Marine cyclotide and methods of synthesis, uses thereof

Abstract

This invention belongs to the field of pharmaceutical research technology, specifically relating to a marine cyclic peptide, its synthesis method, and its uses. The structural formula of the marine cyclic peptide is shown in Formula I. Compared with natural Galaxamide, the Galaxamide analog containing D-leucine provided by this invention exhibits a greater inhibitory effect on human cancer cells, and the more D-leucines introduced, the stronger the anticancer activity. Simultaneously, by replacing one of the L-leucines in Galaxamide with other amino acids, the synthesized Galaxamide derivative is more effective than natural Galaxamide and has the potential to inhibit the proliferation of human cancer cell lines HepG2, U87, and MCF-7.

Description

Technical Field

[0001] This invention belongs to the field of drug research technology, specifically relating to a marine cyclic peptide, its synthesis method, and its uses. Background Technology

[0002] Chemotherapy is a crucial method for treating cancer, but most anti-tumor drugs develop resistance. Drug resistance is the phenomenon where tumor cells become insensitive to anti-tumor drugs during anti-tumor treatment. The emergence of anti-tumor drug resistance is one of the main reasons for chemotherapy failure in cancer and a key issue in cancer treatment. From the perspective of tumor cells, the mechanisms of resistance may include tumor cell heterogeneity, altered drug transport and metabolism, changes in the expression levels of drug targets, activation of alternative pathways, epigenetic alterations, and epithelial-mesenchymal transition. From the perspective of the tumor microenvironment, the occurrence of resistance is also closely related to other cells and components infiltrating the tumor microenvironment, such as immune and inflammatory cells, cancer-associated fibroblasts, and vascular endothelial cells.

[0003] Tumor drug resistance is common among small-molecule targeted anticancer drugs, and designing new small-molecule drugs targeting their resistance mechanisms is quite challenging. Among the many strategies for combating anticancer drug resistance, continuously screening and discovering novel drugs, interfering with specific signaling pathways in tumor growth, inhibiting tumor cell proliferation, and improving treatment efficacy are among the most valuable strategies. Having new drugs available to replace existing ones, to combine or alternate with other drugs after tumor drug resistance develops, and even to reverse drug resistance using new drugs, are important pathways for combating tumors and addressing anticancer drug resistance. Therefore, the discovery of new anticancer drugs is urgently needed.

[0004] Galaxamide is a novel marine cyclic peptide with antitumor activity, showing potential as a novel anticancer drug. Its unique chemical structure and pharmacological properties offer new research ideas and potential drug options for cancer treatment. However, Galaxamide still suffers from drawbacks such as low antitumor activity and a lack of broad-spectrum antitumor activity. Summary of the Invention

[0005] In view of the above technical problems, the present invention provides a marine cyclic peptide.

[0006] The specific technical solution provided by this invention is as follows:

[0007] This invention provides a marine cyclic peptide, the structural formula of which is shown in Formula I:

[0008] ;

[0009] Wherein, R is selected from D-leucine, D-proline, D-phenylalanine, L-valine, glycine, D-alanine, O-methyl-D-serine, O-methyl-D-tyrosine, methyl-D-aspartic acid, 3-fluoro-D-phenylalanine, 2-methyl-D-phenylalanine, 4-chloro-D-phenylalanine, 4-bromo-D-phenylalanine, D-cyclohexyl-glycine, D-phenyl-glycine, D-valine, D-methionine, 3-methyl-D-phenylalanine, and 4-methyl-D-phenylalanine; R2~R5 are each selected from D-leucine or L-leucine.

[0010] As a preferred embodiment of the present invention, the structural formula of the marine cyclic peptide is shown in any of the following:

[0011]

[0012]

[0013]

[0014]

[0015] .

[0016] The present invention also provides a method for synthesizing the aforementioned marine cyclic peptide, comprising the following steps:

[0017] Boc-N-methyl-L-leucine was obtained by nitrogen methylation reaction using N-(tert-butoxycarbonyl)-L-leucine as a starting material, or Boc-N-methyl-D-leucine was obtained by nitrogen methylation reaction using N-(tert-butoxycarbonyl)-D-leucine as a starting material.

[0018] A dipeptide is obtained by condensing Boc-N-methyl-L-leucine and L-leucine benzyl ester p-toluenesulfonate as raw materials; or a dipeptide is obtained by condensing Boc-N-methyl-D-leucine and D-leucine benzyl ester p-toluenesulfonate as raw materials.

[0019] The dipeptide was subjected to hydrogenation reduction to remove its C-terminal benzyl group, yielding product D;

[0020] The dipeptide was subjected to acid hydrolysis to remove its N-terminal tert-butyloxycarbonyl group, yielding product E;

[0021] The product E was mixed with amino acids and subjected to a condensation reaction to obtain a tripeptide. The N-terminal Boc group was removed to obtain product G.

[0022] The product D and the product G are mixed and subjected to a condensation reaction to obtain a linear pentapeptide; the C-terminal benzyl group and the N-terminal Boc group of the linear pentapeptide are removed sequentially to obtain product J;

[0023] The product J was subjected to a macrocyclic lactamation reaction to obtain the marine cyclic peptide.

[0024] In a preferred embodiment of the present invention, the molar ratio of Boc-N-methyl-L-leucine to benzyl leucine p-toluenesulfonate, or the molar ratio of Boc-N-methyl-D-leucine to benzyl leucine p-toluenesulfonate, is 1:1 to 2; the molar ratio of product E to amino acid is 1:1 to 2; and the molar ratio of product D to product G is 1:1 to 2.

[0025] In a preferred embodiment of the present invention, the nitrogen methylation reaction is carried out by stirring at -20 to -10°C for 30 to 60 minutes, followed by stirring at room temperature for 12 to 18 hours; the condensation reaction of Boc-N-methyl-L-leucine and L-leucine benzyl ester with toluenesulfonate, or the condensation reaction of Boc-N-methyl-D-leucine and D-leucine benzyl ester with toluenesulfonate, is carried out at 0 to 10°C for 6 to 12 hours; the hydrogenation of the C-terminus of the dipeptide is also carried out by stirring at -20 to -10°C for 30 to 60 minutes, followed by stirring at room temperature for 12 to 18 hours. The original reaction was carried out at 10℃~20℃ for 6h~12h under the action of palladium catalyst; the acid hydrolysis reaction of the N-terminus of the dipeptide was carried out at -20℃~-10℃ for 6h~12h; the condensation reaction of product E with amino acid was carried out at 0℃~10℃ for 6h~12h; the condensation reaction of product D with product G was carried out at 0℃~10℃ for 6h~12h; the macrocyclic lactamation reaction was carried out at 0℃~10℃ for 12h~24h under the action of coupling agent.

[0026] In a preferred embodiment of the present invention, the coupling agent is selected from 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, propionic anhydride, diphenylphosphide, pentafluorophenyl diphenylphosphinate, or (benzotriazol-1-yloxy)tripyrrolidinephosphine hexafluorophosphate.

[0027] More preferably, the coupling agent is selected from (benzotriazole-1-yloxy)tripyrrolidinephosphine hexafluorophosphate.

[0028] The present invention also provides the use of the marine cyclic peptide in the preparation of anticancer drugs.

[0029] As a preferred embodiment of the present invention, the anticancer effect is the inhibitory effect on non-small cell lung cancer, chronic myeloid leukemia, breast cancer, liver cancer, glioma, and immortalized embryonic kidney cells.

[0030] The present invention also provides an anticancer drug, which uses the marine cyclic peptide as the sole active ingredient.

[0031] In a preferred embodiment of the present invention, the drug is composed of the marine cyclic peptide and pharmaceutically acceptable excipients.

[0032] In a preferred embodiment of the present invention, the drug is an oral preparation or an injectable preparation.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] This invention designs and synthesizes two series of Galaxamide derivatives. The first series mainly involves replacing L-leucine with D-leucine and altering the position and number of D-leucine. The second series involves replacing one L-leucine with other amino acids, such as D-proline, D-phenylalanine, L-valine, glycine, D-alanine, O-methyl-D-serine, O-methyl-D-tyrosine, methyl-D-aspartic acid, 3-fluoro-D-phenylalanine, 2-methyl-D-phenylalanine, 4-chloro-D-phenylalanine, 4-bromo-D-phenylalanine, D-cyclohexyl-glycine, D-phenyl-glycine, D-valine, D-methionine, 3-methyl-D-phenylalanine, and 4-methyl-D-phenylalanine. On the one hand, compared with natural Galaxamide, Galaxamide analogs containing D-leucine exhibit greater inhibitory effects on human cancer cells. The more D-leucine introduced into Galaxamide, the stronger the anticancer activity. Analogs containing four D-leucine residues exhibited the strongest apoptotic activity against various human cancer cell lines such as HepG2, MCF-7, SW480, and U87. Conversely, by replacing one of the L-leucine residues in Galaxamide with other amino acids, synthesized Galaxamide derivatives were more potent than natural Galaxamide and showed the potential to inhibit the proliferation of human cancer cell lines HepG2, U87, and MCF-7.

[0035] The key step in the preparation of Galaxamide derivatives in this invention is macrocyclic lactamation. This invention provides a preliminary analysis of peptide coupling agents for the macrocyclic lactamation of intermediate J to produce Galaxamide derivatives: the reaction conversion of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate and propionic anhydride was poor, while the use of diphenylphosphohydride and pentafluorophenyl diphenylphosphine ester produced a moderate yield of macrocyclic lactamation products. (benzotriazol-1-yloxy)tripyrrolidinephosphine hexafluorophosphate mainly produced the expected product, the marine cyclic peptide. Using a low concentration of acetonitrile as a solvent, the addition of (benzotriazol-1-yloxy)tripyrrolidinephosphine hexafluorophosphate and 4-dimethylaminopyridine reduced the formation of dimer products, achieving a yield of 50%. Furthermore, by slowly adding intermediate J over 4 hours to simulate a highly diluted environment, dimer formation was prevented, and the solvent concentration was made closer to the actual reaction concentration. Detailed Implementation

[0036] The raw materials involved in the embodiments of this invention are abbreviated as follows: HATU: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate; T3P: propionic anhydride; DPPA: diphenylphosphide azide; FDPP: pentafluorophenyl diphenylphosphine ester; PyBOP: (benzotriazol-1-yloxy)tripyrrolidinephosphine hexafluorophosphate; DMAP: 4-dimethylaminopyridine; CH3CN: acetonitrile; EtOAc: ethyl acetate; n-Hexane: n-hexane; Hexane: cyclohexane. The CAS number for 10% Pd / C is 7440-05-3.

