Preparation method of fifteen-membered ring quadruple lactone derivative and application of fifteen-membered ring quadruple lactone derivative in treatment of lung cancer and colon cancer

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a series of 15-membered ring quadruple lactone derivatives with neoantimycin mother nucleus structures, a preparation method of the 15-membered ring quadruple lactone derivatives and application of the 15-membered ring quadruple lactone derivatives in antitumor drugs, wherein the 15-membered ring quadruple lactone derivatives are generated by feeding chemical precursors in the fermentation process of streptomyces S.conglobatusATCC 31005. The compound is a fifteen-membered ring quadruple lactone derivative, the C-2 position of the derivative is connected with a carbonyl group or a hydroxyl group, the C-4 position and the C-9 position are substituted by aliphatic chains with different lengths, and the C-6 position of the derivative is connected with a benzoic acid group containing F atoms or hydroxyl groups. The compounds have obvious tumor cell proliferation inhibition activity on colorectal cancer cells and lung cancer cells, the inhibition activity is equivalent to that of a control drug oxaliplatin, and the compounds have weak toxicity on non-cancer cell strains. A novel lead compound is provided for research and development of novel antitumor drugs, and the compound is expected to be developed into a tumor inhibitor.

Description

[0001] This application is a divisional application of patent application "202410092265.2" a 15-membered cyclic depsipeptide compound and its preparation method and application, and the application date is 2024.01.23. Technical Field

[0002] The present invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a preparation method of a 15-membered ring quadruple lactone derivative and its application in the treatment of lung cancer and colon cancer. Background Art

[0003] Streptomyces is a global resource for novel lead compounds in drug development, producing novel metabolites with diverse scaffolds and complex structures. Neoantimycin (NAT), a depsipeptide natural product produced by Streptomyces, features a 15-membered lactone nucleus with four ester (peptide) groups attached to a 3-N-formamidosalicylic acid group, two alkyl groups, and a benzyl side chain. The structural differences of the reported new antimycin analogs mainly focus on the hydroxylation or ketolation modification of the C1 position of the molecular mother ring, the length of the alkyl side chain at the C4 / C9 position of the ester (peptide) group, and whether the amino group on the salicylic acid acyl group is N-formyl modified or replaced 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.).New antimycin compounds generally have important pharmaceutical activities, such as SW-163A and SW-163B, which 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.), prunustatin A, JBIR-04 and JBIR-05 can negatively regulate the expression of tumor cell target protein GRP78 (glucose-regulated protein) (Izumikawa, M.; et al. Novel GRP78 Molecular Chaperone Expression Down-Regulators JBIR-04and-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 been reported that new antimycin compounds NAT-A, NAT-F, NAT-G and NAT-H have significant inhibitory effects on the cell membrane localization of the key target of carcinogenesis K-Ras and the multidrug resistance of the colon cancer cell line SW620 (Salim, AA; 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, the above compounds all have a major drug-forming defect, with poor selectivity for inhibitory activity on tumor cells and also have significant toxic side effects on normal cells. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a method for preparing a 15-membered ring tetralactone derivative and its application in the treatment of lung cancer and colon cancer.

[0005] The first objective is to disclose eight novel antimycin derivatives, Unantimycin F1-J (UAT-F1-J), produced by fermentation of Streptomyces conglobatus ATCC 31005. Unantimycins generally contain a 3-N-formamidosalicylic acid group at the C-6 position in their molecular structures. However, the 15-membered ring quadruplex lactone derivatives described herein have a non-3-N-formamidosalicylic acid group substituted at the C-6 position. Their structural formulas are as follows:

[0006]

[0007] The second object of the present invention is to provide a method for preparing the novel 15-membered ring tetralactone derivatives. The novel 15-membered ring tetralactone derivatives of the present invention can be obtained by fermentation of Streptomyces conglobatus or by chemical synthesis.

[0008] 1) If prepared by fermentation of Streptomyces conglobatus, preferably, the Streptomyces conglobatus used is preferably the commercially available strain ATCC 31005.

[0009] Preferably, the fermentation preparation comprises the following steps:

[0010] a) Preparation of feeding precursor compounds a-d: The precursor compounds a-d can be prepared by the following routes:

[0011]

[0012] The specific preparation and separation processes of the intermediates in the above preparation routes can refer 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 repeated in the present invention.

