WO2020177568A1 - Novel ll-d49194 α1 analog, preparation method therefor and application thereof - Google Patents

Abstract

The present invention relates to an LL-D49194α1 analog, a preparation method therefor and an application thereof. Specifically, provided by the present invention is a deglycosylated and demethylated LL-D49194α1 analog. The obtained LL-D49194α1 analog has a better antitumor activity than LL-D49194α1.

Description

Novel LL-D49194 α1 analogue, and its preparation method and application Technical field

The present invention belongs to the field of biotechnology engineering and the field of medicine. Specifically, the present invention relates to a new type of LL-D49194 α1 analogue and its preparation method and application.

Background technique

Anthracycline antibiotics have an anthracycline skeleton and also include methylation, glycosyl modification, hydroxylation and other special modifications. Anthracycline antibiotics are mainly used clinically to treat leukemia, melanoma, lymphoma, lung cancer, breast cancer, uterine cancer and ovarian cancer.

LL-D49194s is a type of polyketone compound with an anthraquinone skeleton. The oxygen-containing three-membered spiro ring is the active unit in its structure, which can inhibit the synthesis of DNA and RNA. In the early separation of LL-D49194 α1, other LL-D49194 α1 analogues were obtained, including LL-D49194 β1, LL-D49194 β2, etc., especially LL-D49194α1 has the highest yield and activity, and there are many early pharmaceutical studies Take LL-D49194 α1 as the main research target. Most of the LL-D49194 compounds have good anti-Gram-positive bacteria activity, the cancer cell inhibition ability is equivalent to or better than cisplatin, and the relative cytotoxicity is also lower.

Most anthracycline antibiotics are mainly different in the side chain glycosyl structure, and there are few studies on the modification of anthracycline skeleton. In order to improve the anti-tumor activity and other properties of LL-D49194 α1, the art has been trying to modify the anthracycline skeleton to obtain analogs or derivatives of LL-D49194 α1. However, LL with higher activity and other advantages has not been developed so far. -D49194 α1 analogue. Therefore, the field urgently needs to develop various new types of LL-D49194 α1 analogs.

Summary of the invention

The purpose of the present invention is to provide a new class of LL-D49194 α1 analogs with higher activity.

Another object of the present invention is to provide a preparation method and application of the LL-D49194 α1 analog.

In the first aspect of the present invention, there is provided a novel LL-D49194 α1 analogue, or a pharmaceutically acceptable salt thereof, characterized in that the structure of the compound is as shown in formula I:

In formula I, R _{1 is} selected from: H, hydroxyl, halogen, C _1-4 alkyl, and C _1-4 alkoxy;

R _{2 is} selected from: H, hydroxyl, C _1-4 alkoxy,

R _{3 is} selected from the group consisting of H, C _1-4 alkyl.

In another preferred embodiment, said R _{1 is} selected from: H, hydroxyl, C _1-4 alkoxy; said R _{2 is} selected from: H, hydroxyl,

In another preferred example, the compound has a structure selected from the following group of formula Ia, Ib or Ic:

The second aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising: the LL-D49194 α1 analogue of any one of the first aspect of the present invention, or a pharmaceutically acceptable salt thereof; and Acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is used to treat tumors; preferably, the tumors are selected from the following group: leukemia, melanoma, lymphoma, lung cancer, breast cancer, uterine cancer, and ovarian cancer.

The third aspect of the present invention provides a mutant strain of Streptomyces vinaceusdrappus NRRL15735 that can be used to produce the LL-D49194 α1 analogue as described in the first aspect of the present invention, characterized in that the In the strain, a gene selected from the group of LL-D49194 α1 biosynthetic gene cluster is inactivated and knocked out: cytochrome P450 oxidoreductase gene (lldO10), glycosyltransferase gene (lldB3), or cytochrome P450 Oxidoreductase gene (lldO2).

In another preferred example, the mutant strain is a recombinant strain with lldO10, lldB3 or lldO2 deleted in the same frame.

The fourth aspect of the present invention is a method for preparing the LL-D49194 α1 analog or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention, the method comprising the steps:

(a) Fermenting the strain described in the third aspect of the present invention to obtain a fermentation product; and

(b) Separating the LL-D49194 α1 analog from the fermentation product, and optional step (c)

(c) Converting the compound into its pharmaceutically acceptable salt.

