed anhydride intermediate is formed in situ, and the reaction is completed in one step; according to the method, trimethylacetic anhydride is used as a condensing agent, the structure is simple, the reaction is safe, cheap and non-toxic, a large amount of nitrogen-containing byproducts are not generated in the reaction, and purification is convenient; the method is high in stereoselectivity, and racemization is avoided; according to the method, environment-friendly solvents such as ethyl acetate can be used, the reaction condition is mild, and the reaction time is short.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, and in particular relates to the synthesis of different N-methyl polypeptides. Background Technology

[0002] Peptides and proteins have become increasingly important therapeutic agents in treating a variety of diseases, including infections, metabolic disorders, and malignancies. Among them, N-methylated peptides have attracted widespread attention from the scientific community as highly promising drug candidates, primarily due to their ability to improve oral bioavailability, enhance cell permeability, and increase stability against enzymatic degradation. Despite extensive exploration of coupling agents in peptide synthesis, the synthesis of N-methylated peptides still faces significant challenges: conventional coupling agents are insufficiently reactive to sterically hindered N-methyl amino acids, often leading to low yields and racemization. It has been reported that the amidation reactivity of N-methyl amino acids is approximately 1 / 10 to 1 / 100 that of non-N-methylated amino acids (Angew. Chem., Int. Ed. 2020, 59, 12925-12930.). This long-standing limitation underscores the need for further research into more efficient and selective coupling agents that can meet the unique synthetic requirements.

[0003] The formation of amide bonds via mixed anhydrides is a classic method in the synthesis of N-methylated peptides. Traditionally, in the presence of a tertiary amine, activation of an amino acid derivative with neopentanoyl chloride generates highly reactive neopentanoyl mixed anhydrides, which subsequently react with N-methyl amino acid derivatives to form N-methylated peptides. However, this strategy has inherent limitations: the accumulation of high concentrations of mixed anhydrides can easily induce epimerization. Therefore, anhydride formation and subsequent coupling steps need to be carried out under strictly low-temperature conditions (e.g., -20°C) to mitigate such side reactions (Helv. Chim. Acta 1983, 66, 2672-2702.).

[0004] To address these challenges, several important improvements have been reported. Kurasaki and colleagues used isostearyl bromide (ISTABr) to effectively suppress the formation of anhydride impurities by shortening the activation time and to facilitate product separation (Org. Lett. 2020, 22, 8039-8043.XX). Meanwhile, Fuse's research group developed a milder approach using a mixture of less reactive carbonic anhydrides, aided by N-methylimidazole (NMI) and Brønsted acids (such as hydrochloric acid), combined with microfluidic conditions to accelerate the amidation reaction (Angew. Chem., Int. Ed. 2020, 59, 12925-12930.). However, the above methods all require the prior preparation of mixed anhydrides and a two-step synthesis of dipeptides, which is cumbersome and requires the addition of organic bases, easily leading to product racemization. Kurasaki and colleagues' method requires low temperatures, harsh reaction conditions, and high energy consumption. Fuse's method requires the addition of Brønsted acid, which can corrode equipment in industrial applications. Furthermore, the method relies on microfluidic technology, placing high demands on the equipment. The acyl bromide reagents used are toxic and highly irritating.

[0005] Furthermore, existing technologies for synthesizing N-methylated peptides commonly use condensing agents based on 1-hydroxybenzotriazole (HOBt) structural derivatives, such as PyBop, HBTU, and COMU, which pose a potential explosion risk during large-scale use. The use of such reagents is currently banned in Europe. Since most condensing agents are designed based on the HOBt structure, they also pose an explosion risk, and many companies have stopped using HOBt-based condensing agents. Therefore, there is an urgent need to develop a synthetic method for N-methylated peptides that simultaneously possesses high activity, high stereoselectivity, high atom economy, good safety, low cost, commercial availability, and ease of handling and removal of byproducts during production. Summary of the Invention

[0006] This invention provides a method for synthesizing N-methylated polypeptides, the preparation method comprising the following steps:

[0007] (1) The compound shown in Formula I, the compound shown in Formula II, pentylene anhydride, and a basic substance are dissolved together in an organic solvent to obtain a reaction mixture. The mixture is reacted at 15-80℃ (preferably 15-50℃, more preferably 40℃) for 3-10h (preferably 3-5h, more preferably 5h). The resulting reaction mixture is then post-treated to obtain an N-methyl dipeptide compound shown in Formula III. The molar ratio of the compound shown in Formula I, the compound shown in Formula II, pentylene anhydride, and the basic substance is 1:1.0-1.5:1.1-1.6:0-4.5 (preferably 1:1.0-1.5:1.1-1.6:1-4.5, more preferably 1:1.2:1.3:1).

[0008] The compound represented by formula II is of formula II. 1 Or Formula II 2 ;

[0009]

[0010] Wherein, R in equation I 1 Selected from one of the following groups:

[0011]

[0012] P 1 Selected from the following amino protecting groups: Fmoc, Cbz, or Boc;

[0013] Equation II is Equation II 1 Or Formula II 2 R 2 It is methyl, isopropyl, isobutyl, or benzyl; R in Formula III 1 With R in Equation I 1 Same, R 2 With Equation II 1 Or Formula II 2 R in 2 Same, P 1 With P in Equation I 1 same;

[0014] (2) Take the P from the N-methyl dipeptide compound of formula III obtained in step (1) 1 The N-methyl dipeptide compound is obtained by removing the group or tert-butyl group. The free N-methyl dipeptide compound is then condensed with an amino-protected amino acid or an N-methyl amino acid ester to obtain the N-methylated polypeptide.

[0015] Furthermore, the compound shown in Formula II is of Formula II. 1 The molar ratio of the compound shown in Formula I, the compound shown in Formula II, pentylene anhydride, and the basic substance is 1:1.0-1.5:1.1-1.6:1-4.5 (preferably 1:1.2:1.3:1).

[0016] Furthermore, the compound shown in Formula II is of Formula II. 2 The alkaline substance is 0, that is, when the compound shown in Formula II is of Formula II 2 When using alkaline substances, the closer the mass of the alkaline substance is to zero, the better the effect.

[0017] Further, the post-treatment in step (1) is as follows: the reaction solution is washed sequentially with sodium hydroxide aqueous solution, hydrochloric acid aqueous solution, and saturated brine. The resulting organic phase is dried with anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and the solution is purified by column chromatography to obtain the N-methyl dipeptide compound shown in Formula III. The molar concentration of the sodium hydroxide aqueous solution is 0.5 mol / L-2 mol / L, and the mass concentration of the hydrochloric acid aqueous solution is 0.5 mol / L-3 mol / L.

[0018] Furthermore, the amino protecting group in the amino-protected amino acid in step (2) is Fmoc, Cbz, or Boc.

[0019] Furthermore, the N-methyl amino acid ester mentioned in step (2) is N-methyl amino acid tert-butyl ester.

[0020] Furthermore, in step (2), diethylamine and the N-methyl dipeptide compound of formula III obtained in step (1) are dissolved together in an organic solvent and stirred for 0.5-2 hours to obtain P. 1 Free N-methyl dipeptide compounds with the removal of the radical group.

