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Indolocarbazole alkaloid with cytotoxic activity as well as preparation method and application

A technology of indolocarbazole and alkaloids, which is applied in the fields of biotechnology and active compound preparation, and can solve the structure, activity and preparation method of indolocarbazole alkaloids that have not been studied and reported cytotoxic activity, and no indolocarbazole has been reported. problems such as the test results of the toxicity of azoles, to achieve the effects of easy operation and implementation, reduced toxicity, and broad application prospects

Active Publication Date: 2018-07-20
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] However, the test results of the toxicity of indolecarbazole compounds are not reported in the patents that have been applied for at present.
In addition, there is no research report on the structure, activity and preparation method of the indolecarbazole alkaloid with cytotoxic activity obtained by the present invention

Method used

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  • Indolocarbazole alkaloid with cytotoxic activity as well as preparation method and application
  • Indolocarbazole alkaloid with cytotoxic activity as well as preparation method and application
  • Indolocarbazole alkaloid with cytotoxic activity as well as preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: Construction of M1146 / pWLI628 bacterial strain

[0057] The inventor obtained the cosmid pWLI625 containing the spc gene cluster in the early stage, and used the method of homologous recombination to replace the spcMA gene with the apramycin sulfate resistance gene aac(3)IV, and the aac(3)IV gene was digested by XbaI remove. The spcMB gene was then replaced by the aac(3)IV gene, and the aac(3)IV gene was removed by speI enzyme digestion. Load the obtained cosmid with the heterologous expression element aac(3)IV-oriT- -attP / int, introduced into Streptomyces coelicolor M1146 for heterologous expression, and obtained recombinant strain M1146 / pWLI628.

Embodiment 2

[0058] Embodiment 2: the preparation of compound 1-9

[0059] 1) Inoculate the recombinant strain M1146 / pWLI628 into a 500mL Erlenmeyer flask containing 200mL TSBY medium (3% tryptone bean soup powder, 10.3% sucrose, 0.05% yeast extract, 0.1% tryptone, sterilized at 121°C for 20min) Cultivate in a medium shaker, the culture condition is 30°C, 220rpm for 2 days, and obtain the seed liquid;

[0060] 2) The seed solution obtained in step 1) was transferred to Actinomycetes No. 2 medium (1% soluble starch, 2% glucose, 1% yeast extract, 0.3% beef extract, 0.4% corn extract, 0.05% magnesium sulfate, 0.05% potassium dihydrogen phosphate, 0.2% calcium carbonate, sterilized at 121° C. for 20 min) and cultivated on a shaker at 30° C., 220 rpm for 5 days to obtain a fermentation broth.

[0061] 3) centrifuge the fermented liquid obtained in step 2), soak the cells in acetone and ultrasonically crush them, evaporate the acetone to dryness, combine with the supernatant, extract twice with...

Embodiment 3

[0062] Embodiment 3: the characterization of compound 1 and compound 2

[0063] Compound 1: yellow amorphous powder, molecular formula is C 28 h 24 N 4 o 4 .

[0064] It was identified as 3'-N-acetyl-4'-hydroxylstaurosporine and named as staurosporine M1.

[0065] [α] 21 D +9.8(c 0.05,MeOH); UV(MeOH)λ max (logε)206(4.21),292(4.38),336(3.89),372(3.81)nm; CD(MeOH)λ max (Δε)212(-6.84),247(+1.98),273(-0.75),297.5(+3.93),314.5(+0.28),329(+0.67)nm; HR-ESIMS m / z 481.1864[M+ H] + . See Table 1 for NMR.

[0066] Compound 2: yellow amorphous powder, molecular formula is C 32 h 26 N 4 o 4 .

[0067] It was identified as 3'-[3-methylpyridin-2(1H)-one]-4'-hydroxylstaurosporine and named as staurosporine M3.

[0068] [α] 21 D +198.0(c 0.025,MeOH); UV(MeOH)λ max (logε)244(4.19),292(4.47),336(3.78),372(3.49)nm; CD(MeOH)λ max (Δε)208.5(-10.56),228.5(-2.58),237(-7.48),251.5(+2.21),264(-10.16),298(+39.59),327(+3.84),332.5(+4.05) nm; HR-ESIMS m / z531.2735[M+H] + . See Table...

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Abstract

The invention provides indolocarbazole alkaloid with cytotoxic activity as well as a preparation method thereof, aiming at the problems which exist in indolocarbazole alkaloid in the prior art. The new indolocarbazole alkaloid is obtained through a recombination strain constructed by adopting a biotechnological means and directional transformation carried out on an STA biosynthesized gene clusterspc, thereby opening up a new thought for combinatorial biosynthesis research of the indolocarbazole alkaloid. The indolocarbazole alkaloid with the cytotoxic activity has a good inhibiting effect onall cancer cell strains; compared with STA, the toxicity of the indolocarbazole alkaloid to human normal hepatocyte LO2 is greatly reduced, so that the indolocarbazole alkaloid has potential for developing a medicine for treating diseases relevant to protein kinase. In addition, the method provided by the invention is easy to operate and implement, can be used for realizing industrialized mass production, and has a broad application prospect and tremendous commercial value.

Description

technical field [0001] The invention belongs to the field of biotechnology and preparation of active compounds, and in particular relates to the construction of a recombinant bacterial strain producing indolecarbazole alkaloid staurosporine derivatives by means of biotechnology, and obtains cytotoxic activity from its fermentation product. Indolecarbazole compounds. Background technique [0002] Indolecarbazole alkaloids have diverse chemical structures and a wide range of biological activities, including antibacterial, antifungal, antiviral, inhibition of platelet aggregation, and antitumor. Among them, the anti-tumor effect has been a research hotspot. In 1977, Omura et al. isolated the first indolecarbazole alkaloid staurosporine (STA) from Streptomyces staurosporeus. Studies have shown that STA has a very strong protein kinase C (protein kinase C, PKC) inhibitory activity, IC 50 Reached 2.7nmol / L. PKC is a group of phospholipid-dependent protein serine / threonine kina...

Claims

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Application Information

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IPC IPC(8): C07D498/22A61K31/553A61P35/00A61P35/02C12P17/18C12R1/465
CPCC07D498/22C12P17/188
Inventor 李文利肖菲李花月徐明远李通
Owner OCEAN UNIV OF CHINA
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