In another preferred embodiment, the step (b) includes:

(b1) Centrifuge the fermentation product and discard the supernatant to obtain a mixture of bacteria and HP20 adsorption resin;

(b2) Soak the mixture in an organic solvent, centrifuge to obtain the supernatant;

(b3) Drain the supernatant by vacuum distillation to obtain a paste;

(b4) Column chromatography is performed on the paste with a pre-installed 200-300 mesh silica gel column, the effluent containing the target compound is collected, and then purified by HPLC to obtain the target product.

In another preferred embodiment, the eluent of the column chromatography is selected from the following group: dichloromethane: methanol = 75-50:1 (v/v), dichloromethane: methanol = 20-10:1 ( v/v), dichloromethane: methanol = 30-20:1 (v/v).

In another preferred example, when the mutant strain is a recombinant strain lacking lldO10, the LL-D49194 α1 analog is a compound of formula Ia.

In another preferred example, when the mutant strain is a recombinant strain with a deletion of 11dB3, the LL-D49194 α1 analog is a compound of formula Ib.

In another preferred example, when the mutant strain is a recombinant strain lacking lldO2, the LL-D49194 α1 analog is a compound of formula Ic.

In another preferred embodiment, the fermentation is liquid shake flask fermentation.

In another preferred embodiment, the fermentation includes: first culturing the strain to obtain a fermentation seed culture solution, and then inoculating it into a fermentation medium for secondary culture.

In another preferred example, the inoculation amount is 5%-10%, based on the volume of the fermentation medium.

In another preferred example, the secondary culture further includes: after the strain enters the stable growth phase, adding 2%-3% macroporous resin HP20 for co-cultivation and further fermentation to obtain the LL-D49194 α1 analog.

In another preferred embodiment, the primary culture is carried out in a TSB culture flask without resistance.

The fifth aspect of the present invention provides an application of the LL-D49194 α1 analogue or a pharmaceutically acceptable salt thereof as an antitumor drug as described in the first aspect of the present invention.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

Description of the drawings

The following drawings are used to illustrate the specific embodiments of the present invention, but not to limit the scope of the present invention defined by the claims.

Figure 1 shows the results of electrophoresis of Streptomyces vinaceusdrappus NRRL15735 in-frame knockout mutants. Figure 1i shows the sDL05030 knockout mutant; Figure 1ii shows the sDL05021 knockout mutant; Figure 1iii shows the sDL05027 knockout mutant.

Figure 2 shows the ¹ H NMR (600 MHz, CDCl ₃ ) of LL-D49194 α2.

Figure 3 shows the ¹³ C NMR (600 MHz, CDCl ₃ ) of LL-D49194 α2.

Figure 4 shows the COSY (600MHz, CDCl ₃ ) of LL-D49194 α2.

Figure 5 shows the HSQC (600MHz, CDCl ₃ ) of LL-D49194 α2.

Figure 6 shows the HMBC (600MHz, CDCl ₃ ) of LL-D49194 α2.

Figure 7 shows the NOESY (600MHz, CDCl ₃ ) of LL-D49194 α2.

Figure 8 shows the ¹ H NMR (600 MHz, CDCl ₃ ) of LL-D49194 α3.

Figure 9 shows the ¹³ C NMR (600 MHz, CDCl ₃ ) of LL-D49194 α3.

Figure 10 shows the COSY (600MHz, CDCl ₃ ) of LL-D49194 α3.

Figure 11 shows the HSQC (600MHz, CDCl ₃ ) of LL-D49194 α3.

Figure 12 shows the HMBC (600MHz, CDCl ₃ ) of LL-D49194 α3.

Figure 13 shows the NOESY (600MHz, CDCl ₃ ) of LL-D49194 α3.

Figure 14 shows the ¹ H NMR (600 MHz, CDCl ₃ ) of LL-D49194 α4.

Figure 15 shows the ¹³ C NMR (600 MHz, CDCl ₃ ) of LL-D49194 α4.

Figure 16 shows the COSY (600MHz, CDCl ₃ ) of LL-D49194 α4.

Figure 17 shows the HSQC (600MHz, CDCl ₃ ) of LL-D49194 α4.

Figure 18 shows the HMBC (600MHz, CDCl ₃ ) of LL-D49194 α4.

Figure 19 shows the NOESY (600 MHz, CDCl ₃ ) of LL-D49194 α4.