[0021] Further, in step (2), trifluoroacetic acid and the N-methyl dipeptide compound of formula III obtained in step (1) are dissolved together in an organic solvent and stirred for 0.5-2 hours to obtain a free N-methyl dipeptide compound with tert-butyl removed.

[0022] Furthermore, the alkaline substance is one or more of triethylamine, N-methylimidazole, potassium carbonate, sodium carbonate, sodium bicarbonate, lithium hydroxide monohydrate, or potassium hydroxide (preferably sodium carbonate).

[0023] Further, the organic solvent in step (1) is one or more of acetonitrile, dichloromethane, ethyl acetate or N,N-dimethylformamide (preferably ethyl acetate, and the organic solvent is an organic solvent after drying and dehydration), and the volume of the organic solvent is 5-15 mL / mmol (preferably 12.5 mL / mmol) based on the amount of the compound shown in Formula I.

[0024] In embodiments of the present invention, the N-methyl dipeptide compound represented by Formula III is one of the following:

[0025]

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) Compared with the existing two-step synthesis of N-methyl polypeptides by mixed anhydride method, this method forms mixed anhydride intermediates in situ and completes the reaction in one step.

[0028] (2) Compared with many existing nitrogen-containing polypeptide condensing agents, this method uses pentyl anhydride as a condensing agent, which has a simple structure, safe reaction, low cost and non-toxicity, does not produce a large number of nitrogen-containing byproducts, and is easy to purify.

[0029] (3) This method has strong stereoselectivity and does not cause racemization.

[0030] (4) This method can use environmentally friendly solvents such as ethyl acetate, and the reaction conditions are mild and the reaction time is short. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the examples can be obtained commercially.

[0032] Example 1

[0033]

[0034] Fmoc-L-phenylalanine (0.0775 g, 0.2 mmol), N-methyl-L-alanine tert-butyl hydrochloride (0.047 g, 0.24 mmol), and anhydrous sodium carbonate (0.0254 g, 0.24 mmol) were added sequentially to a reaction flask. 2.5 mL of dried ethyl acetate was added, followed by tertival anhydride (0.0484 g, 0.26 mmol). The reaction was carried out at 40 °C for 3 h. The remaining Fmoc-L-phenylalanine, tertival anhydride, and tertival acid were removed by washing with 2 mol / L sodium hydroxide solution. The remaining amine was removed by washing with 1 mol / L hydrochloric acid. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The product was purified by column chromatography. The product was a colorless oil, 0.0983 g, yield 93%, HPLC purity 99%, dr = 99.9:0.1. The product was characterized as follows: 1 HNMR (400MHz, Chloroform-d): δ7.75(d,J=7.4Hz,2H),7.57(q,J=7.7,7.1Hz,2H),7.39(t,J=7.4Hz,2H),7.34-7.17(m ,8H),5.85(dd,J=41.8,8.6Hz,1H),5.06-4.90(m,2H),4.53-4.08(m,4H),3.18-2.70(m,5H),1.46(s,9H),1.34(s,3H), 13C NMR (101MHz, Chloroform-d): δ171.71,170.67,155.91,144.07,141.47,136.09,129.86,128.62,127.87,127.23, 127.17,125.44,125.36,120.14,81.89,67.20,53.81,52.24,47.31,38.83,31.78,28.21,14.39.HRMS(ESI):calcd for[C 32 H 37 N2O5] + 529.2624, found 529.2625

[0035] Example 2

[0036]

[0037] The other procedures were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Cbz-L-phenylalanine (0.0599 g, 0.2 mmol). The product was a colorless oil, 0.0789 g, with a yield of 90% and an HPLC purity of 99%, dr = 99.6:0.4. The product was characterized as follows: 1 H NMR (400MHz, Chloroform-d): δ7.38-7.27(m,6H),7.25-7.15(m,4H),5.83-5.60(m,1H ),5.11-4.88(m,4H),3.15-2.69(m,6H),1.40(d,J=39.7Hz,9H),1.34(d,J=7.2Hz,3H). 13 C NMR (101MHz, Chloroform-d): δ171.54,170.58,155.81,136.53,136.01,129.72,128.56,128.49,1 28.14,128.01,127.01,81.75,66.81,53.61,52.14,38.74,31.59,28.11,14.27.HRMS(ESI):calcd for[C 32 H 33 N2O5] + 441.2311, found 441.2308.

[0038] Example 3

[0039]

[0040] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Fmoc-O-benzyl-L-tyrosine (0.0987 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a white solid, 0.1081 g, with a yield of 85% and an HPLC purity of 99% (dr = 99.8:0.2). The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.74(dd,J=7.6,3.6Hz,2H),7.61-7.51(m,2H),7.42-7.26(m,9 H),7.11(dd,J=11.3,8.4Hz,2H),6.91-6.79(m,2H),5.89(dd,J=30.3,8.6Hz,1H),5.04-4.94( m,3H),4.38(dt,J=10.3,8.0Hz,1H),4.30-4.09(m,2H),3.07(dd,J=13.9,6.1Hz,1H),2.95(s, 2H), 2.88 (dd, J=13.9, 6.5Hz, 1H), 2.73 (s, 1H), 1.41 (d, J=39.7Hz, 9H), 1.34 (d, J=7.1Hz, 3H). 13 C NMR(101MHz,Chloroform-d)δ171.64,170.55,157.89,155.80,143.95,141.33,137.08,130.79,130.56,128.61,127.98,127.74,127. 56,127.12,125.32,125.24,120.01,114.85,81.73,70.00,67.04,53.72,52.21,47.19,37.74,31.68,28.08,14.25.HRMS(ESI):calcd for[C 39 H 43 N2O6] + 635.3116, found 635.3117.

[0041]

[0042] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Fmoc-L-aspartic-4-tert-butyl ester (0.0823 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was 0.0958 g of colorless oil, with a yield of 87% and an HPLC purity of 98% (dr = 99.8:0.2). The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.59(t,J=6.7Hz,2H),7.39 (t,J=7.4Hz,2H),7.30(tt,J=7.5,1.4Hz,2H),5.90(dd,J=21.6,9.3Hz,1H),5.12 -4.78(m,2H),4.46-4.15(m,3H),3.06(s,2H),2.85(s,1H),2.72(dd,J=15.7,5.4Hz,1H),2.53(dd,J=15.6,7.4Hz,1H),1.48-1.36(m,21H). 13 CNMR(101MHz,Chloroform-d)δ171.24,170.43,169.44,155.79,143.82,141.30,127.73,127.09,125 .19,119.99,81.67,81.43,67.16,53.57,48.35,47.12,38.78,31.61,28.00,14.23.HRMS(ESI):calcd for[C 31 H 41 N2O7] + 553.2836, found 553.2837.

[0043] Example 5

[0044]

[0045] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Boc-L-aspartic acid-1-tert-butyl ester (0.0579 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was 0.084 g of colorless oil, with a yield of 98% and an HPLC purity of 97% (dr = 99.9:0.1). The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ5.73(dd,J=28.6,9.1Hz,1H),5.09(q,J=7.3Hz,1H),4.45-4.28(m,1H),3. 06(dd,J=16.7,4.2Hz,1H),2.87(s,2H),2.79(s,1H),2.69(dd,J=16.7,3.9Hz,1H),1.49-1.33(m,30H). 13CNMR(101MHz,Chloroform-d)δ170.93,170.74,170.06,156.02,81.66,81.62,52.54,50.73,36.10,31.35,28.44,28.07,27.98,14.60.HRMS(ESI):calcd for[C 21 H 39 N2O7] + 431.2679, found 431.2680.

