A 15-membered cyclic depsipeptide compound and its preparation method and application

Abstract

The present invention belongs to the field of pharmaceutical chemistry technology, specifically, a series of 15-membered ring depsipeptide compounds with a new antimycin mother core structure are produced by feeding chemical precursors during the fermentation process of Streptomyces S.conglobatusATCC 31005, and its preparation method and its application in antitumor drugs. This type of compound is a 15-membered ring depsipeptide compound, and its C-2 position is connected to a carbonyl or hydroxyl group, C-4 and C-9 positions have fatty chain substitutions of different lengths, and C-6 is connected to a benzoic acid group containing a F atom or a hydroxyl group. This type of compound has significant tumor cell proliferation inhibitory activity on colorectal cancer cells and lung cancer cells, and the inhibitory activity is comparable to that of the control drug oxaliplatin, and the toxicity to non-cancerous cell lines is weak. The present invention provides a new lead compound for the research and development of new antitumor drugs, which is expected to be developed as a tumor inhibitor.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a fifteen-membered ring cyclopeptide compound, a preparation method thereof, and an application thereof. Background Art

[0002] Streptomyces is an important resource treasure house for novel lead compounds in the world drug research and development, and can produce novel metabolites with diverse scaffolds and complex structures. Neoantimycin (NAT) is a type of phenol peptide natural product produced by Streptomyces. Its structural characteristics are as follows: its parent nucleus skeleton is a fifteen-membered ring lactone, and a 3-N-formylaminosalicylic acid group, two alkyl groups, and a benzyl side chain are respectively connected to the four ester (peptide) groups. The structural differences of the reported neoantimycin analogs mainly focus on the hydroxylation or ketonylation modification at the C1 position of the molecular mother ring, and the ester (peptide) group C 4 / C 9The length of the alkyl side chain at the position, whether the amino group on the salicylic acyl group is N-formylated or substituted by a hydroxyl group (Lin, X.; et al. Applying Molecular Networking for Targeted Isolation of Depsipeptides. RSC Adv. 2021, 11(5), 2774-2782; Lin, X.; et al. Compound Discovery and Structure-Activity Relationship Study of Neoantimycins Against Drug-Resistant Cancer Cells. Front. Chem. 2019, 7, 481.). Neoantimycin compounds generally have important medicinal activities. For example, SW-163A and SW-163B have immunosuppressive and antifungal activities (Takahashi, K.; et al. SW-163A and B, Novel Imimmosuppressants Produced by Streptomyces Sp. J. Antibiot. 2001, 54(11), 867-873.), and prunustatin A, JBIR-04, and JBIR-05 can negatively regulate the expression of the tumor cell target protein GRP78 (glucose-regulated protein) (Izumikawa, M.; et al. Novel GRP78 Molecular Chaperone Expression Down-Regulators JBIR-04 and -05 Isolated from Streptomyces Violaceoniger. J. Antibiot. 2007, 60(10), 640-644; Umeda, Y.; et al. Absolute Structure of Prunustatin A, a Novel GRP78 Molecular Chaperone down-Regulator. Org. Lett. 2007, 9(21), 4239-4242.).Recently, it has also been reported that the new antimycin compounds NAT-A, NAT-F, NAT-G, and NAT-H have significant inhibitory effects on the plasma membrane localization of the key cancer target K-Ras and the multidrug resistance of the colon cancer cell line SW620 (Salim, A.A.; et al. Rare Streptomyces N-Formyl Amino-Salicylamides Inhibit Oncogenic K-Ras. Org. Lett. 2014, 16(19), 5036-5039. https: / / doi.org / 10.1021 / ol502376e.). However, all of the above compounds have a major drug development defect, with poor selectivity for the inhibitory activity against tumor cells and significant toxic side effects on normal cells. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a fifteen-membered cyclic peptide compound, a preparation method thereof, and an application thereof.

[0004] The first object is to disclose eight new structural new antimycin derivatives Unantimycin F1-J (UAT-F1-J) produced by fermentation of Streptomyces conglobatus ATCC 31005. The C-6 position in the new antimycin molecular structure generally contains a 3-N-formylaminosalicylic acid group. The fifteen-membered cyclic peptide compound involved in the present invention is characterized in that the C-6 position is substituted by a non-3-N-formylaminosalicylic acid group, and its structural formula is as follows:

[0005]

[0006] The second object of the present invention is to further provide a preparation method of the above-mentioned novel fifteen-membered cyclic peptide compound. The novel fifteen-membered cyclic peptide compound of the present invention can be obtained by fermentation of Streptomyces conglobatus or by chemical synthesis.