[0013] b) strain fermentation and precursor feeding: Streptomyces conglobatus was inoculated on a solid culture medium and cultured at 27-32° C. for 4-6 days, after which spores were collected; the spores were inoculated into a primary liquid culture medium, and cultured in a shake flask at 27-32° C. for 3-4 days to obtain a seed solution; the seed solution was inoculated into a secondary liquid culture medium, and cultured in a shake flask at 27-32° C. for 3-4 days to obtain a secondary seed solution; the secondary seed solution was inoculated into a fermentation medium, and cultured in a shake flask at 27-32° C. for 2 days, after which compounds a-d were fed at a concentration of 1.0 mmol / L, and the shake flask culture was continued for 3-4 days, after which the fermentation broth was collected; and the novel 15-membered ring quadruple lactone derivatives 1-8 of the present invention were extracted and separated from the fermentation broth.

[0014] Preferably, the solid culture medium contains 1.5-2.5% soy flour, 1.5-2.5% D-mannitol, and 1.5-2.5% agar. The primary liquid culture medium contains 2-4% tryptic soy broth, 9-11% sucrose, and 0.4-0.6% yeast extract. The secondary liquid culture medium contains 2-4% soy flour, 4-6% glucose, 0.4-0.6% CaCO3, 4-6 mg / L CoCl2·6H2O, and 0.15-0.25% (v / v) defoamer. The fermentation medium contains 2-4% soy flour, 4-6% glucose, 0.4-0.6% CaCO3, and 0.15-0.25% (v / v) defoamer.

[0015] c) Compound extraction, separation and purification

[0016] The chromatographic separation includes two vacuum silica gel column chromatography separations, one reverse phase medium pressure ODS column chromatography separation and high performance liquid chromatography separation.

[0017] Preferably, the step of extracting a 15-membered ring tetralactone derivative from the fermentation broth includes: 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, removing the n-hexane layer; after concentrating the methanol solution, separating by column chromatography to obtain compound UAT-F1~J.

[0018] Furthermore, the column chromatography separation includes: separation by a forward silica gel column, the elution solvent is dichloromethane / methanol, and the elution solvent ratio gradient is 50 / 1 (v / v); separation by a forward 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 referring to first eluting with petroleum ether-ethyl acetate (5 / 1, v / v), then gradually increasing the solvent polarity, and gradually increasing the ethyl acetate content; taking the target fraction and separating it by an ODS column, and the elution solvent gradient is 30%-100% acetonitrile; specifically referring to first eluting with an aqueous solution containing 30% acetonitrile, then gradually reducing the solvent polarity, and the acetonitrile content gradient is increased to 100%.

[0019] The target fraction was taken for final separation using preparative HPLC;

[0020] 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). Separation conditions for UAT-G1 and UAT-G2 were: 80% methanol-0.1% formic acid aqueous solution, YMC C 18Chromatographic column, 10×250 mm, 5 μm, 2 mL / min). Separation conditions for UAT-H1 and UAT-H2 were: 82% methanol-0.1% formic acid aqueous solution, YMC C 18 Chromatographic column, 10×250 mm, 5 μm, 2 mL / min). UAT-I separation conditions were: 85% acetonitrile-0.1% formic acid aqueous solution, YMC C 18 Chromatographic column, 10×250 mm, 5 μm, 2 mL / min). UAT-I separation conditions: 70% acetonitrile-0.1% formic acid aqueous solution, cosmosil C 18 Chromatographic column, 10×250 mm, 5 μm, 2 mL / min).

[0021] 2) If prepared by chemical synthesis, the preparation process is as follows:

[0022]

[0023] The target compounds UAT-F1 and UAT-F2 can be obtained by condensing a 15-membered ring quadruple lactone containing two alkyl groups and one benzyl side chain with 2-fluorobenzoic acid;

[0024] The target compounds UAT-G1 and UAT-G2 can be obtained by condensing a 15-membered ring quadruple lactone containing two alkyl groups and one benzyl side chain with 3-fluorobenzoic acid;

[0025] The target compounds UAT-H1 and UAT-H2 can be obtained by condensing a 15-membered ring quadruple lactone containing two alkyl groups and one benzyl side chain with benzoic acid;

[0026] The target compound UAT-I can be obtained by the following method: first, condensing a 15-membered ring quadruple lactone containing two alkyl groups and one benzyl side chain with benzyl-protected 2-hydroxybenzoic acid, followed by hydrogenation and debenzylation to obtain the target compound UAT-I;

[0027] The target compound UAT-J can be obtained by the following method: first, a 15-membered ring quadruple lactone containing two alkyl groups and one benzyl side chain is condensed with benzyl-protected 2-hydroxybenzoic acid, and further hydroboration reduction is performed to obtain the target compounds UAT-C and UAT-J.