Figure 20 shows the HPLC analysis results of the novel LL-D49194 α1 analogue obtained by optimizing the fermentation conditions of the mutant. Figure 20i is the fermentation result of the wild-type Streptomyces vinaceusdrappus NRRL15735; Figure 20ii is the fermentation result of the mutant sDL05030, and sDL05030 is the production LL-D49194 α2 strain; Figure 20iii is the fermentation result of mutant sDL05021, sDL05021 is the strain producing LL-D49194 α3; Figure 20iv is the fermentation result of mutant sDL05027, sDL05027 is the strain that produces LL-D49194 α4.

Symbol Description

In formula Ia, A, B, C, D, E, F represent the number of the ring, the C ring represents the ring numbered C, the numbers 1, 2, 3, 4 represent the numbering sequence of the carbon skeleton of the compound, and the C-2 position is Represents the carbon atom numbered 2.

Specific implementation method

The present inventors studied the biosynthetic mechanism of LL-D49194 α1 and used the method of constructing gene-in-frame deletion mutants, and unexpectedly discovered that when the cytochrome P450 oxidoreductase gene (lldO10) or glycosyltransferase is deleted When the mutant strains of the gene (lldB3) or the cytochrome P450 oxidoreductase gene (lldO2) are subjected to liquid fermentation, the LL-D49194 α1 analogue LL- dehydroxylated at the C-2 position of the C ring and reduced at the C-4 position are prepared respectively. D49194 α2, LL-D49194 α3, LL-D49194 α4, through anti-HL-60 and B16-F10 tumor cell activity experiments, it was found that the inhibitory activity was increased by 2-4 times. The present invention has been completed on this basis.

Active ingredient

As used herein, the terms "compounds of the present invention", "active ingredients of the present invention", "analogs of the present invention" and "LL-D49194 α1 analogs of the present invention" can be used interchangeably, and all refer to LL-D49194 α1 analogs Such as LL-D49194 α2 shown in formula Ia, LL-D49194 α3 shown in Ib and LL-D49194α4 shown in Ic.

It should be understood that the term is also intended to include various crystal forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of the present invention.

In a preferred embodiment of the present invention, the term "C _1-4 alkyl" refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl Group, isobutyl, sec-butyl, tert-butyl, or similar groups; the term C _1-4 alkoxy methoxy, ethoxy, propoxy, isopropyloxy, or similar groups; The term "halogen" refers to F, Cl, Br and I.

A preferred class of LL-D49194 α1 analogs has the chemical structural formula shown in the following formula I:

As used herein, the term "LL-D 49194α2" has the chemical structural formula shown in Formula Ia.

As used herein, the term "LL-D 49194α3" has a chemical structure as shown in Formula Ib.

As used herein, the term "LL-D 49194α4" has a chemical structural formula shown in Formula Ic.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts.

The inventors not only confirmed the structure of the compound through fermentation, separation and purification, and structure analysis of the novel LL-D49194 α1 analog. In addition, it was also confirmed through tumor cell suppression experiments that the LL-D49194 α1 analog of the present invention has a significantly higher tumor cell suppression ability than the LL-D49194 α1 natural product.

Strain

As used herein, the term "the starting strain of the present invention" refers to Streptomyces vinaceusdrappus NRRL 15735 (purchased from the American Agricultural Research Institute (ARS)), which was obtained from the Microorganism Collection of the United States Department of Agriculture. Streptomyces vinaceusdrappus NRRL15735 in Maiese W M. et al. "LL-D49194 Antibiotics, New Anti-tumor Drug Family: Taxonomy, Fermentation and Biological Characteristics[J]. The Journal of Antibiotics, 1990 , 43(3):253-258." and US4626503. It should be understood that the starting strain not only includes the strain of Streptomyces vinaceusdrappus NRRL15735, but also its derivative strains and other strains that produce LL-D49194 α1.

In a preferred embodiment of the present invention, there is provided a mutant strain of Streptomyces vinaceusdrappus NRRL15735, which produces the LL-D49194 α1 analog of the present invention, wherein the LL-D49194 α1 biosynthetic gene cluster is in the strain The cytochrome P450 oxidoreductase gene (lldO10), or glycosyltransferase gene (lldB3) or cytochrome P450 oxidoreductase gene (lldO2) in the cytochrome P450 oxidoreductase gene is inactivated and knocked out.