[0037] Example 1

[0038] Cyclo(Me-L-Leu-L-Leu-D-Leu-Me-L-Leu-L-Leu)

[0039] (1) N-Boc-Me-L-Leu-OH

[0040] Take 9.0 g (38.9 mmol) of L-leucine, 25 g (177.3 mmol) of CH3I, and 5.5 g (137.5 mmol) of NaH dispersed in mineral oil. Mix and stir at -10 °C for 30 min, then stir at room temperature for 18 h. Quench with 50 mL of aqueous solution and wash the mixture with ethyl acetate. Then, acidify the aqueous solution to pH 2 with 1 N HCl solution and extract with ethyl acetate. The resulting ethyl acetate extract is dried over Na2SO4, filtered, and evaporated to give 9.0 g (36.7 mol) of colorless oily B2, namely N-Boc-Me-L-Leu-OH, with a yield of 94%. Its structural formula is as follows:

[0041] .

[0042] (2) N-Boc-Me-L-Leu-L-Leu-OBn.

[0043] C2 was synthesized by reacting 10.0 g (40.8 mmol) of B2, 17.6 g (44.9 mmol) of L-leucine benzyl ester p-toluenesulfonate, 6.0 g (44.9 mmol) of HOBt, and 8.5 g (44.9 mmol) of EDCI at 0 °C for 12 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate in a volume ratio of 90:10 as the elution solvent to give 15.0 g (33.4 mol) of white solid C2, namely N-Boc-Me-L-Leu-L-Leu-OBn, in 82% yield. The structural formula is as follows:

[0044] .

[0045] (3) N-Me-L-Leu-L-Leu-OBn.

[0046] E2 was synthesized by reacting 15.0 g of 33.4 mmol of C2, 10 ml of TFA, and 40 ml of DCM at -20 °C for 12 h. Excess TFA and DCM were then removed under vacuum to produce a colorless oily E2, namely N-Me-L-Leu-L-Leu-OBn, with a yield of 10.0 g and 28.7 mmol, representing a yield of 86%. Its structural formula is as follows:

[0047] .

[0048] (4) N-Boc-Me-L-Leu-L-Leu-OH.

[0049] D2 was synthesized by reacting 15.0 g (33.4 mmol C2), 5 g (4.7 mmol 10% Pd / C), and 1 atm H2 at 10 °C for 12 h. The mixture was filtered and concentrated under vacuum to obtain D2, namely N-Boc-Me-L-Leu-L-Leu-OH, with a yield of 10.0 g (27.9 mmol) and a yield of 84%. The structural formula is as follows:

[0050] .

[0051] (5)N-Boc-D-Leu-Me-L-Leu-L-Leu-OBn

[0052] F2 was synthesized by reacting 10.0 g (28.7 mmol) of E2 with 7.2 g (31.5 mmol) of N-Boc-D-Leu-OH, 4.2 g (31.5 mmol) of HOBT, and 6.0 g (31.5 mmol) of EDCI at 0 °C for 12 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate in a volume ratio of 80:20 as the elution solvent to obtain a white solid, F2, namely N-Boc-D-Leu-Me-L-Leu-L-Leu-OBn, with a yield of 14.0 g (24.9 mmol) and a yield of 86%. The structural formula is as follows:

[0053] .

[0054] (6)ND-Leu-Me-L-Leu-L-Leu-OBn

[0055] G2 was synthesized by reacting 15.0 g, 33.4 mmol F2, 10 ml TFA, and 40 ml DCM at -20 °C for 12 h. Excess TFA and DCM were then removed under vacuum to produce a colorless oily form of G2, ND-Leu-Me-L-Leu-L-Leu-OBn, with a yield of 13.2 g and 28.7 mmol, representing a yield of 86%. The structural formula is as follows:

[0056] .

[0057] (7) N-Boc-Me-L-Leu-L-Leu-D-Leu-Me-L-Leu-L-Leu-OBn.

[0058] H2 was synthesized by reacting 10.0 g (27.9 mmol D2), 14.1 g (30.7 mmol G2), 4.1 g (30.7 mmol HOBT), and 5.9 g (30.7 mmol EDCI) at 10 °C for 6 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate at a volume ratio of 70:30 as the elution solvent, yielding a white solid H2, namely N-Boc-Me-L-Leu-L-Leu-D-Leu-Me-L-Leu-L-Leu-OBn, with yields of 20.0 g and 25.0 mmol, representing a yield of 89%. The structural formula is as follows:

[0059] .

[0060] (8) N-Me-L-Leu-L-Leu-D-Leu-Me-L-Leu-L-Leu-OBn

[0061] I₂ was synthesized by reacting 26.7 g, 33.4 mmol H₂, 10 ml TFA, and 40 ml DCM at -10 °C for 6 h. Subsequently, excess TFA and DCM were removed under vacuum to obtain a colorless oily I₂, namely N-Me-L-Leu-L-Leu-D-Leu-Me-L-Leu-L-Leu-OBn, with a yield of 20.1 g and 28.7 mmol, representing a yield of 86%. The structural formula is as follows:

[0062] .

[0063] (9) N-Me-L-Leu-L-Leu-D-Leu-Me-L-Leu-L-Leu-OH

[0064] J2 was synthesized by reacting 23.4 g of 33.4 mmol I2, 5 g of 4.7 mmol of 10% Pd / C, and 1 atm H2 at 20 °C for 6 h. The mixture was filtered and concentrated under vacuum to obtain J2, namely N-Me-L-Leu-L-Leu-D-Leu-Me-L-Leu-L-Leu-OH, with a yield of 17.0 g and 27.9 mmol, and a yield of 84%. The structural formula is as follows:

[0065] .

[0066] (10) Cyclo(Me-L-Leu-L-Leu-D-Leu-Me-L-Leu-L-Leu)

[0067] 134 mg, 0.22 mmol J2 was dissolved in 4.3 mM MeCN and added dropwise over 10 h to a solution of 229 mg, 0.44 mmol PyBOP and 80 mg, 0.66 mmol DMAP in 1.0 mM MeCN. The mixture was stirred for 3 h. The solvent was removed under reduced pressure, and the residue was suspended in H2O. Cyclohexane and ethyl acetate in a volume ratio of 60:40 were used as the eluent, and the mixture was purified by column chromatography to give compound K2 as a white solid, namely Cyclo(Me-L-Leu-L-Leu-D-Leu-Me-L-Leu-L-Leu), with a yield of 66 mg, 0.11 mmol, and a yield of 50%. The structural formula is as follows:

[0068] .

[0069] 1 H NMR (600 MHz, DMSO- d 6) δ 7.63 (d, J = 8.1 Hz, 1H), 7.39 (d,J = 8.2 Hz, 1H), 7.20 (d, J = 8.8 Hz, 1H), 5.11 (dd, J = 9.5, 6.2 Hz, 1H), 4.77 (td, J = 8.3, 5.6 Hz, 1H), 4.71 (q, J = 7.6 Hz, 1H), 4.49 (dd, J = 10.4, 5.2 Hz, 1H), 4.15(ddd, J = 10.4, 8.1, 5.2 Hz, 1H), 2.98 (s, 3H), 2.73 (s, 3H), 1.75 – 1.32 (m, 15H), 1.02 – 0.61 (m, 30H). 13 C NMR (151 MHz, DMSO- d 6) δ 174.51, 172.25,171.36, 170.83, 170.56, 57.93, 53.65, 51.38, 48.34, 47.70, 41.40, 41.09,38.70, 37.23, 35.17, HRMS (ESI) + ) calcd.for C 32 H 59 N5O5[M+Na] + : 616.4414; found: 616.4392.

[0070] Example 2

[0071] Cyclo(Me-L-Leu-L-Leu-D-Pro-Me-L-Leu-L-Leu)

[0072] The only difference from Example 1 is that N-Boc-D-Leu-OH was replaced with N-Boc-D-Pro-OH, yielding compound K3, a white solid, namely Cyclo(Me-L-Leu-L-Leu-D-Pro-Me-L-Leu-L-Leu), with a yield of 66 mg, 0.112 mmol, and a 51% yield. Its structural formula is as follows:

[0073] 。

[0074] 1 H NMR (600 MHz, DMSO- d 6) δ 7.37 (d, J = 7.3 Hz, 1H), 6.89 (d, J = 7.5 Hz,1H), 4.91 (t, J = 7.8 Hz, 1H), 4.75 (td, J = 8.5, 8.1, 5.3 Hz, 2H), 4.60 (dd, J =10.5, 5.8 Hz, 1H), 4.33 (ddd, J = 11.6, 7.5, 4.2 Hz, 1H), 3.51 (t, J = 8.5 Hz,1H), 3.45 (q, J = 5.3, 3.8 Hz, 1H), 3.02 (d, J = 11.7 Hz, 6H), 2.17 (q, J = 6.4 Hz,1H), 2.04 – 1.91 (m, 1H), 1.88 (dd, J = 12.5, 6.6 Hz, 1H), 1.81 – 1.74 (m, 1H),1.74 – 1.66 (m, 1H), 1.65 – 1.29 (m, 11H), 0.99 – 0.69 (m, 24H). 13 C NMR (151MHz, DMSO- d 6) δ 173.85, 172.95, 171.28, 170.24, 169.51, 57.55, 56.01, 53.83,50.16, 47.93, 46.42, 42.25, 36.75, 36.33, 31.00, 30.79, 28.46, 25.41, 25.21,25.08, 24.87, 24.69, 23.69, 23.43, 23.25, 23.20, 22.73, 22.37, 21.95, 21.81.HRMS (ESI + ) calcd. for C 31 H 55 N5O5[M+Na] +: 600.4101; found: 600.4223.

