Preparation method of cyclic peptide Lagunamide D

Abstract

The invention discloses a preparation method of cyclic peptide Lagunamide D. The method comprises the following steps: by taking a compound 12 as a raw material, carrying out Corey-Bakshii-Shibata reduction reaction, hydroxyl protection reaction, debenzylation reaction, Pinnick oxidation reaction, Yamagguchi esterification reaction, Fmoc protecting group removal and condensation reaction, and then carrying out allyl ester deprotection, Boc group deprotection and condensation reaction to obtain the cyclic peptide Lagunamide D. According to the invention, a compound 12 is used as a raw material, and Lagunamide D is synthesized for the first time; the synthesis route is short; the total yield is far higher than the extraction efficiency from the nature.

Description

Technical Field

[0001] The invention relates to the technical field of organic synthesis, and in particular to a preparation method of cyclic peptide Lagunamide D. Background Art

[0002] Cyclic peptides are polypeptides formed by the cyclization of terminal amino acids. Compared with linear peptides, cyclic peptides have absolute advantages in structural rigidity, biochemical stability, binding affinity, and membrane permeability, which greatly improves their biological activity. Because natural cyclic peptides have these excellent properties, they are often considered to be lead compounds for new drug research and development.

[0003] The marine natural product Lagunamide D is a cyclic peptide ester natural product isolated by American scientists from Marine Cyanobacteria in Florida in 2019. Studies have found that Lagunamide D has significant anti-proliferative activity against human A549 lung cancer cells and HCT116 colorectal cancer cells. Its IC 50 The concentrations of Lagunamide D and Lagunamide D were 7.1nM and 5.1nM, respectively, which are of great value for drug development and are expected to become lead compounds. However, the extraction efficiency of Lagunamide D from the isolated source is only 0.003%, which cannot meet the subsequent drug activity research. So far, no total synthesis research on Lagunamide D has been reported.

[0004]

[0005] Therefore, it is urgent to design an efficient, concise and stereospecific synthetic route. The artificial synthesis of Lagunamide D will be of great significance for its subsequent activity screening and structure-activity relationship research. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the first aspect of the present invention provides a preparation method of cyclic peptide Lagunamide D, which has a short synthesis route and a high yield.

[0007] According to the first aspect of the present invention, a method for preparing a cyclic peptide Lagunamide D is provided, comprising the following steps:

[0008] (1) subjecting compound 12 to Corey-Bakshi-Shibata reduction reaction to obtain compound 14;

[0009] (2) Compound 14 and tert-butyldimethylsilyl trifluoromethanesulfonate are subjected to a hydroxyl protection reaction to obtain compound 15;

[0010] (3) subjecting compound 15 to a debenzylation reaction to obtain compound 16;

[0011] (4) reacting Fmoc-N-methyl-L-alanine with oxalyl chloride to obtain intermediate I; and then reacting intermediate I with compound 16 to obtain compound 17;

[0012] (5) Compound 17 is subjected to olefin metathesis reaction with methacrolein to obtain compound 19;

[0013] (6) subjecting compound 19 to Pinnick oxidation reaction to obtain compound 20;

[0014] (7) Compound 20 and compound 21 are subjected to a Yamaguchi esterification reaction to obtain compound 22;

[0015] (8) removing the Fmoc protecting group from compound 22 to obtain compound 24;

[0016] (9) in the presence of a condensing agent, subjecting compound 23 and compound 24 to a condensation reaction to obtain compound 25;

[0017] (10) Compound 25 is subjected to allyl ester deprotection, Boc group deprotection and condensation reaction to obtain Lagunamide D;

[0018] Wherein, the structural formulas of Compound 12, Compound 14, Compound 15, Compound 16, Compound 17, Compound 19, Compound 20, Compound 21, Compound 22, Compound 23, Compound 24, Compound 25 and Lagunamide D are as follows:

[0019]

[0020] The preparation method according to the embodiment of the present invention has at least the following beneficial effects:

[0021] The present invention uses compound 12 as a raw material to synthesize Lagunamide D for the first time; the synthesis route is short; and the total yield is much higher than the efficiency of extraction from nature.

[0022] According to a preferred embodiment of the present invention, in the Corey-Bakshi-Shibata reduction reaction, the CBS catalyst used includes at least one of (R)-2-methyl-CBS-oxazaborolidine, (R)-2-phenyl-CBS-oxazaborolidine, and (R)-2-n-butyl-CBS-oxazaborolidine.

[0023] According to a preferred embodiment of the present invention, the compound 12 is prepared by the following method:

[0024] Compound 11 was subjected to Weinreb ketone synthesis reaction to obtain compound 12;

[0025] The structural formula of the compound 11 is as follows:

[0026]

[0027] According to a preferred embodiment of the present invention, the compound 11 is prepared by the following method:

[0028] The compound 10, N,O-dimethylhydroxylamine hydrochloride and isopropylmagnesium chloride are reacted to obtain the product;

[0029] The structural formula of the compound 10 is as follows:

[0030]

[0031] According to a preferred embodiment of the present invention, the compound 10 can be purchased commercially.

[0032] According to a preferred embodiment of the present invention, in step (9), the condensing agent includes HATU and HOAt.

[0033] According to a preferred embodiment of the present invention, in step (5), the reaction temperature is 35°C to 60°C; and the reaction time is 12h to 36h.

[0034] According to a preferred embodiment of the present invention, in step (6), the Pinnick oxidation reaction step comprises:

[0035] Compound 19, sodium chlorite, sodium dihydrogen phosphate and a solvent are mixed and reacted to obtain the product.

