Polyether compounds and methods for their preparation

Abstract

The present application aims to provide a compound shown in formula (I), pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein the definitions of each group are described in the specification. In addition, the present application also discloses the preparation method and application of the compound.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a polyether compound and its preparation method. Background Technology

[0002] Polyether compounds are an important class of natural microbial products, characterized by a carboxyl group and 2 to 5 ether oxygen atoms, typically located in tetrahydrofuran and tetrahydropyran structures. This structure, with an external lipophilic alkyl backbone and an internal abundance of oxygen atoms, allows polyether compounds to readily carry basic metal ions, cross the cell membrane, and enter the cell, leading to cell depolarization and death. Therefore, polyether compounds possess a wide range of biological activities, such as antibacterial, antifungal, antiparasitic, and antitumor activities. Lassamycin, monensin, salinomycin, and other polyether compounds are widely used in veterinary and animal husbandry. Furthermore, recent studies have discovered that some polyether compounds also exhibit broad-spectrum antiviral activity. It is evident that polyether compounds have enormous potential as pharmaceutical molecules and have attracted widespread attention from researchers. However, naturally occurring polyether compounds still fall short of meeting human pharmaceutical needs, and many molecules cannot be used clinically due to insufficient drug-likeness. Therefore, further structural optimization and modification of bioactive polyether compounds to obtain derivatives with significantly improved activity or better drug-like properties has gradually become a research trend, which is more targeted than randomly screening new natural products from nature.

[0003] Given the complex chemical and stereostructure of polyether compounds, many modifications exhibit stereoselectivity and regioselectivity. Furthermore, due to their chemical properties, chemical modification methods are only applicable to the modification of active functional groups (such as hydroxyl and amino groups) on the backbone, and are largely ineffective for inert backbone components. Therefore, chemical modification of polyether compounds presents a significant challenge. In recent years, with the rapid development of genome sequencing and bioinformatics analysis technologies, and the increasing characterization of biosynthetic pathways and enzyme functions of natural products, methods for obtaining novel structural derivatives by modifying the biosynthetic pathways of natural products have become increasingly common. This method can modify the structure of natural products while maintaining their original complexity and efficacy, yielding new polyether compounds. Activity screening of these new compounds holds promise for obtaining polyether molecules with greater pharmaceutical value.

[0004] Streptomyces strains hold promise for discovering more polyether compounds with novel structures and good activity. Summary of the Invention

[0005] The purpose of this invention is to provide a new polyether compound, its biological preparation method, and its uses.

[0006] In one aspect, the present invention provides a compound of formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a solvent compound thereof, or a prodrug thereof.

[0007]

[0008] in,

[0009] R1, R2, R3, R4, R5, R7, R9, R 10 R 11 R 12 R 13 Each is independently selected from H, -CH3, and -CH2CH3;

[0010] R6 and R8 are independently selected from H, -OH, -CH3, -OCH3, and -CH2CH3, respectively.

[0011] R 14 R 15 The values ​​are H, -OH, -CH3, -OCH3, and -CH2CH3.

[0012] The compound of formula (I) is not

[0013] In one implementation scheme, R6 and R8 are not simultaneously...

[0014] This invention provides the following compounds, their pharmaceutically acceptable salts, stereoisomers, solvent compounds, or prodrugs:

[0015]

[0016] In another aspect, the present invention provides a method for preparing a compound of formula (I) or a stereoisomer thereof.

[0017]

[0018] in,

[0019] R1, R2, R3, R4, R5, R7, R9, R 10 R 11 R 12 R 13 Each is independently selected from H, -CH3, and -CH2CH3;

[0020] R6 and R8 are independently selected from H, -OH, -CH3, -OCH3, and -CH2CH3, respectively.

[0021] R 14 R 15The possible values ​​are H, -OH, -CH3, -OCH3, and -CH2CH3.

[0022] The method involves knocking out the 3G5 gene, 3P3 gene, 3P1 gene, or 3M gene. [1] The internal Streptomyces endus subsp. aureus mutant strain;

[0023] Alternatively, knock out the 2P1 gene or the 2M gene. [2] The mutant strain of Streptomyces hygroscopicus A-130;

[0024] Alternatively, a Streptomyces endocarpus mutant strain with the 3G5 gene knocked out and the 2G5 gene introduced;

[0025] Alternatively, the compound of formula (I) can be prepared by fermentation of a Streptomyces endocarpus mutant strain with the 3P1 gene knocked out and the 2G5 gene introduced.

[0026] In one embodiment, the compound of formula (I) is a compound of formula (End-3), (End-4), (End-5), (End-16), (Len-10), or Len-11, or a compound of formula (End-2), one of which is a reported compound X-14931A (named End-2).