[0075] Example 3

[0076] Cyclo(Me-L-Leu-L-Leu-D-Phe-Me-L-Leu-L-Leu)

[0077] The only difference from Galaxamide is the replacement of N-Boc-D-Leu-OH with N-Boc-D-Phe-OH, yielding compound K4, a white solid, namely Cyclo(Me-L-Leu-L-Leu-D-Phe-Me-L-Leu-L-Leu), with a yield of 70 mg, 0.110 mmol, and a 51% yield. Its structural formula is as follows:

[0078] .

[0079] 1 H NMR (600 MHz, DMSO- d 6) δ 7.60 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.35 – 7.04 (m, 5H), 5.06 (dd, J = 9.8, 6.0 Hz, 1H), 4.87 (q, J = 7.9 Hz, 1H), 4.76 (q, J = 7.5 Hz, 1H), 4.58 (dd, J = 10.1, 5.6Hz, 1H), 4.12 (td, J = 8.8, 5.7 Hz, 1H), 3.05 (dd, J = 13.1, 8.4 Hz, 1H), 2.97(s, 3H), 2.76 (dd, J = 13.2, 6.2 Hz, 1H), 2.63 (s, 3H), 1.86 – 1.45 (m, 7H), 1.40 (q, J = 8.2, 7.3 Hz, 4H), 1.24 – 1.07 (m, 1H), 0.97 – 0.59 (m, 24H). 13 C NMR (151 MHz, DMSO-) d6) δ 174.22, 171.82, 171.28, 170.86, 170.38, 138.17, 129.65,128.43, 126.63, 57.70, 53.65, 51.56, 51.17, 48.23, 41.40, 38.72, 37.83,37.20, 35.23, 31.20, 29.84, 25.24, 24.84, 24.75, 24.65, 23.47, 23.38, 23.18,22.16, 22.00, 21.88. HRMS (ESI + ) calcd. for C 35 H 57 N5O5[M+Na] + : 650.4257; found:650.4556.

[0080] Example 4

[0081] Cyclo(Me-L-Leu-L-Leu-L-Val-Me-L-Leu-L-Leu)

[0082] The only difference from Example 1 is that N-Boc-D-Leu-OH was replaced with N-Boc-L-Val-OH, yielding compound K5, a white solid, namely Cyclo(Me-L-Leu-L-Leu-L-Val-Me-L-Leu-L-Leu), with a yield of 67 mg, 0.110 mmol, and a 51% yield. Its structural formula is as follows:

[0083] .

[0084] 1 H NMR (600 MHz, DMSO- d 6) δ 8.39 – 7.83 (m, 1H), 7.83 – 7.61 (m, 1H), 7.53 – 6.64 (m, 1H), 5.31 – 4.84 (m, 1H), 4.81 – 4.62 (m, 1H), 4.60 – 4.40(m, 1H), 4.20 – 3.93 (m, 1H), 3.93 – 3.52 (m, 1H), 3.36 (s, 1H), 3.17 (s,1H), 2.90 (s, 1H), 2.78 (s, 1H), 2.58 (d, J= 16.2 Hz, 1H), 2.42 (s, 1H), 1.73– 1.24 (m, 12H), 0.87 (dtdd, J = 37.4, 24.0, 13.2, 6.4 Hz, 30H). 13 C NMR (151MHz, DMSO- d 6) δ 174.07, 171.13, 170.83, 169.38, 167.86, 60.20, 57.76, 55.22,53.52, 50.98, 47.69, 42.98, 41.48, 38.17, 37.00, 29.07, 28.95, 25.29, 25.13,24.88, 24.75, 24.60, 24.55, 24.41, 23.25, 23.22, 23.18, 23.13, 23.08, 23.00,22.97, 22.81, 22.37, 22.35, 22.16. HRMS (ESI + ) calcd. for C 31 H 57 N5O5[M+H] + :602.4257; found: 602.4375.

[0085] Example 5

[0086] Cyclo(Me-L-Leu-L-Leu-Gly-Me-L-Leu-L-Leu)

[0087] The only difference from Example 1 was the replacement of N-Boc-D-Leu-OH with N-Boc-Gly-OH, resulting in the preparation of compound K6, a white solid, namely Cyclo(Me-L-Leu-L-Leu-Gly-Me-L-Leu-L-Leu), with a yield of 62 mg, 0.112 mmol, and a 52% yield. Its structural formula is as follows:

[0088] .

[0089] 1 H NMR (600 MHz, DMSO- d 6) δ 7.92 (dd, J = 65.6, 8.3 Hz, 1H), 7.76 – 7.38(m, 1H), 7.22 (d, J = 4.3 Hz, 1H), 5.04 (t,J = 7.7 Hz, 1H), 4.83 (td, J = 9.0, 4.6Hz, 1H), 4.60 (dd, J = 13.8, 9.1 Hz, 1H), 4.45 (dd, J = 9.7, 5.3 Hz, 1H), 4.09(ddd, J = 10.8, 8.1, 4.8 Hz, 1H), 3.25 (d, J = 13.7 Hz, 1H), 3.01 (s, 3H), 2.64(s, 3H), 1.78 – 1.30 (m, 12H), 1.03 – 0.62 (m, 24H). 13 C NMR (151 MHz, DMSO- d 6)δ 174.67, 171.72, 171.31, 170.27, 170.18, 58.09, 53.80, 51.83, 48.33, 43.17,41.45, 38.29, 37.36, 35.06, 31.62, 31.45, 30.35, 25.28, 24.84, 24.81, 24.75,23.62, 23.49, 23.35, 23.05, 22.26, 22.13, 22.00, 21.38. HRMS (ESI + ) calcd.for C 28 H 51 N5O5[M+Na] + : 560.3788; found: 560.3422.

[0090] Example 6

[0091] Cyclo(Me-L-Leu-L-Leu-D-Ala-Me-L-Leu-L-Leu)

[0092] The only difference from Example 1 is that N-Boc-D-Leu-OH was replaced with N-Boc-D-Ala-OH, yielding compound K7, namely Cyclo(Me-L-Leu-L-Leu-D-Ala-Me-L-Leu-L-Leu), a white solid, with a yield of 59 mg, 0.110 mmol, and a 49% yield. Its structural formula is as follows:

[0093] .

[0094] 1 1H NMR (600 MHz, DMSO- d 6) δ 7.78 (d, J J = 8.3 Hz, 1H), 7.21 (d, J J = 8.4 Hz,1H), 7.10 (d, J J = 8.8 Hz, 1H), 5.13 (t, J J = 7.6 Hz, 1H), 4.83 (td, J J = 8.7, 5.0 Hz,1H), 4.74 (dd, J J = 8.7, 6.1 Hz, 1H), 4.40 (dd, J J = 10.1, 5.1 Hz, 1H), 4.12 (ddd, J J = 12.1, 8.2, 4.3 Hz, 1H), 3.01 (s, 3H), 2.63 (s, 3H), 1.80 – 1.65 (m, 2H),1.65 – 1.29 (m, 10H), 1.12 (d, J J = 6.3 Hz, 3H), 1.04 – 0.60 (m, 24H). 13 13C NMR(151 MHz, DMSO- d 6) δ 174.88, 172.24, 171.62, 170.87, 170.38, 58.31, 53.56,51.46, 48.43, 45.25, 41.50, 38.45, 37.38, 35.00, 31.48, 29.57, 25.30, 24.84,24.81, 23.68, 23.55, 23.31, 23.06, 22.28, 22.07, 21.92, 21.31, 17.85. HRMS(ESI + ) calcd. for C 29 H 53 N5O5[M+Na] + : 574.3944; found: 574.3212.

[0095] Example 7

[0096] Cyclo(Me-L-Leu-L-Leu-O-Me-D-Ser-Me-L-Leu-L-Leu)

[0097] The only difference from Example 1 is that N-Boc-D-Leu-OH was replaced with N-Boc-O-methyl-D-serine, yielding compound K8, a white solid, namely Cyclo(Me-L-Leu-L-Leu-O-Me-D-Ser-Me-L-Leu-L-Leu), with a yield of 67 mg, 0.112 mmol, and 52% yield. Its structural formula is as follows:

[0098] .

[0099] 1 H NMR (600 MHz, DMSO- d 6) δ 7.80 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.09 (d, J = 9.0 Hz, 1H), 5.10 (t, J = 7.7 Hz, 1H), 4.82 (td, J = 8.2, 4.1 Hz, 2H), 4.45 (dd, J = 10.1, 4.9 Hz, 1H), 4.13 (ddd, J = 10.8, 8.2, 4.6 Hz, 1H), 3.63(t, J = 8.6 Hz, 1H), 3.28 (dd, J = 9.4, 5.7 Hz, 1H), 3.23 (s, 3H), 2.99 (s, 3H), 2.67 (s, 3H), 1.78 – 1.65 (m, 2H), 1.64 – 1.31 (m, 10H), 1.05 – 0.57 (m, 24H). 13 C NMR (151 MHz, DMSO- d 6) δ 174.72, 171.52, 171.25, 170.71, 170.27,71.70, 58.77, 58.04, 53.60, 51.35, 48.59, 48.36, 41.40, 38.44, 37.26, 35.05,31.39, 29.72, 25.27, 24.95, 24.80, 23.63, 23.51, 23.27, 23.12, 22.21, 22.01,21.98, 21.48. HRMS (ESI +) calcd. for C 30 H 55 N5O6[M+Na] + : 604.4050; found: 604.4212.

[0100] Example 8

[0101] Cyclo(Me-L-Leu-L-Leu-O-Tyr-Me-L-Leu-L-Leu)

[0102] The only difference from Example 1 is that N-Boc-D-Leu-OH is replaced with N-Boc-O-methyl-D-tyrosine, yielding compound K9, a white solid, namely Cyclo(Me-L-Leu-L-Leu-O-Tyr-Me-L-Leu-L-Leu), with a yield of 77 mg, 0.112 mmol, and a 53% yield. Its structural formula is as follows:

[0103] .