[0036] According to a preferred embodiment of the present invention, in step (10), the step of allyl ester deprotection reaction comprises:

[0037] Compound 22, N-methylaniline, a palladium catalyst and a solvent are mixed to react.

[0038] According to a preferred embodiment of the present invention, in step (10), the step of deprotecting the Boc group comprises:

[0039] The product after the allyl ester deprotection reaction is completed is mixed with trifluoroacetic acid for reaction to obtain the product.

[0040] According to a preferred embodiment of the present invention, in step (10), the condensation reaction step comprises:

[0041] The intermediate after deprotection of the Boc group is mixed with HATU, HOAt and 2,4,6-trimethylpyridine to react to obtain the product.

[0042] According to a preferred embodiment of the present invention, in step (3), the reaction conditions of the debenzylation reaction include mixing compound 15 and 2,3-dichloro-5,6-dicyanobenzoquinone for reaction, preferably, the reaction temperature is 30 to 60° C.; the reaction time is 30 to 180 min.

[0043] According to a preferred embodiment of the present invention, in step (5), compound 17, methacrolein, Grubbs II catalyst and solvent are mixed and reacted to obtain compound 19; preferably, the reaction temperature is 25 to 60° C.; and the reaction time is 12 to 36 hours.

[0044] According to a preferred embodiment of the present invention, in step (8), the method for removing the Fmoc protecting group can be selected from conventional methods used by those skilled in the art, such as removal under alkaline conditions.

[0045] According to a preferred embodiment of the present invention, the compound 23 is prepared by the following method:

[0046] Compound 9 is reacted under alkaline conditions to obtain the compound.

[0047] The structural formula of the compound 9 is as follows:

[0048]

[0049] According to a preferred embodiment of the present invention, the compound 9 is prepared by the following method:

[0050] The Boc group of compound 7 is removed; the compound 7 is then mixed with compound 8, HOAT, DIPEA and HATU and stirred for reaction; the product is obtained.

[0051] The structural formulas of the compounds 7 and 8 are as follows:

[0052]

[0053] According to a preferred embodiment of the present invention, the compound 7 is prepared by the following method:

[0054] The Boc group of compound 5 is removed; the compound 5 is then mixed with compound 6, HOAT, DIPEA and HATU for reaction to obtain the product.

[0055] The structural formulas of the compounds 5 and 6 are as follows:

[0056]

[0057] According to a preferred embodiment of the present invention, the compound 5 is prepared by the following method:

[0058] Boc-N-methyl-L-phenylalanine, sarcosine methyl ester hydrochloride and DMF were mixed, and HOAT, DIPEA and HATU were added in sequence at room temperature with stirring to react.

[0059] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. DETAILED DESCRIPTION

[0060] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0061] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0062] Example

[0063] This example provides a method for preparing cyclic peptide Lagunamide D, comprising the following steps:

[0064] Synthesis of compound 5 , the reaction equation and preparation steps are as follows:

[0065]

[0066] Sarcosine methyl ester hydrochloride (0.65 g, 4.7 mmol) and Boc-N-methyl-L-phenylalanine (1.00 g, 3.6 mmol) were dissolved in anhydrous DMF (5.0 mL), and HOAT (0.1 g, 0.72 mmol), DIPEA (3.1 mL, 17.9 mmol) and HATU (2.72 g, 7.2 mmol) were added in sequence at room temperature and stirred overnight. The reaction was quenched with saturated aqueous ammonium chloride solution, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, washed with saturated aqueous ammonium chloride solution, water, and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:2) to obtain compound 5 (1.20 g, 93%).

[0067] The data of compound 5 are as follows:

[0068] 1H NMR (500MHz, CDCl3) δ7.31–7.11(m,5H),5.33(dd,J=8.4,6.7Hz,0.33H),5.21(dd,J=9.1,6.0Hz,0.17H),5.02(dd,J=9.6,5.1Hz ,0.33H),4.85(dd,J=9.2,5.3Hz,0.17H),4.39–4.14(m,1H),4.04–3.90(m,0.67H),3.78(d,J=17.2Hz,0.33H),3.72(s,1H),3.71 (s,1H),3.68(s,0.5H),3.67(s,0.5H),3.14(dd,J=13.9,6.7Hz,0.5H),3.07(dd,J=22.3,5.7Hz,0.5H),3.04–2.98(m,3H),2.98 –2.90(m,1H),2.84(s,1H),2.81(s,1H),2.73(s,0.5H),2.70(s,0.5H),1.31(s,3H),1.25(s,1.5H),1.17(s,3H),1.12(s,1.5H);

[0069] 13 C NMR (126MHz, CDCl3) δ170.9,170.6,170.5,170.1,169.5,169.5,169.4,169.1,155.2,15 5.2,154.3,153.9,138.1,138.0,137.6,137.5,129.5,129.5,129.5,128.4,128.4,128. 2,126.4,126.3,80.4,80.1,80.1,80.0,58.1,57.9,55.8,55.6,52.3,52.2,52.1,52.1,50.7,50.5,50.0,49.9,36.0,35.8,35.1,35.1,29.6,28.9,28.9,28.2,28.1,27.9,27.7;

[0070] HRMS(ESI)m / z:C 19 H 28 N2NaO5 + [M+Na] + :calcd:387.1890; found:387.1896.