[0027]

[0028] The compound of formula (End-3) was prepared by fermentation of an internally coated Streptomyces mutant strain with the 3G5 gene knocked out;

[0029] The compound of formula (End-4) was prepared by fermentation of an internally coated Streptomyces mutant strain with the 3P3 gene knocked out;

[0030] The compound of formula (End-5) was prepared by fermentation of an internal Streptomyces mutant strain with the 3M gene knocked out;

[0031] The compound of formula (Len-10) was prepared by fermentation of a Streptomyces hygroscopicus mutant strain with the 2P1 gene knocked out;

[0032] The compound of formula (Len-11) was prepared by fermentation of a Streptomyces hygroscopicus mutant strain with the 2M gene knocked out;

[0033] The compound of formula (End-16) is prepared by fermentation of a Streptomyces endocarpus mutant strain with the 3G5 gene knocked out and the 2G5 gene introduced, or a Streptomyces endocarpus mutant strain with the 3P1 gene knocked out and the 2G5 gene introduced.

[0034] The compound of formula (End-2) is prepared by fermentation from a Streptomyces endocarpus mutant strain with the 3P1 gene knocked out, a Streptomyces endocarpus mutant strain with the 3G5 gene knocked out, a Streptomyces endocarpus mutant strain with the 3G5 gene knocked out and the 2G5 gene introduced, or a Streptomyces endocarpus mutant strain with the 3P1 gene knocked out and the 2G5 gene introduced.

[0035] In one embodiment, the method involves introducing a knockout plasmid containing the target gene into Streptomyces, followed by relaxation and double screening to obtain a Streptomyces mutant strain with the corresponding gene successfully knocked out. The obtained mutant strain is then fermented, cultured, extracted, and separated.

[0036] In one embodiment, the method involves introducing a knockout plasmid of the target gene into Streptomyces, followed by relaxation and double screening to obtain Streptomyces mutant strains with the corresponding gene successfully knocked out. Subsequently, heterologous expression plasmids are introduced into some of the Streptomyces mutant strains, and all obtained mutant strains are fermented, cultured, extracted, and isolated.

[0037] In one embodiment, the knockout plasmid is obtained by using genomic DNA of Streptomyces endothelioides or Streptomyces hydrophila as a template, amplifying the upstream and downstream homologous arms of the target gene using primers, and then ligating the upstream and downstream homologous arms of the target gene to a vector by yeast assembly or enzyme digestion and ligation methods.

[0038] In one embodiment, the knockout plasmid is obtained by using Streptomyces endothelioides genomic DNA as a template, amplifying the upstream and downstream homologous arms of the 3P3 gene using primers, and then ligating the upstream and downstream homologous arms of the target gene to a vector using a yeast assembly method.

[0039] Preferably, the primer nucleic acid sequences for obtaining the upstream homologous arm of the 3P3 gene are shown in SEQ ID NO:15 and SEQ ID NO:16.

[0040] Preferably, the primer nucleic acid sequences for obtaining the downstream homologous arm of the 3P3 gene are shown in SEQ ID NO:17 and SEQ ID NO:18.

[0041] Preferably, the primer nucleic acid sequences for obtaining yeast expression elements in the yeast assembly method are shown in SEQ ID NO:19 and SEQ ID NO:20.

[0042] In one embodiment, the knockout plasmid is obtained by using Streptomyces endothelioides genomic DNA as a template, amplifying the upstream and downstream homologous arms of the 3G5 gene using primers, and then ligating the upstream and downstream homologous arms of the target gene to a vector using enzyme digestion and ligation methods.

[0043] Preferably, the primer nucleic acid sequences for obtaining the upstream homologous arm of the 3G5 gene are shown in SEQ ID NO:21 and SEQ ID NO:22.

[0044] Preferably, the primer nucleic acid sequences for obtaining the downstream homologous arm of the 3G5 gene are shown in SEQ ID NO:23 and SEQ ID NO:24.

[0045] In one embodiment, the knockout plasmid is obtained by using Streptomyces endothelioides genomic DNA as a template, amplifying the upstream and downstream homologous arms of the 3P1 gene using primers, and then ligating the upstream and downstream homologous arms of the target gene into a vector using enzyme digestion and ligation methods.

[0046] Preferably, the primer nucleic acid sequences for obtaining the upstream homologous arm of the 3P1 gene are shown in SEQ ID NO:25 and SEQ ID NO:26.

[0047] Preferably, the primer nucleic acid sequences for obtaining the downstream homologous arm of the 3P1 gene are shown in SEQ ID NO:27 and SEQ ID NO:28.

[0048] In one embodiment, the knockout plasmid is obtained by using Streptomyces endothelioides genomic DNA as a template, amplifying the upstream and downstream homologous arms of the 3M gene using primers, and then ligating the upstream and downstream homologous arms of the target gene to a vector using enzyme digestion and ligation methods.

[0049] Preferably, the primer nucleic acid sequences for obtaining the upstream homologous arm of the 3M gene are shown in SEQ ID NO:29 and SEQ ID NO:30.

