Preparation method and application of neoantimycin derivative containing fluorine element

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a series of fluorine-containing neoantimycin derivatives with a neoantimycin mother nucleus structure, a preparation method of the fluorine-containing neoantimycin derivatives and application of the fluorine-containing neoantimycin derivatives in antitumor drugs, wherein the fluorine-containing neoantimycin derivatives are generated by feeding chemical precursors in the fermentation process of streptomyces S.conglobatusATCC 31005. The compound is a neoantimycin derivative containing a fluorine element, the C-2 site of the compound is connected with a carbonyl group or a hydroxyl group, the C-4 site and the C-9 site are substituted by aliphatic chains with different lengths, and the C-6 site is connected with a benzoic acid group containing an F atom or a hydroxyl group. 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 the patent application "202410092265.2" for a fifteen-membered cyclic peptide compound, its preparation method and application. The application date is January 23, 2024. Technical Field

[0002] The present invention belongs to the technical field of medicinal chemistry, and specifically relates to a preparation method and application of a novel antimycin derivative containing fluorine elements. Background Art

[0003] Streptomyces is an important resource treasure house for new leading compounds in the world's drug research and development, capable of producing novel metabolites with diverse scaffolds and complex structures. Neoantimycin (NAT) is a depsipeptide natural product produced by Streptomyces, and its structural characteristics are as follows: its parent nucleus skeleton is a 15-membered lactone ring, 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 parent ring, the length of the alkyl side chain at the C4 / C9 position of the ester (peptide) group, whether the amino group on the salicylic acyl group is N-formylated 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 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 drawback in drug development, with poor selectivity for the inhibitory activity against tumor cells and significant toxic side effects on normal cells. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method and application of a new antimycin derivative containing fluorine elements.

[0005] The first objective is to disclose eight new structural antifungal antibiotic derivatives Unantimycin F1-J (UAT-F1-J) produced by fermentation of Streptomyces conglobatus ATCC 31005. The C-6 position in the molecular structure of the antifungal antibiotic generally contains a 3-N-formylaminosalicylic acid group, while for the fluorine-containing antifungal antibiotic derivatives involved in the present invention, the C-6 position is substituted by a non-3-N-formylaminosalicylic acid group, and their structural formulas are as follows:

[0006]

[0007] The second objective of the present invention is to further provide a preparation method for the novel fluorine-containing antifungal antibiotic derivatives as described above. The novel fluorine-containing antifungal antibiotic derivatives described in 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 used Streptomyces conglobatus is preferably the commercially available strain ATCC 31005.

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

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

[0011]

[0012] 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 the present invention.

[0013] b) Strain fermentation and precursor feeding: Inoculate Streptomyces conglobatus on a solid medium, culture at 27-32 °C for 4-6 days and then collect the spores; take the spores and inoculate them into a primary liquid medium, shake flask culture at 27-32 °C for 3-4 days to obtain a seed solution; take the seed solution and inoculate it into a secondary liquid medium, shake flask culture at 27-32 °C for 3-4 days to obtain a secondary seed solution; take the secondary seed solution and inoculate it into a fermentation medium, shake flask culture at 27-32 °C for 2 days and then feed compounds a-d at a concentration of 1.0 mmol / L, continue to shake flask culture for 3-4 days and then collect the fermentation broth; extract and separate the novel fluorine-containing antifungal antibiotic derivatives 1-8 described in the present invention from the fermentation broth.

[0014] Preferably, the solid medium contains 1.5 - 2.5% soybean powder, 1.5 - 2.5% D-mannitol, and 1.5 - 2.5% agar. The first-stage liquid medium contains 2 - 4% tryptic soy broth, 9 - 11% sucrose, and 0.4 - 0.6% yeast extract. The second-stage liquid medium contains 2 - 4% soybean powder, 4 - 6% glucose, 0.4 - 0.6% CaCO3, 4 - 6 mg / L CoCl2·6H2O, and 0.15 - 0.25% (v / v) antifoaming agent. The fermentation medium contains 2 - 4% soybean powder, 4 - 6% glucose, 0.4 - 0.6% CaCO3, and 0.15 - 0.25% (v / v) antifoaming agent.