[0104] 1 H NMR (600 MHz, DMSO- d 6) δ 7.57 (t, J = 7.9 Hz, 2H), 7.45 (d, J = 8.2 Hz,1H), 7.25 – 6.99 (m, 2H), 6.91 – 6.66 (m, 2H), 5.04 (dd, J = 9.9, 6.0 Hz, 1H), 4.78 (dtd, J = 39.0, 8.5, 6.1 Hz, 2H), 4.57 (dd, J = 10.2, 5.6 Hz, 1H), 4.11(ddd, J = 9.6, 7.9, 5.6 Hz, 1H), 3.70 (s, 3H), 2.96 (s, 3H), 2.94 (s, 1H), 2.69(dd, J = 13.1, 6.4 Hz, 1H), 2.62 (s, 3H), 1.78 – 1.44 (m, 7H), 1.39 (qd, J =13.1, 10.8, 5.0 Hz, 5H), 0.96 – 0.69 (m, 24H). 13 C NMR (151 MHz, DMSO- d6) δ174.14, 171.96, 171.27, 170.89, 170.38, 158.26, 130.68, 129.96, 113.89,57.69, 55.39, 53.61, 51.53, 51.33, 48.19, 41.37, 38.77, 37.15, 36.93, 35.26,31.18, 29.87, 25.23, 24.85, 24.75, 24.63, 23.47, 23.41, 23.38, 23.19, 22.19,22.01, 21.92. HRMS (ESI + ) calcd. for C 36 H 59 N5O6[M+Na] + : 680.4363; found: 680.4532.

[0105] Example 9

[0106] Cyclo(Me-L-Leu-L-Leu-Methylester-D-Asp-Me-L-Leu-L-Leu)

[0107] The only difference from Example 1 is that N-Boc-D-Leu-OH is replaced with N-Boc-methylester-D-aspartic acid, yielding compound K10, a white solid, namely Cyclo(Me-L-Leu-L-Leu-Methylester-D-Asp-Me-L-Leu-L-Leu), with a yield of 71 mg, 0.110 mmol, and a 52% yield. Its structural formula is as follows:

[0108] .

[0109] 1 H NMR (600 MHz, DMSO- d 6) δ 7.88 (d, J = 8.0 Hz, 1H), 7.28 (dd, J = 8.7, 6.4 Hz, 2H), 5.09 (dd, J = 9.6, 6.0 Hz, 1H), 4.99 (td, J = 9.6, 4.3 Hz, 1H), 4.80(td, J = 8.9, 4.8 Hz, 1H), 4.47 (dd, J= 9.9, 5.0 Hz, 1H), 4.05 (ddd, J = 11.4,8.0, 4.4 Hz, 1H), 3.55 (s, 3H), 3.00 (s, 3H), 2.78 (dd, J = 16.1, 10.1 Hz, 1H), 2.67 (s, 3H), 2.47 (dd, J = 16.1, 4.3 Hz, 1H), 1.78 – 1.28 (m, 13H), 0.88(dddd, J = 47.8, 27.7, 10.8, 6.1 Hz, 24H). 13 C NMR (151 MHz, DMSO- d 6) δ 174.66,171.57, 171.15, 171.05, 170.84, 170.33, 57.80, 53.77, 51.81, 51.64, 48.37,46.57, 41.30, 38.24, 37.35, 36.25, 34.91, 31.40, 29.62, 25.25, 24.80, 24.78,23.64, 23.49, 23.43, 23.11, 22.07, 22.01, 21.94, 21.45. + ) calcd.for C 31 H 55 N5O7[M+Na] + : 632.3999; found: 632.3621.

[0110] Example 10

[0111] Cyclo(Me-L-Leu-L-Leu-3-Flu-D-Phe-Me-L-Leu-L-Leu)

[0112] The only difference from Example 1 is that N-Boc-D-Leu-OH is replaced with N-Boc-3-fluoro-D-phenylalanine, yielding compound K11, a white solid, namely Cyclo(Me-L-Leu-L-Leu-3-Flu-D-Phe-Me-L-Leu-L-Leu), with a yield of 75 mg, 0.120 mmol, and a 53% yield. Its structural formula is as follows:

[0113] .

[0114] 1 H NMR (600 MHz, DMSO- d 6) δ 7.59 (t, J = 9.1 Hz, 2H), 7.45 (d, J = 8.1 Hz,1H), 7.27 (q, J = 7.4 Hz, 1H), 7.10 – 6.83 (m, 3H), 5.07 (dd, J = 10.0, 5.9 Hz,1H), 4.89 (q, J = 7.8 Hz, 1H), 4.75 (q, J = 7.8 Hz, 1H), 4.58 (dd, J = 10.1, 5.6Hz, 1H), 4.09 (td, J = 8.7, 5.6 Hz, 1H), 3.05 (dd, J = 13.2, 7.8 Hz, 1H), 2.97(s, 3H), 2.79 (dd, J = 13.2, 6.8 Hz, 1H), 2.66 (s, 3H), 1.71 – 1.15 (m, 13H),1.00 – 0.58 (m, 24H). 13 C NMR (151 MHz, DMSO- d 6) δ 174.24, 171.70, 171.31,170.84, 170.42, 163.27, 141.20, 141.15, 130.25, 130.20, 125.85, 116.49,116.35, 60.18, 57.71, 53.72, 51.57, 50.91, 48.26, 41.32, 38.68, 37.41, 37.20,35.25, 31.21, 29.88, 25.23, 24.85, 24.76, 24.69, 23.46, 23.40, 23.31, 23.17,22.16, 22.00, 21.88, 21.80. HRMS (ESI + ) calcd. for C 35 H 56 FN5O5[M+Na] + : 668.4163;found: 668.4134.

[0115] Example 11

[0116] Cyclo(Me-L-Leu-L-Leu-2-Me-D-Phe-Me-L-Leu-L-Leu)

[0117] The only difference from Example 1 is that N-Boc-D-Leu-OH is replaced with N-Boc-2-methyl-D-phenylalanine, yielding compound K12, a white solid, namely Cyclo(Me-L-Leu-L-Leu-2-Me-D-Phe-Me-L-Leu-L-Leu), with a yield of 76 mg, 0.120 mmol, and a 53% yield. Its structural formula is as follows:

[0118] .

[0119] 1 H NMR (600 MHz, DMSO- d 6) δ 7.66 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.18 – 6.85 (m, 4H), 5.04 (dd, J = 9.9, 6.1 Hz, 1H), 4.90 (td, J = 8.8, 5.6 Hz, 1H), 4.76 (q, J = 7.6 Hz, 1H), 4.57 (dd, J = 10.3, 5.4 Hz, 1H), 4.12 (td, J = 8.8, 5.3 Hz, 1H), 3.08 (dd, J = 13.5, 9.0 Hz, 1H), 2.96 (s, 3H), 2.75 (dd, J = 13.5, 5.6 Hz, 1H), 2.59 (s, 3H), 2.32 (s, 3H), 1.77– 1.29 (m, 11H), 1.18 – 1.04 (m, 1H), 1.00 – 0.53 (m, 24H). 13 C NMR (151 MHz, DMSO- d6) δ 174.18, 171.72, 171.26, 171.04, 170.25, 136.70, 136.12, 130.35,130.08, 126.74, 125.88, 57.71, 53.61, 51.44, 49.88, 48.21, 41.43, 38.65,37.15, 35.14, 34.92, 31.18, 29.80, 25.24, 24.81, 24.75, 24.61, 23.48, 23.39,23.19, 22.14, 22.00, 21.88, 21.80, 19.54. HRMS (ESI + ) calcd. for C 36 H 59 N5O5[M+H] + : 664.4414; found: 664.4438.

[0120] Example 12

[0121] Cyclo(Me-L-Leu-L-Leu-4-Chl-D-Phe-Me-L-Leu-L-Leu)

[0122] The only difference from Example 1 is that N-Boc-D-Leu-OH is replaced with N-Boc-4-chloro-D-phenylalanine, yielding compound K13, a white solid, namely Cyclo(Me-L-Leu-L-Leu-4-Chl-D-Phe-Me-L-Leu-L-Leu), with a yield of 80 mg, 0.120 mmol, and a 55% yield. Its structural formula is as follows:

[0123] .

[0124] 1 H NMR (600 MHz, DMSO- d 6) δ 7.58 (dd, J = 27.1, 8.2 Hz, 2H), 7.42 (dd, J =20.0, 8.1 Hz, 1H), 7.30 – 7.16 (m, 4H), 5.04 (dt, J = 10.5, 5.2 Hz, 1H), 4.95 –4.80 (m, 1H), 4.80 – 4.68 (m, 1H), 4.57 (dt, J= 11.6, 5.8 Hz, 1H), 4.18 – 3.81(m, 1H), 3.03 (ddd, J = 20.0, 12.9, 8.0 Hz, 1H), 2.97 (s, 3H), 2.76 (dd, J =13.3, 6.4 Hz, 1H), 2.64 (d, J = 12.2 Hz, 3H), 1.76 – 1.20 (m, 12H), 1.03 – 0.53 (m, 24H). 13 C NMR (151 MHz, DMSO- d 6) δ 174.22, 171.83, 171.30, 170.87, 170.40,138.18, 131.65, 129.66, 128.45, 128.35, 57.75, 57.71, 53.66, 51.53, 51.15,48.23, 41.36, 38.72, 37.18, 35.22, 31.21, 29.86, 25.23, 24.84, 24.76, 24.66,23.47, 23.39, 23.35, 23.18, 22.17, 22.01, 21.90, 21.87. HRMS (ESI + ) calcd.for C 35 H 56 ClN5O5[M+H] + : 684.3868; found: 684.3854.

[0125] Example 13

[0126] Cyclo(Me-L-Leu-L-Leu-4-Bro-D-Phe-Me-L-Leu-L-Leu)

[0127] The only difference from Example 1 is that N-Boc-D-Leu-OH is replaced with N-Boc-4-bromo-D-phenylalanine, yielding compound K14, a white solid, namely Cyclo(Me-L-Leu-L-Leu-4-Bro-D-Phe-Me-L-Leu-L-Leu), with a yield of 87 mg, 0.122 mmol, and a 56% yield. Its structural formula is as follows:

[0128] .