[0028] Preferably, the 15-membered ring quadruple lactone can be prepared by the following route:

[0029]

[0030] The specific preparation and separation processes of the intermediates in the above preparation routes can refer 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 repeated in the present invention.

[0031] The third object of the present invention is to disclose the use of the above-mentioned new antimycin derivatives UAT-F1 to UAT-J in the preparation of antitumor drugs.

[0032] Furthermore, the anti-tumor drug can be used to prevent and / or treat one or more of lung cancer and colon cancer.

[0033] Anti-tumor cell tests revealed that the compounds UAT-F1 to UAT-J all exhibited anti-tumor cell activity, and this activity was also selective. Their half-maximum inhibitory strength against human lung cancer and colorectal cancer cells was comparable to or significantly superior to that of the control drug oxaliplatin. In addition, except for UAT-I, UAT-J also exhibited inhibitory activity against human non-cancerous small intestinal cells NCM460 (IC 50 UAT-F1~H2 generally had weak inhibitory activity against non-cancer cell lines (IC 50 >40μM). Furthermore, compounds UAT-F1 to UAT-J exhibited selective inhibitory effects against colorectal cancer cells harboring K-RAS target mutations. Therefore, UAT-F1 to UAT-J have the potential to be developed as targeted drugs for colorectal and lung cancer.

[0034] The present invention also provides a pharmaceutical composition comprising the 15-membered ring quadruple lactone derivatives (UAT-F1 to UAT-J) described above.

[0035] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0036] During the research on secondary metabolites of the actinomycete S. conglobatus ATCC 31005, the present invention isolated and obtained a series of novel 15-membered ring tetralactone derivatives (UAT-F1 to UAT-J) with similar structures. Antitumor activity tests showed that the inhibitory activity of compounds UAT-F1 to UAT-J on human lung cancer cells and colorectal cancer cells was comparable to or significantly superior to that of the control drug oxaliplatin. In addition, with the exception of UAT-I, UAT-J had weaker inhibitory activity on human small intestinal cells NCM460 (IC 50 =6.61,4.75μM), UAT-F1~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 anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 2D NMR (DMSO-d6) correlation diagram of compounds UAT-F1~J of the present invention.

[0038] Figure 2 This is the HR-ESI-MS result of the compounds UAT-F1~J of the present invention.

[0039] Figures 3a-3f is the NMR spectrum of the compound UAT-F1 of the present invention;

[0040] Figures 4a-4f is the NMR spectrum of the compound UAT-F2 of the present invention;

[0041] Figures 5a-5f is the NMR spectrum of the compound UAT-G1 of the present invention;

[0042] Figures 6a-6f is the nuclear magnetic spectrum of the compound UAT-G2 of the present invention;

[0043] Figures 7a-7f is the NMR spectrum of the compound UAT-H1 of the present invention;

[0044] Figures 8a-8f is the NMR spectrum of the compound UAT-H2 of the present invention;

[0045] Figures 9a-9f is the NMR spectrum of compound UAT-1 of the present invention;

[0046] Figures 10a-10f is the NMR spectrum of compound UAT-J of the present invention;

[0047] Where af represents 1 H-NMR spectrum, 13 C-NMR spectrum, HSQC spectrum, COSY spectrum, HMBC spectrum, REOSY spectrum.

[0048] Figure 11a This is a comparative analysis result of LC-MS of the reaction of compound 1-8 of the present invention with S-MTPA-Cl, and the standard 2S-hydroxyisovaleric acid with R-MTPA-Cl and S-MTPA-Cl.

[0049] Figure 11b This is a comparative analysis result of LC-MS of the reaction of compound 1-7 of the present invention with S-MTPA, and the standard L-isoleucic acid with R-MTPA-Cl and S-MTPA-Cl.