The present invention also provides a method for constructing mutant strains that can produce LL-D49194 α1 analogues, which includes the construction of in-frame knockout plasmids, the plasmids with screening resistance are introduced into wild-type strains, and the plasmids carry the same The source arm segment is recombined with the homologous segment of the wild-type strain genome, thereby replacing the corresponding gene from the genome, thereby obtaining mutant strains with genes knocked out or deleted in frame. Mutants that lack the cytochrome P450 oxidoreductase gene (lldO10), glycosyltransferase gene (lldB3) or cytochrome P450 oxidoreductase gene (lldO2) in the LL-D49194 α1 biosynthetic gene cluster cannot synthesize LL- normally D49194 α1.

In a preferred embodiment of the present invention, a method for constructing a mutant strain and fermenting the mutant strain to prepare an analog of LL-D49194 α1 is provided, which includes:

1. Construction of lldO10, lldB3 and lldO2 in-frame deletion plasmids

The gene sequence of lldO10, lldB3 and lldO2 and the corresponding protein sequence

PCR was used to amplify the left arm and right arm fragments used for gene knockout of lldO10, lldB3 and lldO2 genes, and the two fragments of the left and right arms were connected into the temperature-sensitive shuttle plasmid pKC1139 (US5,955,319) to obtain the recombinant plasmid. The recombinant plasmids were transformed into E. coli DH5α, and the monoclonal colonies were selected for amplification and culture, and then the verified plasmids were transformed into E. coli S17-1 respectively.

2. Preparation of recombinant strains

Fresh spores of wild-type Streptomyces vinaceusdrappus NRRL15735 were collected and washed three times with TES buffer solution. The washed spores were resuspended in 500uL TES buffer and placed in a 50℃ water bath for 10 minutes. The heat-shocked spores were transferred to 37°C to germinate for about 4 hours, mixed and smeared with E.coli S17-1 containing recombinant plasmid on the IWL-4 medium plate according to a certain ratio, and incubated at 30°C for 16 hours before using Anpu Cover the plate with mycin. The zygote grows after 5 to 7 days of incubation, and the monoclonal zygote is selected and cultured on an apramycin resistant plate at 37°C for 2 to 3 days. Select the well-growing zygote to passage more than 8 times in the anti-TSB culture solution, and then streak a single colony on the IWL-4 medium plate. Through apramycin resistance screening, strains without apramycin resistance were selected for genotype PCR verification to obtain recombinant strains with lldO10, lldB3 and lldO2 deleted in frame.

3. Optimize liquid fermentation program

The present invention provides a culturing plan for strains producing LL-D49194α1 and its analogues, which adopts the method of liquid shake flask for fermentation, and at the same time optimizes the amount of oxygen, temperature, inoculum, etc., and adopts the first-level seed fermentation method , Firstly incubate in a TSB culture flask without resistance for 36 hours to prepare a fermentation seed culture solution, and then inoculate 5%-10% (based on the volume of the fermentation medium) into the fermentation medium, and the strain will stabilize in 2-3 days After the growth period, 2%-3% macroporous resin HP20 is added for co-cultivation, and LL-D49194α1 and its analogs can be obtained stably after 6 days of fermentation.

4. Preparation of active ingredients

The present invention also provides a method for preparing the compound of formula I, which includes the steps of: centrifuging and discarding the supernatant to obtain a mixture of bacteria and HP20 adsorption resin; after immersing the bacteria and HP20 adsorption resin in 2 times the volume of acetone, centrifugation to take the supernatant; After the supernatant was distilled and drained under reduced pressure, the obtained paste was crudely fractionated with a pre-installed 200-300 mesh silica gel column, and then further prepared and purified by HPLC. The effluent containing the compound of formula I was collected and drained. Finally get the target product.

The present inventors not only confirmed the structure of the compound through a large amount of fermentation and separation and purification of the compound of formula I, but also confirmed that the active product of the present invention is significantly improved compared with the tumor cell suppression ability of LL-D49194α1 through tumor cell suppression experiments.

The main advantages of the present invention include:

(a) A novel LL-D49194 α1 analogue with the structure of formula I and the advantages of higher activity is provided.

(b) Based on the LL-D49194 α1 analog of the present invention, it is helpful to prepare other anthracycline derivatives.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not specify specific conditions in the following examples usually follow the conventional conditions such as those described in Sambrook et al., Molecular Cloning Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described by the manufacturer. Suggested conditions. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the content described can be applied to the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only, but cannot limit the content of the present invention.