[0015] c) Compound extraction, separation, and purification

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

[0017] Preferably, the steps for extracting the new antimycin derivative containing fluorine element from the fermentation broth include: adding 0.1 - 0.2% (v / v) formic acid to the fermentation broth, then extracting with ethyl acetate. The ethyl acetate extract is concentrated under reduced pressure at 30 - 45 °C to obtain an extract: the extract is redissolved with methanol, filtered to remove residues, and then extracted with n-hexane to remove the n-hexane layer; after the methanol solution is concentrated, compounds UAT-F1 - J are obtained through column chromatography separation.

[0018] 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 ethyl acetate content; 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 acetonitrile solution containing 30%, and then gradually decreasing the solvent polarity and increasing the acetonitrile content gradient to 100%.

[0019] Take the target fraction and perform final separation by preparative HPLC;

[0020] The separation conditions for 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). The separation conditions for UAT-G1 and UAT-G2 are: 80% methanol - 0.1% formic acid aqueous solution, YMC C 18Chromatographic column, 10×250 mm, 5 μm, 2 mL / min). The separation conditions for UAT-H1 and UAT-H2 are: 82% methanol - 0.1% formic acid aqueous solution, YMC C 18 Chromatographic column, 10×250 mm, 5 μm, 2 mL / min). The separation conditions for UAT-I are: 85% acetonitrile - 0.1% formic acid aqueous solution, YMC C 18 Chromatographic column, 10×250 mm, 5 μm, 2 mL / min). The separation conditions for UAT-I are: 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 the following method: condensation of a fifteen-membered ring tetra-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 the following method: condensation of a fifteen-membered ring tetra-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 the following method: condensation of a fifteen-membered ring tetra-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, condense a fifteen-membered ring 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;

[0027] The target compound UAT-J can be obtained by the following method: First, condense a fifteen-membered ring 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.

[0028] Preferably, the fifteen-membered ring tetra-lactone can be prepared by the following route,

[0029]

[0030] For the specific preparation and separation processes of the intermediates 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), and they will not be elaborated in the present invention.

[0031] 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.

[0032] Furthermore, the anti-tumor drug can be used for preventing and / or treating one or more of lung cancer and colon cancer.

[0033] 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-inhibitory intensity against human lung cancer cells and colorectal cancer cells is 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 being 6.61 and 4.75 μM respectively), UAT-F1 to H2 generally have weak inhibitory activity against non-cancer cell lines (IC 50 > 40 μM); in addition, the compounds UAT-F1 to UAT-J have selective inhibitory effects on colorectal cancer cells with K-RAS target mutations. Therefore, UAT-F1 to J have the potential to be developed into targeted drugs for colorectal cancer and lung cancer.

[0034] The present invention also provides a pharmaceutical composition, comprising the novel antimycin derivatives containing fluorine elements (UAT-F1 to UAT-J) as described above.

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

[0036] During the research process of the secondary metabolites of actinomycete S. conglobatus ATCC 31005, a series of novel antimycin derivatives containing fluorine elements with similar structures (UAT-F1 to UAT-J) were isolated. Anti-tumor activity tests found that the compounds UAT-F1 to UAT-J have equivalent 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 have weak inhibitory activity against human small intestinal cells NCM460 (IC 50 = 6.61, 4.75 μM), UAT-F1 to H2 generally have weak inhibitory activity against non-cancer cell lines (IC 50 > 40 μM). The present invention provides new lead compounds for the research and development of new anti-tumor drugs. Description of the Drawings

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

[0038] Figure 2 HR-ESI-MS result diagrams of compounds UAT-F1 to J of the present invention.

[0039] Figures 3a - 3f 1H NMR spectrum of compound UAT-F1 of the present invention;

[0040] Figures 4a - 4f 1H NMR spectrum of compound UAT-F2 of the present invention;

[0041] Figures 5a - 5f 1H NMR spectrum of compound UAT-G1 of the present invention;

[0042] Figures 6a - 6f 1H NMR spectrum of compound UAT-G2 of the present invention;

[0043] Figures 7a - 7f 1H NMR spectrum of compound UAT-H1 of the present invention;

[0044] Figures 8a - 8f 1H NMR spectrum of compound UAT-H2 of the present invention;

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

[0046] Figures 10a - 10f 1H NMR spectrum of compound UAT-J of the present invention;

[0047] where a-f respectively represent 1 1H-NMR spectrum, 13 13C-NMR spectrum, HSQC spectrum, COSY spectrum, HMBC spectrum, ROESY spectrum.