[0007] 1) If prepared by fermentation of Streptomyces conglobatus, preferably, the used Streptomyces conglobatus is preferably the commercially available strain ATCC 31005.

[0008] Preferably, the fermentation preparation includes the following steps:

[0009] a) Preparation of the precursor compounds a-d: The precursor compounds a-d can be prepared by the following route:

[0010]

[0011] For the specific preparation and separation processes of each intermediate in the above preparation route, reference can be made to the preparation and purification processes of compounds with the same or similar structures in the existing literature (Kusebauch, Bjoern et al ChemBioChem, 12(15), 2284-2288; 2011), which will not be elaborated in this invention.

[0012] b) Strain fermentation and precursor feeding: Inoculate Streptomyces S.conglobatus on a solid medium and culture at 27-32 °C for 4-6 days to collect spores; inoculate the spores into a primary liquid medium and culture in a shaking flask at 27-32 °C for 3-4 days to obtain a seed solution; inoculate the seed solution into a secondary liquid medium and culture in a shaking flask at 27-32 °C for 3-4 days to obtain a secondary seed solution; inoculate the secondary seed solution into a fermentation medium and culture in a shaking flask at 27-32 °C for 2 days, then feed compounds a-d at a concentration of 1.0 mmol / L, and continue to culture in a shaking flask for 3-4 days to collect the fermentation broth; extract and separate the novel 15-membered cyclic peptide compounds 1-8 described in this invention from the fermentation broth.

[0013] Preferably, the solid medium contains 1.5-2.5% soybean powder, 1.5-2.5% D-mannitol, and 1.5-2.5% agar. The primary liquid medium contains 2-4% tryptic soy broth, 9-11% sucrose, and 0.4-0.6% yeast extract. The secondary liquid medium contains 2-4% soybean powder, 4-6% glucose, 0.4-0.6% CaCO 3 , 4-6 mg / L CoCl 2 ·6H 2 O, 0.15-0.25% (v / v) antifoaming agent. The fermentation medium contains 2-4% soybean powder, 4-6% glucose, 0.4-0.6% CaCO 3 , 0.15-0.25% (v / v) antifoaming agent.

[0014] c) Compound extraction, separation, and purification

[0015] The chromatographic separation includes two times of reduced-pressure silica gel column chromatography separation, one time of reversed-phase medium-pressure ODS column chromatography separation, and high-performance liquid chromatography separation.

[0016] Preferably, the steps for extracting the 15-membered cyclic peptide compounds from the fermentation broth include: adding 0.1-0.2% (v / v) formic acid to the fermentation broth, then extracting with ethyl acetate, and concentrating the ethyl acetate extract under reduced pressure at 30-45 °C to obtain an extract; redissolving the extract with methanol, filtering to remove the residue, and then extracting with n-hexane to remove the n-hexane layer; after concentrating the methanol solution, separating the compounds UAT-F1-J by column chromatography.

[0017] Further, the column chromatography separation includes: separation through a normal-phase silica gel column, with the elution solvent being dichloromethane / methanol and the elution solvent ratio gradient being 50 / 1 (v / v); separation through a normal-phase silica gel column, with the elution solvent being petroleum ether / ethyl acetate and the elution solvent ratio gradient being 5 / 1 to 0 / 1 (v / v), specifically referring to first eluting with petroleum ether - ethyl acetate (5 / 1, v / v), and then gradually increasing the solvent polarity and gradually increasing the content of ethyl acetate; taking the target fraction and separating it through an ODS column, with the elution solvent gradient being 30% - 100% acetonitrile; specifically referring to first eluting with an aqueous solution of acetonitrile containing 30%, and then gradually decreasing the solvent polarity and increasing the acetonitrile content gradient to 100%.

[0018] Take the target fraction and perform final separation by preparative HPLC;

[0019] The separation conditions for UAT-F1 and UAT-F2 are: 78% acetonitrile - 0.1% formic acid aqueous solution, YMC C 18 chromatographic column, 10×250mm, 5μm, 2mL / min). The separation conditions for UAT-G1 and UAT-G2 are: 80% methanol - 0.1% formic acid aqueous solution, YMC C 18 chromatographic column, 10×250mm, 5μm, 2mL / min). The separation conditions for UAT-H1 and UAT-H2 are: 82% methanol - 0.1% formic acid aqueous solution, YMC C 18 chromatographic column, 10×250mm, 5μm, 2mL / min). The separation conditions for UAT-I are: 85% acetonitrile - 0.1% formic acid aqueous solution, YMC C 18 chromatographic column, 10×250mm, 5μm, 2mL / min). The separation conditions for UAT-I are: 70% acetonitrile - 0.1% formic acid aqueous solution, cosmosil C 18 chromatographic column, 10×250mm, 5μm, 2mL / min).