[0046] Example 6

[0047]

[0048] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Fmoc-O-tert-butyl-L-serine (0.0767 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0946 g, with a yield of 90% and an HPLC purity of 98% (dr = 99.7:0.3). The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.61(dd,J=7.5,4.6Hz,2H),7.39(t,J=7.5Hz,2H),7.34-7.27(m,2H),5.92(dd,J=13.1,8.2Hz,1H),5 .00-4.81(m,2H),4.35(dd,J=7.4,5.0Hz,2H),4.21(q,J=7.9Hz,1H),3.64-3. 35(m,2H),2.99(d,J=91.2Hz,3H),1.50-1.34(m,12H),1.16(d,J=5.9Hz,9H). 13 C NMR(101MHz,Chloroform-d)δ171.38,156.01,144.04,141.37,127.14,125.31,120.04,81. 45,73.69,67.16,63.13,54.08,51.48,47.22,32.32,28.07,27.36,14.34.HRMS(ESI):calcd for[C 30 H 41 N2O6] + 525.2886, found 525.2887.

[0049] Example 7

[0050]

[0051] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Fmoc-N-methyl-L-aspartic acid 4-tert-butyl ester (0.0851 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.1024 g, with a yield of 90% and an HPLC purity of 98% (dr = 99.4:0.6). The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.56(dt,J=11.4,4.7Hz,2H),7.43-7.3 5(m,2H),7.35-7.27(m,2H),5.49-5.38(m,1H),4.99-4.56(m,2H),4.47(qd,J=10.6,6.4Hz, 1H),4.27-4.13(m,2H),2.88(d,J=34.0Hz,4H),2.74(t,J=15.2Hz,3H),2.59(d,J=23.6Hz,1 H),2.44(td,J=15.6,6.2Hz,1H),1.44(s,9H),1.40(d,J=2.5Hz,9H),1.34(d,J=2.3Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ170.55,170.29,169.82,169.62,169.50,168.87,155.98,155.89,143.83,141.46,127.82,127.14,124. 97,120.09,81.83,81.54,80.95,67.68,55.48,54.33,47.38,35.37,31.81,29.50,28.02,27.97,27.12,14.41.HRMS(ESI):calcdfor[C 32 H 43 N2O7] + 567.2992, found 567.2994.

[0052] Example 8

[0053]

[0054] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Fmoc-L-valine (0.0679 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was 0.0882 g of colorless oil, with a yield of 92%, HPLC purity of 97%, and dr = 99.5:0.5. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.62(dd,J=7.5,4.5Hz,2H),7.39(tt,J=7.5,1.5 Hz,2H),7.30(tdd,J=7.4,2.9,1.2Hz,2H),5.92(d,J=9.3Hz,1H),5.20(q,J=7.2Hz,1H),4.56(dd,J=9 .3,6.2Hz,1H),4.45–4.27(m,2H),4.22(t,J=7.2Hz,1H),2.93(d,J=76.3Hz,3H),2.07(h,J=6.7Hz,1H ), 1.41 (d, J = 23.2Hz, 9H), 1.35 (d, J = 7.2Hz, 3H), 1.04 (d, J = 6.8Hz, 3H), 0.95 (dd, J = 15.1, 6.7Hz, 3H). 13 C NMR(101MHz,Chloroform-d)δ170.63,156.64,141.35,127.75,127.13,125.28,120.02,81. 76,67.12,55.91,53.05,47.25,38.50,31.36,28.04,19.56,17.51,14.15.HRMS(ESI):calcd for[C 28 H 37 N2O5] + 481.2624, found 481.2825.

[0055] Example 9

[0056]

[0057] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Boc-L-alanine (0.0378 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0637 g, with a yield of 96%, HPLC purity of 99%, and dr > 99.9:0.1. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ5.51(dd,J=34.8,8.0Hz,1H),5.13(q,J=7.3Hz,1H), 4.66-4.53(m,1H),2.85(d,J=54.6Hz,3H),1.45-1.30(m,21H),1.30-1.23(m,4H). 13 C NMR(101MHz,Chloroform-d)δ173.13,170.68,155.21,81.66,52.78,46.55,30.88,28.43,28.05,18.73,14.14.HRMS(ESI):calcd for[C 16 H 31 N2O5] + 331.2155, found 331.2157.

[0058] Example 10

[0059]

[0060] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Boc-L-holyalanine (0.0407 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was 0.0623 g of colorless oil, with a yield of 91%, HPLC purity of 99%, and dr > 99.9:0.1. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ5.39(s,1H),5.03(q,J=7.3Hz,1H),4.03-3.90(m,1H),2.83( d,J=58.4Hz,3H),2.68-2.35(m,2H),1.34(dd,J=38.9,7.0Hz,21H),1.21(d,J=6.9Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ171.36,171.09,155.46,81.52,52.83,43.79,39.30,31.90,28.53,28.10,20.44,14.48.HRMS(ESI):calcd for[C 17 H 33 N2O5] + 345.2311, found 345.2310.

[0061] Example 11

[0062]

[0063] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Fmoc-L-leucine (0.0707 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0942 g, with a yield of 95%, HPLC purity of 99%, and dr = 99.9:0.1. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.6Hz,2H),7.61(t,J=6.8Hz,2H),7.39(tt,J=7.5,1.5Hz ,2H),7.30(tt,J=7.5,1.6Hz,2H),5.90(d,J=8.9Hz,1H),5.18(q,J=7.3Hz,1H),4.76(td,J=9.5,4 .0Hz,1H),4.42-4.28(m,2H),4.21(t,J=7.3Hz,1H),2.93(d,J=62.2Hz,3H),1.85-1.73(m,1H),1. 55(tdd,J=13.9,9.7,4.4Hz,2H),1.47-1.34(m,12H),1.03(d,J=6.6Hz,3H),0.97(d,J=6.7Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ173.24,170.61,156.40,144.01,143.85,141.31,127.69,127.08,125.25,119.96 ,81.70,67.01,52.91,49.55,47.21,42.20,30.99,28.02,27.19,24.61,23.49,21.72,14.19.HRMS(ESI):calcd for[C 29 H 39 N2O5] + 495.2781, found 495.2782.

[0064] Example 12

[0065]

[0066] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Fmoc-L-methionine (0.0743 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0909 g, with a yield of 89%, HPLC purity of 99%, and dr = 99.8:0.2. The product was characterized as follows: 1H NMR (400MHz, Chloroform-d) δ7.75 (d, J=7.6Hz, 2H), 7.60 (dd, J=7.6, 4.7Hz, 2H), 7.39 (tt, J= 7.5,1.5Hz,2H),7.30(tdd,J=7.4,3.1,1.2Hz,2H),5.94(d,J=8.7Hz,1H),5.16(q,J=7.3Hz,1 H),4.89(td,J=8.5,4.4Hz,1H),4.43-4.30(m,2H),4.21(t,J=7.0Hz,1H),2.94(d,J=77.1Hz, 3H), 2.58 (t, J = 7.7Hz, 2H), 2.17-1.82 (m, 5H), 1.42 (d, J = 19.2Hz, 9H), 1.36 (d, J = 7.3Hz, 3H). 13 C NMR(101MHz,Chloroform-d)δ172.08,170.48,156.26,143.97,141.34,127.76,127.12,125.22,120 .03,81.83,67.09,53.05,50.13,47.21,32.64,31.11,30.06,28.05,15.68,14.16.HRMS(ESI):calcd for[C 28 H 37 N2O5S] + 513.2345, found 513.2346.