[0048] Figure 11a LC-MS comparative analysis result diagrams of the reactions of compounds 1-8 of the present invention with S-MTPA-Cl, the standards 2S-hydroxyisovaleric acid with R-MTPA-Cl and S-MTPA-Cl.

[0049] Figure 11b LC-MS comparative analysis result diagrams of the reactions of compounds 1-7 of the present invention with S-MTPA, the standards L-isoleucic acid with R-MTPA-Cl and S-MTPA-Cl.

[0050] Figure 11c LC-MS comparative analysis result diagrams of the reaction of compound 8 of the present invention with S-MTPA-Cl, the standards

[0051] LC-MS Comparative Analysis Results Diagram of the Reaction of 5-benzyl-4-hydroxy-3,3-dimethyldihydrofuran-2-one with R-MTPA-Cl and S-MTP-Cl

[0052] Figure 12 LC-MS Comparative Analysis Results Diagram of the Reaction of Compounds 1-8, L-threonine, and L-allo-threonine of the Present Invention with FDLA

[0053] Figure 13 CD Data Diagram 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 Modes

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

[0055] 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

[0056] The Streptomyces S. conglobatus used in the following experiments is preferably the commercially available strain ATCC 31,005

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

[0058] 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 sterilized separately and then added to 1 L of SGC medium), using the method of liquid shaking flask fermentation, ferment Streptomyces S. conglobatus ATCC 31,005 at 30 °C and 220 r on a shaking flask for 7 days. Feed 2-fluoroacetylcysteine benzoate thioester to the strain on the 3rd day of fermentation and then continue fermentation culture, with a total fermentation volume of 36 L. After fermentation, add 0.1% formic acid to the fermentation broth, extract it three times with an equal volume of ethyl acetate, combine the extracts, and concentrate the extracts under reduced pressure to obtain an ethyl acetate extract. Suspend the ethyl acetate layer extract in a methanol solution, extract it 3 times with an equal volume of n-hexane, combine the lower layer solution, and concentrate it under reduced pressure to obtain a methanol extract

[0059] 44.7 g of the above-mentioned methanol extract was subjected to normal-phase vacuum column chromatography, eluted with dichloromethane-methanol 50:1, gradient eluted with ethyl acetate-petroleum ether, and tracked by mass spectrometry. The fractions containing the peak of mass-to-charge ratio m / z 656.3 were combined. Separated by ODS medium-pressure column chromatography, eluted with a MeCN / H2O gradient (20%-80%, 180 min), and analyzed by mass spectrometry tracking and localization to obtain fine fractions containing the 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 novel antimycin derivatives 1-2 (UAT-F1-F2) containing fluorine element of the present invention.

[0060] Preparation and separation 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 were sterilized separately and then added to 1 L of SGC medium), and Streptomyces 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 benzoate 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 the mixture 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 a methanol solution and extracted 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.

[0061] 43.5 g of the above-mentioned methanol extract was subjected to normal-phase vacuum column chromatography, eluted with dichloromethane-methanol 50:1, gradient eluted with ethyl acetate-petroleum ether, and tracked by mass spectrometry. The fractions containing the peak of mass-to-charge ratio m / z 656.3 were combined. Separated by ODS medium-pressure column chromatography, eluted with a MeCN / H2O gradient (20%-80%, 180 min), and analyzed by mass spectrometry tracking and localization to obtain fine fractions 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 novel antimycin derivatives 3-4 (UAT-G1-G2) containing fluorine element of the present invention.

[0062] 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 are sterilized separately and then added to 1 L of SGC medium), using the method of liquid shaker fermentation, ferment the actinomycete S. conglobatus ATCC31005, at 30 °C, shake at 220 r for 7 days, feed the strain with acetylcysteine benzoate thioester on the 3rd day of fermentation and then continue fermentation culture, with a total of 12 L of fermentation. After fermentation, add 0.1% formic acid to the fermentation broth, and extract three times with an equal volume of ethyl acetate, combine the extracts, and concentrate the extract under reduced pressure to obtain an ethyl acetate extract. Suspend the ethyl acetate layer extract in methanol solution, extract three times with an equal volume of n-hexane, combine the lower layer solution, and concentrate under reduced pressure to obtain a methanol extract.