[0020] 2) If prepared by chemical synthesis, the preparation process is as follows:

[0021]

[0022] The target compounds UAT-F1 and UAT-F2 can be obtained in the following way: by condensing a fifteen-membered ring tetra-lactone containing two alkyl groups and one benzyl side chain with 2-fluorobenzoic acid;

[0023] The target compounds UAT-G1 and UAT-G2 can be obtained in the following way: by condensing a fifteen-membered ring tetra-lactone containing two alkyl groups and one benzyl side chain with 3-fluorobenzoic acid;

[0024] The target compounds UAT-H1 and UAT-H2 can be obtained in the following manner: by condensing a fifteen-membered cyclic tetra-lactone containing two alkyl groups and one benzyl side chain with benzoic acid;

[0025] The target compound UAT-I can be obtained in the following manner: First, condense a fifteen-membered cyclic tetra-lactone containing two alkyl groups and one benzyl side chain with benzyl-protected 2-hydroxybenzoic acid, and then remove the benzyl protection by hydrogenation to obtain the target compound UAT-I;

[0026] The target compound UAT-J can be obtained in the following manner: First, condense a fifteen-membered cyclic tetra-lactone containing two alkyl groups and one benzyl side chain with benzyl-protected 2-hydroxybenzoic acid, and further reduce it by hydroboration to obtain the target compounds UAT-C and UAT-J.

[0027] Preferably, the fifteen-membered cyclic tetra-lactone can be prepared by the following route,

[0028]

[0029] For the specific preparation and separation processes of each intermediate in the above preparation route, reference can be made to the preparation and purification processes of compounds with the same or similar structures in existing literature (including but not limited to the literature mentioned in the background art), which will not be elaborated in the present invention.

[0030] The third object of the present invention is to disclose the application of the above new antimycin derivatives UAT-F1 to UAT-J in the preparation of anti-tumor drugs.

[0031] Furthermore, the anti-tumor drug can be used for preventing and / or treating one or more of lung cancer and colon cancer.

[0032] Anti-tumor cell tests found that the compounds UAT-F1 to UAT-J of the present invention all have anti-tumor cell activity, and this activity also has a certain selectivity. That is, the half-maximal inhibitory concentration (IC 50 values) against human lung cancer cells and colorectal cancer cells are equivalent to or significantly better than that of the control drug oxaliplatin; and except for UAT-I and UAT-J which have inhibitory activity against human non-cancerous small intestinal cells NCM460 (IC 50 values are 6.61 and 4.75 μM respectively), UAT-F1 to H2 generally have weak inhibitory activity against non-cancer cell lines (IC

[0033] The present invention also provides a pharmaceutical composition, comprising the fifteen-membered cyclic peptide compounds (UAT-F1 to UAT-J) as described above.

[0034] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0035] During the research process of the secondary metabolites of Actinomyces S. conglobatus ATCC 31005 of the present invention, a series of novel fifteen-membered ring cyclopeptide compounds (UAT-F1 to UAT-J) with similar structures were isolated. Antitumor activity tests found that compounds UAT-F1 to UAT-J had comparable or significantly better inhibitory activity against human lung cancer cells and colorectal cancer cells than the control drug oxaliplatin. And except for UAT-I and UAT-J which had weak inhibitory activity (IC 50 = 6.61, 4.75 μM) against human small intestinal cells NCM460, UAT-F1 to H2 generally had weak inhibitory activity against non-cancer cell lines (IC 50 > 40 μM). The present invention provides a new lead compound for the research and development of new antitumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a 2D NMR (DMSO-d6) correlation diagram of compounds UAT-F1 to J of the present invention.

[0037] Figure 2 It is an HR-ESI-MS result diagram of compounds UAT-F1 to J of the present invention.

[0038] Figures 3a - 3f It is the NMR spectrum of compound UAT-F1 of the present invention;

[0039] Figures 4a - 4f It is the NMR spectrum of compound UAT-F2 of the present invention;

[0040] Figures 5a - 5f It is the NMR spectrum of compound UAT-G1 of the present invention;

[0041] Figures 6a - 6f It is the NMR spectrum of compound UAT-G2 of the present invention;

[0042] Figures 7a - 7f It is the NMR spectrum of compound UAT-H1 of the present invention;

[0043] Figures 8a - 8f It is the NMR spectrum of compound UAT-H2 of the present invention;

[0044] Figures 9a - 9f It is the NMR spectrum of compound UAT-I of the present invention;

[0045] Figures 10a - 10f It is the NMR spectrum of compound UAT-J of the present invention;

[0046] wherein a-f respectively represent 1 H-NMR spectrum, 13 C-NMR spectrum, HSQC spectrum, COSY spectrum, HMBC spectrum, ROESY spectrum.