[0067] Example 13

[0068]

[0069] The other procedures were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Fmoc-L-tryptophan (0.0853 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a white solid, 0.1082 g, with a yield of 95%, HPLC purity of 99%, and dr = 99.8:0.2. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ8.40(d,J=9.1Hz,1H),7.77(t,J=8.4Hz,2H),7.66(d d,J=29.5,7.6Hz,2H),7.55(t,J=8.2Hz,1H),7.45-7.27(m,5H),7.21-7.04(m,3H), 5.98(dd,J=57.3,8.5Hz,1H),5.17-4.98(m,2H),4.45-4.22(m,2H),4.20-4.04(m,1 H),3.35-3.13(m,2H),2.77(d,J=84.4Hz,3H),1.48(d,J=11.6Hz,9H),1.35(s,3H). 13 C NMR(101MHz,Chloroform-d)δ172.32,170.63,156.06,143.95,141.31,136.21,127.73,127.13,125.33,123.45,122.08,119.99, 119.67,118.58,111.35,110.03,81.70,67.10,53.77,51.58,47.17,31.72,28.67,28.12,28.08,27.79,14.27.HRMS(ESI):calcd for[C 34 H 38 N3O5] + 568.2806, found 568.2808.

[0070] Example 14

[0071]

[0072] The other procedures were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Fmoc-L-proline (0.0675 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0704 g, with a yield of 74%, HPLC purity of 98%, and dr = 99.7:0.3. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ7.74(t,J=7.1Hz,2H),7.68-7.53(m,2H),7.38(td,J=7.5,4.1H z,2H),7.3-7.26(m,2H),5.22(dq,J=19.1,7.3Hz,1H),5.08(s,1H),4.5-4.36(m,2H),4.33-4 .22(m,1H),3.81-3.64(m,1H),3.55(ddt,J=24.8,10.6,7.4Hz,1H),2.88(d,J=89.9Hz,3H),2 .18(dddt,J=32.3,17.1,12.2,7.6Hz,2H),2.06-1.81(m,3H),1.47(s,3H),1.46-1.38(m,9H). 13 C NMR(101MHz,Chloroform-d)δ172.24,172.16,171.20,171.02,154.97,154.41,144.61,1 44.32,144.00,143.81,141.34,141.30,127.66,127.09,125.36,125.19,125.04,124.98 ,119.95,119.88,119.84,81.46,67.47,66.74,58.20,57.40,57.00,52.67,52.38,47.59 ,47.28,46.81,30.76,29.98,29.09,28.04,27.26,24.15,22.99,14.26.HRMS(ESI):calcd for[C 28 H 35 N2O5] + 479.2468, found 479.2467.

[0073] Example 15

[0074]

[0075] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with N-α-Fmoc-N-epsilon-tert-butoxycarbonyl-L-lysine (0.0937 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a colorless oil of 0.1146 g, with a yield of 94%, HPLC purity of 98%, and dr = 99.8:0.2. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ7.74(d,J=7.6Hz,2H),7.59(dd,J=7.6,4.0Hz,2H),7.38(t,J=7.6Hz ,2H),7.29(td,J=7.7,3.1Hz,2H),5.88(d,J=8.8Hz,1H),5.14(q,J=7.2Hz,1H),4.73(t,J=5.7Hz,1 H),4.67(td,J=8.3,4.3Hz,1H),4.35(d,J=7.3Hz,2H),4.20(t,J=7.0Hz,1H),3.09(dq,J=13.9,6.6 Hz,2H),2.90(d,J=59.0Hz,3H),1.70-1.47(m,4H),1.42(d,J=10.8Hz,18H),1.35(d,J=7.3Hz,5H). 13 C NMR(101MHz,Chloroform-d)δ172.33,170.58,156.24,156.08,144.00,141.33,127.74,127.11,125.23,120.01,8 1.78,67.06,53.01,50.88,47.20,40.21,32.46,31.12,29.61,28.47,28.06,22.25,14.20.HRMS(ESI):calcdfor[C 34 H 48 N3O7] + 610.3414, found 610.3415.

[0076] Example 16

[0077]

[0078] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with Fmoc-L-glutamic acid-gamma-methyl ester (0.0768 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.1004 g, with a yield of 99%, HPLC purity of 99%, and dr = 99.9:0.1. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.60(t,J=6.7Hz,2H),7.39(td,J=7.4,2.4Hz,2H) ,7.30(td,J=7.6,3.2Hz,2H),5.96(d,J=8.5Hz,1H),5.15(q,J=7.3Hz,1H),4.79(tt,J=9.2,4.9Hz,1H) ,4.35(d,J=7.4Hz,2H),4.20(t,J=7.1Hz,1H),3.68(s,3H),2.95(d,J=88.4Hz,3H),2.46(ddt,J=30.1, 17.5,7.5Hz,2H),2.22-2.08(m,1H),1.78(dq,J=14.8,6.7Hz,1H),1.45(s,9H),1.36(d,J=7.3Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ173.53,172.11,170.56,156.51,144.07,141.42,127.84,127.22,125.32, 120.11,81.92,67.22,53.20,51.87,50.41,47.29,31.16,29.40,28.08,27.26,14.16.HRMS(ESI):calcd for[C 29 H 37 N2O7] + 525.2523, found 525.2523.

[0079] Example 17

[0080]

[0081] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with N-Fmoc-N'-Boc-L-histidine (0.0955 g, 0.2 mmol), and the reaction time was extended from 3 h to 5 h. The product was a white solid, 0.11064 g, with a yield of 86%, HPLC purity of 99%, and dr = 99.9:0.1. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ8.05(d,J=8.6Hz,1H),7.78(d,J=7.5Hz,2H),7.62( t,J=7.0Hz,2H),7.42(t,J=7.5Hz,2H),7.37-7.32(m,2H),7.31(d,J=1.4Hz,1H),6 .03(d,J=8.8Hz,1H),5.12(ddt,J=14.3,8.1,4.7Hz,2H),4.40-4.26(m,3H),3.11 (s,3H),3.01-2.82(m,2H),1.59(s,9H),1.51(d,J=1.4Hz,3H),1.50-1.42(m,9H). 13 C NMR(101MHz,Chloroform-d)δ171.84,170.58,156.07,146.95,143.94,141.33,138.46,137.00,127.14,125. 36,120.00,115.06,85.48,81.62,67.19,58.66,50.86,47.17,34.00,28.07,27.90,14.26.HRMS(ESI):calcd for[C 34 H 43 N4O7] + 619.3126, found 619.3127.