[0063] 17.2 g of the above methanol extract was subjected to normal-phase vacuum column chromatography, eluted with dichloromethane-methanol gradient and ethyl acetate-petroleum ether gradient, and the fractions containing the peak of mass-to-charge ratio m / z 638.3 were combined by mass spectrometry positioning and tracking. Separation was carried out by ODS medium-pressure column chromatography, eluted with a MeCN / H2O gradient (20%-80%, 180 min), and fine fractions containing large molecular weight linear peptide compounds were obtained by mass spectrometry tracking and positioning analysis; 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 cyclopeptide compounds 5-6 (UAT-H1-H2) of the present invention.

[0064] Preparation and isolation 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 are sterilized separately and then added to 1 L of SGC medium), using the method of liquid shaker fermentation, ferment the actinomycete S. conglobatus ATCC31005, at 30 °C, shake at 220 r for 7 days, feed the strain with acetylcysteine salicylate thioester on the 3rd day of fermentation and then continue fermentation culture, with a total of 12 L of fermentation. After fermentation, add 0.1% formic acid to the fermentation broth, and extract three times with an equal volume of ethyl acetate, combine the extracts, and concentrate the extract under reduced pressure to obtain an ethyl acetate extract. Suspend the ethyl acetate layer extract in methanol solution, extract three times with an equal volume of n-hexane, combine the lower layer solution, and concentrate under reduced pressure to obtain a methanol extract.

[0065] 36.1 g of the above-mentioned methanol extract was subjected to normal-phase vacuum column chromatography, eluted with a gradient of dichloromethane-methanol and a gradient of ethyl acetate-petroleum ether, and tracked by mass spectrometry. The fractions containing the peaks of m / z 654.3 and m / z 642.3 were combined. Separated by ODS medium-pressure column chromatography, eluted with a gradient of MeCN / H2O (20%-80%, 180 min), and analyzed by mass spectrometry tracking and positioning to obtain a fine fraction containing large-molecular-weight linear peptide compounds; finally, the fraction was 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, CosmosilC 18 chromatographic column, 10×250 mm, 5 μm, 2 mL / min), to obtain the 15-membered ring cyclopeptide compounds 7-8 (UAT-I-J) of the present invention.

[0066] Example 2: Obtaining compounds UAT-F1-J by chemical synthesis

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

[0068]

[0069] Compounds UAT-I-J can be obtained through multiple chemical synthesis steps, and the synthesis process is 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 by using Example 1 or Example 2 can be determined by 1D- and 2D-NMR data analysis.

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

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

[0083] Figure 1 Show the 2D NMR (DMSO-d6) correlations of compounds UAT-F1 to J.

[0084] Figure 2 Show the HR-ESI-MS of compounds UAT-F1 to J.

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

[0086] Figures 11a~11c Are the LC-MS analysis charts of the Mosher reactions of compounds 1 to 8 and the reference standard

[0087] Figure 12 Are the LC-MS analysis charts of the Marfey reactions of compounds 1 - 8, threonine, and allothreonine

[0088] Figure 13 Are the ECD data of compounds 1 - 7 and compounds UAT-B1, UAT-B2 with known chiral configurations.

[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: NMR data of UAT-I

[0107]

[0108]

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

[0110]

[0111]

[0112] Example 3: In vitro anti-tumor activity experiment of novel antimycin derivatives UAT-B1 to UAT-J containing fluorine element of the present invention

[0113] The cell proliferation was detected by CCK8 assay. The sample was dissolved in DMSO to prepare a stock solution of 10 mM 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 serially diluted to a working concentration of 3 nM - 20 μM. The above cancer cells in the logarithmic growth phase were used to prepare a single cell suspension of 1×10 6cells / mL. Add this suspension to a 96-well plate, 100 μL per well. Incubate in an incubator with 5% CO2 at 37 °C for 24 h, then add the test drugs at various concentrations (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. Set 3 replicates for each sample. The negative control is the same volume of medium and the corresponding DMSO concentration as the solvent control to eliminate the influence of DMSO on cell growth. The positive control drug is oxaliplatin. After incubating in an incubator with 5% CO2 at 37 °C for 72 h, add 10 μL of CCK8 solution to each well. After incubating (at 37 °C, 5% CO2) for 40 - 60 min, measure the absorbance value (O.D.) at 450 nm using an enzyme-linked immunosorbent assay reader. Calculate the inhibition rate from the measured O.D. value and fit the IC 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 without K-RAS target mutations (HT-29), human non-small cell lung cancer cells with K-RAS mutations (A549, NCIH460), human non-small cell lung cancer without K-RAS mutations (H1299), and normal human small intestine cells (NCM460) as experimental cell lines.

