A method for preparing the cyclic peptide Lagunamide D

Abstract

This invention discloses a method for preparing the cyclic peptide Lagunamide D. The method uses compound 12 as a starting material and proceeds through Corey-Bakshi-Shibata reduction, hydroxyl protection, debenzylation, Pinnick oxidation, Yamaguchi esterification, removal of the Fmoc protecting group, condensation, followed by allyl ester deprotection, Boc group deprotection, and condensation to obtain Lagunamide D. This invention represents the first synthesis of Lagunamide D using compound 12 as a starting material; the synthetic route is relatively short; and the overall yield is significantly higher than that obtained from natural sources.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to a method for preparing the cyclic peptide Lagunamide D. Background Technology

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

[0003] Lagunamide D, a marine natural product, is a cyclic peptide ester isolated in 2019 by American scientists from marine cyanobacteria in Florida. Studies have found that Lagunamide D exhibits significant anti-proliferative activity against human A549 lung cancer cells and HCT116 colorectal cancer cells, with an IC50 value of [missing information]. 50 The molecular weights (MW) reached 7.1 nM and 5.1 nM respectively, indicating significant potential for drug development and making them promising lead compounds. However, the extraction efficiency of isolated Lagunamide D was only 0.003%, which is insufficient for subsequent drug activity studies. To date, no total synthesis studies of Lagunamide D have been reported.

[0004]

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

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

[0007] A method for preparing the cyclic peptide Lagunamide D according to a first aspect of the present invention includes the following steps:

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

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

[0010] (3) Compound 15 was subjected to a debenzylation reaction to obtain compound 16;

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

[0012] (5) Compound 17 was reacted with methacrolein in an olefin metathesis reaction to obtain compound 19;

[0013] (6) Compound 19 was subjected to Pinnick oxidation to obtain compound 20;

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

[0015] (8) Compound 22 was deprotected by the Fmoc protecting group to obtain compound 24;

[0016] (9) Compound 23 and compound 24 were condensed in the presence of a condensing agent to obtain compound 25;

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

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

[0019]

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

[0021] This invention uses compound 12 as a raw material to synthesize Lagunamide D for the first time; the synthetic route is relatively short; and the overall yield is much higher than that extracted from nature.

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

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

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

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

[0026]

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

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

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

[0030]

[0031] According to a preferred embodiment of the present invention, the compound 10 is commercially available.

[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, step (6) of the Pinnick oxidation reaction includes:

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

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

[0037] Compound 22, N-methylaniline, palladium catalyst, and solvent were mixed and reacted.

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

[0039] The product obtained after the deprotection reaction of the allyl ester is completed is reacted with trifluoroacetic acid to obtain the final product.

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

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

[0042] According to a preferred embodiment of the present invention, in step (3), the reaction conditions for the debenzylation reaction include mixing compound 15 and 2,3-dichloro-5,6-dicyanobenzoquinone for reaction, preferably, the reaction temperature is 30-60°C; and the reaction time is 30-180 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 25-60°C; the reaction time is 12-36 h.

[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 means used by those skilled in the art, such as removal under alkaline conditions.

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

[0046] Compound 9 is obtained by reacting it under alkaline conditions.

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

[0048]

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

[0050] Compound 7 was deactivated by removing the Boc group; then it was mixed with compound 8, HOAT, DIPEA and HATU and stirred to react; the result was obtained.

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

[0052]

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

[0054] Compound 5 is debonded by removing the Boc group; then it is mixed with compound 6, HOAT, DIPEA and HATU and reacted to obtain the final product.

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

[0056]

[0057] According to a preferred embodiment of the present invention, 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 sequentially at room temperature and stirred to carry out the reaction.

[0059] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Detailed Implementation

[0060] The following are specific embodiments of the present invention, and the technical solutions of the present invention will be 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 this invention are all conventional reagents, methods and equipment in this technical field.

[0062] Example

[0063] This example provides a method for preparing the cyclic peptide Lagunamide D, including 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 together in anhydrous DMF (5.0 mL). HOAT (0.1 g, 0.72 mmol), DIPEA (3.1 mL, 17.9 mmol), and HATU (2.72 g, 7.2 mmol) were added sequentially at room temperature, and the mixture was stirred overnight. The reaction was quenched with saturated ammonium chloride aqueous solution. The aqueous phase was extracted with ethyl acetate. The combined organic phases were washed successively with saturated ammonium chloride aqueous solution, water, and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:2) to give compound 5 (1.20 g, 93%).