[0082] Example 18

[0083]

[0084] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine and N-methylalanine tert-butyl hydrochloride were used instead of Fmoc-L-leucine (0.0707 g, 0.2 mmol) and N-methylphenylalanine tert-butyl hydrochloride (0.0652 g, 0.24 mmol), respectively, and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0954 g, with a yield of 84%, HPLC purity of 99%, and dr > 99.9:0.1. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ7.76(d,J=7.6Hz,2H),7.58(dt,J=11.3,5.5Hz,2H),7.39(t,J=7.4Hz,2H),7.31(t,J=7.6Hz, 2H),7.23-7.12(m,4H),7.11-7.05(m,1H),5.43(t,J=7.1Hz,1H),5.14(dd,J=10.3,5.6Hz,1H),4.63(dtd,J=18.7,9.8,9.3, 4.5Hz,1H),4.45-4.28(m,2H),4.19(q,J=7.0Hz,1H),3.32(dd,J=14.5,5.5Hz,1H),2.98(dd,J=14.6,10.3Hz,1H),2.93(d, J=15.7Hz,3H),1.71(ddt,J=11.4,8.8,5.7Hz,1H),1.48(dd,J=18.7,4.6Hz,2H),1.41(d,J=19.2Hz,9H),1.01-0.66(m,6H). 13 C NMR(101MHz,Chloroform-d)δ173.07,169.66,156.12,144.07,143.93,141.39,137.14,128.98,128.47,127.78,127.14,126 .74,125.29,120.05,81.97,66.98,59.58,49.36,47.30,42.45,34.63,32.89,28.07,24.64,23.44,21.88.HRMS(ESI):calcd for[C 35 H 43 N2O5] + 571.3094, found 571.3095.

[0085] Example 19

[0086]

[0087] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine and N-methylalanine tert-butyl hydrochloride were used instead of Fmoc-L-leucine (0.0707 g, 0.2 mmol) and N-methylleucine tert-butyl hydrochloride (0.0571 g, 0.24 mmol), respectively, and sodium carbonate was not added. The reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0989 g, with a yield of 92%, HPLC purity of 98%, and dr = 99.5:0.5. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ7.76(d,J=7.5Hz,2H),7.60(dd,J=7.7,3.2Hz,2H),7. 40(t,J=7.5Hz,2H),7.31(tt,J=7.4,1.6Hz,2H),5.60(d,J=9.1Hz,1H),5.21(dd,J= 10.5,5.1Hz,1H),4.74(td,J=9.0,4.9Hz,1H),4.36(d,J=7.3Hz,2H),4.22(t,J=7.3 Hz,1H),2.97(s,3H),1.83-1.47(m,6H),1.42(d,J=19.7Hz,9H),1.05-0.85(m,12H). 13 C NMR (101MHz, Acetonitrile-d3) δ173.46,170.77,156.36,144.10,143.88,141.37,127.75,127.13,125.30,120. 03,81.71,67.05,55.41,49.67,47.28,42.37,37.18,31.00,28.12,25.00,24.70,21.77,21.60.HRMS(ESI):calcd for[C 32 H 45 N2O5] + 537.3250, found 537.3252.

[0088] Example 20

[0089]

[0090] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine and N-methylalanine tert-butyl hydrochloride were used instead of Fmoc-L-leucine (0.0707 g, 0.2 mmol) and N-methylvaline tert-butyl hydrochloride (0.0537 g, 0.24 mmol), respectively, and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0646 g, with a yield of 61%, HPLC purity of 98%, and dr = 99.7:0.3. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.60(dt,J=7.0,3.0Hz,2H),7.44–7.35(m,2H),7.3 0(tdd,J=7.5,2.3,1.2Hz,2H),5.68(dd,J=9.2,4.3Hz,1H),4.97–4.60(m,2H),4.43–4.26(m,2H),4.21(t ,J=7.1Hz,1H),2.98(d,J=45.6Hz,3H),2.19(dp,J=10.3,6.7Hz,1H),1.75(dtd,J=11.6,6.8,5.5,3.5Hz ,1H),1.55(ddd,J=14.2,9.6,4.7Hz,1H),1.44(d,J=10.5Hz,10H),1.09–0.80(m,12H).HRMS(ESI):calcd for[C 31 H 43 N2O5] + 522.31, found. 13 C NMR(101MHz,Chloroform-d)δ173.72,169.99,156.35,144.07,143.91,141.39,127.78,127.16,125.28,120.05,81.61 ,67.06,62.76,49.65,47.28,42.41,31.25,28.12,27.87,27.25,24.71,23.53,21.96,19.97,19.06.HRMS(ESI):calcd for[C 31 H 43 N2O5] + 523.3094, found 523.3095.

[0091] Example 21

[0092]

[0093] The other operations were the same as in Example 1, except that Fmoc-L-valine (0.0679 g, 0.2 mmol) and N-methylleucine tert-butyl hydrochloride (0.0537 g, 0.24 mmol) were replaced with Fmoc-L-phenylalanine and N-methylalanine tert-butyl hydrochloride, respectively, and the reaction time was extended from 3 h to 5 h. The product was 0.092 g of colorless oil, with a yield of 88%, HPLC purity of 98%, and dr = 99.8:0.2. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.61(d,J=7.5Hz,2H),7.39(ddd,J=9.2,4.8,2.0Hz,2H),7.30(t t,J=7.5,1.9Hz,2H),5.91(d,J=9.3Hz,1H),5.23(dd,J=10.3,5.2Hz,1H),4.55(dd,J=9.3,6.4Hz,1H),4.35(dd,J=7. 6,1.9Hz,2H),4.22(t,J=7.3Hz,1H),3.02(s,3H),2.08(dq,J=13.7,6.9Hz,1H),1.72(ddd,J=14.5,9.4,5.2Hz,1H),1 .62(ddd,J=14.5,10.4,4.8Hz,1H),1.44(s,11H),1.04(d,J=6.8Hz,3H),0.97(d,J=6.8Hz,3H),0.90(t,J=6.2Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ172.91,170.62,156.60,144.00,143.86,141.32,127.70,127.09,125.21,119.96,8 1.72,67.09,55.98,55.50,47.20,38.48,37.12,31.19,28.03,24.89,23.23,21.62,19.55,17.50.RMS(ESI):calcd for[C 31 H 42 N2O5] + 523.3094, found 523.3094.

[0094] Example 22

[0095]

[0096] The other operations were the same as in Example 1, except that Fmoc-D-alanine (0.0623 g, 0.2 mmol) and N-methylleucine tert-butyl hydrochloride were replaced with Fmoc-L-phenylalanine and N-methylalanine tert-butyl hydrochloride, respectively, and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.088 g, with a yield of 89%, HPLC purity of 98%, and dr = 99.9:0.1. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ7.76(d,J=7.5Hz,2H),7.61(d,J=7.6Hz,2H),7.40(t,J=7.4H z,2H),7.31(td,J=7.5,1.3Hz,2H),5.93(d,J=7.6Hz,1H),5.10(dd,J=9.6,6.3Hz,1H),4.73 (p,J=6.7Hz,1H),4.35(d,J=7.3Hz,2H),4.21(t,J=7.3Hz,1H),2.94(d,J=39.6Hz,3H),1.8 3-1.51(m,3H),1.46(d,J=10.5Hz,9H),1.37(d,J=6.8Hz,3H),0.94(dd,J=16.9,6.5Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ170.61,155.57,144.11,141.39,127.78,127.17,125.32,120.07,81 .90,67.04,55.76,47.41,47.26,37.49,31.51,28.11,25.25,23.42,21.36,19.21.RMS(ESI):calcd for[C 29 H 39 N2O5] + 495.2781, found 495.2782.