[0050] Figure 11c The compound 8 of the present invention and S-MTPA-Cl, standard

[0051] Comparative analysis results of the LC-MS reaction of 5-benzyl-4-hydroxy-3,3dimethyldihydrofuran-2-one with R-MTPA-Cl and S-MTP-Cl.

[0052] Figure 12 This is a comparative analysis result of LC-MS of the reaction of compounds 1-8, L-threonine and L-allothreonine with FDLA.

[0053] Figure 13 The graph shows the CD data of compounds 1-7 of the present invention and the reference substance (standard substance 1 is UAT-B1, whose C-2 position is S configuration; standard substance 2 is UAT-B2, whose C-2 position is R configuration). DETAILED DESCRIPTION

[0054] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0055] Any feature disclosed in this specification (including any accompanying claims and abstract), unless otherwise stated, is merely one example of a series of equivalent or similar features.

[0056] The Streptomyces conglobatus used in the following experiments is preferably the commercially available strain ATCC 31005.

[0057] Example 1: Preparation of Compounds 1-8 (UAT-F1-J) by Fermentation with Streptomyces conglobatus

[0058] Preparation and isolation of compounds 1-2 (UAT-F1-F2): Actinomycete S. conglobatus ATCC31005 was fermented in a liquid shaker in SGC medium (1 L of medium contained: 30 g soybean meal, 3 g calcium carbonate, 800 mL distilled water, 5 mg cobalt chloride hexahydrate. 200 mL of 25% glucose solution and 2-3 mL of defoamer were sterilized separately and added to 1 L of SGC medium). Fermentation was carried out at 30°C and 220 rpm for 7 days. On the third day of fermentation, the strain was fed 2-fluoroacetylcysteamine benzoic acid thioester and continued to ferment for a total of 36 L. After fermentation, the fermentation broth was supplemented with 0.1% formic acid and extracted three times with equal volumes of ethyl acetate. The extracts were combined and concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate extract was suspended in methanol and extracted three times with equal volumes of n-hexane. The lower layers were combined and concentrated under reduced pressure to obtain a methanol extract.

[0059] 44.7 g of the methanol extract was subjected to normal phase vacuum column chromatography, eluted with dichloromethane-methanol 50:1, ethyl acetate-petroleum ether gradient elution, and mass spectrometry tracking was used to combine the fractions containing the mass-to-charge ratio m / z 656.3 peak. ODS medium-pressure column chromatography was used for separation, eluted with MeCN / H2O gradient (20%-80%, 180 min), and mass spectrometry tracking and positioning analysis was used to obtain fine fractions containing target compounds 1 and 2; finally, the fractions were separated by semi-preparative high performance liquid chromatography (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 quadruple lactone derivatives 1 to 2 (UAT-F1 to F2) of the present invention.

[0060] Preparation and isolation of compounds 3-4 (UAT-G1-G2): Actinomycete S. conglobatus ATCC31005 was fermented in a liquid shaker in SGC medium (1 L of medium contained: 30 g soybean meal, 3 g calcium carbonate, 800 mL distilled water, 5 mg cobalt chloride hexahydrate. 200 mL of 25% glucose solution and 2-3 mL of defoamer were sterilized separately and added to 1 L of SGC medium). Fermentation was carried out at 30°C and 220 rpm for 7 days. On the third day of fermentation, the strain was fed 3-fluoroacetylcysteamine benzoic acid thioester and continued to ferment for a total of 24 L. After fermentation, the fermentation broth was supplemented with 0.1% formic acid and extracted three times with equal volumes of ethyl acetate. The extracts were combined and concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate extract was suspended in methanol and extracted three times with equal volumes of n-hexane. The lower layers were combined and concentrated under reduced pressure to obtain a methanol extract.

[0061] 43.5 g of the methanol extract was subjected to normal phase vacuum column chromatography, eluted with dichloromethane-methanol 50:1, ethyl acetate-petroleum ether gradient elution, and mass spectrometry tracking was used to combine the fractions containing the mass-to-charge ratio m / z 656.3 peak. ODS medium-pressure column chromatography was used for separation, eluted with MeCN / H2O gradient (20%-80%, 180 min), and mass spectrometry tracking was used for positioning analysis to obtain a fine fraction containing large molecular weight linear peptide compounds; finally, the fractions were separated by semi-preparative high performance liquid chromatography (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 ring quadruple lactone derivatives 3 to 4 (UAT-G1 to G2) of the present invention.