[0067] The data for 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 THF / MeOH / H₂O (2:2:1) (25.0 mL) mixture and cooled to 0 °C. LiOH·H₂O (0.39 g, 16.5 mmol) was added. The reaction mixture was stirred at 0 °C to room temperature for two hours, then the pH was adjusted to 3 with 1 M hydrochloric acid solution. The low-boiling organic solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated acid was pumped under high vacuum for 3 hours. It was then dissolved together with compound 6 (3.62 g, 7.5 mmol) in DMF (10.0 mL). HOAT (1.03 g, 7.5 mmol), DIPEA (9.2 mL, 52.7 mmol), and HATU (35.7 g, 15.1 mmol) were added sequentially at room temperature, and the mixture was stirred overnight. The reaction was quenched by saturated ammonium chloride aqueous solution. The aqueous phase was extracted with ethyl acetate. The organic phases were combined and washed successively with saturated ammonium chloride aqueous solution and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:2) to give compound 7 (3.70 g, 68%).

[0074] The data for 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), and TFA (5.0 mL) was added at room temperature. The mixture was stirred for 0.5 hours, the solvent was removed under reduced pressure, and the mixture was pumped under high vacuum for 3 hours. Then, it was dissolved together with compound 8 in DMF. HOAT (0.68 g, 5.0 mmol), DIPEA (6.07 mL, 34.9 mmol), and HATU (3.79 g, 10.0 mmol) were added sequentially at room temperature, and the mixture was stirred overnight. The reaction was quenched with saturated ammonium chloride aqueous solution. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined and washed successively with saturated ammonium chloride aqueous solution and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 2:3) to give 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 together in tetrahydrofuran and cooled to -30°C in a cryogenic reactor. Isopropylmagnesium chloride (26.6 mL, 53.3 mmol) was slowly added dropwise, and the mixture was stirred for 1 hour. The reaction mixture was then slowly heated from -30°C to -20°C and stirred for 1 hour, followed by a slow heating from -20°C to 0°C and stirring for 1 hour. The reaction was quenched with saturated ammonium chloride aqueous solution, extracted with ethyl acetate, and the combined organic phases were washed successively with saturated ammonium chloride aqueous solution and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:4) to give compound 11 (0.95 g, 78%).

[0087] The data for 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 12The reaction equation and preparation steps are as follows:

[0092]

[0093] Compound 11 (1.50 g, 5.4 mmol) was dissolved in tetrahydrofuran and cooled to 0 °C. Allyl magnesium 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 h. The reaction was quenched with saturated ammonium chloride aqueous solution, extracted with ethyl acetate, and the combined organic phases were washed successively with saturated ammonium chloride aqueous solution and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:30) to give 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. (R)-Me-CBS compound 13 (2.3 mL, 2.3 mmol, 1.0 M in THF) was added at room temperature and stirred for 10 minutes. The mixture was then cooled to -78°C under nitrogen protection, and a boron dimethyl sulfide complex (0.20 mL, 2.0 mmol, 10.0 M in THF) was added. The resulting reaction mixture was stirred at -78°C for 3 hours, then slowly heated to -20°C and stirred for 0.5 hours. Finally, the mixture was heated to 0°C, quenched with methanol, and then heated to room temperature. The mixture was extracted with ethyl acetate, and the organic phases were combined and washed successively with saturated ammonium chloride aqueous solution and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:10) to give compound 14 (0.40 g, 89%).

[0099] The data for 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, and triethylamine (4.7 mL, 33.8 mmol) and tert-butyldimethylsilyltrifluoromethanesulfonate (3.9 mL, 16.9 mmol) were added sequentially at room temperature, and the mixture was stirred for 1 hour. The reaction was quenched with saturated ammonium chloride aqueous solution, and the mixture was extracted with ethyl acetate. The combined organic phases were washed successively with saturated ammonium chloride aqueous solution and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether) to give compound 15 (2.11 g, 67%).