[0129] 11H NMR (600 MHz, DMSO- d 6) δ 7.58 (dd, J J = 17.6, 8.3 Hz, 2H), 7.45 (d, J J =8.1 Hz, 1H), 7.20 (ddt, J J = 22.1, 14.2, 7.5 Hz, 5H), 5.05 (t, J J = 7.9 Hz, 1H),4.87 (q, J J = 8.0 Hz, 1H), 4.75 (q, J J = 7.6 Hz, 1H), 4.57 (dd,​​​​​​​​​​​​​​​​​​​​​​​​​Cyclo(Me-L-Leu-L-Leu-D-cyclohexyl-Gly-Me-L-Leu-L-Leu)

[0132] The only difference from Example 1 is that N-Boc-D-Leu-OH is replaced with N-Boc-D-cyclohexyl-glycine, yielding compound K15, namely Cyclo(Me-L-Leu-L-Leu-D-cyclohexyl-Gly-Me-L-Leu-L-Leu), a white solid, with a yield of 70 mg, 0.110 mmol, and a 51% yield. Its structural formula is as follows:

[0133] .

[0134] 1 H NMR (600 MHz, DMSO- d 6) δ 7.63 (d, J = 7.8 Hz, 1H), 7.45 (d, J = 9.1 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 5.10 (dd, J = 10.2, 6.0 Hz, 1H), 4.71 (t, J = 7.4Hz, 1H), 4.69 – 4.54 (m, 1H), 4.43 (t, J = 9.5 Hz, 1H), 4.24 (q, J = 7.6 Hz, 1H), 2.95 (s, 3H), 2.81 (s, 3H), 1.77 (d, J = 11.1 Hz, 1H), 1.73 – 1.56 (m, 8H), 1.57 – 1.22 (m, 10H), 1.13 (p, J = 12.9, 12.4 Hz, 3H), 0.97 – 0.73 (m, 26H). 13 CNMR (151 MHz, DMSO- d6) δ 173.99, 172.50, 171.08, 171.02, 170.70, 57.54,54.13, 53.69, 51.32, 48.22, 41.24, 37.04, 35.61, 31.12, 30.27, 29.82, 28.58,26.45, 25.91, 25.20, 24.96, 24.72, 23.46, 23.24, 22.24, 22.14, 21.90, 21.74.HRMS (ESI + ) calcd. for C 34 H 61 N5O5[M+Na] + : 642.4570; found: 642.5435.

[0135] Example 15

[0136] Cyclo(Me-L-Leu-L-Leu-D-phenyl-Gly-Me-L-Leu-L-Leu)

[0137] The only difference from Example 1 was the replacement of N-Boc-D-Leu-OH with N-Boc-D-phenyl-glycine, yielding compound K16, a white solid, namely Cyclo(Me-L-Leu-L-Leu-D-phenyl-Gly-Me-L-Leu-L-Leu), with a yield of 72 mg, 0.112 mmol, and a 52% yield. Its structural formula is as follows:

[0138] .

[0139] 1 H NMR (600 MHz, DMSO- d 6) δ 7.89 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.31 (dt, J = 29.4, 7.8 Hz, 5H), 5.88 (d, J = 8.8 Hz, 1H), 5.08 (t, J = 7.8Hz, 1H), 4.86 (td, J = 9.4, 4.0 Hz, 1H), 4.47 (dd, J = 9.9, 5.4 Hz, 1H), 4.20(td, J= 9.4, 8.8, 4.7 Hz, 1H), 3.03 (s, 3H), 2.80 (s, 3H), 1.84 – 1.13 (m,13H), 0.89 (tdd, J = 59.3, 14.5, 6.6 Hz, 24H). 13 C NMR (151 MHz, DMSO- d 6) δ174.79, 171.80, 171.29, 170.19, 138.81, 128.25, 128.09, 127.63, 58.46, 53.86,53.44, 51.38, 48.46, 41.42, 38.36, 37.45, 35.01, 31.47, 30.12, 25.29, 24.85,24.81, 24.78, 24.57, 23.64, 23.56, 23.31, 23.04, 22.25, 22.18, 21.90, 21.29.HRMS (ESI + ) calcd. for C 34 H 55 N5O5[M+Na] + : 636.4101; found: 636.4421.

[0140] Example 16

[0141] Cyclo(Me-L-Leu-L-Leu-D-Val-Me-L-Leu-L-Leu)

[0142] The only difference from Example 1 is that N-Boc-D-Leu-OH is replaced with N-Boc-D-Val-OH, yielding compound K17, namely Cyclo(Me-L-Leu-L-Leu-D-Val-Me-L-Leu-L-Leu), a white solid, with a yield of 66 mg, 0.110 mmol, and a 51% yield. Its structural formula is as follows:

[0143] .

[0144] 1 H NMR (600 MHz, DMSO- d 6) δ 7.62 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 9.1 Hz, 1H), 7.37 (d, J= 7.7 Hz, 1H), 5.12 (dd, J = 10.3, 5.7 Hz, 1H), 4.70 (d, J = 7.4Hz, 1H), 4.63 (dd, J = 10.4, 5.7 Hz, 1H), 4.38 (t, J = 9.4 Hz, 1H), 4.24 (d, J =7.4 Hz, 1H), 2.95 (s, 3H), 2.83 (s, 3H), 2.04 (dt, J = 9.7, 6.7 Hz, 1H), 1.76 –1.18 (m, 12H), 0.94 – 0.77 (m, 26H). 13 C NMR (151 MHz, DMSO- d 6) δ 173.92,172.68, 171.07, 170.99, 170.71, 57.49, 55.29, 53.65, 51.45, 48.19, 41.21,39.12, 37.01, 35.66, 31.08, HRMS (ESI) + )calcd. for C 31 H 57 N5O5[M+H] + : 602.4257; found: 602.4375.

[0145] Example 17

[0146] Cyclo(Me-L-Leu-L-Leu-D-Met-Me-L-Leu-L-Leu)

[0147] The only difference from Example 1 is that N-Boc-D-Leu-OH is replaced with N-Boc-D-Met-OH, yielding compound K18, a white solid, namely Cyclo(Me-L-Leu-L-Leu-D-Met-Me-L-Leu-L-Leu), with a yield of 70 mg, 0.110 mmol, and a 52% yield. Its structural formula is as follows:

[0148] .

[0149] 1 H NMR (600 MHz, DMSO- d 6) δ 7.71 (d, J = 8.1 Hz, 1H), 7.33 (d, J = 8.2 Hz,1H), 7.26 (d, J = 8.9 Hz, 1H), 5.12 (dd, J = 9.5, 6.3 Hz, 1H), 4.81 (dq, J = 22.6,7.3 Hz, 2H), 4.47 (dd, J = 10.1, 5.0 Hz, 1H), 4.16 (ddd, J = 10.7, 8.0, 5.0 Hz,1H), 2.99 (s, 3H), 2.70 (s, 3H), 2.44 – 2.18 (m, 2H), 2.03 (s, 4H), 1.70(dtd, J = 19.1, 9.6, 9.1, 5.4 Hz, 3H), 1.66 – 1.44 (m, 7H), 1.39 (dt, J = 29.3,7.0 Hz, 3H), 0.88 (dddd, J = 48.4, 35.5, 14.9, 6.5 Hz, 24H). 13 C NMR (151 MHz,DMSO- d 6) δ 174.59, 171.62, 171.47, 171.11, 170.42, 58.02, 53.59, 51.41,48.49, 48.36, 41.37, 38.55, 37.27, 35.06, 31.48, 31.37, 29.96, 29.78, 25.26,24.86, 24.83, 24.78, 23.56, 23.52, 23.41, 23.12, 22.03, 22.00, 21.57,14.91.HRMS (ESI + ) calcd. for C 31 H 57 N5O5S [M+Na] + : 634.3978; found: 634.3990.

[0150] Example 18

[0151] Cyclo(Me-D-Leu-L-Leu-L-Leu-Me-D-Leu-L-Leu)

[0152] (1) N-Boc-Me-D-Leu-OH

[0153] 9.0 g (38.9 mmol) of N-Boc-D-leucine, 25 g (177.3 mmol) of CH3I, and 5.5 g (137.5 mmol) of NaH dispersed in mineral oil were mixed and stirred at -20 °C for 60 min, then stirred at room temperature for 12 h. The mixture was quenched with 100 mL of aqueous solution and washed with ethyl acetate. Subsequently, the aqueous solution was acidified to pH 5 with 1 N HCl solution and extracted with ethyl acetate. The resulting ethyl acetate extract was dried over Na2SO4, filtered, and evaporated to give a colorless oily B3, namely N-Boc-Me-D-Leu-OH, with a yield of 8.8 g (35.8 mol), 92% yield.

[0154] (2)N-Boc-Me-D-Leu-L-Leu-OBn

[0155] C3 was synthesized by reacting 10.0 g (40.8 mmol) of B3, 31.5 g (44.9 mmol) of L-leucine benzyl ester p-toluenesulfonate, 6.0 g (44.9 mmol) of HOBt, and 8.5 g (44.9 mmol) of EDCI at 10 °C for 6 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate in a volume ratio of 90:10 as the elution solvent to give C3 as a white solid, namely N-Boc-Me-D-Leu-L-Leu-OBn, in a yield of 14.6 g (32.6 mol), representing 80% yield.

[0156] (2) N-Me-D-Leu-L-Leu-OBn

[0157] E3 was synthesized by reacting 15.0 g of 33.4 mmol of C3, 10 ml of TFA, and 40 ml of DCM at -10 °C for 6 h. Excess TFA and DCM were then removed under vacuum to produce a colorless oily E3, namely N-Me-D-Leu-L-Leu-OBn, with a yield of 9.9 g (28.4 mmol) and a yield of 85%.

[0158] (4)N-Boc-Me-D-Leu-L-Leu-OH

[0159] D3 was synthesized by reacting 15.0 g (33.4 mmol C3), 5 g (4.7 mmol 10% Pd / C), and 1 atm H2 at 20 °C for 6 h. The mixture was filtered and concentrated under vacuum to obtain D3, namely N-Boc-Me-D-Leu-L-Leu-OH, with a yield of 9.6 g (26.7 mmol) and an 80% yield.