[0097] Example 23

[0098]

[0099] The other operations were the same as in Example 1, except that Fmoc-N-methyl-L-leucine (0.0735 g, 0.2 mmol) and N-methylvaline tert-butyl ester hydrochloride were replaced with Fmoc-N-methyl-L-leucine (0.0735 g, 0.2 mmol) and N-methylvaline tert-butyl ester (0.045 g, 0.24 mmol), respectively, and sodium carbonate was not added. The reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0966 g, with a yield of 90%, HPLC purity of 99%, and dr = 99.8:0.2. The product was characterized as follows: 1H NMR(400MHz,Chloroform-d)δ7.74(dd,J=15.2,7.5Hz,2H),7.64-7.51(m,2H) ,7.43-7.34(m,2H),7.34-7.27(m,2H),5.13(dt,J=8.1,6.3Hz,1H),4.82-4.39 (m,2H),4.30-4.17(m,2H),2.98-2.68(m,6H),2.17(m,J=13.3,6.6,2.9Hz,1H) ,1.72-1.50(m,2H),1.46-1.34(m,10H),1.03-0.91(m,7H),0.82-0.50(m,5H).

[0100] Example 24

[0101]

[0102] The other operations were the same as in Example 1, except that Fmoc-L-phenylalanine and N-methylalanine tert-butyl hydrochloride were used instead of Fmoc-L-aminobutyric acid (0.0651 g, 0.2 mmol) and N-methylglycine tert-butyl ester (0.0348 g, 0.24 mmol), respectively, and sodium carbonate was not added. The reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0805 g, with a yield of 89%, HPLC purity of 99%, and dr > 99.9:0.1. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.61(dd,J=7.5,3.9Hz,2H),7.39(t,J=7.5 Hz,2H),7.30(t,J=7.4Hz,2H),5.82(d,J=8.5Hz,1H),4.60(dtd,J=86.4,7.8,5.1Hz,1H),4.43-4 .28(m,3H),4.21(dd,J=9.5,4.6Hz,1H),3.81(dd,J=74.8,17.6Hz,1H),3.06(d,J=53.8Hz,3H), 1.88(tq,J=17.5,6.6,5.7Hz,1H),1.73-1.56(m,1H),1.47(s,9H),0.96(dt,J=24.1,7.4Hz,3H). 13CNMR(101MHz,Chloroform-d)δ172.44,167.97,156.09,144.08,143.95,141.38,127.78,127.17, 125.29,120.06,82.15,67.07,51.85,50.52,47.25,36.47,28.16,26.08,9.47.HRMS(ESI):calcd for[C 26 H 33 N2O5] + 453.2311, found 453.2313.

[0103] Example 25

[0104]

[0105] The other operations were the same as in Example 1, except that Fmoc-N-methyl-L-leucine (0.0735 g, 0.2 mmol) and L-valine tert-butyl hydrochloride (0.0504 g, 0.24 mmol) were replaced with Fmoc-L-phenylalanine and N-methylalanine tert-butyl hydrochloride, respectively, and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0805 g, with a yield of 77%, HPLC purity of 98%, and dr = 99.9:0.1. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.77(d,J=7.5Hz,2H),7.58(dt,J=7.6,2.0Hz,2H),7.4 0(t,J=7.5Hz,2H),7.34-7.27(m,2H),6.48(d,J=8.8Hz,1H),4.76(t,J=7.8Hz,1H),4 .58-4.43(m,2H),4.39(dd,J=8.8,4.5Hz,1H),4.27(t,J=6.7Hz,1H),2.83(s,3H),2. 18-2.07(m,1H),1.74(s,1H),1.69(t,J=7.5Hz,2H),1.45(s,9H),0.99-0.79(m,12H). 13 C NMR(101MHz,Chloroform-d)δ170.8,157.4,144.0,141.5,127.9,127.2,125.0,120.2,82.0,6 8.0,57.4,57.0,47.4,36.4,31.4,29.7,28.1,24.8,23.3,22.0,19.1,17.6.HRMS(ESI):calcd for[C 31 H43 N2O5] + 523.3166, found 523.3167.

[0106] Example 26

[0107]

[0108] The other operations were the same as in Example 1, except that Fmoc-N-methyl-L-leucine (0.0735 g, 0.2 mmol) and L-alanine tert-butyl hydrochloride (0.0437 g, 0.24 mmol) were replaced with Fmoc-L-phenylalanine and N-methylalanine tert-butyl hydrochloride, respectively, and the reaction time was extended from 3 h to 5 h. The product was a colorless oil, 0.0929 g, with a yield of 94%, HPLC purity of 99%, and dr = 99.8:0.2. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.8(d,J=7.5Hz,2H),7.6(d,J=7.5Hz,2H),7.4(t,J=7.4Hz,2H),7.3(td,J=7.4,1.2Hz,2H),6.5(d,J=7.3Hz,1H), 4.8-4.5(m,1H),4.53-4.35(m,2H),4.3(t,J=6.9Hz,1H),2.8(s,3H),1. 7-1.5(m,2H),1.46(d,J=7.1Hz,10H),1.4-1.3(m,3H),1.0-0.6(m,6H). 13 C NMR(101MHz,Chloroform-d)δ171.9,170.5,157.3,144.0,141.5,127.8,127.2,125.0,120. 1,82.0,67.8,56.9,48.8,47.4,36.7,29.7,28.1,24.8,23.3,22.0,18.5.HRMS(ESI):calcd for[C 29 H 39 N2O5] + 495.2853, found 495.2854.

[0109] Example 27

[0110]

[0111] The product obtained in Example 22 (0.2473 g, 0.5 mmol), trifluoroacetic acid (1.8 mL), and dichloromethane (3 mL) were stirred in a reaction flask at room temperature for 1 h. After the reaction was completed, the product was washed three times with water, and the aqueous phase was washed three times with dichloromethane. The organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 0.204 g of the product Fmoc-D-Ala-L-MeLeu-OH as a white solid, with a yield of 93%. The obtained Fmoc-D-Ala-L-MeLeu-OH (0.0877 g, 0.2 mmol), N-methylleucine tert-butyl ester (0.0483 g, 0.24 mmol), and ethyl acetate (2.5 mL) were placed in a reaction flask and heated to 40 °C with stirring for 4 h. The remaining Fmoc-D-Ala-L-MeLeu-OH, tertival anhydride, and tertival acid were removed from the reaction system by washing with 2 mol / L sodium hydroxide solution. The remaining amine was removed by washing with 1 mol / L hydrochloric acid. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The product was purified by column chromatography. The product was a light yellow oil, 0.0933 g, with a yield of 75% and an HPLC purity of 98%. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.76(d,J=7.5Hz,2H),7.60(dd,J=7.7,3.6Hz,2H),7.39(t,J=7.5Hz,2H),7.31(tt,J=7.5,1.5Hz,2H),6.05-5.73(m,1H),5.57 -5.41(m,1H),5.12(ddd,J=15.5,10.3,5.5Hz,1H),4.78-4.56(m,1H),4.46-4.14(m,3H),3.0 5-2.90(m,3H),2.91-2.74(m,3H),1.89-1.46(m,6H),1.44-1.31(m,12H),1.02-0.83(m,12H). 13 C NMR(101MHz,Chloroform-d)δ172.84,171.99,170.87,155.64,144.03,143 .94,141.41,127.82,127.17,125.23,120.10,81.65,67.06,55.59,51.66,4 7.42,47.24,37.99,37.33,31.24,30.48,29.80,28.13,27.93,25.12,24.97 ,24.59,23.42,23.10,22.42,21.99,21.48,19.19,18.78.HRMS(ESI):calcd for[C 36 H52 N3O6] + 622.3778, found 622.3776.