[0047] Figure 11a It is a LC-MS comparative analysis result graph of the reaction of compounds 1-8 of the present invention with S-MTPA-Cl, the standard 2S-hydroxyisovaleric acid with R-MTPA-Cl and S-MTPA-Cl.

[0048] Figure 11b It is a LC-MS comparative analysis result graph of the reaction of compounds 1-7 of the present invention with S-MTPA, the standard L-isoleucic acid with R-MTPA-Cl and S-MTPA-Cl.

[0049] Figure 11c It is for compound 8 of the present invention and S-MTPA-Cl, the standard

[0050] LC-MS comparative analysis result graph of the reaction of 5-benzyl-4-hydroxy-3,3-dimethyldihydrofuran-2-one with R-MTPA-Cl and S-MTP-Cl.

[0051] Figure 12 It is a LC-MS comparative analysis result graph of the reaction of compounds 1-8 of the present invention, L-threonine and L-allo-threonine with FDLA.

[0052] Figure 13 It is a graph of the CD data of compounds 1-7 of the present invention and reference substances (reference substance 1 is UAT-B1 with an S configuration at C-2; reference substance 2 is UAT-B2 with an R configuration at C-2). Detailed implementation manners

[0053] All features disclosed in this specification, or all steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0054] Any feature disclosed in this specification (including any additional claims, abstract), unless specifically stated, each feature is only an example of a series of equivalent or similar features.

[0055] The Streptomyces S. conglobatus used in the following experiments is preferably the commercially available strain ATCC 31005.

[0056] Example 1: Preparation of Compounds 1-8 (UAT-F1-J) by Fermentation of Streptomyces conglobatus

[0057] Preparation and Isolation of Compounds 1-2 (UAT-F1-F2): SGC medium (1 L of medium contains: 30 g of soybean cake powder, 3 g of calcium carbonate, 800 mL of distilled water, 5 mg of cobalt chloride hexahydrate. 200 mL of 25% glucose solution and 2-3 mL of antifoaming agent are sterilized separately and then added to 1 L of SGC medium). The actinomycete Streptomyces conglobatus ATCC31005 is fermented by liquid shaking culture at 30 °C and 220 r on a shaker for 7 days. On the 3rd day of fermentation, 2-fluoroacetylcysteine benzoate thioester is fed to the strain and then fermentation is continued for a total of 36 L. After fermentation, 0.1% formic acid is added to the fermentation broth, and it is extracted three times with an equal volume of ethyl acetate. The extracts are combined, and the extract is concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate layer extract is suspended in a methanol solution and extracted three times with an equal volume of n-hexane. The lower layer solutions are combined and concentrated under reduced pressure to obtain a methanol extract.

[0058] 44.7 g of the above methanol extract is subjected to normal-phase vacuum column chromatography, eluted with dichloromethane-methanol 50:1, and gradient eluted with ethyl acetate-petroleum ether. By mass spectrometry tracking, the fractions containing the peak with a mass-to-charge ratio of m / z 656.3 are combined. ODS medium-pressure column chromatography is used for separation, eluted with a MeCN / H 2 O gradient (20%-80%, 180 min), and mass spectrometry tracking and localization analysis are used to obtain the fine fractions containing the target compounds 1 and 2; finally, semi-preparative high-performance liquid chromatography is used to separate the fractions (elution system: 78% acetonitrile-0.1% formic acid aqueous solution, YMC C 18 chromatographic column, 10×250 mm, 5 μm, 2 mL / min) to obtain the 15-membered ring cyclopeptide compounds 1-2 (UAT-F1-F2) of the present invention.

[0059] Preparation and isolation of compounds 3 - 4 (UAT - G1 - G2): SGC medium (1 L of medium contains: 30 g of soybean cake powder, 3 g of calcium carbonate, 800 mL of distilled water, 5 mg of cobalt chloride hexahydrate, 200 mL of 25% glucose solution and 2 - 3 mL of antifoaming agent, which are sterilized separately and then added to 1 L of SGC medium). The actinomycete S. conglobatus ATCC31005 was fermented by liquid shaking culture at 30 °C and 220 r on a shaker for 7 days. On the 3rd day of fermentation, 3 - fluoroacetylcysteine benzoic acid thioester was fed to the strain and then fermentation was continued for a total of 24 L. After fermentation, 0.1% formic acid was added to the fermentation broth, and it was extracted three times with an equal volume of ethyl acetate. The extracts were combined and concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate layer extract was suspended in methanol solution and extracted three times with an equal volume of n - hexane. The lower layer solutions were combined and concentrated under reduced pressure to obtain a methanol extract.