[0071] Synthesis of compound 7 : The reaction equation and preparation steps are as follows:

[0072]

[0073] Compound 5 (2.75 g, 8.2 mmol) was dissolved in a mixed solution of THF / MeOH / H2O (2:2:1) (25.0 mL), cooled to 0°C, and LiOH·H2O (0.39 g, 16.5 mmol) was added. The reaction mixture was stirred at 0°C to room temperature for two hours, and then the pH value was adjusted to 3 with 1M hydrochloric acid aqueous solution. The low boiling point organic solvent was evaporated on a rotary evaporator, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated acid was pumped on a high vacuum oil pump for 3 hours. Then it was dissolved in DMF (10.0 mL) together with compound 6 (3.62 g, 7.5 mmol), and HOAT (1.03 g, 7.5 mmol), DIPEA (9.2 mL, 52.7 mmol) and HATU (35.7 g, 15.1 mmol) were added in sequence at room temperature and stirred overnight. The reaction was quenched with saturated aqueous ammonium chloride solution, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, washed with saturated aqueous ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:2) to give compound 7 (3.70 g, 68%).

[0074] The data of compound 7 are as follows:

[0075] 1H NMR(500MHz,CDCl3)δ7.85–7.74(m,2H),7.66–7.55(m,2H),7.46–7.38(m,2H),7.38–7.31(m,2H),7.30–7.14(m,5H),6.82(d,J=8.1Hz,0.15H),6.63(d,J=8.6Hz,0.3H),6.56(d,J=8.6Hz,0.45H),6.21(d,J=8.6Hz,0.1H),5.37(t,J=7.6Hz,0.45H),5.15(t,J=7.7Hz,0.15H),5.06(dd,J=9.2,5.4Hz,0.3H),5.00(dd,J=10.4,4.2Hz,0.1H),4.64–4.51(m,3H),4.28–4.17(m,1.5H),4.09–3.95(m,0.8H),3.95–3.80(m,0.3H),3.75(d,J=15.3Hz,0.4H),3.17(dd,J=14.0,7.1Hz,0.45H),3.12–3.06(m,0.45H),3.07(s,1H),3.06–3.02(m,1.55H),3.02–2.93(m,1.55H),2.89–2.81(m,3H),1.88–1.75(m,1H),1.42–1.32(m,1H),1.35(s,3H),1.31–1.26(m,1H),1.25–1.13(m,1H),1.20(s,3H),1.11(s,1H),1.03–0.91(m,1H),0.88–0.75(m,6H);

[0076] 13 C NMR(126MHz,CDCl3)δ171.6,171.5,171.5,171.2,168.5,168.3,155.4,154.3,143.6,143.4,143.4,141.4,141.4,137.9,137.4,129.5,129.4,128.5,128.3,128.3,127.9,127.2,126.5,126.5,124.9,124.8,120.1,80.3,80.1,66.7,66.6,57.8,56.5,56.5,55.6,53.5,52.7,52.5,46.8,37.6,37.5,36.4,36.1,35.3,29.7,29.1,28.2,28.1,28.0,27.7,24.9,24.9,24.8,15.3,15.3,11.6;

[0077] HRMS(ESI)m / z:C 38 H 48 N3O6 + [M+H] + :calcd:642.3538; found:642.3542.

[0078] Synthesis of compound 9 : The reaction equation and preparation steps are as follows:

[0079]

[0080] Compound 7 (3.58 g, 5.0 mmol) was dissolved in dichloromethane (25.0 mL), TFA (5.0 mL) was added at room temperature, stirred for 0.5 hours, the solvent was evaporated under reduced pressure, and the mixture was pumped on a high vacuum oil pump for 3 hours, then dissolved in DMF together with compound 8, HOAT (0.68 g, 5.0 mmol), DIPEA (6.07 mL, 34.9 mmol) and HATU (3.79 g, 10.0 mmol) were added in sequence at room temperature, and stirred overnight. The reaction was quenched with saturated aqueous ammonium chloride solution, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, washed with saturated aqueous ammonium chloride solution and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 2:3) to obtain compound 9 (2.40 g, 61%).

[0081] 1H NMR(500MHz,CDCl3)δ7.85–7.75(m,2H),7.65–7.58(m,2H),7.45–7.38(m,2H),7.33(q,J=7.8Hz,2H),7.30–7.12(m,5H),6.83(d,J=8.3Hz,0.3H),6.64(d,J=8.7Hz,0.7H),5.78(dd,J=9.2,6.7Hz,0.7H),5.62(dd,J=9.8,6.2Hz,0.3H),5.38(d,J=8.2Hz,0.7H),5.30(d,J=7.8Hz,0.3H),4.67–4.50(m,3H),4.49–4.36(m,1H),4.22(t,J=6.1Hz,1H),4.17(d,J=17.0Hz,0.3H),4.13(d,J=15.5Hz,0.7H),3.87(d,J=15.5Hz,0.7H),3.72(d,J=17.0Hz,0.3H),3.23–3.14(m,1H),3.14–3.06(m,1H),3.04(s,3H),3.00(s,3H),1.90–1.77(m,1H),1.44–1.34(m,9H),1.22–1.13(m,1H),1.04–0.95(m,1H),0.88(d,J=6.8Hz,3H),0.85–0.75(m,6H);

[0082] 13 C NMR(126MHz,CDCl3)δ173.9,173.3,171.7,171.5,171.1,170.2,168.2,155.3,143.5,143.3,141.4,141.3,136.4,129.3,129.2,128.4,127.9,127.9,127.2,127.2,126.8,124.9,124.8,120.1,79.6,79.5,66.7,66.7,56.7,56.4,53.9,53.4,52.5,52.4,46.8,46.8,46.6,37.6,37.6,36.6,35.5,35.4,35.2,30.4,29.7,28.3,28.3,25.0,24.8,17.7,17.5,15.3,15.3,11.7,11.6;

[0083] HRMS(ESI)m / z:C 41 H 52 N4NaO7 + [M+Na] +:calcd:735.3728; found:735.3726.