[0062] Compounds 5-6 (UAT-H1-H2) were prepared and isolated using SGC medium (1 L of medium containing: 30 g soybean meal, 3 g calcium carbonate, 800 mL of distilled water, 5 mg of cobalt chloride hexahydrate. 200 mL of 25% glucose solution and 2-3 mL of defoamer were sterilized separately and added to 1 L of SGC medium). Actinomycete S. conglobatus ATCC31005 was fermented in a liquid shaker at 30°C and 220 rpm for 7 days. On the third day of fermentation, the strain was fed with acetylcysteamine benzoate thioester and continued to ferment for a total of 12 L. After fermentation, the fermentation broth was supplemented with 0.1% formic acid and extracted three times with equal volumes of ethyl acetate. The extracts were combined and concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate extract was suspended in methanol and extracted three times with equal volumes of n-hexane. The lower layers were combined and concentrated under reduced pressure to obtain a methanol extract.

[0063] 17.2 g of the methanol extract was subjected to normal phase vacuum column chromatography, using a dichloromethane-methanol gradient elution and an ethyl acetate-petroleum ether gradient elution, and the fractions containing the mass-to-charge ratio m / z 638.3 peak were combined by mass spectrometry tracking. ODS medium-pressure column chromatography was used for separation, using a MeCN / H2O gradient (20%-80%, 180 min) for elution, and mass spectrometry tracking and positioning analysis were used to obtain a fine fraction containing a large molecular weight linear peptide compound; finally, the fractions were separated by semi-preparative high performance liquid chromatography (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 the 15-membered ring quadruple lactone derivatives 5 to 6 (UAT-H1 to H2) of the present invention.

[0064] Preparation and isolation of compounds 7-8 (UAT-I-J): Actinomycete S. conglobatus ATCC31005 was fermented in a liquid shaker in SGC medium (1 L of medium containing: 30 g soybean meal, 3 g calcium carbonate, 800 mL distilled water, 5 mg cobalt chloride hexahydrate. 200 mL of 25% glucose solution and 2-3 mL of defoamer were sterilized separately and added to 1 L of SGC medium). Fermentation was carried out at 30°C and 220 rpm for 7 days. On the third day of fermentation, the strain was fed with acetylcysteamine salicylate thioester and continued to ferment for a total of 12 L. After fermentation, the fermentation broth was supplemented with 0.1% formic acid and extracted three times with equal volumes of ethyl acetate. The extracts were combined and concentrated under reduced pressure to obtain an ethyl acetate extract. The ethyl acetate extract was suspended in methanol and extracted three times with equal volumes of n-hexane. The lower layers were combined and concentrated under reduced pressure to obtain a methanol extract.

[0065] 36.1 g of the methanol extract was subjected to normal phase vacuum column chromatography using a dichloromethane-methanol gradient elution and an ethyl acetate-petroleum ether gradient elution. The fractions containing the mass-to-charge ratios m / z 654.3 and m / z 642.3 peaks were combined by mass spectrometry tracking. ODS medium-pressure column chromatography was used for separation using a MeCN / H2O gradient (20%-80%, 180 min) and mass spectrometry tracking and positioning analysis to obtain a fine fraction containing high molecular weight linear peptide compounds. Finally, the fractions were separated by semi-preparative high performance liquid chromatography (UAT-I elution system: 85% acetonitrile-0.1% formic acid aqueous solution, YMC C 18 Chromatographic column, 10×250 mm, 5 μm, 2 mL / min; UAT-J elution system: 70% acetonitrile-0.1% formic acid aqueous solution, Cosmosil C 18 chromatographic column, 10×250 mm, 5 μm, 2 mL / min), to obtain the 15-membered ring quadruple lactone derivatives 7 to 8 (UAT-I to J) of the present invention.