[0160] (5)N-Boc-L-Leu-Me-D-Leu-L-Leu-OBn

[0161] F3 was synthesized by reacting 10.0 g (28.7 mmol) of E3 with 13.3 g (57.5 mmol) of N-Boc-L-Leu-OH, 4.2 g (31.5 mmol) of HOBT, and 6.0 g (31.5 mmol) of EDCI at 10 °C for 6 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate in a volume ratio of 80:20 as the elution solvent to obtain a white solid, F3, namely N-Boc-L-Leu-Me-D-Leu-L-Leu-OBn, with a yield of 13.2 g (23.5 mmol), representing an 82% yield.

[0162] (6)NL-Leu-Me-D-Leu-L-Leu-OBn

[0163] G3 was synthesized by reacting 15.0 g, 33.4 mmol F3, 10 ml TFA, and 40 ml DCM at -10 °C for 6 h. Excess TFA and DCM were then removed under vacuum to produce a colorless oily form of G3, NL-Leu-Me-D-Leu-L-Leu-OBn, with a yield of 13.0 g (28.1 mmol) and an 84% yield.

[0164] (7) N-Boc-Me-D-Leu-L-Leu-L-Leu-Me-D-Leu-L-Leu-OBn

[0165] H3 was synthesized by reacting 10.0 g (27.9 mmol D3), 25.7 g (55.8 mmol G3), 4.1 g (30.7 mmol HOBT), and 5.9 g (30.7 mmol EDCI) at 0 °C for 12 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate at a volume ratio of 70:30 as the elution solvent, yielding a white solid H3, namely N-Boc-Me-D-Leu-L-Leu-L-Leu-Me-D-Leu-L-Leu-OBn, with a yield of 20.0 g (25.0 mmol) and an 88% yield.

[0166] (8) N-Me-D-Leu-L-Leu-L-Leu-Me-D-Leu-L-Leu-OBn

[0167] I3 was synthesized by reacting 26.7 g, 33.4 mmol H3, 10 ml TFA, and 40 ml DCM at -20 °C for 12 h. Subsequently, excess TFA and DCM were removed under vacuum to obtain a colorless oily I3, namely N-Me-D-Leu-L-Leu-L-Leu-Me-D-Leu-L-Leu-OBn, with a yield of 20.1 g and 28.7 mmol, representing an 85% yield.

[0168] (9) N-Me-D-Leu-L-Leu-L-Leu-Me-D-Leu-L-Leu-OH

[0169] J3 was synthesized by reacting 23.4 g of 33.4 mmol I3, 5 g of 4.7 mmol of 10% Pd / C, and 1 atm H2 at 10 °C for 12 h. The mixture was filtered and concentrated under vacuum to obtain J3, namely N-Me-D-Leu-L-Leu-L-Leu-Me-D-Leu-L-Leu-OH, with a yield of 17.0 g of 27.9 mmol and an 85% yield.

[0170] 134 mg, 0.22 mmol J3 was dissolved in 4.3 mM MeCN and added dropwise over 10 h to a solution of 229 mg, 0.44 mmol PyBOP and 80 mg, 0.66 mmol DMAP in 1.0 mM MeCN. The crude residue was purified by column chromatography using a mixture of cyclohexane and ethyl acetate at a volume ratio of 60:40 as the eluent to give compound K03, i.e., Cyclo(Me-D-Leu-L-Leu-L-Leu-Me-D-Leu-L-Leu), as a white solid, with a yield of 67 mg, 0.113 mmol, and a 51% yield. Its structural formula is as follows:

[0171] .

[0172] 1 H NMR (600 MHz, DMSO- d 6) δ 8.29 (d, J = 8.7 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.01 (d, J = 9.4 Hz, 1H), 5.06 (t, J = 7.8 Hz, 1H), 4.80 (td, J= 9.1, 5.5 Hz,1H), 4.73 – 4.50 (m, 2H), 4.16 (td, J = 9.5, 5.1 Hz, 1H), 2.95 (s, 3H), 2.54(s, 3H), 1.75 – 1.31 (m, 15H), 1.09 – 0.55 (m, 30H). 13 C NMR (151 MHz, DMSO- d 6)δ 173.74, 171.69, 171.50, 171.16, 169.72, 55.88, 53.48, 52.31, 47.62, 47.52,41.65, 41.26, 40.92, 37.10, 34.91, HRMS(ESI) + ) calcd. for C 32 H 59 N5O5[M+Na] + : 616.4414; found: 616.4393.

[0173] Example 19

[0174] Cyclo(Me-D-Leu-L-Leu-D-Leu-Me-D-Leu-L-Leu)

[0175] The only difference from Example 18 is in steps (2) and (5), specifically:

[0176] (2)N-Boc-Me-D-Leu-L-Leu-OBn

[0177] C1 was synthesized by reacting 10.0 g (40.8 mmol) of N-Boc-Me-D-Leu-OH with 17.6 g (44.9 mmol) of D-leucine benzyl ester p-toluenesulfonate, 6.0 g (44.9 mmol) of HOBt, and 8.5 g (44.9 mmol) of EDCI at 5 °C for 8 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate in a volume ratio of 90:10 as the elution solvent to give C1 as a white solid, namely N-Boc-Me-D-Leu-L-Leu-OBn, in a yield of 14.6 g (32.6 mol), or 80% yield.

[0178] (5)N-Boc-D-Leu-Me-D-Leu-L-Leu-OBn

[0179] F1 was synthesized by reacting 10.0 g (28.7 mmol) of N-Me-D-Leu-L-Leu-OBn, 7.2 g (31.5 mmol) of N-Boc-D-Leu-OH, 4.2 g (31.5 mmol) of HOBT, and 6.0 g (31.5 mmol) of EDCI prepared according to the aforementioned steps at 5 °C for 8 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate at a volume ratio of 80:20 as the elution solvent to obtain a white solid, F1, namely N-Boc-D-Leu-Me-D-Leu-L-Leu-OBn, with a yield of 13.7 g (24.4 mmol) and an 85% yield.

[0180] Compound K01, a white solid, namely Cyclo(Me-D-Leu-L-Leu-D-Leu-Me-D-Leu-L-Leu), was prepared using F1 following the same steps as in Example 18, with a yield of 71 mg, 0.120 mmol, and a 55% yield. Its structural formula is as follows:

[0181] .

[0182] 1 H NMR (600 MHz, DMSO- d 6) δ 8.29 (d, J = 8.7 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.01 (d, J = 9.4 Hz, 1H), 5.06 (t, J = 7.8 Hz, 1H), 4.80 (q, J = 3.6 Hz, 1H),4.69 – 4.52 (m, 2H), 4.16 (q, J = 4.3 Hz, 1H), 2.95 (s, 3H), 2.54 (s, 3H), 1.82– 1.29 (m, 15H), 1.06 – 0.61 (m, 30H). 13 C NMR (151 MHz, DMSO- d6) δ 173.74,171.69, 171.50, 171.16, 169.72, 55.88, 53.48, 52.31, 47.62, 47.52, 41.65,41.26, 40.92, 37.10, 34.91, HRMS (ESI) + )calcd. for C 32 H 59 N5O5[M+Na] + : 616.4414; found: 616.4393.

[0183] Example 20

[0184] Cyclo(Me-L-Leu-D-Leu-L-Leu-Me-L-Leu-D-Leu)

[0185] The only difference from Example 18 is in steps (1) and (5); specifically:

[0186] (1) N-Boc-Me-L-Leu-OH.

[0187] Take 9.0 g (38.9 mmol) of L-leucine, 25 g (177.3 mmol) of CH3I, and 5.5 g (137.5 mmol) of NaH dispersed in mineral oil. Mix and stir at -20 °C for 60 min, then stir at room temperature for 12 h. Quench with 100 mL of aqueous solution and wash the mixture with ethyl acetate. Subsequently, acidify the aqueous solution to pH=5 with 1N HCl solution and extract with ethyl acetate. The resulting ethyl acetate extract is dried over Na2SO4, filtered, and evaporated to give colorless oily B21, i.e., N-Boc-Me-L-Leu-OH, with a yield of 9.0 g (36.7 mol), 94% yield.

[0188] (5)N-Boc-L-Leu-Me-L-Leu-D-Leu-OBn

[0189] The 10.0 g, 28.7 mmol N-Me-L-Leu-D-Leu-OBn, 7.2 g, 31.5 mmol N-Boc-L-Leu-OH, 4.2 g, 31.5 mmol HOBT, and 6.0 g, 31.5 mmol EDCI prepared by the aforementioned steps were reacted at 10 °C for 6 h to synthesize F21. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate at a volume ratio of 80:20 as the elution solvent, yielding a white solid F21, namely N-Boc-L-Leu-Me-L-Leu-D-Leu-OBn, with a yield of 13.7 g, 24.4 mmol, and an 85% yield.

[0190] Using F21, compound K21, namely Cyclo(Me-L-Leu-D-Leu-L-Leu-Me-L-Leu-D-Leu), was finally obtained as a white solid according to the method in Example 18, with a yield of 68 mg, 0.115 mmol, and 52% yield. Its structural formula is as follows:

[0191] .

[0192] 1 H NMR (600 MHz, DMSO- d 6) δ 8.30 (d, J = 8.7 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.01 (d, J = 9.4 Hz, 1H), 5.06 (t, J = 7.7 Hz, 1H), 4.79 (dd, J = 9.2, 5.8 Hz, 1H), 4.64 (t, J = 8.4 Hz, 2H), 4.15 (dt, J = 9.5, 4.7 Hz, 1H), 2.95 (s, 3H), 2.54(s, 3H), 1.88 – 1.15 (m, 15H), 1.15 – 0.41 (m, 30H). 13 C NMR (151 MHz, DMSO- d6)δ 173.73, 171.69, 171.50, 171.15, 169.72, 55.87, 53.46, 52.30, 47.61, 47.51,41.65, 41.24, 40.92, 37.09, 34.90, HRMS(ESI) + ) calcd. for C 32 H 59 N5O5[M+Na] + : 616.4414; found: 616.4393.