[0050] Preferably, the primer nucleic acid sequences for obtaining the downstream homologous arm of the 3M gene are shown in SEQ ID NO:31 and SEQ ID NO:32.

[0051] In one embodiment, the knockout plasmid is obtained by using Streptomyces hygroscopicus genomic DNA as a template, amplifying the upstream and downstream homologous arms of the 2P1 gene using primers, and then ligating the upstream and downstream homologous arms of the target gene to a vector using enzyme digestion and ligation methods.

[0052] Preferably, the primer nucleic acid sequences for obtaining the upstream homologous arm of the 2P1 gene are shown in SEQ ID NO:33 and SEQ ID NO:34.

[0053] Preferably, the primer nucleic acid sequences for obtaining the downstream homologous arm of the 2P1 gene are shown in SEQ ID NO:35 and SEQ ID NO:36.

[0054] In one embodiment, the knockout plasmid is obtained by using Streptomyces hygroscopicus genomic DNA as a template, amplifying the upstream and downstream homologous arms of the 2M gene using primers, and then ligating the upstream and downstream homologous arms of the target gene to a vector using enzyme digestion and ligation methods.

[0055] Preferably, the primer nucleic acid sequences for obtaining the upstream homologous arm of the 2M gene are shown in SEQ ID NO:37 and SEQ ID NO:38.

[0056] Preferably, the primer nucleic acid sequences for obtaining the downstream homologous arm of the 2M gene are shown in SEQ ID NO:39 and SEQ ID NO:40.

[0057] In one embodiment, the method involves introducing a heterologous expression plasmid containing the 2G5 gene into Streptomyces strains that have either knocked out the 3G5 or 3P1 gene, and then fermenting, culturing, extracting, and separating the resulting mutant strains.

[0058] In one embodiment, the heterologous expression plasmid is obtained by amplifying the 2G5 gene using primers with Streptomyces hygroscopicus genomic DNA as a template, and then ligating the 2G5 gene into a vector using an enzyme digestion and ligation method.

[0059] Preferably, the primer nucleic acid sequences for obtaining the 2G5 gene are shown in SEQ ID NO:41 and SEQ ID NO:42. In one embodiment, the knockout plasmid is constructed using a yeast assembly method.

[0060] In one embodiment, the knockout plasmid is constructed by an enzyme digestion and ligation method.

[0061] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned compound, a compound prepared by the above-described preparation method, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a solvent compound thereof or a prodrug thereof, and a pharmaceutically acceptable carrier thereof.

[0062] In another aspect, the present invention provides a mutant strain obtained by individually knocking out the 3G5 gene, 3P3 gene, 3P1 gene, and 3M gene in the genome of Streptomyces endocarpus.

[0063] In one embodiment, the mutant strain is used to prepare the following compounds:

[0064]

[0065] In another aspect, the present invention provides a mutant strain obtained by knocking out the 2P1 and 2M genes in the genome of Streptomyces hygroscopicus, or by knocking out the 3G5 and 3P1 genes in the genome of Streptomyces endothelioides and then introducing the 2G5 gene into each gene.

[0066] In one embodiment, the mutant strain is used to prepare the following compounds:

[0067]

[0068] In one embodiment, the 3G5 gene encodes the amino acid sequence shown in SEQ ID NO:8; the 3P3 gene encodes the amino acid sequence shown in SEQ ID NO:9; the 3P1 gene encodes the amino acid sequence shown in SEQ ID NO:10; the 3M gene encodes the amino acid sequence shown in SEQ ID NO:11; the 2P1 gene encodes the amino acid sequence shown in SEQ ID NO:12; the 2M gene encodes the amino acid sequence shown in SEQ ID NO:13; and the 2G5 gene encodes the amino acid sequence shown in SEQ ID NO:14.

[0069] In one embodiment, the nucleic acid sequence of the 3G5 gene is shown in SEQ ID NO: 1; the nucleic acid sequence of the 3P3 gene is shown in SEQ ID NO: 2; the nucleic acid sequence of the 3P1 gene is shown in SEQ ID NO: 3; the nucleic acid sequence of the 3M gene is shown in SEQ ID NO: 4; the nucleic acid sequence of the 2P1 gene is shown in SEQ ID NO: 5; the nucleic acid sequence of the 2M gene is shown in SEQ ID NO: 6; and the nucleic acid sequence of the 2G5 gene is shown in SEQ ID NO: 7.

[0070] In another aspect, the present invention provides the use of the aforementioned compounds, pharmaceutically acceptable salts thereof, stereoisomers thereof, solvent compounds thereof or prodrugs thereof, or the aforementioned pharmaceutical compositions, compounds thereof or stereoisomers thereof prepared by the aforementioned preparation methods, and the aforementioned mutant strains in the preparation of antitumor drugs.