[0060] 43.5 g of the above - mentioned methanol extract was subjected to normal - phase vacuum column chromatography, eluted with dichloromethane - methanol 50:1, and gradient - eluted with ethyl acetate - petroleum ether. By using mass spectrometry for positioning and tracking, the fractions containing the peak with a mass - to - charge ratio of m / z 656.3 were combined. ODS medium - pressure column chromatography was used for separation, eluted with a MeCN / H 2 O gradient (20% - 80%, 180 min), and mass spectrometry was used for tracking and positioning analysis to obtain fine fractions containing large - molecular - weight linear peptide compounds. Finally, semi - preparative high - performance liquid chromatography was used to separate the fractions (elution system: 80% methanol - 0.1% formic acid aqueous solution, YMC C 18 chromatographic column, 10×250 mm, 5 μm, 2 mL / min) to obtain the 15 - membered cyclic peptide compounds 3 - 4 (UAT - G1 - G2) of the present invention.

[0061] Preparation and isolation of compounds 5 - 6 (UAT - H1 - H2): SGC medium (1 L of medium contains: 30 g of soybean cake powder, 3 g of calcium carbonate, 800 mL of distilled water, 5 mg of cobalt chloride hexahydrate, 200 mL of 25% glucose solution and 2 - 3 mL of antifoaming agent, which are sterilized separately and then added to 1 L of SGC medium). The actinomycete S. conglobatus ATCC31005 was fermented by liquid shaking culture at 30 °C and 220 r on a shaker for 7 days. On the 3rd day of fermentation, acetylcysteine benzoic acid thioester was fed to the strain and then fermentation was continued for a total of 12 L. After fermentation, 0.1% formic acid was added to the fermentation broth, and it was extracted three times with an equal volume of ethyl acetate. The extracts were combined and concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate layer extract was suspended in methanol solution and extracted three times with an equal volume of n - hexane. The lower layer solutions were combined and concentrated under reduced pressure to obtain a methanol extract.

[0062] 17.2 g of the above-mentioned methanol extract was subjected to normal-phase vacuum column chromatography, with gradient elution using dichloromethane-methanol and ethyl acetate-petroleum ether, and mass spectrometry was used for positioning and tracking. The fractions containing the peak with a mass-to-charge ratio of m / z 638.3 were combined. ODS medium-pressure column chromatography was used for separation, and elution was carried out with a MeCN / H 2 O gradient (20%-80%, 180 min). Mass spectrometry was used for tracking and positioning analysis to obtain fine fractions containing large-molecular-weight linear peptide compounds. Finally, semi-preparative high-performance liquid chromatography was used to separate the fractions (elution system: 82% methanol-0.1% formic acid aqueous solution, YMC C 18 chromatographic column, 10×250 mm, 5 μm, 2 mL / min), to obtain 5-6 cyclic pentadecapeptide compounds (UAT-H1-H2) of the present invention.

[0063] Preparation and separation of compounds 7-8 (UAT-I-J): SGC medium (1 L of medium contains: 30 g of soybean cake powder, 3 g of calcium carbonate, 800 mL of distilled water, 5 mg of cobalt chloride hexahydrate. 200 mL of 25% glucose solution and 2-3 mL of antifoaming agent were sterilized separately and then added to 1 L of SGC medium). The actinomycete S. conglobatus ATCC31005 was fermented by liquid shaking culture at 30 °C and 220 r on a shaker for 7 days. On the 3rd day of fermentation, acetylcysteine salicylate thioester was fed to the strain and then fermentation was continued for a total of 12 L. After fermentation, 0.1% formic acid was added to the fermentation broth, and extraction was carried out three times with an equal volume of ethyl acetate. The extracts were combined, and the extract was concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate layer extract was suspended in a methanol solution, and extraction was carried out 3 times with an equal volume of n-hexane. The lower-layer solutions were combined and concentrated under reduced pressure to obtain a methanol extract.