Example 1. Construction of mutant strains sDL05030, sDL05021 and sDL05027

1. Construction of deletion plasmids for lldO10, lldB3 and lldO2 genes

The primer sequence of the left arm of clone lldO10 in-frame deletion is as follows:

lldO10 left arm forward primer (SEQ ID NO: 1)

5’-ATAGAATTCACGAGGGTGCGGTCGTCT-3’

lldO10 left arm reverse primer (SEQ ID NO: 2)

5’-ATATCTAGAATCGGCGACCAGGGCCTC-3’

The primer sequence of the right arm of clone lldO10 in-frame deletion is as follows:

lldO10 right arm forward primer (SEQ ID NO: 3)

5’-ATATCTAGACAACGCCTGCCGGTCCGGTTC-3’

lldO10 right arm reverse primer (SEQ ID NO: 4)

5’-ATAAAGCTTACGTTCGCGCTGCCCCGGTTC-3’

The primer sequence of the left arm of clone lldB3 in-frame deletion is as follows:

lldB3 left arm forward primer (SEQ ID NO: 5)

5’-ATAGAATTCCCTGTTCCAGCTCCGCCTGG-3’

lldB3 left arm reverse primer (SEQ ID NO: 6)

5’-ATATCTAGAACCCTGGAGAGGCTCACCGC-3’

The primer sequence of the right arm of clone lldB3 in-frame deletion is as follows:

lldB3 Right arm forward primer (SEQ ID NO: 7)

5’-ATATCTAGAGTGAGCCTGGACGGGCCACA-3’

lldB3 right arm reverse primer (SEQ ID NO: 8)

5’-ATAAAGCTTGGAGCAGCCGCCTCATCAAG-3’

The primer sequence of the left arm of clone lldO2 in-frame deletion is as follows:

lldO2 left arm forward primer (SEQ ID NO: 9)

5’-ATAGAATTCCGCCGGCGTCTGCGGCCA-3’

lldO2 left arm reverse primer (SEQ ID NO: 10)

5’-ATATCTAGAGGTGTCGACCTCAGTGGCCAT-3’

The primer sequence of the right arm of clone lldO2 in-frame deletion is as follows:

lldO2 right arm forward primer (SEQ ID NO: 11)

5’-ATATCTAGACCGGTCCGGATCCGCTGAC-3’

lldO2 right arm reverse primer (SEQ ID NO: 12)

5’-ATAAAGCTTCGTGCCGGACGGCCCCGA-3’

Take the total DNA of Streptomyces vinaceusdrappus NRRL15735 as a template, use DMSO, dNTP, enzyme-free water, forward and reverse primers, high-fidelity KOD DNA polymerase and buffer to form a PCR reaction system, which is amplified for lldO10, lldB3 and lldO2 respectively The left and right arm fragments of the gene knockout. The cloned left and right arm fragments were separated by gel electrophoresis, gel cut, recovered and purified, and the restriction enzymes EcoRI and XbaI and XbaI and HindIII were added to digest the recovered fragments, which were connected to the restriction enzymes EcoRI and HindIII digested vector plasmid pKC1139, and then the ligation system was transformed into E.coli DH5α, and a single colony was picked and cultured in an LB culture tube (containing apramycin antibiotic) overnight with shaking. The extracted plasmid is verified by restriction digestion and sent to sequencing for further verification. Transform the verified plasmid into E.coli S17-1.

2. Obtaining of recombinant strains sDL05030, sDL05021 and sDL05027

Fresh spores of wild-type Streptomyces vinaceusdrappus NRRL15735 were collected and washed three times with TES buffer solution. The washed spores were resuspended in 500uL TES buffer and placed in a 50℃ water bath for 10 minutes. The heat-shocked spores were transferred to 37°C to germinate for about 4 hours, mixed and smeared with E.coli S17-1 containing the recombinant plasmid on the IWL-4 medium plate in a certain proportion, and cultured at 30°C for 16 hours. Cover the plate with pramycin (50 μg/mL). The zygote grows after 5 to 7 days of incubation, and the monoclonal zygote is selected and cultured on an apramycin-resistant plate at 37°C for 2 to 3 days. Select the well-growing zygote to passage more than 8 times in the non-resistant TSB culture solution, and then streak a single colony on the plate. Through apramycin resistance screening, the strains without apramycin resistance were selected for genotype PCR verification to obtain recombinant strains sDL05030, sDL05021 and sDL05027 with the same frame deletion of lldO10, lldB3 and lldO2 genes. The experimental results are shown in Figure 1.