[0193] Example 21

[0194] Cyclo(Me-L-Leu-D-Leu-D-Leu-Me-L-Leu-D-Leu)

[0195] The only difference from Example 18 is in steps (1) and (5), specifically:

[0196] (1) N-Boc-Me-L-Leu-OH

[0197] 9.0 g (38.9 mmol) of L-leucine, 25 g (177.3 mmol) of CH3I, and 5.5 g (137.5 mmol) of NaH were dispersed in mineral oil. The mixture was stirred at -20 °C for 60 min, then stirred at room temperature for 12 h. The mixture was quenched with 100 mL of aqueous solution and washed with ethyl acetate. Subsequently, the aqueous solution was acidified to pH 5 with 1 N HCl solution and extracted with ethyl acetate. The ethyl acetate extract was dried over Na2SO4, filtered, and evaporated to give a colorless oily B22, namely N-Boc-Me-L-Leu-OH, in a yield of 9.0 g (36.7 mol), 94% yield.

[0198] (5)N-Boc-D-Leu-Me-L-Leu-D-Leu-OBn

[0199] F22 was synthesized by reacting 10.0 g (28.7 mmol) of N-Me-L-Leu-D-Leu-OBn, 7.2 g (31.5 mmol) of N-Boc-D-Leu-OH, 4.2 g (31.5 mmol) of HOBT, and 6.0 g (31.5 mmol) of EDCI at 10 °C for 6 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate at a volume ratio of 80:20 as the elution solvent to obtain a white solid, F22, i.e., N-Boc-D-Leu-Me-L-Leu-D-Leu-OBn, with a yield of 13.2 g (23.5 mmol), representing an 82% yield.

[0200] Using F22, compound K22, namely Cyclo(Me-L-Leu-D-Leu-D-Leu-Me-L-Leu-D-Leu), was finally obtained as a white solid according to the method in Example 18, with a yield of 67 mg, 0.113 mmol, and 51% yield. Its structural formula is as follows:

[0201] .

[0202] 1 H NMR (600 MHz, DMSO- d 6) δ 8.30 (d, J = 8.7 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.01 (d, J = 9.4 Hz, 1H), 5.06 (t, J = 7.7 Hz, 1H), 4.79 (dd, J = 9.2, 5.8 Hz, 1H), 4.64 (t, J = 8.4 Hz, 2H), 4.15 (dt, J = 9.5, 4.7 Hz, 1H), 2.95 (s, 3H), 2.54(s, 3H), 1.88 – 1.15 (m, 15H), 1.15 – 0.41 (m, 30H). 13 C NMR (151 MHz, DMSO- d6)δ 173.73, 171.69, 171.50, 171.15, 169.72, 55.87, 53.46, 52.30, 47.61, 47.51,41.65, 41.24, 40.92, 37.09, 34.90, HRMS(ESI) + ) calcd. for C 32 H 59 N5O5[M+Na] + : 616.4414; found: 616.4393.

[0203] Example 22

[0204] Cyclo(Me-D-Leu-D-Leu-L-Leu-Me-D-Leu-D-Leu)

[0205] The only difference from Example 18 is that steps (2) and (5) are different, specifically:

[0206] (2)N-Boc-Me-D-Leu-D-Leu-OBn

[0207] C23 was synthesized by reacting 10.0 g (40.8 mmol) of N-Boc-Me-D-Leu-OH, 17.6 g (44.9 mmol) of D-leucine benzyl ester p-toluenesulfonate, 6.0 g (44.9 mmol) of HOBt, and 8.5 g (44.9 mmol) of EDCI at 10 °C for 6 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate in a volume ratio of 90:10 as the eluent to give C23, namely N-Boc-Me-D-Leu-D-Leu-OBn, as a white solid, with a yield of 15.0 g (33.4 mol), representing an 81% yield.

[0208] (5)N-Boc-L-Leu-Me-D-Leu-D-Leu-OBn

[0209] 10.0 g, 28.7 mmol N-Me-D-Leu-D-Leu-OBn, 7.2 g, 31.5 mmol N-Boc-D-Leu-OH, 4.2 g, 31.5 mmol HOBT, and 6.0 g, 31.5 mmol EDCI prepared by the aforementioned steps were reacted at 10 °C for 6 h to synthesize F23. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate at a volume ratio of 80:20 as the elution solvent to obtain a white solid F23, namely N-Boc-L-Leu-Me-D-Leu-D-Leu-OBn, with a yield of 13.7 g, 24.4 mmol, and an 85% yield.

[0210] Using F23, compound K23, namely Cyclo(Me-D-Leu-D-Leu-L-Leu-Me-D-Leu-D-Leu), was finally obtained as a white solid according to the method in Example 18, with a yield of 72 mg, 0.122 mmol, and 56% yield. Its structural formula is as follows:

[0211] .

[0212] 1 H NMR (600 MHz, DMSO- d 6) δ 7.63 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 5.11 (t, J = 7.6 Hz, 1H), 4.77 (d, J = 7.4 Hz, 1H), 4.71 (d, J = 7.8 Hz, 1H), 4.49 (dd, J = 10.1, 5.2 Hz, 1H), 4.27 – 3.96 (m, 1H), 2.99 (s, 3H), 2.73 (s, 3H), 1.73 – 1.26 (m, 15H), 0.87 (tdd, J = 33.3, 12.5, 6.6 Hz, 30H). 13 C NMR (151 MHz, DMSO- d6) δ 174.53, 172.27, 171.37, 170.84,170.57, 57.94, 53.64, 51.37, 48.34, 47.69, 41.40, 41.09, 38.70, 37.23, 35.17,31.34, 29.88, 25.26, 24.92, 24.87, 24.81, 24.77, 23.50, 23.40, 23.15, 23.02,22.98, 22.06, 21.97, 21.91, 21.70. HRMS (ESI + ) calcd. for C 32 H 59 N5O5[M+Na] + :616.4414; found: 616.4393.

[0213] Example 23

[0214] Cyclo(Me-D-Leu-D-Leu-D-Leu-Me-D-Leu-D-Leu)

[0215] The only difference from Example 18 is in steps (2) and (5), specifically:

[0216] (2)N-Boc-Me-D-Leu-D-Leu-OBn

[0217] C24 was synthesized by reacting 10.0 g (40.8 mmol B3), 17.6 g (44.9 mmol D-leucine benzyl ester p-toluenesulfonate), 6.0 g (44.9 mmol HOBt), and 8.5 g (44.9 mmol EDCI) at 10 °C for 6 h. The crude product was purified by silica gel chromatography using a 90:10 volume ratio of n-hexane and ethyl acetate as the elution solvent to give C24 (N-Boc-Me-D-Leu-D-Leu-OBn) as a white solid, with a yield of 15.0 g (33.4 mol), representing 81% yield.

[0218] (5)N-Boc-D-Leu-Me-D-Leu-D-Leu-OBn

[0219] F24 was synthesized by reacting 10.0 g (28.7 mmol) of N-Me-D-Leu-D-Leu-OBn, 7.2 g (31.5 mmol) of N-Boc-D-Leu-OH, 4.2 g (31.5 mmol) of HOBT, and 6.0 g (31.5 mmol) of EDCI at 10 °C for 6 h. The crude product was purified by silica gel chromatography using a mixture of n-hexane and ethyl acetate at a volume ratio of 80:20 as the elution solvent to obtain a white solid, F24, i.e., N-Boc-D-Leu-Me-D-Leu-D-Leu-OBn, with a yield of 13.7 g (24.4 mmol) and an 85% yield.

[0220] Using F24, compound K24, namely Cyclo(Me-D-Leu-D-Leu-D-Leu-Me-D-Leu-D-Leu), was finally obtained as a white solid according to the method in Example 18, with a yield of 71 mg, 0.120 mmol, and a yield of 55%. Its structural formula is as follows:

[0221] .

[0222] 1 H NMR (600 MHz, DMSO- d 6) δ 7.63 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 5.11 (t, J = 7.6 Hz, 1H), 4.77 (d, J = 7.4 Hz, 1H), 4.71 (d, J = 7.8 Hz, 1H), 4.49 (dd, J = 10.1, 5.2 Hz, 1H), 4.27 – 3.96 (m, 1H), 2.99 (s, 3H), 2.73 (s, 3H), 1.73 – 1.26 (m, 15H), 0.87 (tdd, J = 33.3, 12.5, 6.6 Hz, 30H). 13 C NMR (151 MHz, DMSO- d6) δ 174.53, 172.27, 171.37, 170.84,170.57, 57.94, 53.64, 51.37, 48.34, 47.69, 41.40, 41.09, 38.70, 37.23, 35.17,31.34, 29.88, 25.26, 24.92, 24.87, 24.81, 24.77, 23.50, 23.40, 23.15, 23.02,22.98, 22.06, 21.97, 21.91, 21.70. HRMS (ESI + ) calcd. for C 32 H 59 N5O5[M+Na] + :616.4414; found: 616.4393.

[0223] Example 24

[0224] Cyclo(Me-L-Leu-L-Leu-3-Me-D-Phe-Me-L-Leu-L-Leu)

[0225] The only difference from Example 1 is that N-Boc-L-leucine was replaced with N-Boc-3-methyl-D-phenylalanine, yielding compound K19, or Cyclo(Me-L-Leu-L-Leu-3-Me-D-Phe-Me-L-Leu-L-Leu), a white solid, with a yield of 75 mg, 0.12 mmol, and a 53% yield. Its structural formula is as follows:

[0226] .