[0064] 36.1 g of the above-mentioned methanol extract was subjected to normal-phase vacuum column chromatography, with gradient elution using dichloromethane-methanol and ethyl acetate-petroleum ether, and mass spectrometry was used for positioning and tracking. The fractions containing the peaks with mass-to-charge ratios of m / z 654.3 and m / z 642.3 were combined. ODS medium-pressure column chromatography was used for separation, and elution was carried out with a MeCN / H 2 O gradient (20%-80%, 180 min). Mass spectrometry was used for tracking and positioning analysis to obtain fine fractions containing large-molecular-weight linear peptide compounds. Finally, semi-preparative high-performance liquid chromatography was used to separate the fractions (elution system for UAT-I: 85% acetonitrile-0.1% formic acid aqueous solution, YMC C 18 chromatographic column, 10×250 mm, 5 μm, 2 mL / min; elution system for UAT-J: 70% acetonitrile-0.1% formic acid aqueous solution, CosmosilC 18Chromatographic column, 10×250 mm, 5 μm, 2 mL / min), to obtain the 15-membered cyclic peptide compounds 7-8 (UAT-I-J) of the present invention.

[0065] Example 2: Obtaining compounds UAT-F1-J by chemical synthesis

[0066] Compounds UAT-F1-H2 can be obtained through multiple chemical synthesis steps, and the synthesis process is as follows:

[0067]

[0068] Compounds UAT-I-J can be obtained through multiple chemical synthesis steps, and the synthesis process is as follows:

[0069]

[0070] The physical and chemical properties and nuclear magnetic resonance data of the compounds UAT-F1-J of the present invention are as follows:

[0071] Compound 1, UAT-F1: Molecular formula C 35 H 43 NO 10 F, light yellow amorphous powder; [α] 20 D +7.60912 (c 0.10, MeOH).

[0072] Compound 2, UAT-F2: Molecular formula C 35 H 43 NO 10 F, light yellow amorphous powder; [α] 20 D +10.0902 (c 0.10, MeOH).

[0073] Compound 3, UAT-G1: Molecular formula C 35 H 43 NO 10 F, light yellow amorphous powder; [α] 20 D +9.65397 (c 0.10, MeOH).

[0074] Compound 4, UAT-G2: Molecular formula C 35 H 43 NO 10 F, light yellow amorphous powder; [α] 20 D +3.05422 (c 0.10, MeOH).

[0075] Compound 5, UAT-H1: Molecular formula C 35 H44 NO 10 ,light yellow amorphous powder; [α] 20 D +31.2834 (c 0.10, MeOH).

[0076] Compound 6, UAT-H2: Molecular formula C 35 H 44 NO 10 ,light yellow amorphous powder; [α] 20 D +79.5253 (c 0.10, MeOH).

[0077] Compound 7, UAT-I: Molecular formula C 35 H 44 NO 11 ,light yellow amorphous powder; [α] 20 D +6.21296 (c 0.10, MeOH).

[0078] Compound 8, UAT-J: Molecular formula C 34 H 42 NO 11 ,light yellow amorphous powder; [α] 20 D +31.8150 (c 0.10, MeOH).

[0079] The planar structures of the compounds UAT-F1-J of the present invention obtained in Example 1 or Example 2 can be determined by 1D- and 2D-NMR data analysis.

[0080] The stereoconfigurations of the compounds UAT-F1-J of the present invention obtained in Example 1 or Example 2 can be determined by comparing the results of the Marfey method, Mosher method and ECD spectra.

[0081] Tables 1-8 are the NMR analyses of the compounds UAT-F1-J

[0082] Figure 1 Show the 2D NMR (DMSO-d6) correlations of the compounds UAT-F1-J.

[0083] Figure 2 Show the HR-ESI-MS of the compounds UAT-F1-J.

[0084] Figures 3a~3f 、 Figures 4a~4f 、 Figures 5a~5f 、 Figures 6a~6f 、 Figures 7a~7f 、 Figures 8a~8f 、 Figures 9a~9f 、Figures 10a~10f Respectively for the 1 1H-NMR, 13 13C-NMR, HSQC, 1 1H- 1 1H COSY, HMBC, ROESY spectra of compounds UAT-F1 to J.

[0085] Figures 11a~11c LC-MS analysis diagrams of the Mosher reactions of compounds 1 to 8 and the reference standard respectively

[0086] Figure 12 LC-MS analysis diagrams of the Marfey reactions of compounds 1 - 8, threonine, and allothreonine

[0087] Figure 13 ECD data of compounds 1 - 7 and compounds UAT-B1, UAT-B2 with known chiral configurations.