Figure 1i shows the verification result of the sDL05030 knockout mutant. From the electropherogram in Figure 1i, it can be seen that the sDL05030 gene in the sDL05030 knockout mutant is deleted by about 1.15kb, indicating that the sDL05030 knockout mutation was successfully constructed in this example. Strain.

Figure 1ii shows the verification result of the sDL05021 knockout mutant. From the electropherogram in Figure 1ii, it can be seen that the sDL05021 gene in the sDL05021 knockout mutant is missing about 1.16 kb, indicating that the sDL05021 knockout mutation was successfully constructed in this example. Strain.

Figure 1iii shows the verification result of the sDL05027 knockout mutant. From the electropherogram of Figure 1iii, it can be seen that the sDL05027 gene in the sDL05027 knockout mutant is deleted by approximately 1.18kb, indicating that the sDL05027 knockout mutation was successfully constructed in this example Strain.

Example 2. Fermentation, detection, separation and purification and structure identification of LL-D49194 α1 analog

The mutant strains sDL05030, sDL05021, and sDL05027 were respectively inoculated into 100mL seed culture medium and cultured with shaking (including TSB 30g/L), rotating at 220 rpm and temperature at 30°C for 36 hours. Inoculate 5-10mL seed shake flask bacteria liquid in 100mL fermentation medium (containing soluble starch 60g/L, glucose 10g/L, yeast extract 10g/L, sodium chloride 3g/L, dipotassium hydrogen phosphate 1g/L , Magnesium sulfate heptahydrate 1g/L, calcium carbonate 2g/L, trace salt solution 0.1mL (1000 times storage solution: copper sulfate pentahydrate 70g/L, ferrous sulfate heptahydrate 10g/L, manganese chloride tetrahydrate 8g/ L, zinc sulfate heptahydrate 2g/L, cobalt chloride heptahydrate 0.06g/L), pH=7.3±0.2), cultivate at 30℃, 220rpm, add HP20 resin (3g/100mL) after 2-3 days of inoculation, continue The fermentation broth was processed after cultivation until the 6th day after inoculation.

Separately centrifuge the sDL05030, sDL05021 and sDL05027 fermentation broths to collect the precipitate, soak the precipitate twice with 2 times the volume of acetone, spin off the acetone on a rotary evaporator, extract four times with ethyl acetate, and drain the ethyl acetate by vacuum distillation to obtain Dark brown paste. Separate the pastes respectively, and use 200-300 mesh silica gel pre-packed normal phase column for crude separation. The gradient elution conditions are shown in Table 1.

Table 1

LL-D49194 α1 analogue LL-D49194 α2 (structure shown in formula Ia) was obtained by separating the fermentation product of sDL05030, and appeared in the elution fraction of 75:1 and 50:1 (dichloromethane: methanol).

LL-D49194 α3 (structure shown in formula Ib) was obtained by separating the fermentation product of sDL05021, and it appeared in the elution fraction of 20:1 and 10:1 (dichloromethane: methanol).

LL-D49194 α4 (structure shown in formula Ic) is obtained by separating the fermentation product of sDL05027, which appears in the elution part of 30:1 and 20:1 (dichloromethane: methanol), and collects containing LL-D49194 α2, LL-D49194 The eluents of α3 and LL-D49194 α4 were drained under reduced pressure, dissolved in 3ml methanol, and then purified by HPLC.

The semi-preparative conditions of HPLC are:

Instrument: Shimadzu LC-20-AT (Shimadzu Corporation, Japan)

Detection wavelength: UV=400nm

Column: YMC-Pack ODS-AQ, C18column, 10×250mm, 5μM (Japan YMC company)

Mobile phase: A=H ₂ O; B=CH ₃ CN

Flow rate: 2mL/min

See Table 2 for the mobile phase gradient ratio.