[0084] Synthesis of compound 11 : The reaction equation and preparation steps are as follows:

[0085]

[0086] Compound 10 (2.32 g, 4.4 mmol) and N,O-dimethylhydroxylamine hydrochloride (3.03 g, 31.1 mmol) were dissolved in tetrahydrofuran and cooled to -30° in a low temperature reactor. Isopropylmagnesium chloride (26.6 mL, 53.3 mmol) was slowly added dropwise, and after stirring for 1 hour, the reaction mixture was slowly heated from -30° to -20°, stirred for 1 hour, and then slowly heated from -20° to 0°, and stirred for 1 hour. The reaction was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, and the organic phases were combined and washed with saturated aqueous ammonium chloride solution and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:4) to obtain compound 11 (0.95 g, 78%).

[0087] The data of compound 11 are as follows:

[0088] 1 H NMR (500MHz, CDCl3) δ7.43–7.25(m,5H),4.54(s,2H),3.75(ddd,J=8.7,6.8,3.1Hz,1H),3.65(s,3H),3.29–3 .17(m,1H),3.22(s,3H),1.69–1.58(m,1H),1.58–1.40(m,3H),1.10(d,J=6.9Hz,3H),0.95(t,J=7.1Hz,3H);

[0089] 13 C NMR (126MHz, CDCl3) δ176.4,139.0,128.2,127.8,127.3,81.0,72.8,61.4,39.1,33.5,32.0,18.0,14.4,13.5;

[0090] HRMS(ESI)m / z:C 16 H 25 NNaO3 + [M+Na] + :calcd:302.1727; found:302.1737.

[0091] Synthesis of compound 12: The reaction equation and preparation steps are as follows:

[0092]

[0093] Compound 11 (1.50 g, 5.4 mmol) was dissolved in tetrahydrofuran, cooled to 0°C, and allylmagnesium bromide (13.6 mL, 13.6 mmol, 1.0 M in THF) was added. The reaction mixture was heated to room temperature and stirred for 0.5 hours. The reaction was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, and the organic phases were combined, washed with saturated aqueous ammonium chloride solution and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:30) to obtain compound 12 (1.23 g, 86%).

[0094] 1 H NMR (500MHz, CDCl3) δ7.40–7.27(m,5H),5.95(ddt,J=17.1,10.2,6.9Hz,1H),5.19(d d,J=10.2,1.5Hz,1H),5.10(dd,J=17.2,1.6Hz,1H),4.52(d,J=11.1Hz,1H),4.43(d,J =11.0Hz,1H),3.71(ddd,J=8.2,6.0,3.6Hz,1H),3.28(dd,J=6.9,1.7Hz,2H),2.93(d q,J=8.3,7.0Hz,1H),1.58–1.39(m,4H),1.05(d,J=7.0Hz,3H),0.95(t,J=7.0Hz,3H); 13 C NMR (126MHz, CDCl3) δ211.8,138.4,130.7,128.3,127.9,127.6,118.6,81.0,72.3,49.0,48.3,33.1,17.7,14.3,12.6;

[0095] HRMS(ESI)m / z:C 17 H 24 NaO2 + [M+Na] + :calcd:283.1669; found:283.1677.

[0096] (1) Synthesis of compound 14: The reaction equation and preparation steps are as follows:

[0097]

[0098] Compound 12 (0.40 g, 1.5 mmol) was dissolved in tetrahydrofuran, and (R)-Me-CBS compound 13 (2.3 mL, 2.3 mmol, 1.0 M in THF) was added at room temperature, stirred for 10 minutes, cooled to -78 under nitrogen protection, and borane dimethyl sulfide complex (0.20 mL, 2.0 mmol, 10.0 M in THF) was added. The resulting reaction solution was stirred at -78 ° C for 3 hours, then slowly heated to -20 ° C and stirred for 0.5 hours, and finally heated to 0 ° C, methanol was added to quench, and then heated to room temperature, extracted with ethyl acetate, the organic phases were combined, washed with saturated ammonium chloride aqueous solution and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:10) to obtain compound 14 (0.40 g, 89%).

[0099] The data of compound 14 are as follows:

[0100] 1 H NMR (500MHz, CDCl3) δ7.42–7.28(m,5H),6.00–5.86(m,1H),5.25–5.12(m,2H),4.62–4.46(m,2H),3.68–3.57(m,2H),2.49–2.38(m,1H),2. 22–2.08(m,1H),2.00–1.89(m,1H),1.68–1.57(m,1H),1.57–1.49(m,2H),1.48–1.40(m,1H),0.95(t,J=7.2Hz,3H),0.88(d,J=7.0Hz,3H);

[0101] 13 C NMR (126MHz, CDCl3) δ138.5,135.2,128.4,127.9,127.6,117.8,82.2,73.17,7.35,4.55,39.2,32.3 18.0,14.4,11.9;

[0102] HRMS(ESI)m / z:C 17 H 26 NaO2 + [M+Na] + :calcd:285.1825; found:285.1832.

[0103] (2) Synthesis of compound 15: The reaction equation and preparation steps are as follows:

[0104]

[0105] Compound 14 (2.22 g, 8.5 mmol) was dissolved in dichloromethane, triethylamine (4.7 mL, 33.8 mmol) and tert-butyldimethylsilyl trifluoromethanesulfonate (3.9 mL, 16.9 mmol) were added successively at room temperature, and stirred for 1 hour. The reaction was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, and the organic phases were combined, washed with saturated aqueous ammonium chloride solution and saturated brine successively, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether) to obtain compound 15 (2.11 g, 67%).