[0071] Preferably, the tumor is a solid tumor or a hematoma;

[0072] Preferably, the tumor is cervical cancer or leukemia, and the leukemia is preferably acute T-cell leukemia.

[0073] In another aspect, the present invention provides a method for treating tumor diseases, comprising the steps of administering to a patient in need the aforementioned compound, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a solvent compound thereof or a prodrug thereof, or the aforementioned pharmaceutical composition, or a compound prepared by the aforementioned preparation method.

[0074] Preferably, the dosage is a therapeutically effective dosage. Attached Figure Description

[0075] Figure 1 The HPLC chromatograms of fermentation products from wild-type and mutant S. endus subsp. aureus are shown.

[0076] Figure 2The HPLC chromatograms of fermentation products from wild-type S. hygroscopicus A-130 and its mutant strains are shown.

[0077] Figure 3 The HPLC chromatogram of the fermentation product of the 2G5 heterologous expression mutant is shown.

[0078] Figure 4 The HR-ESI-MS spectrum of compound End-3 is shown.

[0079] Figure 5 The compound End-3 is shown. 1 H NMR spectrum.

[0080] Figure 6 The compound End-3 is shown. 13 C10 NMR spectrum.

[0081] Figure 7 The DEPT135 NMR spectrum of compound End-3 is shown.

[0082] Figure 8 The DEPT90 NMR spectrum of compound End-3 is shown.

[0083] Figure 9 The HSQC spectrum of compound End-3 is shown.

[0084] Figure 10 The HMBC spectrum of compound End-3 is shown.

[0085] Figure 11 The compound End-3 is shown. 1 H- 1 H shows the COSY spectrum.

[0086] Figure 12 The NOESY spectrum of compound End-3 is shown.

[0087] Figure 13 The HR-ESI-MS spectrum of compound End-4 is shown.

[0088] Figure 14 The compound End-4 is shown. 1 H NMR spectrum.

[0089] Figure 15 The compound End-4 is shown. 13 C10 NMR spectrum.

[0090] Figure 16 The DEPT135 NMR spectrum of compound End-4 is shown.

[0091] Figure 17 The DEPT90 NMR spectrum of compound End-4 is shown.

[0092] Figure 18 The HSQC spectrum of compound End-4 is shown.

[0093] Figure 19 The HMBC spectrum of compound End-4 is shown.

[0094] Figure 20 The compound End-4 is shown. 1 H- 1 H COSY spectrum.

[0095] Figure 21 The NOESY spectrum of compound End-4 is shown.

[0096] Figure 22 The HR-ESI-MS spectrum of compound End-5 is shown.

[0097] Figure 23 The compound End-5 is shown. 1 H NMR spectrum.

[0098] Figure 24 The compound End-5 is shown. 13 C10 NMR spectrum.

[0099] Figure 25 The DEPT135 NMR spectrum of compound End-5 is shown.

[0100] Figure 26 The DEPT90 NMR spectrum of compound End-5 is shown.

[0101] Figure 27 The HSQC spectrum of compound End-5 is shown.

[0102] Figure 28 The HMBC spectrum of compound End-5 is shown.

[0103] Figure 29 The compound End-5 is shown. 1 H- 1 H COSY spectrum.

[0104] Figure 30 The NOESY spectrum of compound End-5 is shown.

[0105] Figure 31 The HR-ESI-MS spectrum of compound Len-10 is shown.

[0106] Figure 32 The compound Len-10 is shown. 1 H NMR spectrum.

[0107] Figure 33 The compound Len-10 is shown. 13 C10 NMR spectrum.

[0108] Figure 34 The DEPT135 NMR spectrum of compound Len-10 is shown.

[0109] Figure 35 The DEPT90 NMR spectrum of compound Len-10 is shown.

[0110] Figure 36 The HSQC spectrum of compound Len-10 is shown.

[0111] Figure 37 The HMBC spectrum of compound Len-10 is shown.

[0112] Figure 38 The compound Len-10 is shown. 1 H- 1 H COSY spectrum.

[0113] Figure 39 The NOESY spectrum of compound Len-10 is shown.

[0114] Figure 40 The HR-ESI-MS spectrum of compound Len-11 is shown.

[0115] Figure 41 The compound Len-11 is shown. 1 H NMR spectrum.

[0116] Figure 42 The compound Len-11 is shown. 13 C10 NMR spectrum.

[0117] Figure 43 The DEPT135 NMR spectrum of compound Len-11 is shown.

[0118] Figure 44 The DEPT90 NMR spectrum of compound Len-11 is shown.

[0119] Figure 45 The HSQC spectrum of compound Len-11 is shown.

[0120] Figure 46 The HMBC spectrum of compound Len-11 is shown.

[0121] Figure 47 The compound Len-11 is shown. 1 H- 1 H COSY spectrum.