[0106] The data for 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, and 2,3-dichloro-5,6-dicyanobenzoquinone (3.89 g, 17.2 mmol) was added. The mixture was stirred for 1 hour. Extraction was performed with dichloromethane, followed by washing with 1 M sodium hydroxide / 1 M sodium thiosulfate / water (1:1:1) (15 mL) and saturated brine, drying over anhydrous sodium sulfate, filtration, and concentration under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:20) to give compound 16 (1.20 g, 97%).

[0113] The data for 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). Oxaloyl chloride (2.8 mL, 32.5 mmol) and catalytic amount of DMF (32 μL, 0.41 mmol) were added sequentially at 0 °C. After stirring at room temperature for 1 hour, the mixture was concentrated under reduced pressure in a rotary evaporator. The resulting acyl chloride was then evacuated under high vacuum for 0.5 hours 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 together in anhydrous dichloromethane (15.0 mL). Triethylamine (4.5 mL, 32.5 mmol) was added under nitrogen protection. The mixture was cooled to 0 °C in an ice bath, and the prepared acyl chloride (dissolved in 15.0 mL of anhydrous dichloromethane) was added. The mixture was brought to room temperature and stirred for 1 hour. The reaction was then quenched with methanol (3 mL). The reaction solution was diluted with ethyl acetate (150.0 mL), followed by washing with 1 M hydrochloric acid solution, saturated ammonium chloride aqueous solution, and saturated brine. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (Qingdao Marine) (ethyl acetate: petroleum ether = 1:20) to give compound 17 (1.26 g, 66%).

[0121] The data for 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). α-Methacrolein compound 18 (11.1 mL, 0.13 mmol) and Grubbs II catalyst (8.9 mg, 0.010 mmol) were added sequentially at room temperature, followed by nitrogen purging for 30 minutes. The mixture was then heated from room temperature to 50 °C and stirred overnight. The reaction solution was concentrated under reduced pressure using a rotary evaporator. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:10) to give compound 19 (1.06 g, 81%).

[0128] The data for 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-butanol / isoprene / water (2:2:1) (40.0 mL), and sodium chlorite (4.33 g, 49.5 mmol) and sodium dihydrogen phosphate (9.53 g, 61.1 mmol) were added at room temperature, and the mixture was stirred overnight. The reaction mixture was acidified to pH 3 with 1 M hydrochloric acid, extracted with ethyl acetate, and the combined organic phases were washed successively with saturated ammonium chloride aqueous solution and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:1) to give 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 together in xylene, cooled to -20°C, and 2,4,6-trichlorobenzoyl chloride (0.30 mL, 1.7 mmol) was added. The mixture was stirred for 0.5 hours, then heated from -20°C to room temperature and stirred overnight. The reaction mixture was acidified to pH 3 with 1 M hydrochloric acid, extracted with ethyl acetate, and the combined organic phases were washed successively with 1 M hydrochloric acid, saturated sodium bicarbonate aqueous solution, and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 3:1) to give compound 22 (318 mg, 68%).

[0138] The data for 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] Synthesis of compounds (8) and (9), and compound 25: The reaction equations and preparation steps are as follows:

[0143]

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

[0145] The acid obtained by removing the Fm protecting group from compound 9 and the free ammonia obtained by removing the Fmoc protecting group from 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 heated to room temperature and stirred for 14 hours. The reaction was quenched with saturated ammonium chloride aqueous solution (5.0 mL), extracted with ethyl acetate (30.0 mL × 3), and the organic phases were combined and washed successively with water (15 mL × 3) and saturated brine (15 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 3:2) to give compound 25 (8.0 mg, 44%).

[0146] The data for 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 tetraphenylphosphine 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°C, 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 sequentially quenched with 1 M hydrochloric acid (…). Wash with 3.0 mL × 2) and saturated brine (3.0 mL), dry to anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The resulting acid is then evacuated under high vacuum for 3 hours and dissolved in trifluoroacetic acid-dichloromethane (v:v = 1:5, 3.0 mL) solvent. The mixture is stirred at room temperature for 0.5 hours and then concentrated under reduced pressure on a rotary evaporator to obtain the ring-closing precursor. The precursor is then evacuated under high vacuum for 3 hours and dissolved in N,N-dimethylformamide (10.0 mL) solvent. Ring-closing reagents HATU (90 mg, 0.24 mmol), HOAt (16 mg, 0.12 mmol), and 2,4,6-trimethylpyridine (94 μL, 0.72 mmol) are added. The reaction mixture is stirred at room temperature for 48 hours. Solvents and other volatile substances are removed by high vacuum distillation. The residue was dissolved in ethyl acetate (100.0 mL) and washed successively with 1M hydrochloric acid (5.0 mL), water (15.0 mL × 3), and saturated brine (15.0 mL). After drying with anhydrous sodium sulfate, the residue was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (Qingdao Marine) (ethyl acetate: petroleum ether = 3:2) to obtain the natural product Lagunamide D (13 mg, 67%).