[0066] Example 2: Obtaining Compound UAT-F1~J by Chemical Synthesis

[0067] Compounds UAT-F1-H2 can be obtained through multiple chemical synthesis steps, as follows:

[0068]

[0069] Compounds UAT-I to UAT-J can be obtained through multiple chemical synthesis steps, as follows:

[0070]

[0071] The physicochemical properties and nuclear magnetic resonance data of the compounds UAT-F1-J of the present invention are as follows:

[0072] 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).

[0073] 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).

[0074] Compound 3, UAT-G1: Molecular formula C35 H 43 NO 10 F, light yellow amorphous powder; [α] 20 D +9.65397 (c 0.10, MeOH).

[0075] 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).

[0076] Compound 5, UAT-H1: Molecular formula C 35 H 44 NO 10 , light yellow amorphous powder; [α] 20 D +31.2834 (c 0.10, MeOH).

[0077] 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).

[0078] 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).

[0079] 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).

[0080] 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.

[0081] The stereo configuration of the compound UAT-F1-J of the present invention obtained in Example 1 or Example 2 can be determined by comparing the Marfey method, the Mosher method and the results of the ECD spectrum.

[0082] Tables 1 to 8 are the NMR analyses of compounds UAT-F1 to J

[0083] Figure 1 The 2D NMR (DMSO-d6) correlations of compounds UAT-F1~J are shown.

[0084] Figure 2 HR-ESI-MS of compound UAT-F1~J is shown.

[0085] Figures 3a to 3f 、 Figures 4a to 4f 、 Figures 5a to 5f 、 Figures 6a to 6f 、 Figures 7a to 7f 、 Figures 8a to 8f 、 Figures 9a to 9f 、 Figures 10a to 10f are compounds UAT-F1~J respectively. 1 H-NMR, 13 C-NMR, HSQC, 1 H- 1 H COSY, HMBC, and ROESY spectra.

[0086] Figures 11a to 11c LC-MS analysis of Mosher reaction of compounds 1 to 8 and standard products

[0087] Figure 12 LC-MS analysis of the Marfey reaction of compound 1-8, threonine, and allothreonine

[0088] Figure 13 The ECD data of compounds 1-7 and compounds UAT-B1 and UAT-B2 with known chiral configurations are shown.

[0089] Table 1: NMR data of UAT-F1

[0090]

[0091]

[0092] Table 2: NMR data of UAT-F2

[0093]

[0094]

[0095] Table 3: NMR data of UAT-G1

[0096]

[0097]

[0098] Table 4: NMR data of UAT-G2

[0099]

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

[0101]

[0102]

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

[0104]

[0105]

[0106] Table 7: UAT-I NMR data

[0107]

[0108]

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

[0110]

[0111]

[0112] Example 3: In vitro antitumor activity test of the 15-membered ring quadruple lactone derivatives UAT-B1 to UAT-J of the present invention

[0113] The cell proliferation was detected by CCK8 assay. The sample was dissolved in DMSO to a 10 mM stock solution and stored at low temperature. The concentration of DMSO in the final system was controlled within the range that did not affect the detection activity, and the dilution ratio was 3 nM-20 μM. The above cancer cells in the logarithmic growth phase were cultured in L-15 (SW620) or RPMI-1640 (DLD1, HT-29, NCIH460, A549, H1299, NCM460) containing 10% calf serum to prepare a single cell suspension of 1×10 6 / mL, the suspension was added to a 96-well plate, and 100 μL was added to each well. After culturing in a 5% CO2, 37°C incubator for 24 hours, the test drugs (the fifteen-membered cyclic depsipeptide compound UAT-B1-UAT-J of the present invention and the positive control drug oxaliplatin) of various concentrations were added respectively to make their final concentrations 0.3nM-20μM, and 3 replicates were set up for each sample. The negative control was an equal volume of culture medium and the corresponding DMSO concentration as a solvent control to eliminate the effect of DMSO on cell growth. The positive control drug was oxaliplatin. After incubation in a 37°C, 5% CO2 incubator for 72 hours, 10 μL of CCK8 solution was added to each well. After incubation (37°C, 5% CO2) for 40 to 60 minutes, the absorbance value (OD) at 450nm was detected with a microplate reader. The inhibition rate was calculated using the measured OD value, and the IC value of the compound was fitted using GraphpadPrism 8 software. 50 value.