[0106] The data of compound 15 are as follows:

[0107] 1 H NMR(500MHz, CDCl3)δ7.45–7.24(m,5H),5.95–5.83(m,1H),5.11–5.01(m,2H),4.56–4.44 (m,2H),3.82(td,J=6.1,4.5Hz,1H),3.53(ddd,J=8.6,5.7,3.2Hz,1H),2.39–2.26(m,1H), 2.26–2.18(m,1H),2.11–1.99(m,1H),1.63–1.54(m,1H),1.54–1.42(m,2H),1.42–1.28(m ,1H),0.94(t,J=6.9Hz,3H),0.92(s,9H),0.86(d,J=7.0Hz,3H),0.08(s,3H),0.05(s,3H);

[0108] 13 C NMR (126MHz, CDCl3) δ139.2,135.4,128.3,127.8,127.3,116.7,79.8,72.8,71.0,40.2,38.1,32.3,25.9,18.9,18.1,14.5,10.1,-4.0,-4.7;

[0109] HRMS(ESI)m / z:C 23 H 40 NaO2Si + [M+Na] + :calcd:399.2690; found:399.2699.

[0110] (3) Synthesis of compound 16: The reaction equation and preparation steps are as follows:

[0111]

[0112] Compound 15 (1.62 g, 4.3 mmol) was dissolved in 1,2-dichloroethane (20.0 mL), heated from room temperature to 50°C, 2,3-dichloro-5,6-dicyanobenzoquinone (3.89 g, 17.2 mmol) was added, and stirred for 1 hour. The mixture was extracted with dichloromethane, washed with 1M sodium hydroxide / 1M sodium thiosulfate / water (1:1:1) (15 mL) and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:20) to obtain compound 16 (1.20 g, 97%).

[0113] The data of compound 16 are as follows:

[0114] 1 H NMR(500MHz, CDCl3)δ5.94–5.82(m,1H),5.18–4.99(m,2H),3.80(q,J=5.4Hz,1H),3.61–3.54(m,1H),2.74(s,1H),2.41–2.33(m,1H),2.32–2 .23(m,1H),1.76–1.68(m,1H),1.58–1.49(m,2H),1.43–1.33(m,2H),0 .95(t,J=6.6Hz,3H),0.93(s,9H),0.84(d,J=7.0Hz,3H),0.11(s,6H);

[0115] 13 C NMR (126MHz, CDCl3) δ135.0,117.1,75.9,73.8,43.1,39.3,36.5,25.9,18.6,18.0,14.2,12.8,-4.2,-4.7;

[0116] HRMS(ESI)m / z:C 16 H 34 NaO2Si + [M+Na] + :calcd:309.2220; found:309.2226.

[0117] (4) Synthesis of compound 17: The reaction equation and preparation steps are as follows:

[0118]

[0119] Fmoc-N-methyl-L-alanine (3.17 g, 9.8 mmol) was dissolved in anhydrous dichloromethane (15.0 mL). Oxalyl chloride (2.8 mL, 32.5 mmol) and catalytic amount of DMF (32 μL, 0.41 mmol) were added in sequence at 0°C. After stirring at room temperature for 1 hour, the mixture was concentrated under reduced pressure on a rotary evaporator. The obtained acid chloride was pumped on a high vacuum oil pump for 0.5 hour and used directly in the next step.

[0120] Compound 16 (932 mg, 3.3 mmol) and DMAP (0.99 g, 8.1 mmol) were dissolved in anhydrous dichloromethane (15.0 mL). Triethylamine (4.5 mL, 32.5 mmol) was added under nitrogen protection, cooled to 0 ° C in an ice bath, and the above-prepared acyl chloride (dissolved in 15.0 mL of anhydrous dichloromethane) was added. Warm to room temperature, stir for 1 hour, and then quench the reaction with methanol (3 mL). The reaction solution was diluted with ethyl acetate (150.0 mL), then washed with 1M hydrochloric acid solution, saturated ammonium chloride aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column (Qingdao Ocean) chromatography (ethyl acetate: petroleum ether = 1:20) to obtain compound 17 (1.26 g, 66%).

[0121] The data of compound 17 are as follows:

[0122] 1 H NMR (500MHz, CDCl3) δ7.85–7.77(m,2H),7.70–7.57(m,2H),7.46–7.37(m,2H),7.37–7.30(m,2H),5.91 –5.78(m,1H),5.20–4.97(m,3H),4.97–4.91(m,0.7H),4.85–4.79(m,0.3H),4.49–4.35(m,2H),4.33–4. 23(m,1H),3.72–3.63(m,1H),2.97–2.91(m,3H),2.34–2.24(m,1H),2.22–2.13(m,1H),1.99–1.90(m,1 H),1.64–1.48(m,2H),1.45(d,J=7.3Hz,3H),1.40–1.20(m,2H),1.00–0.82(m,15H),0.12–0.01(m,6H);

[0123] 13C NMR (126MHz, CDCl3) δ171.5,171.3,156.5,155.9,144.1,144.9,141.3,135.4,135.0,127.7,127.1,125.1,125.1,120.0,119.8,116.9,76.1, 76.0,72.9,67.9,67.8,54.2,47.3,41.6,41.5,37.9,37.7,32.8,32.5, 30.4,30.2,25.9,18.8,18.7,18.1,15.4,14.9,14.1,10.4,-4.1,-4.7;

[0124] HRMS(ESI)m / z:C 35 H 51 NNaO5Si + [M+Na] + :calcd:616.3429; found:616.3436.