[0122] Figure 48 The NOESY spectrum of compound Len-11 is shown.

[0123] Figure 49 The HR-ESI-MS spectrum of compound End-16 is shown.

[0124] Figure 50 The 1H NMR spectrum of compound End-16 is shown.

[0125] Figure 51 The compound End-16 is shown. 13 C10 NMR spectrum.

[0126] Figure 52 The DEPT135 NMR spectrum of compound End-16 is shown.

[0127] Figure 53 The DEPT90 NMR spectrum of compound End-16 is shown.

[0128] Figure 54 The HSQC spectrum of compound End-16 is shown.

[0129] Figure 55 The HMBC spectrum of compound End-16 is shown.

[0130] Figure 56 The compound End-16 is shown. 1 H- 1 H COSY spectrum.

[0131] Figure 57 The NOESY spectrum of compound End-16 is shown.

[0132] Figure 58 The results of the in vitro anti-HeLa cell activity test of the compound in Example 6 are shown.

[0133] Figure 59 The results of the in vitro anti-Jurkat cell activity test of the compound in Example 6 are shown. Detailed Implementation

[0134] I. Definition

[0135] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0136] As described herein, the term "LA plate" refers to Luria-Bertani agar plates, which are formulated with 1% peptone, 0.5% yeast extract, 1% NaCl and 1.6% agar.

[0137] As described in this article, the term "homologous arm" refers to the flanking sequences on a knockout plasmid that are completely identical to the sequences flanking the knockout gene. These sequences are used to identify and allow recombination to occur. Specifically, the upstream homologous arm refers to a DNA segment that is identical to the upstream sequence of the knockout gene, and the downstream homologous arm refers to a DNA segment that is identical to the downstream sequence of the knockout gene.

[0138] As described in this article, the term "mutant strain" refers to recombinant Streptomyces with the modified genes knocked out, and mutant strain fermentation refers to inoculating the mutant strain into a culture medium for fermentation culture so that the mutant strain produces secondary metabolites.

[0139] II. Detailed Implementation

[0140] III. Examples

[0141] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The provided embodiments are merely illustrative of the method of the present invention and do not limit the rest of the content disclosed herein in any way.

[0142] Example 1: Construction of knockout plasmid

[0143] 1.1 Yeast assembly method to construct pYH7-3P3 for knocking out the post-biosynthetic modification gene 3P3 of endusamycin in S. endus subsp. aureus.

[0144] The vector for the knockout plasmid pYH7-3P3 is pYH7. Using S. endus subsp. aureus (Streptomycesendus subsp. aureus) genomic DNA as a template, approximately 2 kb of upstream and downstream homologous arms of 3P3 were amplified using primers. These homologous arms were then recombined with the enzyme-digested linear pYH7 fragment via yeast assembly to obtain the plasmid pYH7-3P3 containing the upstream and downstream homologous arms of 3P3. The primer sequences are shown in Table 2.

[0145] 1.2 Enzyme digestion and ligation methods were used to construct pYH7-3G5, pYH7-3P1, pYH7-3M, pYH7-2P1, and pYH7-2M, which were used to knock out the post-biosynthetic modification genes 3G5, 3P1, and 3M of endusamycin in S. endus subsp. aureus, and the post-biosynthetic modification genes 2P1 and 2M of lenoremycin in S. hygroscopicus A-130 (Streptomyces hygroscopicus A-130), respectively.

[0146] The knockout plasmid vector was pYH7. Using *S. endus subsp. aureus* genomic DNA as a template, approximately 2kb upstream and downstream homologous arms of 3G5, 3P1, and 3M were amplified using primers. Using *S. hygroscopicus A-130* genomic DNA as a template, approximately 2kb upstream and downstream homologous arms of 2P1 and 2M were amplified using primers. The upstream and downstream homologous arms were treated with double enzyme digestion. The digested upstream and downstream homologous arms were ligated with digested pYH7 to obtain plasmids pYH7-3G5, pYH7-3P1, pYH7-3M, pYH7-2P1, and pYH7-2M containing upstream and downstream homologous arms of 3G5, 3P1, 3M, 2P1, and 2M, respectively. The knockout gene, restriction enzymes used, and other relevant parameters are shown in Table 1, and the primer sequences are shown in Table 2.

[0147] Table 1. Parameters related to the construction of knockout plasmids using the enzyme digestion and ligation method in Example 1.

[0148]

[0149] Table 2 shows the primer sequences for constructing plasmids in Example 1.

[0150]

[0151]

[0152] Table 3. Sequence numbers of knockout genes and heterologous expression genes.