Table 2

Time (min)	A%		B%
0	80	20
5	80	20
20	35	65
twenty one	10	90
twenty four	10	90
26	80	20
30	80	20

According to the above-mentioned HPLC elution conditions, the effluents of LL-D49194 α2, LL-D49194 α3 and LL-D49194 α4 were collected, and finally the target products LL-D49194 α2, LL-D49194 α3 and LL-D49194 α4 were obtained. The target product LL-D49194 α2 was identified, and the NMR assignment results are shown in Table 3 (CD ₃ Cl, 600MHz); the target product LL-D49194 α3 was identified, and the NMR assignment results were shown in Table 4 (CD ₃ Cl, 600MHz); The product LL-D49194 α4 was identified, and the NMR assignment results are shown in Table 5 (CD ₃ Cl, 600MHz).

table 3

HR-ESI-MS (m/z): [M+H] ⁺ measured value is 457.1129 (calculated value for C ₂₃ H ₂₁ O ₁₀ is 457.1129).

Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7 are the ¹ H NMR (CD ₃ Cl, 600MHz), ¹³ C NMR (CD ₃ Cl, LL-D49194 α2 (structure shown in formula Ia), 600MHz), COSY (CD ₃ Cl, 600MHz), HSQC (CD ₃ Cl, 600MHz), HMBC (CD ₃ Cl, 600MHz) and NOESY (CD ₃ OD, 600MHz) spectra. Structure identification showed that the inventors successfully prepared LL-D49194 α2 (structure shown in Ia).

Table 4

HR-ESI-MS (m/z): [M+H] ⁺ measured value is 487.1235 (calculated value for C ₂₄ H ₂₃ O ₁₁ is 487.1235).

Figure 8, Figure 9, Figure 10, Figure 11, Figure 12 and Figure 13 are the ¹ H NMR (CD ₃ Cl, 600MHz), ¹³ C NMR (CD ₃ Cl, LL-D49194 α3 (structure shown in formula Ib) 600MHz), COSY (CD ₃ Cl, 600MHz), HSQC (CD ₃ Cl, 600MHz), HMBC (CD ₃ Cl, 600MHz) and NOESY (CD ₃ OD, 600MHz) spectra. Structure identification showed that the inventors successfully prepared LL-D49194 α3 (structure shown in Ib).

table 5

HR-ESI-MS (m/z): [M+Na] ⁺ measured value is 811.2786 (calculated value for C ₃₉ H ₄₈ O ₁₇ is 811.2784).

Figure 14, Figure 15, Figure 16, Figure 17, Figure 18 and Figure 19 are the ¹ H NMR (CD ₃ Cl, 600MHz), ¹³ C NMR (CD ₃ Cl, LL-D49194 α4 (structure shown in formula Ic) 600MHz), COSY (CD ₃ Cl, 600MHz), HSQC (CD ₃ Cl, 600MHz), HMBC (CD ₃ Cl, 600MHz) and NOESY (CD ₃ OD, 600MHz) spectra. Structural identification showed that the inventors successfully prepared LL-D49194 α4 (structure shown in Ic).

Example 3. HPLC analysis of fermentation products of original strain and modified strain

The detection conditions of HPLC (Figure 20) are:

Instrument: Dionex Ultimate 3000 system

Detection wavelength: UV=400nm

Column: Acclaim PolarAdvantage II, C18,

4.6×250mm, 5μm, (ThermoScientific company);

Mobile phase: A=H ₂ O (containing 1‰HCOOH); B=CH ₃ CN (containing 1‰HCOOH)

Flow rate: 1mL/min

See Table 6 for the mobile phase gradient ratio.

Table 6

Time (min)	A%		B%
0	10	90
5	10	90
twenty four	90	10
26	90	10
27	10	90
31	10	90

The fermentation products were analyzed by HPLC, and the results are as follows:

Figure 20 shows the HPLC analysis results of the novel LL-D49194 α1 analogue obtained by optimizing the fermentation conditions of the mutant. Figure 20i is the fermentation result of the wild-type Streptomyces vinaceusdrappus NRRL15735; Figure 20ii is the HPLC analysis of the mutant strain sDL05030, the fermentation results indicate The mutant strain sDL05030 can produce LL-D49194 α2 (shown in formula Ia); Figure 20iii is the HPLC analysis of the mutant strain sDL05021, and the fermentation results show that the mutant strain sDL05021 can produce LL-D49194 α3 (shown in formula Ib); Figure 20iv is the mutant strain HPLC analysis of sDL05027, fermentation results showed that the mutant strain sDL05027 can produce LL-D49194 α4 (shown in formula Ic). The results show that the wild-type strain can normally produce LL-D49194 α1, and the mutant strain does not have the ability to synthesize LL-D49194 α1, but has the ability to produce LL-D49194 α1 analogs.