[0125] (5) Synthesis of compound 19: The reaction equation and preparation steps are as follows:

[0126]

[0127] Compound 17 (1.22 g, 2.1 mmol) was dissolved in anhydrous dichloromethane (10.0 mL), and α-methylacrolein compound 18 (11.1 mL, 0.13 mmol) and Grubbs II catalyst (8.9 mg, 0.010 mmol) were added in sequence at room temperature, nitrogen was blown for 30 minutes, and then the temperature was raised from room temperature to 50° C. and stirred overnight. The reaction solution was concentrated under reduced pressure on a rotary evaporator, and the obtained residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:10) to obtain compound 19 (1.06 g, 81%).

[0128] The data of compound 19 are as follows:

[0129] 1H NMR(500MHz, CDCl3)δ9.38(s,0.73H),9.35(s,0.27H),7.87–7.74(m,2H),7.68–7.55(m,2H),7.52–7.41(m ,2H),7.41–7.30(m,2H),6.67–6.48(m,1H),5.14–5.00(m,1H),4.94–4.81(m,1H),4.52–4.33(m,2H),4.33 –4.20(m,1H),3.95–3.79(m,1H),2.96(s,2.2H),2.92(s,0.8H),2.59–2.42(m,2H),2.02–1.93(m,1H),1.7 4(s,3H),1.65–1.51(m,2H),1.49–1.42(m,3H),1.41–1.22(m,2H),0.99–0.84(m,15H),0.13–-0.02(m,6H);

[0130] 13 C NMR (126MHz, CDCl3) δ195.3,195.0,171.9,156.5,152.0,151.0,144.0,143.9,141.3,140.3,127.7,127.1,127.1,125.0,120.0,120.0,76.0,75 .8,71.6,71.4,68.0,67.8,54.2,47.2,42.6,42.3,33.4,32.2,30.2,25. 8,18.6,18.5,18.0,15.3,14.9,14.2,14.1,10.3,10.2,9.4.-4.4,-4.7;

[0131] HRMS(ESI)m / z:C 37 H 53 NNaO6Si + [M+Na] + :calcd:658.3534; found:658.3541.

[0132] (6) Synthesis of compound 20: The reaction equation and preparation steps are as follows:

[0133]

[0134] Compound 19 (1.05 g, 1.7 mmol) was dissolved in tert-butyl alcohol / isoamylene / water (2:2:1) (40.0 mL), sodium chlorite (4.33 g, 49.5 mmol) and sodium dihydrogen phosphate (9.53 g, 61.1 mmol) were added at room temperature and stirred overnight. The reaction mixture was acidified to pH = 3 with 1M hydrochloric acid, extracted with ethyl acetate, the organic phases were combined, washed with saturated aqueous ammonium chloride solution and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:1) to obtain compound 20 (1.05 g, 98%).

[0135] (7) Synthesis of compound 22: The reaction equation and preparation steps are as follows:

[0136]

[0137] Compound 20 (378 mg, 0.58 mmol), compound 21 (300 mg, 1.7 mmol) and 4-dimethylaminopyridine (0.35 g, 2.9 mmol) were dissolved in xylene, cooled to -20°C, 2,4,6-trichlorobenzoyl chloride (0.30 mL, 1.7 mmol) was added, stirred for 0.5 hours, then warmed from -20°C to room temperature and stirred overnight. The reaction mixture was acidified to pH = 3 with 1M hydrochloric acid, extracted with ethyl acetate, the organic phases were combined, washed with 1M hydrochloric acid, saturated sodium bicarbonate aqueous solution, and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 3:1) to obtain compound 22 (318 mg, 68%).

[0138] The data of compound 22 are as follows:

[0139] 1H NMR(600MHz,CDCl3)δ7.84–7.76(m,2H),7.72–7.56(m,2H),7.48–7.39(m,2H),7.39–7.31(m,2H),7.00–6.88(m,1H),5.91(ddt,J=16.5,10.9,5.6Hz,1H),5.33(d,J=17.4Hz,1H),5.24(d,J=10.3Hz,1H),5.08(d,J=3.5Hz,1H),5.07–5.03(m,1H),4.95–4.86(m,0.66H),4.83–4.79(m,0.34H),4.75–4.60(m,2H),4.49–4.36(m,2H),4.32–4.23(m,1H),3.86–3.79(m,1H),2.93(s,2H),2.91(s,1H),2.42–2.30(m,2H),2.10–2.00(m,1H),2.00–1.91(m,1H),1.88(s,2.5H),1.85(s,0.5H),1.63–1.51(m,3H),1.51–1.39(m,4H),1.37–1.23(m,2H),0.99(d,J=6.9Hz,3H),0.98–0.81(m,18H),0.10–0.00(m,6H);

[0140] 13 C NMR(151MHz,CDCl3)δ171.6,171.2,169.9,167.4,156.4,155.8,144.1,143.9,141.3,141.3,140.6,140.2,131.8,127.7,127.1,125.1,125.1,120.0,118.5,76.2,76.0,74.8,71.7,71.6,67.9,67.8,65.5,54.3,54.1,47.2,42.5,42.4,36.8,33.4,33.1,32.4,32.3,30.2,29.7,26.1,25.8,18.5,17.9,15.3,14.9,14.5,14.1,12.7,11.7,10.4.-4.3,-4.7;

[0141] HRMS(ESI)m / z:C 46 H 67 NNaO9Si + [M+Na] + :calcd:828.4477;found:828.4487.