[0088] Table 1: NMR data of UAT-F1

[0089]

[0090]

[0091] Table 2: NMR data of UAT-F2

[0092]

[0093]

[0094] Table 3: NMR data of UAT-G1

[0095]

[0096]

[0097] Table 4: NMR data of UAT-G2

[0098]

[0099]

[0100] Table 5: NMR data of UAT-H1

[0101]

[0102]

[0103] Table 6: NMR data of UAT-H2

[0104]

[0105]

[0106] Table 7: NMR data of UAT-I

[0107]

[0108]

[0109] Table 8: NMR data of UAT-J

[0110]

[0111] Example 3: In vitro anti-tumor activity experiment of the 15-membered cyclic peptide compounds UAT-B1 to UAT-J of the present invention

[0112] The CCK8 assay was used to detect cell proliferation. The samples were dissolved in DMSO to form a 10 mM stock solution and stored at low temperature. The concentration of DMSO in the final system was controlled within the range that does not affect the detection activity, and it was serially diluted to a working concentration of 3 nM - 20 μM. Take the above cancer cells in the logarithmic growth phase and use L-15 (SW620) or RPMI-1640 (DLD1, HT-29, NCIH460, A549, H1299, NCM460) culture medium containing 10% calf serum to prepare a single-cell suspension of 1×10 6 cells / mL. Add this suspension to a 96-well plate, 100 μL per well. Incubate in a 5% CO 2 2, 37 °C incubator for 24 h, then add each concentration of the test drug (the 15-membered cyclic peptide compounds UAT-B1 - UAT-J of the present invention and the positive control drug oxaliplatin) to make their final concentrations 0.3 nM - 20 μM respectively. Each sample was set with 3 replicates, and the negative control was an equal volume of culture medium and the corresponding DMSO concentration as the solvent control to eliminate the influence of DMSO on cell growth. The positive control drug was oxaliplatin. Incubate in a 37 °C, 5% CO 2 2 incubator for 72 h, then add 10 μL of CCK8 solution to each well. Incubate (37 °C, 5% CO 2 ) for 40 - 60 min, and then measure the absorbance value (O.D.) at 450 nm with an enzyme-linked immunosorbent assay reader. Calculate the inhibition rate from the measured O.D. value, and use Graphpad Prism 8 software to fit the IC 50 value of the compound.

[0113] Experimental cell lines: The tumor cells used included: human colorectal cancer cells with K-RAS target mutations (SW620, DLD1), colorectal cancer cells without K-RAS target mutations (HT-29), human non-small cell lung cancer cells with K-RAS mutations (AA549, NCIH460), human non-small cell lung cancer without K-RAS mutations (H1299), and normal human small intestine cells (NCM460) as experimental cell lines.

[0114] The test results are shown in Table 9. The results show that compounds UATF1-UAT-J all have selective anti-tumor activities. Compared with human colorectal cancer cells without K-RAS target mutations, compounds UAT-F1-UAT-J all show stronger inhibitory activities against human colorectal cancer cells with K-RAS target mutations. However, for lung cancer cells, compounds UAT-F1-UAT-J do not show this property. In addition, compounds UAT-F1-UAT-H2 have good inhibitory effects on both human colorectal cancer and lung cancer cells (0.04 μM - 11.2 μM) and have low toxicity to normal cells.

[0115] Table 9 Inhibitory rates and half-maximal inhibitory concentration IC 50 values (μM) (n = 3) of Compounds 1-8 against different tumor cells and normal cells

[0116] Compound DLD1 SW620 HCT116 HCT116 / 5 - fu HT - 29 NCIH460 A549 H1299 NCM460 UAT - F1 2.59 0.56 3.67 ＞20.0 9.40 ＞20.0 >20.0 >20.0 >20.0 UAT - F2 0.60 0.14 0.82 ＞20.0 >20.0 1.30 4.30 3.31 >20.0 UAT - G1 3.51 1.12 9.09 ＞20.0 >20.0 >20.0 >20.0 >20.0 >20.0 UAT - G2 2.66 0.33 2.53 ＞20.0 >20.0 8.80 11.20 >20.0 >20.0 UAT - H1 1.90 0.36 4.24 ＞20.0 >20.0 >20.0 >20.0 >20.0 >20.0 UAT - H2 0.07 0.04 0.11 8.63 >20.0 1.56 0.33 4.50 >20.0 UAT - I 2.85 0.33 1.35 ＞20.0 >20.0 4.40 9.70 >20.0 6.61 UAT - J 2.86 1.45 2.57 ＞20.0 >20.0 >20.0 >20.0 3.49 4.75 oxaliplatin 4.80 2.50 4.66 ＞20.0 7.85 6.70 1.40 0.65 1.80

[0117] As can be seen from Table 9, Compounds 1-8 in the present invention have significant inhibitory activities against some or all of the tumor cells of DLD1, SW620, NCIH460, HCT116, HCT116 / 5-fu, HT-29, A549, and H1299. The efficacy of some compounds against the tumor cells DLD1, SW620, and A549 is better than that of the positive drug oxaliplatin, and the toxicity to normal small intestine cells is much lower than that of the positive drug. The compounds of the present invention are potential anti-tumor targeted drugs and provide new lead compounds for the development of new anti-tumor drugs.