[0115] 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 exhibit this property. In addition, compounds UAT-F1 - UAT-H2 have good inhibitory effects (0.04 μM - 11.2 μM) on both human colorectal cancer and lung cancer cells and have low toxicity to normal cells.

[0116] Table 9 Inhibition rates and half-maximal inhibitory concentration IC 50 values (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 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 intestinal cells is much lower than that of the positive drug. The compounds of the present invention are potential anti-tumor targeted drugs, providing new lead compounds for the development of new anti-tumor drugs.

[0119] Although the present invention has been described in detail with general descriptions and specific embodiments above, 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 required by the present invention.

Claims

1. A new antimycin derivative containing a fluorine element, characterized in that, It is one of the following two compounds:

2. The preparation method of the novel antimycin derivative containing fluorine element according to claim 1, characterized in that, The compound as described in Claim 1 is obtained by fermentation of Streptomyces conglobatus or by chemical synthesis.

3. The method according to claim 2, characterized in that, It includes the steps of: preparation of precursor compound b; inoculating Streptomyces conglobatus on a solid medium, culturing at 27 - 32 °C for 4 - 6 days and then collecting spores; Taking the spores and inoculating them into a first-stage liquid medium, culturing in a shaking flask at 27 - 32 °C for 3 - 4 days to obtain a seed solution; Taking the seed solution and inoculating it into a second-stage liquid medium, culturing in a shaking flask at 27 - 32 °C for 3 - 4 days to obtain a second-stage seed solution; Taking the second-stage seed solution and inoculating it into a fermentation medium, culturing in a shaking flask at 27 - 32 °C for 2 days, then feeding precursor compound b at a concentration of 1.0 mmol / L, and continuing to culture in a shaking flask for 3 - 4 days and then collecting the fermentation broth; Extracting and separating the novel antimycin derivative containing fluorine element as described in Claim 1 from the fermentation broth; The structural formula of the precursor compound b is as follows:

4. The method according to claim 3, wherein The solid medium contains 1.5% - 2.5% soybean powder, 1.5% - 2.5% D-mannitol, 1.5% - 2.5% agar; the first-stage liquid medium contains 2% - 4% tryptic soy broth, 9% - 11% sucrose, 0.4% - 0.6% yeast extract; The second-stage liquid medium contains 2% - 4% soybean powder, 4% - 6% glucose, 0.4% - 0.6% CaCO3, 4 - 6 mg / L CoCl2·6H2O, 0.15% - 0.25% (v / v) antifoaming agent; the fermentation medium contains 2% - 4% soybean powder, 4% - 6% glucose, 0.4% - 0.6% CaCO3, 0.15% - 0.25% (v / v) antifoaming agent.

5. The method according to claim 3, characterized in that The precursor compound b is prepared by the following route:

6. The method according to claim 3, wherein The steps for extracting and separating the novel antimycin derivative containing fluorine element as described in Claim 1 from the fermentation broth are: 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, and removing the n-hexane layer; After concentrating the methanol solution, separating by column chromatography to obtain the novel antimycin derivative containing fluorine element as described in Claim 1.

7. The method according to claim 6, characterized in that Column chromatography separation includes: separating through a normal-phase silica gel column, the elution solvent is dichloromethane / methanol, and the elution solvent ratio is 50 / 1 (v / v); Separating through a normal-phase 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), and then gradually increasing the solvent polarity and gradually increasing the ethyl acetate content; Taking the target fraction and separating it through an ODS column, and the elution solvent gradient is 30% - 100% acetonitrile; Taking the target fraction and performing final separation by preparative HPLC; The separation conditions for UAT-G1 and UAT-G2 are: 80% 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 a novel antimycin derivative containing a fluorine element according to claim 1, characterized in that, Its preparation process is as follows: The target compounds UAT-G1 and UAT-G2 are obtained by the following method: condensing a fifteen-membered ring tetra-lactone containing two alkyl groups and one benzyl side chain with 3-fluorobenzoic acid.

9. Use of the novel antimycin derivative containing a fluorine element as claimed in claim 1 in the preparation of an anti-tumor drug, characterized in that, The tumor is selected from one or more of lung cancer and colon cancer.

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