[0153]

[0154] The nucleotide and amino acid sequences corresponding to the genes 3G5, 3P3, 3P1, 3M2P1, 2M, and 2G5 are SEQ ID NO:1–7 and SEQ ID NO:8–14, respectively (Table 3), and the specific sequences are as follows:

[0155] SEQ ID NO:1(3G5)

[0156]

[0157]

[0158] SEQ ID NO:2(3P3)

[0159]

[0160] SEQ ID NO:3(3P1)

[0161]

[0162] SEQ ID NO:4(3M)

[0163]

[0164] SEQ ID NO:5(2P1)

[0165]

[0166]

[0167] SEQ ID NO:6(2M)

[0168]

[0169] SEQ ID NO:7(2G5)

[0170]

[0171] SEQ ID NO:8(3G5)

[0172]

[0173] SEQ ID NO:9(3P3)

[0174]

[0175] SEQ ID NO:10(3P1)

[0176]

[0177]

[0178] SEQ ID NO:11(3M)

[0179]

[0180] SEQ ID NO:12(2P1)

[0181]

[0182] SEQ ID NO: 13(2M)

[0183]

[0184] SEQ ID NO: 14(2G5)

[0185]

[0186] Example 2: Construction of mutant strains

[0187] 2.1 The knockout plasmid was transformed into E. coli ET12567 / pUZ8002

[0188] E. coli ET12567 / pUZ8002 was cultured and grown to OD. 600 =0.4-0.6, take 1 mL of bacterial culture into a 1.5 mL EP tube, centrifuge in a pre-cooled centrifuge at 4℃ to collect the bacterial cells, and wash twice with pre-cooled sterile water at 4℃, discard the supernatant, so that the final volume is about 50 μL. The plasmids obtained in Example 1 were added to the bacterial cells, mixed well, and transferred to a 1 mm electroporation cuvette. Electroporation was performed at 1.8 kV, and 1 mL of pre-cooled LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) was added as soon as possible. The mixture was incubated at 37°C and 220 rpm for 1 h. The incubated transformants were then plated on LA plates containing kanamycin (50 μg / mL), chloramphenicol (34 μg / mL), and apopramycin (50 μg / mL). PCR verification of the transformed transformants yielded E. coli ET12567 / pUZ8002 containing pYH7-3G5, pYH7-3P3, pYH7-3P1, pYH7-3M, pYH7-2P1, and pYH7-2M, respectively.

[0189] 2.2 Conjugation and transfer of knockout plasmids between E. coli ET12567 / pUZ8002 and S. endus subsp. aureus and S. hygroscopicus A-130

[0190] Ecoli ET12567 / pUZ8002 samples containing pYH7-3G5, pYH7-3P3, pYH7-3P1, pYH7-3M, pYH7-2P1, and pYH7-2M, obtained in Example 2.1, were transferred to LB medium and cultured at 37°C and 220 rpm until OD200. 600 =0.6-0.8, centrifuge to collect bacterial cells, and wash the bacterial cells twice with antibiotic-free LB.

[0191] Spores of *S. endus subsp. aureus* and *S. hygroscopicus* A-130 were collected separately and placed in 0.05 M MTES buffer (pH 8.0). The mixture was incubated at 50°C for 10 min under heat shock. An equal volume of 2× spore pre-germination medium (10 g / L Difco yeast extract, 10 g / L Difco casein amino acids, containing 0.01 M CaCl2) was added, and the mixture was pre-germinated at 37°C and 220 rpm for 2.5 h. The spores were then collected and suspended in LB medium. *E. coli* ET12567 / pUZ8002 and *S. endus subsp. aureus* spores containing pYH7-3G5, pYH7-3P3, pYH7-3P1, and pYH7-3M were thoroughly mixed in specific proportions. *E. coli* spores containing pYH7-2P1 and pYH7-2M were also mixed separately. ET12567 / pUZ8002 and Shygroscopicus A-130 spores were thoroughly mixed in a specific ratio and then spread separately onto SFM plates (20 g / L soybean meal, 20 g / L sorbitol, 16 g / L agar, pH 7.2). After drying in a clean bench, the plates were incubated at 30°C for 16 h. Then, the plates were covered with apopramycin (15 μg / mL) and trimethoprim (50 μg / mL) and incubated at 30°C for another 5-10 days until single colonies appeared. These single colonies were then inoculated onto SFM plates containing apopramycin (15 μg / mL) and trimethoprim (50 μg / mL) for further culture and PCR verification to obtain single-exchange strains.

[0192] 2.3 Screening of double crossover mutants

[0193] Using the streak plating method, the single-exchange strains obtained in Example 2.2 were cultured on antibiotic-free SFM medium to allow for the loss of apopramine resistance. After incubation at 30°C for 4-5 days, single colonies were selected and cultured on antibiotic-free SFM plates and SFM plates containing apopramine (15 μg / mL) for resistance verification. Candidate mutants sensitive to apopramine (15 μg / mL) were selected for PCR verification, thereby obtaining S. endus subsp. aureus mutants with 3G5, 3P3, 3P1, and 3M knockouts (S. endus subsp. aureus Δ3G5, S. endus subsp. aureus Δ3P3, S. endus subsp. aureus Δ3P1, S. endus subsp. aureus Δ3M), and S. hygroscopicus A-130 mutants with 2P1 and 2M knockouts (S. endus subsp. aureus Δ3M). HPLC chromatograms of fermentation products from wild-type (wt) S. endus subsp. Aureus, wild-type hygroscopicus A-130, and its mutant strains are shown below. Figure 1 and Figure 2 .