[0112] Example 28

[0113]

[0114] The product of Example 23 (0.5367 g, 1 mmol), diethylamine (3.3 mL), and methanol (7 mL) were placed in a reaction flask and stirred at room temperature for 1 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the product was purified by column chromatography to obtain N-Me-L-Leu-L-MeLeu-O. t Bu is a pale yellow oil, 0.274 g, yield 88%. The obtained N-Me-L-Leu-L-MeLeu-O t Bu (0.0755 g, 0.24 mmol), Fmoc-N-methyl-L-leucine (0.0735 g, 0.2 mmol), and ethyl acetate (2.5 mL) were placed in a reaction flask and heated to 40 °C with stirring for 5 h. The remaining Fmoc-N-methyl-L-leucine, pentylene anhydride, and pentylene acid were removed by washing with 2 mol / L sodium hydroxide solution. The remaining amine was removed by washing with 1 mol / L hydrochloric acid. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The product was purified by column chromatography. The product was a colorless oil, 0.1182 g, with a yield of 89% and an HPLC purity of 98%. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.73(dd,J=22.8,7.5Hz,2H),7.60-7.51(m,2H),7.43-7.27(m,4H),5.55- 5.33(m,1H),5.10(td,J=9.9,9.2,5.6Hz,1H),4.89-4.68(m,2H),4.64-4.40(m,1H),4.30-4.15(m,1H), 3.01-2.61(m,9H),2.18-2.08(m,1H),1.75-1.49(m,4H),1.43(t,J=3.6Hz,9H),1.38-1.24(m,2H),1.01 (dt,J=6.5,2.5Hz,3H),0.95-0.86(m,10H),0.76(dd,J=20.0,6.8Hz,3H),0.53(dd,J=11.5,6.2Hz,2H).

[0115] Example 29

[0116]

[0117] The product of Example 28 (0.3983 g, 0.6 mmol), diethylamine (2.0 mL), and methanol (4.6 mL) were placed in a reaction flask and stirred at room temperature for 1 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was purified by column chromatography to obtain N-Me-L-Leu-L-MeLeu-L-MeLeu-O. t Bu was a pale yellow oil, 0.2359 g, yield 89%. The obtained N-Me-L-Leu-L-MeLeu-O t Bu (0.106 g, 0.24 mmol), Fmoc-D-alanine (0.0623 g, 0.2 mmol), and ethyl acetate (2.5 mL) were placed in a reaction flask and heated to 40 °C with stirring for 5 h. The remaining Fmoc-N-methyl-L-leucine, pentylamino acid anhydride, and pentylamino acid were removed by washing with 2 mol / L sodium hydroxide solution. The remaining amines were removed by washing with 1 mol / L hydrochloric acid. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The product was purified by column chromatography. The product was a white solid, 0.0806 g, yield 84%, HPLC purity 99%. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.76(d,J=7.5Hz,2H),7.59(dd,J=7.4,3.5Hz,2H),7.39(t,J=7.5Hz,2H),7.30(t,J=7.5Hz,2H),5.90-5.66(m,1H),5.61-5.3 8(m,2H),4.80-4.58(m,2H),4.28(dt,J=59.5,7.3Hz,3H),3.22-2.73(m,9H), 2.17(ddt,J=13.9,10.4,7.2Hz,1H),1.83-1.29(m,18H),1.04-0.72(m,18H).

[0118] Example 30

[0119]

[0120] The product of Example 25 (0.1208 g, 0.23 mmol), trifluoroacetic acid (0.82 mL), and dichloromethane (1.6 mL) were stirred in a reaction flask at room temperature for 1 h. After the reaction was complete, the mixture was washed three times with water, and the aqueous phase was washed three times with dichloromethane. The organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 0.0985 g of Fmoc-L-MeLeu-L-Val-OH as a white solid, with a yield of 92%, which was used in the next step. The product of Example 26 (1.484 g, 3.0 mmol), diethylamine (9.9 mL), and methanol (20 mL) were stirred in a reaction flask at room temperature for 1 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was purified by column chromatography to obtain N-Me-L-Leu-L-Ala-O. t Bu was a pale yellow oil, 0.6425 g, yield 92%, used in the next step. Other procedures were the same as in Example 1, except that Fmoc-L-phenylalanine was replaced with the obtained Fmoc-N-methyl-L-leucine-L-valine (0.0467 g, 0.1 mmol), and N-methylalanine tert-butyl hydrochloride was replaced with N-methyl-L-leucine-L-alanine tert-butyl ester (0.0327 g, 0.12 mmol). No alkali was added, and the reaction time was extended from 3 h to 4 h. The product was a colorless oil, 0.0318 g, yield 44%, HPLC purity 99%. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.76(d,J=7.6Hz,2H),7.58(d,J=7.4Hz,2H),7.40(t,J=7.5Hz,2H),7. 31(ddd,J=9.8,4.8,2.0Hz,2H),6.63(d,J=9.4Hz,1H),6.47(d,J=7.4Hz,1H),5.19–5.08(m,1H),4.73 (dd,J=9.0,6.6Hz,2H),4.59-4.30(m,3H),4.26(t,J=6.5Hz,1H),3.05-2.76(m,6H),2.03-1.77(m,2 H),1.65(t,J=7.6Hz,4H),1.44(s,10H),1.29(d,J=6.6Hz,3H),0.91(ddq,J=32.7,19.4,6.6Hz,18H). 13C NMR(101MHz,Chloroform-d)δ172.65,171.78,170.90,169.85,141.48,127.86,127.22,125.04,120.15,82.12,67.91,56.94,54.58 ,53.91,48.72,47.41,36.69,36.16,31.23,30.77,29.81,28.05,24.88,24.85,23.18,22.09,19.68,18.54,17.68.HRMS(ESI):calcd for[C 41 H 61 N4O7] + 721.4462, found 721.4464.

[0121] Example 31

[0122]

[0123] The product of Example 24 (0.6344 g, 1.4 mmol), trifluoroacetic acid (5 mL), and dichloromethane (7 mL) were stirred in a reaction flask at room temperature for 1 h. After the reaction was complete, the product was washed three times with water, and the aqueous phase was washed three times with dichloromethane. The organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 0.504 g of Fmoc-L-Abu-L-MeGly-OH as a white solid, with a yield of 91%, which was used in the next step. The product of Example 30 (0.2163 g, 0.3 mmol), diethylamine (1.0 mL), and methanol (2.6 mL) were stirred in a reaction flask at room temperature for 1 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was purified by column chromatography to obtain the product.