[0118] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A 15-membered cyclic depsipeptide compound, characterized in that: It is one of the following 2 compounds: 。 2. The method for preparing a 15-membered cyclic depsipeptide compound according to claim 1, wherein: The compound as claimed in claim 1 is obtained by fermentation of Streptomyces S. conglobatus or by chemical synthesis.

3. The method according to claim 2, characterized in that The method comprises the following steps: preparing a precursor compound a; inoculating Streptomyces conglobatus on a solid culture medium, culturing at 27 to 32° C. for 4 to 6 days, and collecting spores; Take the spores and inoculate them into the first-level liquid culture medium, and culture them in a shake flask at 27-32°C for 3-4 days to obtain the seed liquid; Take the seed solution and inoculate it into the secondary liquid culture medium, and culture it in a shake flask at 27-32°C for 3-4 days to obtain the secondary seed solution; The secondary seed solution was inoculated into the fermentation medium, and the precursor compound a was fed at a concentration of 1.0 mmol / L after 2 days of shaking flask culture at 27-32°C, and the fermentation broth was collected after 3-4 days of shaking flask culture. Extracting and separating the 15-membered cyclic depsipeptide compound according to claim 1 from the fermentation broth; The structural formula of the precursor compound a is as follows: 。 4. The method according to claim 3, characterized in that The solid culture medium contains 1.5% to 2.5% soybean powder, 1.5% to 2.5% D-mannitol, and 1.5% to 2.5% agar; the primary liquid culture medium contains 2% to 4% tryptic soy broth, 9% to 11% sucrose, and 0.4% to 0.6% yeast extract; The secondary liquid culture medium contains 2% to 4% soybean powder, 4% to 6% glucose, 0.4% to 0.6% CaCO3, 4 to 6 mg / L CoCl2·6H2O, and 0.15% to 0.25% (v / v) defoamer; the fermentation culture medium contains 2% to 4% soybean powder, 4% to 6% glucose, 0.4% to 0.6% CaCO3, and 0.15% to 0.25% (v / v) defoamer.

5. The method according to claim 3, characterized in that The precursor compound a is prepared by the following route: 。 6. The method according to claim 3, characterized in that The step of extracting and separating the 15-membered cyclic depsipeptide compound as claimed in claim 1 from the fermentation broth is as follows: adding 0.1% to 0.2% (v / v) formic acid to the fermentation broth, then extracting with ethyl acetate, and concentrating the ethyl acetate extract under reduced pressure at 30 to 45° C. to obtain an extract: Redissolve the extract with methanol, filter out the residue, extract with n-hexane, and remove the n-hexane layer; After the methanol solution is concentrated, it is separated by column chromatography to obtain the 15-membered cyclic depsipeptide compound as claimed in claim 1.

7. The method according to claim 6, characterized in that Column chromatography separation includes: separation via a normal silica gel column, the elution solvent is dichloromethane / methanol, and the elution solvent ratio is 50 / 1 (v / v); Separating by a normal silica gel column, the elution solvent is petroleum ether / ethyl acetate, and the elution solvent ratio gradient is 5 / 1 to 0 / 1 (v / v), specifically, first eluting with petroleum ether-ethyl acetate 5 / 1 (v / v), then gradually increasing the solvent polarity and gradually increasing the ethyl acetate content; The target fraction was separated by ODS column, and the elution solvent gradient was 30%-100% acetonitrile; The target fraction was taken for final separation using preparative HPLC; The separation conditions of UAT-F1 and UAT-F2 are: 78% acetonitrile-0.1% formic acid aqueous solution, YMC C 18 Chromatographic column, 10 × 250 mm, 5 μm, 2 mL / min.

8. The method for preparing the 15-membered cyclic depsipeptide compound according to claim 1, wherein: The preparation process is as follows: ； The target compounds UAT-F1 and UAT-F2 were obtained by condensing a 15-membered ring quadruple lactone containing two alkyl groups and one benzyl side chain with 2-fluorobenzoic acid; The structural formula of the 15-membered ring quadruple lactone containing two alkyl groups and one benzyl side chain is as follows: 。 9. The use of the 15-membered cyclic depsipeptide compound as claimed in claim 1 in the preparation of anti-tumor drugs, characterized in that: The tumor is selected from one or more of lung cancer and colon cancer.

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