[0142] (8) and (9), Synthesis of Compound 25: The reaction equation and preparation steps are as follows:

[0143]

[0144] Compound 9 (26 mg, 0.033 mmol) was dissolved in anhydrous acetonitrile (2.0 mL) solvent, diethylamine (1.0 mL) was added, and the mixture was stirred at room temperature for 2 hours. The organic solvent was then evaporated on a rotary evaporator, extracted with ethyl acetate (30.0 mL × 3), and the organic phases were combined and washed with 1M hydrochloric acid (3.0 mL × 2) and saturated brine (3.0 mL) in turn, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained acid was pumped on a high vacuum oil pump for 3 hours. Compound 22 (13 mg, 0.017 mmol) was dissolved in anhydrous acetonitrile (2.0 mL) solvent, diethylamine (1.0 mL) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure on a rotary evaporator to obtain free ammonia, which was pumped on a high vacuum oil pump for 3 hours.

[0145] The acid obtained by removing the Fm protecting group of compound 9 and the free ammonia obtained by removing the Fmoc protecting group of compound 22 were dissolved in DMF (3.0 mL), cooled to 0°C, and HATU (20 mg, 0.050 mmol), HOAt (5.0 mg, 0.034 mmol) and 2,4,6-trimethylpyridine (11 μL, 0.085 mmol) were added under nitrogen protection. The reaction mixture was slowly warmed to room temperature and stirred for 14 hours. The reaction was quenched with saturated aqueous ammonium chloride solution (5.0 mL), extracted with ethyl acetate (30.0 mL×3), and the organic phases were combined and washed with water (15 mL×3) and saturated brine (15 mL) in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 3:2) to obtain compound 25 (8.0 mg, 44%).

[0146] The data of compound 25 are as follows:

[0147] 1H NMR(600MHz,CDCl3)δ7.39–7.13(m,5H),7.13–7.03(m,1H),7.02–6.91(m,1H),6.81–6.66(m,1H),5.98–5.87(m,1H),5.78–5.68(m,1H),5.67–5.59(m,1H),5.38–5.30(m,1H),5.30–5.22(m,1H),5.13–5.07(m,1H),5.07–4.96(m,1H),4.96–4.82(m,1H),4.71–4.61(m,2H),4.59–4.36(m,2H),4.34–4.26(m,1H),3.94–3.84(m,1H),3.84–3.72(m,1H),3.32–3.11(m,2H),3.11–2.80(m,8H),2.42–2.27(m,2H),2.10–2.01(m,1H),1.97–1.80(m,4H),1.79–1.38(m,18H),1.38–1.06(m,15H),1.04–0.82(m,18H),0.09–-0.02(m,6H);

[0148] 13 C NMR(151MHz,CDCl3)δ173.1,172.0,171.3,170.9,170.7,169.8,167.9,167.5,155.3,140.8,136.5,131.8,129.9,129.4,128.4,128.3,126.8,118.6,79.4,76.1,74.9,71.8,65.5,54.2,53.2,52.2,51.9,46.6,42.8,37.6,36.8,36.4,35.3,33.4,32.4,31.9,31.3,30.4,29.7,29.7,29.4,28.3,28.3,26.1,25.8,24.0,22.7,18.7,18.0,17.7,15.5,14.5,14.5,14.1,14.1,12.6,11.7,11.2,10.4,-4.3,-4.7,-4.7;

[0149] HRMS(ESI)m / z:C 58 H 97 N5NaO 13 Si + [M+Na] + :calcd:1122.6744;found:1122.6726.

[0150] (10) Synthesis of cyclic peptide Lagunamide D: The reaction equation and preparation steps are as follows:

[0151]

[0152] Compound 25 (26 mg, 0.024 mmol) and tetrakistriphenylphosphine palladium (2.7 mg, 0.0024 mmol) were dissolved in tetrahydrofuran (2.0 mL) solvent, N-methylaniline (5 μL, 0.048 mmol) was added at 0°, and the mixture was stirred at room temperature for 0.5 hours. The reaction was quenched with trifluoroacetic acid (10 μL, 0.14 mmol), and extracted with ethyl acetate (30.0 mL×3). The organic phases were combined and washed with 1 M hydrochloric acid ( The mixture was washed with saturated sodium chloride (3.0 mL, 3.0 mL × 2) and saturated sodium chloride (3.0 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained acid was pumped on a high vacuum oil pump for 3 hours, then dissolved in trifluoroacetic acid-dichloromethane (v:v=1:5, 3.0 mL) solvent, stirred at room temperature for 0.5 hours, and then concentrated under reduced pressure on a rotary evaporator to obtain a ring-closing precursor, which was pumped on a high vacuum oil pump for 3 hours and then dissolved in N, N-dimethylformamide (10.0 mL) solvent. The ring-closing reagent HATU (90 mg, 0.24 mmol), HOAt (16 mg, 0.12 mmol) and 2,4,6-trimethylpyridine (94 μL, 0.72 mmol) were added. The reaction mixture was stirred at room temperature for 48 hours. The solvent and other volatile substances were removed by reduced pressure distillation under high vacuum. The residue was dissolved in ethyl acetate (100.0 mL), washed with 1 M hydrochloric acid (5.0 mL), water (15.0 mL × 3) and saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (Qingdao Ocean) chromatography (ethyl acetate: petroleum ether = 3:2) to obtain the natural product Lagunamide D (13 mg, 67%).