Example 4. Anti-tumor activity of LL-D49194 α1 analog

The anti-tumor activity of LL-D49194 α1 and its analogues was determined by the following method: adjust the concentration of cells to 3×10 ⁴ cells/mL, and inoculate 100 μL/well in 96-well flat-bottomed clear cell culture plate, respectively after 24 hours Add drugs containing different concentrations for an appropriate period of time (for example: 24 or 48 hours). After adding 10μl of CCK-8 reagent to each well for 2 hours, the absorbance OD value at 450nm (reference wavelength 630nm) was measured with a microplate reader. The blank group is a cell-free medium, the control group is added with the same volume of DMSO as the drug, and the cell survival rate is calculated = (the OD value of the experimental group-the OD value of the blank group)/(the OD value of the control group-the OD value of the blank group). After calculating the compound of the cells was determined semi-inhibitory concentration IC _50.

HL-60 and B16-F10 cells were selected for tumor cell inhibitory activity experiments. The half-inhibitory concentration (IC ₅₀ ) test results showed that the IC ₅₀ value of LL-D49194 α1 analogue was lower than the corresponding original natural product LL-D49194 α1 -4 times (Table 7), which shows that the C-ring C-2 position is dehydroxylated and the C-4 position reduced LL-D49194 α1 analog has higher anti-tumor activity, which inhibits HL-60 and B16-F10 The cell activity is 2-4 times higher than that of the corresponding original LL-D49194 α1 natural product.

Table 7

Compound	IC50(HL60, nM)	IC50(B16-F10, nM)
LL-D49194 α1	5.45±0.01	23.88±0.09
LL-D49194 α2	2.84±0.01	10.05±0.08
LL-D49194 α3	3.93±0.01	6.35±0.01
LL-D49194 α4	6.32±0.01	8.19±0.02

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A novel LL-D49194 α1 analogue, or a pharmaceutically acceptable salt thereof, characterized in that the structure of the compound is as shown in formula I:

   

   In formula I, R _{1 is} selected from: H, hydroxyl, halogen, C _1-4 alkyl, and C _1-4 alkoxy;

   R _{2 is} selected from: H, hydroxyl,

   

   R _{3 is} selected from the group consisting of H, C _1-4 alkyl.
2. The LL-D49194α1 analogue according to claim 1, or a pharmaceutically acceptable salt thereof, wherein said R _{1 is} selected from: H, hydroxyl, and C _1-4 alkoxy; and said R _{2 is} selected from: H, hydroxyl,

   
3. The LL-D49194α1 analogue according to any one of claims 1-2, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure selected from the group consisting of formula Ia, Ib or Ic:

   

   
4. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: the LL-D49194 α1 analog of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable Carrier.
5. A mutant strain of Streptomyces vinaceusdrappus NRRL15735 (Streptomyces vinaceusdrappus NRRL15735) that can be used to produce the LL-D49194 α1 analogue of any one of claims 1-3, characterized in that, among the strains, LL -D49194 In the α1 biosynthesis gene cluster, a gene selected from the following group is inactivated and knocked out: cytochrome P450 oxidoreductase gene (lldO10), glycosyltransferase gene (lldB3), or cytochrome P450 oxidoreductase gene ( lldO2).
6. A method for preparing the LL-D49194 α1 analog or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized by comprising the steps:

   (a) Fermenting the strain of claim 5 to obtain a fermentation product; and

   (b) Separating the LL-D49194 α1 analog from the fermentation product, and optional step (c)

   (c) Converting the compound into its pharmaceutically acceptable salt.
7. The method according to claim 6, wherein the fermentation is liquid shake flask fermentation.
8. 8. The method according to claim 6, wherein the fermentation comprises: first culturing the strains to obtain a fermentation seed culture solution, and then inoculating the fermentation medium to perform the second culturing.
9. The method according to claim 8, wherein the secondary culture further comprises: after the strain enters a stable growth phase, adding 2%-3% macroporous resin HP20 for co-cultivation and further fermentation to obtain LL- D49194 α1 analogue.
10. The use of the LL-D49194 α1 analog or a pharmaceutically acceptable salt thereof according to claims 1-3 as an anti-tumor drug.

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