[0194] Example 3: Construction of heterologous expression plasmids

[0195] The vector for the heterologous expression plasmid pSET152-2G5 is pSET152 containing the SPL42 promoter. Using S. hygroscopicus A-130 genomic DNA as a template, the 2G5 gene was amplified using primers. The 2G5 gene was obtained by double digestion with NdeI and KpnI. The digested 2G5 gene was then ligated with pSET152 that had been digested with NdeI and KpnI to obtain the plasmid pSET152-2G5 containing the 2G5 gene, with the SPL42 promoter upstream of the 2G5 gene.

[0196] Example 4: Construction of Heterologous Expression Mutant

[0197] 4.1 The heterologous expression plasmid pSET152-2G5 was transformed into E. coli ETi2567 / pUZ8002

[0198] The method is the same as that described in Example 2.1. After PCR verification of the grown transformants, E. coli ET12567 / pUZ8002 containing pSET152-2G5 was obtained.

[0199] 4.2 Conjugation transfer of knockout plasmids between E. coli ET12567 / pUZ8002 and S. endus subsp. aureus Δ3P1 or S. endus subsp. aureus Δ3G5

[0200] The E. coli ET12567 / pUZ8002 containing pSET152-2G5 obtained in Example 4.1 was transferred to LB medium and cultured at 37°C and 220 rpm until OD152-2G5 was reached. 600 =0.6-0.8, centrifuge to collect bacterial cells, and wash the cells twice with antibiotic-free LB. Collect spores of *S. endus subsp. aureus* Δ3P1 and *S. endus subsp. aureus* Δ3G5 separately in 0.05 MTE buffer (pH 8.0), heat shock at 50°C for 10 min, add an equal volume of 2× spore pre-germination medium, pre-germinate at 37°C and 220 rpm for 2.5 h, then collect the spores and suspend them in LB medium. Add *E. coli* ET12567 / pUZ8002 containing pSET152-2G5 to *S. endus subsp. aureus* Δ3P1 and *S. endus* Δ3G5 separately. The spores of subsp. aureus Δ3G5 were thoroughly mixed in a certain proportion, then spread separately on SFM plates. After drying in a clean bench, the plates were incubated at 30°C for 16 hours. Then, the plates were covered with apopramycin (15 μg / mL) and trimethoprim (50 μg / mL) and incubated at 30°C for another 5-10 days. Single colonies grew. Single colonies were inoculated onto SFM plates containing apopramine (15 μg / mL) and trimethoprim (50 μg / mL) for expansion culture, and PCR verification was performed to obtain the S. endus subsp. aureus Δ3P1 mutant containing pSET152-2G5 (S. endus subsp. aureus Δ3P1::2G5) and the S. endus subsp. aureus Δ3G5 mutant containing pSET152-2G5 (S. endus subsp. aureus Δ3G5::2G5). Their HPLC chromatograms are shown in [Figure 1]. Figure 3 .

[0201] Example 5: Fermentation of mutant strains and product isolation and purification