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

[0154] 1H 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)

[0155] 13 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;

[0156] HRMS(ESI)m / z:C 44 H 69 N5NaO 10 + [M+Na] +:calcd:850.4937; found:850.4909.

[0157] The present invention has been described in detail above with reference to the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A process for the preparation of the cyclic peptide Lagunamide D, characterized in that, Comprise the following steps: (1) Corey-Bakshi-Shibata reduction reaction is carried out to compound 12 to obtain compound 14; (2) hydroxyl protection reaction is carried out to compound 14 and tert-butyl dimethyl silyl trifluoromethanesulfonate to obtain compound 15; (3) debenzyl reaction is carried out to compound 15 to obtain compound 16; (4) acyl chloride is obtained by reaction of Fmoc-N-methyl-L-alanine, oxalyl chloride and a catalytic amount of DMF; then the acyl chloride and compound 16 are reacted to obtain compound 17; (5) olefin metathesis reaction is carried out to compound 17 and methacrolein to obtain compound 19; (6) Pinnick oxidation reaction is carried out to compound 19 to obtain compound 20; (7) Yamaguchi esterification reaction is carried out to compound 20 and compound 21 to obtain compound 22; (8) Fmoc protecting group is removed from compound 22 to obtain compound 24; (9) condensation reaction is carried out to compound 23 and compound 24 in the presence of a condensing agent to obtain compound 25; (10) allyl ester deprotection, Boc group deprotection and condensation reaction are carried out to compound 25 to obtain Lagunamide D; The structural formula of the 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 process for the preparation of 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, R -2-n-butyl-CBS-oxazaborolidine.

3. Process for the preparation of the cyclic peptide Lagunamide D according to claim 1 or 2, characterized in that, The compound 12 is prepared by the following method: Weinreb ketone synthesis reaction is carried out to compound 11 to obtain compound 12; The structural formula of the compound 11 is as follows: 。 4. The process for the preparation of the cyclic peptide Lagunamide D according to claim 3, characterized in that, The compound 11 is prepared by the following method: Reaction is carried out to compound 10, N, O-dimethylhydroxylamine hydrochloride and isopropyl magnesium chloride to obtain compound 11. The structural formula of the compound 10 is as follows: 。 5. The process for the preparation of the cyclic peptide Lagunamide D according to claim 1, characterized by, In step (9), the condensing agent comprises HATU and HOAt.

6. The process for the preparation of the cyclic peptide Lagunamide D according to claim 1, characterized by, In step (5), the temperature of the reaction is 35℃-60℃; and the reaction time is 12 h-36 h.

7. The method of claim 1, wherein the ring peptide Lagunamide D is prepared by the steps of, In step (6), the steps of the Pinnick oxidation reaction comprise: Reaction is carried out to compound 19, sodium chlorite, sodium dihydrogen phosphate and a solvent to obtain compound 20.

8. The method of claim 1, wherein the ring peptide Lagunamide D is prepared by the method comprising the steps of, In step (10), the steps of the allyl ester deprotection reaction comprise: Reaction is carried out to compound 25, N-methylaniline, a palladium catalyst and a solvent.

9. The method for preparing the cyclic peptide Lagunamide D according to claim 1, characterized in that, In step (10), the steps of the Boc group deprotection comprise: After the allyl ester deprotection reaction is completed, the product is mixed with trifluoroacetic acid to obtain compound 25.

10. The method of claim 1, wherein the ring peptide Lagunamide D is prepared by the method comprising the steps of, In step (10), the steps of the condensation reaction comprise: After the Boc group deprotection, the intermediate is mixed with HATU, HOAt and 2,4,6-trimethylpyridine to obtain compound 25.

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