[0114] Experimental cell lines: The tumor cells used include: human colorectal cancer cells with K-RAS target mutations (SW620, DLD1), colorectal cancer cells with non-K-RAS target mutations (HT-29), human non-small cell lung cancer cells with K-RAS mutations (A549, NCIH460), human non-K-RAS mutations (H1299), and normal human small intestinal cells (NCM460) as experimental cell lines.

[0115] The test results are shown in Table 9, which show that compounds UATF1 to UAT-J all have selective anti-tumor activity. Compared with human colorectal cancer cells with non-K-RAS target mutations, compounds UAT-F1 to UAT-J all exhibit stronger inhibitory activity against human colorectal cancer cells with K-RAS target mutations, while compounds UAT-F1 to UAT-J did not exhibit this property for lung cancer cells. In addition, compounds UAT-F1 to UAT-H2 have good inhibitory effects on human colorectal cancer and lung cancer cells (0.04 μM-11.2 μM) and have low toxicity to normal cells.

[0116] Table 9 Inhibitory rates and half effective inhibitory concentrations (IC) of compounds 1-8 on different tumor cells and normal cells 50 Value (μM) (n=3)

[0117] 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

[0118] As shown in Table 9, compounds 1-8 of the present invention exhibit significant inhibitory activity against some or all of the tumor cells of DLD1, SW620, NCIH460, HCT116, HCT116 / 5-fu, HT-29, A549, and H1299. Some compounds exhibited superior efficacy against DLD1, SW620, and A549 tumor cells compared to the active agent oxaliplatin, and exhibited significantly lower toxicity against normal intestinal cells than the active agent oxaliplatin. The compounds of the present invention are potential targeted antitumor drugs and provide novel lead compounds for the development of novel antitumor drugs.

[0119] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A 15-membered ring tetralactone derivative, characterized in that: It is one of the following two compounds:

2. The method for preparing the 15-membered ring tetralactone derivative according to claim 1, wherein: The compound according to claim 1 is obtained by fermentation of Streptomyces conglobatus or by chemical synthesis.

3. The method according to claim 2, wherein The method comprises the following steps: preparing a precursor compound c; inoculating Streptomyces conglobatus on a solid culture medium, culturing at 27-32° C. for 4-6 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℃ for 3-4 days to obtain the seed liquid; Take the seed liquid and inoculate it into the secondary liquid culture medium, and culture it in a shake flask at 27-32℃ for 3-4 days to obtain the secondary seed liquid; The secondary seed solution was inoculated into the fermentation medium, and the precursor compound c was fed at a concentration of 1.0 mmol / L after culturing in a shake flask at 27-32°C for 2 days. The fermentation broth was collected after continuing the shake flask culture for 3-4 days. Extracting and separating the 15-membered ring quadruple lactone derivative according to claim 1 from the fermentation broth; The structural formula of the precursor compound c is as follows:

4. The method according to claim 3, wherein The solid culture medium contains 1.5% to 2.5% soybean flour, 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) defoaming agent; and 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) defoaming agent.

5. The method according to claim 3, wherein The precursor compound c is prepared by the following route:

6. The method according to claim 3, wherein The steps of extracting and separating the 15-membered ring quadruple lactone derivative according to claim 1 from the fermentation broth are 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, and extract with n-hexane to remove the n-hexane layer; The methanol solution is concentrated and then separated by column chromatography to obtain the 15-membered ring quadruple lactone derivative as claimed in claim 1.

7. The method according to claim 6, wherein Column chromatography separation includes: separation on a normal silica gel column, the elution solvent is dichloromethane / methanol, and the elution solvent ratio gradient is 50 / 1 (v / v); Separation was performed on a normal silica gel column, and the elution solvent was petroleum ether / ethyl acetate, and the elution solvent ratio gradient was 5 / 1 to 0 / 1 (v / v), specifically, petroleum ether-ethyl acetate (5 / 1, v / v) was first used for elution, and then the solvent polarity was gradually increased, and the ethyl acetate content was gradually increased; 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-H1 and UAT-H2 are: 82% methanol-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 ring tetralactone derivative according to claim 1, wherein: The preparation process is as follows: The target compounds UAT-H1 and UAT-H2 were obtained by condensing a 15-membered ring quadruple lactone containing two alkyl groups and one benzyl side chain with benzoic acid.

9. Use of the 15-membered ring tetralactone derivative according to 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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