[0202] The *S. endus subsp. aureus* mutants obtained in Example 2.3 with 3G5, 3P3, 3P1, and 3M knocked out (*S. endus subsp. aureus Δ3G5, *S. endus subsp. aureus Δ3P3, *S. endus subsp. aureus Δ3P1, *S. endus subsp. aureus Δ3M*), and the *S. hygroscopicus* A-130 mutants with 2P1 and 2M knocked out (*S. hygroscopicus A-130 Δ2P1, *S. hygroscopicus A-130 Δ2M*), and the *S. endus subsp. aureus* Δ3P1 mutant containing pSET152-2G5 obtained in Example 4.2 (*S. endus subsp. aureus Δ3P1::2G5*), and the *S. endus* Δ3P1 mutant containing pSET152-2G5, were also included. The *S. endus* subsp. *aureus* Δ3G5 mutant strain (*S. endus* subsp. *aureus* Δ3G5: 2G5) was subjected to liquid fermentation culture. First, the mutant strain was transferred to SFM plates and incubated at 30°C for 4-5 days. Then, it was transferred to 50 mL of TSB medium (30 g / L tryptone soybean broth) and incubated at 30°C and 220 rpm for 2 days. Finally, it was inoculated at a 5% inoculum into fermentation medium (30 g / L soluble starch, 10 g / L soybean meal, 2.5 g / L yeast extract, 3 g / L CaCO3, pH 7.2) and incubated at 30°C for 8 days. After fermentation, the cells were soaked in methanol and extracted using ultrasound three to four times at room temperature. The extracts were combined, and the methanol was removed by vacuum concentration to obtain an aqueous suspension. The extract was extracted with ethyl acetate to obtain a total extract, which was then purified by silica gel column chromatography, reverse gel column chromatography and semi-preparative HPLC. Finally, six new polyether compounds (End-3, End-4, End-5, End-16, Len-10, Len-11) and one previously reported compound x-14931A (named End-2) were obtained. The NMR data of the six new polyether compounds are shown in Tables 4-6, and the NMR data of End-2 are shown in Table 7.Among them, End-3 was prepared by fermentation of *Streptomyces endus subsp. aureus* Δ3G5 with the 3G5 gene knocked out; End-4 was prepared by fermentation of *Streptomyces endus subsp. aureus* Δ3P3 with the 3P3 gene knocked out; End-5 was prepared by fermentation of *Streptomyces endus subsp. aureus* Δ3M with the 3M gene knocked out; Len-10 was prepared by fermentation of *Streptomyces hygroscopicus* A-130Δ2P1 with the 2P1 gene knocked out; Len-11 was prepared by fermentation of *Streptomyces hygroscopicus* A-130Δ2M with the 2M gene knocked out; and End-16 was prepared by fermentation of *Streptomyces endus* Δ3M with the 3G5 gene knocked out and the 2G5 gene introduced. End-2 was prepared by fermentation of either a *Streptomyces endus* mutant strain with the 3P1 gene knocked out and the 2G5 gene introduced (*S. endus subsp. aureus Δ3G5::2G5*) or a *Streptomyces endus* mutant strain with the 3P1 gene knocked out and the 2G5 gene introduced (*S. endus subsp. aureus Δ3G5::2G5*) or a *Streptomyces endus* mutant strain with the 3P1 gene knocked out and the 2G5 gene introduced (*S. endus subsp. aureus Δ3G5::2G5*).

[0203] Table 4. NMR data of compounds End-3 and End-4

[0204]

[0205]

[0206] Table 5 NMR data for compounds End-5 and Len-10

[0207]

[0208]

[0209] Table 6. NMR data of compounds Len-11 and End-16

[0210]

[0211]

[0212] Table 7 NMR data for compound End-2

[0213]

[0214]

[0215] Example 6: In vitro anti-tumor cell activity test

[0216] The six novel polyether compounds obtained in Example 5, as well as structurally similar compounds endusamycin, lenoremycin, and X-14931A (named End-2), were tested for in vitro antitumor cell proliferation activity using the CCK-8 assay (Cell Counting Kit-8), including HeLa cells (human cervical cancer cells) and Jurkat cells (human acute T-cell leukemia cells). Tumor cells were cultured in culture dishes until the logarithmic growth phase, then digested, and the cells were collected and counted using a cell counting chamber. Cells were then seeded at a density of 3000 cells per well into 96-well plates and pre-cultured at 37°C for 24 hours in a constant temperature incubator containing 5% carbon dioxide. The culture medium was then replaced with the corresponding culture medium containing the test drugs (End-3, End-4, End-5, End-16, Len-10, Len-11) at concentrations of 0, 0.5, 5, and 50 μmol / L, with three replicates for each concentration of each drug. Incubate the culture plate in an incubator for 48 hours, add 10 μL of CCK-8 solution (containing substrate WST-8) to each well, and incubate for another 1-4 hours in an incubator. Measure the absorbance at 450 nm using a microplate reader.

[0217] Cell viability was calculated using the following formula: [(As-Ab) / (Ac-Ab)]×100%. As: Absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution); Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding drug); Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drug). The IC50 was calculated based on the change in cell viability with drug concentration. 50 .

[0218] The results showed that End-4, End-5, End-16, Len-10, and Len-11 had significant inhibitory effects on HeLa cells (human cervical cancer cells) and Jurkat cells (human acute T-cell leukemia cells), and their inhibitory activity was comparable to that of endusamycin and lenoremycin.

[0219]

[0220] References:

[0221] [1] Liu Ran. Biosynthetic gene clusters of compounds and their applications [P]. Hubei Province: CN106916836A.

[0222] [2] Liu Ran. Biosynthetic gene clusters of compounds and their applications [P]. Hubei Province: CN106916834A.

[0223] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. The following compounds, their pharmaceutically acceptable salts, and stereoisomers: 、 、 。 2. A pharmaceutical composition, characterized in that, This includes the compound of claim 1, its pharmaceutically acceptable salt, stereoisomer, and pharmaceutically acceptable carrier.

3. Use of the compound of claim 1, its pharmaceutically acceptable salt, stereoisomer, or the composition of claim 2 in the preparation of an antitumor drug; The tumor is either cervical cancer or leukemia.

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