[0227] 1 H NMR (600 MHz, DMSO- d 6) δ 7.62 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.05 – 6.87 (m, 3H), 5.06 (dd, J = 9.8, 6.1 Hz, 1H), 4.84 (d, J = 7.7 Hz, 1H), 4.76 (t, J= 7.5 Hz, 1H), 4.57 (dd, J = 10.4, 5.5 Hz, 1H), 4.13 (d, J = 7.6 Hz, 1H), 3.01 (dd, J = 13.1, 8.5Hz, 1H), 2.97 (s, 3H), 2.71 (dd, J = 13.3, 6.2 Hz, 1H), 2.62 (s, 3H), 2.25 (s,3H), 1.64 – 1.31 (m, 10H), 1.31 – 1.10 (m, 2H), 1.00 – 0.50 (m, 24H). 13 C NMR (151 MHz, DMSO-) d 6) δ 174.22, 171.85, 171.30, 170.85, 170.37, 138.09, 137.42,130.23, 128.36, 127.29, 126.69, 57.73, 53.64, 51.51, 51.12, 48.23, 41.40,38.69, 37.75, 37.19, 35.22, 31.21, 29.84, 26.82, 25.24, 24.84, 24.75, 24.67,23.48, 23.41, 23.19, 22.16, 21.98, 21.85, 21.38. HRMS (ESI + ) calcd. forC 36 H 59 N5O5[M+Na] + : 664.4414; found: 664.4423.

[0228] Example 25

[0229] Cyclo(Me-L-Leu-L-Leu-3-Me-D-Phe-Me-L-Leu-L-Leu)

[0230] The only difference from Example 1 is that N-Boc-L-leucine was replaced with N-Boc-4-methyl-D-phenylalanine, yielding compound K20, a white solid, namely Cyclo(Me-L-Leu-L-Leu-3-Me-D-Phe-Me-L-Leu-L-Leu), with a yield of 77 mg, 0.122 mmol, and a 54% yield. Its structural formula is as follows:

[0231] .

[0232] 1 H NMR (600 MHz, DMSO- d 6) δ 7.58 (t, J = 9.2 Hz, 2H), 7.45 (d, J = 8.1 Hz,1H), 7.31 – 6.75 (m, 4H), 5.03 (dd, J = 9.8, 5.9 Hz, 1H), 4.83 (q, J = 7.9 Hz, 1H), 4.74 (q, J = 7.6 Hz, 1H), 4.57 (dd, J = 10.3, 5.6 Hz, 1H), 4.11 (q, J = 7.6Hz, 1H), 3.02 – 2.97 (m, 1H), 2.96 (s, 3H), 2.71 (dd, J = 13.2, 6.2 Hz, 1H), 2.62 (s, 3H), 2.24 (s, 3H), 1.70 – 1.12 (m, 12H), 1.00 – 0.53 (m, 24H). 13 C NMR (151 MHz, DMSO-) d 6) δ 174.13, 171.88, 171.27, 170.92, 170.36, 135.57, 135.01,129.52, 129.06, 57.67, 53.62, 51.55, 51.18, 48.19, 41.37, 38.78, 37.39,37.15, 35.26, 31.17, 29.87, 25.23, 24.85, 24.75, 24.63, 23.47, 23.41, 23.39,23.20, 22.19, 22.01, 21.91, 21.09. HRMS (ESI + ) calcd. for C 36 H 59 N5O5[M+Na] + :664.4414; found: 664.4423.

[0233] Experimental Example 1

[0234] Uses of marine cyclic peptides in anticancer

[0235] 1. Cell Culture

[0236] A549, K562, MDA-MB-231, HepG2, U251, U87MG, and 293T cells were all purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. A549 and K562 cells were cultured in RPMI 1640 medium with 1% p / s. MDA-MB-231, HepG2, U251, U87MG, and 293T cells were cultured in DMEM medium. All cells were cultured at 37°C in an incubator containing 5% CO2. The DMEM medium contained 10% FBS and 1% p / s. p / s stands for penicillin-streptomycin.

[0237] 2. CCK-8 Experiment

[0238] Various cell types were seeded into 96-well plates. After the cells recovered to normal conditions, different concentrations of K01-K25 and Galaxamide were added for 48 hours. The relative cell viability was then measured according to the CCK-8 assay instructions, and the relative cell viability was analyzed and calculated using GraphPad Prism software. CCK-8 assay kit catalog number: MA0218, Meilunbio, China.

[0239] 3. Results

[0240] Galaxamide and its analogues on the IC50 of seven cell lines 50 The values ​​are shown in Table 1.

[0241] Table 1. Effects of Galaxamide and its analogues on different human cancer cell lines.

[0242]

[0243] Table 1 shows that various human tumor cell lines exhibit cytotoxicity against Galaxamide and K01-K25. All evaluated compounds showed significant cytotoxic activity against HepG2 cells. Comparing Galaxamide and its 25 analogues, K09 showed the greatest cytotoxicity against the MDA-MB231 cell line. Furthermore, the anticancer efficacy of Galaxamide analogues against tumor cell lines was significantly influenced by the number of D-leucine residues. K20 showed IC50 activity against K562, MDA-MB-231, HepG2, and U87MG cell lines. 50The concentrations were 14.36, 2.775, 1.446, and 0.075 µM, exhibiting significant broad-spectrum anticancer activity. These activities were >5.57, 4.26, 55.32, and 11.47 times higher than those of Galaxamide, respectively. Furthermore, each Galaxamide analog showed potent anticancer activity against HepG2 cells, suggesting that Galaxamide and its analogs may act more effectively on HepG2 than other tumor cell lines. K20 has five D-amino acids at positions 1–5. In contrast, K19 has two D-amino acids at positions 3 and 5, and K22 has three D-amino acids at positions 1, 2, and 4. K20 is more effective than other analogs, indicating that compounds containing five D-amino acids have superior activity compared to analogs containing fewer D-amino acids (K19–K24).

[0244] Furthermore, alterations in the position of D-leucine significantly affect the anticancer efficacy of analogs. The best analog for preventing the proliferation of all cancer cells is K01, which has D-leucine residues at positions 1, 3, and 5. Its IC50... 50 The values ​​are half that of Galaxamide; K562 is 24.24, MDA-MB-231 is 0.1230, HepG2 is 1.705, and 293T is 14.14. Except for K01, K02 has a D-amino acid at position 1 and exhibits strong inhibitory activity against K562, MDA-MB-231, HepG2, and U87MG, with an IC50 value of 14.14. 50 The values ​​were 8.6, 0.3979, 5.0, and 0.6945, respectively. The reason behind this may be that Galaxamide acquires a more stable and restricted cyclic structure when the D-amino acids are located at positions 1, 3, and 5 (K02, K01). K01 exhibits the most stable cyclic conformation, with the lowest energy and the most active amino acid residues against cancer cells. The Galaxamide analog K21 showed lower antitumor activity than analogs K01 and K02, as it has D-amino acids at positions 2 and 4. Specifically, K01 has twice the anticancer potential of K21, with an IC50 of 8.6 for HepG2 and K562 cell lines. 50 The values ​​were 6.368 and 75.24, respectively. Compared with Galaxamide, K22 showed slightly altered antitumor efficacy against A549, K562, and U87MG, with IC50 values ​​of 6.368 and 75.24, respectively. 50 The values ​​were >22, >80, and 0.5878, respectively. Since K22 and K21 showed relatively low anticancer activity against the examined cancer cells, the restriction structure of Galaxamide analogs might be disrupted when the D-amino acids are located at positions 2 and 4.

Claims

1. A marine cyclic peptide, characterized in that, Its structural formula is as follows: 。 2. A method for synthesizing the marine cyclic peptide according to claim 1, characterized in that, Includes the following steps: Using N-(tert-butoxycarbonyl)-L-leucine as a starting material, a nitrogen methylation reaction was carried out to obtain Boc-N-methyl-L-leucine; A dipeptide was obtained by condensation reaction using the Boc-N-methyl-L-leucine and L-leucine benzyl ester p-toluenesulfonate as raw materials. The dipeptide was subjected to hydrogenation reduction to remove its C-terminal benzyl group, yielding product D; The dipeptide was subjected to acid hydrolysis to remove its N-terminal tert-butyloxycarbonyl group, yielding product E; The product E was mixed with N-Boc-D-Pro-OH and subjected to a condensation reaction to obtain a tripeptide. The N-terminal Boc group was removed to obtain product G. The product D and the product G are mixed and subjected to a condensation reaction to obtain a linear pentapeptide; the C-terminal benzyl group and the N-terminal Boc group of the linear pentapeptide are removed sequentially to obtain product J; The product J was subjected to a macrocyclic lactamation reaction to obtain the marine cyclic peptide.

3. The synthesis method according to claim 2, characterized in that, The molar ratio of Boc-N-methyl-L-leucine to L-leucine benzyl ester p-toluenesulfonate is 1:1~2; The molar ratio of product E to N-Boc-D-Pro-OH is 1:1~2; The molar ratio of product D to product G is 1:1~2.

4. The synthesis method according to claim 3, characterized in that, The nitrogen methylation reaction was carried out by stirring at -20 to -10°C for 30 to 60 minutes, followed by stirring at room temperature for 12 to 18 hours. The condensation reaction of Boc-N-methyl-L-leucine and L-leucine benzyl ester with toluenesulfonate was carried out at 0℃~10℃ for 6h~12h. The hydrogenation reduction reaction at the C-terminus of the dipeptide is carried out at 10℃~20℃ for 6h~12h under the action of palladium catalyst; The acid hydrolysis reaction at the N-terminus of the dipeptide was carried out at -20℃ to -10℃ for 6 to 12 hours. The condensation reaction of product E with N-Boc-D-Pro-OH is carried out at 0℃~10℃ for 6h~12h. The condensation reaction of product D and product G is carried out at 0℃~10℃ for 6h~12h. The macrocyclic intra-amidation reaction was carried out at 0℃~10℃ for 12h~24h under the action of a coupling agent.

5. The synthesis method according to claim 4, characterized in that, The coupling agent is selected from 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, propionic anhydride, diphenylphosphide, pentafluorophenyl diphenylphosphinate, or (benzotriazol-1-yloxy)tripyrrolidinephosphine hexafluorophosphate.

6. The use of the marine cyclic peptide of claim 1 in the preparation of an anticancer drug, characterized in that, The anticancer effects described are the inhibitory effects on non-small cell lung cancer, chronic myeloid leukemia, breast cancer, glioma, and immortalized embryonic kidney cells.

7. An anticancer drug, characterized in that, It uses the marine cyclic peptide described in claim 1 as its sole active ingredient.

8. The anticancer drug according to claim 7, characterized in that, The drug is formulated by combining the marine cyclic peptide with pharmaceutically acceptable excipients.

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