[0124] N-Me-L-Leu-L-Val-L-MeLeu-L-Ala-O t Bu, a colorless oil, was prepared in 0.1418 g form (95% yield) and used in the next step. The resulting Fmoc-L-Abu-L-MeGly-OH (0.0793 g, 0.2 mmol), N-Me-L-Leu-L-Val-L-MeLeu-L-Ala-O tBu (0.1194 g, 0.24 mmol) and ethyl acetate (2.5 mL) were placed in a reaction flask, heated to 40 °C and stirred for 5 h. The reaction mixture was washed with 2 mol / L sodium hydroxide solution to remove residual Fmoc-L-Abu-L-MeGly-OH, pentylene anhydride, and pentylene acid. The remaining amine was removed by washing with 1 mol / L hydrochloric acid. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The product was purified by column chromatography. The product was a white solid, 0.1126 g, yield 63%, HPLC purity 99%. The product was characterized as follows: 1 H NMR(400MHz,Chloroform-d)δ7.74(d,J=7.5Hz,2H),7.66-7.55(m,2H),7.37(td,J=7.6,3.5Hz,2H), 7.30(dd,J=7.5,5.0Hz,2H),6.49(d,J=8.0Hz,1H),6.01(d,J=8.4Hz,1H),5.26-4.86(m,2H),4.76-4. 48(m,2H),4.43-4.28(m,3H),4.23(t,J=7.1Hz,1H),3.92(d,J=15.6Hz,1H),3.38-2.73(m,9H),2.34- 2.04(m,3H),1.94-1.46(m,6H),1.44(d,J=7.5Hz,9H),1.27(s,1H),1.24(s,2H),1.01-0.79(m,21H). 13 C NMR(101MHz,Chloroform-d)δ172.64,172.55,171.66,169.74,168.85,156.11,144 .04,143.87,141.30,127.66,127.08,125.24,119.93,81.95,67.03,54.95,54.52, 54.03,51.86,49.77,48.59,47.19,37.04,36.39,36.07,30.99,30.71,30.27,27.9 2,25.93,24.98,24.76,23.05,21.92,19.45,18.36,17.89,9.46.HRMS(ESI):calcd for[C 48 H 73 N6O9] + 877.5361, found 877.5362.

[0125] Conditional optimization

[0126]

[0127]

[0128] Currently, traditional methods for synthesizing dipeptides involve using various condensing agents, such as EDCi, HOBt / HOAt, HATU, HBTU, and PyBop. These methods often suffer from problems such as easy racemization of the product, long reaction times, low yields, and high agent costs. In addition, the mixed anhydride method is also commonly used for dipeptide synthesis; however, currently published methods all require the amino acids to be prepared into mixed anhydrides before the next condensation step, which is cumbersome. This method, however, can achieve in-situ synthesis of dipeptides from amino acids and N-methyl amino acids in a short time, with high yields and no obvious racemization detected.

Claims

1. A method for preparing an N-methylated polypeptide, characterized in that, The preparation method is carried out according to the following steps: (1) The compound shown in Formula I, the compound shown in Formula II, pivalic anhydride, and a basic substance are dissolved together in an organic solvent to obtain a reaction mixture. The mixture is reacted at 15-80℃ for 3-10 hours. The resulting reaction solution is post-treated to obtain an N-methyl dipeptide compound shown in Formula III. The molar ratio of the compound shown in Formula I, the compound shown in Formula II, pivalic anhydride, and the basic substance is 1:1.0-1.5:1.1-1.6:0-4.

5. The compound shown in Formula II is a compound of Formula II. 1 Or Formula II 2 ; Wherein, R in equation I 1 Selected from one of the following groups: P 1 Selected from the following amino protecting groups: Fmoc, Cbz, or Boc; Equation II is Equation II 1 Or Formula II 2 R 2 It is methyl, isopropyl, isobutyl, or benzyl; R in Formula III 1 With R in Equation I 1 Same, R 2 With Equation II 1 Or Formula II 2 R in 2 Same, P 1 With P in Equation I 1 same; (2) Take the P from the N-methyl dipeptide compound of formula III obtained in step (1) 1 The N-methyl dipeptide compound is obtained by removing the group or tert-butyl group. The free N-methyl dipeptide compound is then condensed with an amino-protected amino acid or an N-methyl amino acid ester to obtain the N-methylated polypeptide.

2. The method for preparing the N-methylated polypeptide according to claim 1, characterized in that, The compound shown in Formula II is of Formula II 1 The molar ratio of the compound shown in Formula I, the compound shown in Formula II, pentylene anhydride, and the basic substance is 1:1.0-1.5:1.1-1.6:1-4.

5.

3. The method for preparing the N-methylated polypeptide according to claim 1, characterized in that, The compound shown in Formula II is of Formula II 2 The alkaline substance is 0.

4. The method for preparing the N-methylated polypeptide according to claim 1, characterized in that, The post-treatment in step (1) is as follows: the reaction solution is washed sequentially with sodium hydroxide aqueous solution, hydrochloric acid aqueous solution, and saturated brine. The resulting organic phase is dried with anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and the solution is purified by column chromatography to obtain the N-methyl dipeptide compound shown in Formula III. The molar concentration of the sodium hydroxide aqueous solution is 0.5 mol / L-2 mol / L, and the mass concentration of the hydrochloric acid aqueous solution is 0.5 mol / L-3 mol / L.

5. The method for preparing the N-methylated polypeptide according to claim 1, characterized in that, The amino protecting group in the amino-protected amino acid in step (2) is Fmoc, Cbz or Boc.

6. The method for preparing the N-methylated polypeptide according to claim 1, characterized in that, The N-methyl amino acid ester mentioned in step (2) is N-methyl amino acid tert-butyl ester.

7. The method for preparing the N-methylated polypeptide according to claim 1, characterized in that, In step (2), diethylamine and the N-methyl dipeptide compound of formula III obtained in step (1) are dissolved together in an organic solvent and stirred for 0.5-2 hours to obtain P. 1 Free N-methyl dipeptide compounds with the removal of the radical group.

8. The method for preparing the N-methylated polypeptide as described in claim 1, characterized in that, In step (2), trifluoroacetic acid and the N-methyl dipeptide compound of formula III obtained in step (1) are dissolved together in an organic solvent and stirred for 0.5-2 hours to obtain the free N-methyl dipeptide compound with tert-butyl removed.

9. The method for preparing the N-methylated polypeptide as described in claim 1, characterized in that, The alkaline substance is one or more of triethylamine, N-methylimidazole, potassium carbonate, sodium carbonate, sodium bicarbonate, lithium hydroxide monohydrate, or potassium hydroxide.

10. The method for preparing the N-methylated polypeptide as described in claim 1, characterized in that, The organic solvent in step (1) is one or more of acetonitrile, dichloromethane, ethyl acetate or N,N-dimethylformamide, and the volume of the organic solvent is 5-15 mL / mmol based on the amount of the compound shown in Formula I.

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