[0153] The data of Lagunamide D are as follows:

[0154] 1<h2 style=";text-align:left;direction:ltr">H NMR(600MHz,DMSO-d6)δ8.49(d,J=6.4Hz,0.3H),8.34(d,J=8.3Hz,0.1H),7.23–7 .05(m,5.1H),6.92–6.74(m,0.2H),6.74–6.54(m,0.2H),5.81–5.67(m,0.2H),5.3 7–5.23(m,0.6H),5.23–4.99(m,0.6H),4.99–4.90(m,0.6H),4.90–4.81(m,1.0H) ,4.81–4.72(m,0.7H),4.72–4.44(m,0.9H),4.43–4.25(m,0.6H),4.25–4.07(m,0. 8H),4.07–3.87(m,1.1H),3.87–3.72(m,0.3H),3.72–3.38(m,0.7H),3.21(s,2.4 H),3.16–3.06(m,0.6H),3.06–2.88(m,1.9H),2.85(s,2.7H),2.80–2.76(m,0.8H) 2.73(s,2.2H),2.64(s,1.2H),2.45–2.18(m,1.2H),2.15–1.95(m,0.9H),1.95–1.85(m,1.2H),1.85–1.64(m,4.9H),1.62–1.08(m,11.0H),1.06–0.41(m,21.0H);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0155] <h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> C NMR(151MHz,DMSO-d6)δ172.45,170.8,170.14,170.11,169.52,168.23,168.01,1 44.45,137.17,129.18,127.48,126.83,125.84,75.36,74.62,69.3,58.07,52.45 ,51.1,50.4,44.65,40.89,37.29,37.1,36.27,35.79,34.42,33.86,29.6,29.36,25.83,23.19,17.05,14.89,14.69,14.06,13.92,12.93,11.98,11.4,10.27,9.6;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0156] <h2 style=";text-align:left;direction:ltr"> HRMS(ESI)m / z:C<h2 style=";text-align:left;direction:ltr"> 44 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 69 <h2 style=";text-align:left;direction:ltr"> N5NaO<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> + <h2 style=";text-align:left;direction:ltr"> [M+Na]<h2 style=";text-align:left;direction:ltr"> +:calcd:850.4937; found:850.4909.

[0157] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A method for preparing cyclic peptide Lagunamide D, characterized in that: The steps include: (1) subjecting compound 12 to Corey-Bakshi-Shibata reduction reaction to obtain compound 14; (2) Compound 14 and tert-butyldimethylsilyl trifluoromethanesulfonate are subjected to a hydroxyl protection reaction to obtain compound 15; (3) subjecting compound 15 to a debenzylation reaction to obtain compound 16; (4) reacting Fmoc-N-methyl-L-alanine with oxalyl chloride to obtain intermediate I; and then reacting intermediate I with compound 16 to obtain compound 17; (5) Compound 17 is subjected to olefin metathesis reaction with methacrolein to obtain compound 19; (6) subjecting compound 19 to Pinnick oxidation reaction to obtain compound 20; (7) Compound 20 and compound 21 are subjected to a Yamaguchi esterification reaction to obtain compound 22; (8) removing the Fmoc protecting group from compound 22 to obtain compound 24; (9) in the presence of a condensing agent, subjecting compound 23 and compound 24 to a condensation reaction to obtain compound 25; (10) Compound 25 is subjected to allyl ester deprotection, Boc group deprotection and condensation reaction to obtain Lagunamide D; Wherein, the structural formulas of Compound 12, Compound 14, Compound 15, Compound 16, Compound 17, Compound 19, Compound 20, Compound 21, Compound 22, Compound 23, Compound 24, Compound 25 and Lagunamide D are as follows:

2. The method for preparing the cyclic peptide Lagunamide D according to claim 1, characterized in that: In the Corey-Bakshi-Shibata reduction reaction, the CBS catalyst used includes at least one of (R)-2-methyl-CBS-oxazaborolidine, (R)-2-phenyl-CBS-oxazaborolidine, and (R)-2-n-butyl-CBS-oxazaborolidine.

3. The method for preparing the cyclic peptide Lagunamide D according to claim 1 or 2, characterized in that: The compound 12 was prepared by the following method: Compound 11 was subjected to Weinreb ketone synthesis reaction to obtain compound 12; The structural formula of the compound 11 is as follows:

4. The method for preparing the cyclic peptide Lagunamide D according to claim 3, characterized in that: The compound 11 was prepared by the following method: The compound 10, N,O-dimethylhydroxylamine hydrochloride and isopropylmagnesium chloride are reacted to obtain the product; The structural formula of the compound 10 is as follows:

5. The method for preparing the cyclic peptide Lagunamide D according to claim 1, characterized in that: In step (9), the condensing agent includes HATU and HOAt.

6. The method for preparing the cyclic peptide Lagunamide D according to claim 1, characterized in that: In step (5), the reaction temperature is 35°C to 60°C; and the reaction time is 12h to 36h.

7. The method for preparing the cyclic peptide Lagunamide D according to claim 1, characterized in that: In step (6), the Pinnick oxidation reaction comprises: Compound 19, sodium chlorite, sodium dihydrogen phosphate and a solvent are mixed and reacted to obtain the product.

8. The method for preparing the cyclic peptide Lagunamide D according to claim 1, characterized in that: In step (10), the step of allyl ester deprotection reaction comprises: Compound 22, N-methylaniline, a palladium catalyst and a solvent are mixed to react.

9. The method for preparing the cyclic peptide Lagunamide D according to claim 1, characterized in that: In step (10), the step of deprotecting the Boc group comprises: The product after the allyl ester deprotection reaction is completed is mixed with trifluoroacetic acid for reaction to obtain the product.

10. The method for preparing the cyclic peptide Lagunamide D according to claim 1, characterized in that: In step (10), the condensation reaction comprises: The intermediate after deprotection of the Boc group is mixed with HATU, HOAt and 2,4,6-trimethylpyridine to react to obtain the product.

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