CN104804020A - Sulfodionepiperazine compound, and preparation method and use thereof - Google Patents
Sulfodionepiperazine compound, and preparation method and use thereof Download PDFInfo
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- CN104804020A CN104804020A CN201410042650.2A CN201410042650A CN104804020A CN 104804020 A CN104804020 A CN 104804020A CN 201410042650 A CN201410042650 A CN 201410042650A CN 104804020 A CN104804020 A CN 104804020A
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Abstract
The invention relates to a microbe metabolite technology, and concretely relates to a sulfodionepiperazine compound, and a preparation method and a use thereof. The sulfodionepiperazine compound is a compound 1 or a compound 2 represented by formula I, and is prepared through fermentation of a penicillium fungus Penicillium brocae. The above fungus is preserved in China General Microbiological Culture Collection Center on Jan. 17, 2014, and has a preservation number of CGMCC 8736. Experiments show that the compound has antitumor activity, and can be used as a cell proliferation inhibitor or an antitumor preparation.
Description
Technical field
The present invention relates to microbial metabolites technology, specifically sulfo-diketopiperazine compound and its production and use.
Background technology
Tumour is one of principal disease threatening human health.Find from natural product, find that antitumor drug is one of the focus in study of pharmacy field always.Marine microorganism has aboundresources, miscellaneous feature.Ocean has the features such as high salt, high pressure, low temperature, oligotrophic, impart the pathways metabolism of marine microorganism uniqueness, the chemical structure of its meta-bolites has complexity, various feature, therefore, from marine microorganism and meta-bolites thereof, research finds that chemical structure is novel, the significant natural active matter of biological activity provides favourable conditions.On the other hand, marine microorganism is easy to artificial large scale culturing, and metabolic process can artificial regulatory, not by the season of growth and natural biology quantitative limitation, is comparatively easy to solve medicine source problem.
Summary of the invention
This object is to provide a kind of sulfo-diketopiperazine compound and its production and use.
For achieving the above object, the technical solution used in the present invention is: 1. a sulfo-diketopiperazine compound, is characterized in that: sulfo-diketopiperazine compound is the compound 1 shown in formula I or compound 2
Formula I.
2. a preparation method for sulfo-diketopiperazine compound according to claim 1, is characterized in that comprising the steps:
1) by mould Penicillium broca through fermentation culture, be then extracted with ethyl acetate 3-4 time by fermented liquid, united extraction liquid concentrates, and obtains fermentation broth coarse extract and mycelium;
Mycelium acetone-water (80-20) ultrasonication is extracted 3-4 time, and revolve after united extraction liquid and steam removing acetone, be then extracted with ethyl acetate 3-4 time, combining extraction liquid concentrates again, obtains mycelium crude extract;
Described mould (Penicillium brocae) is preserved in China General Microbiological culture presevation administrative center, preservation date: on January 17th, 2014, preserving number is: CGMCC8736;
2) after the fermentation broth coarse extract in step 1) and mycelium crude extract being merged, carry out decompression silica gel column chromatography, the petroleum ether-ethyl acetate of the elutriant of employing to be gradient be successively 20:1 to 1:1 (v/v) and gradient are the chloroform-methanol of 20:1 to 1:1 (v/v);
3) by step 2) in carry out reversed phase column chromatography using methanol-water (ratio of methanol-water) as elutriant with the component under chloroform-methanol 10:1 (v/v) gradient elution, collect the component that methanol-water 40:60 (v/v) wash-out obtains, recycle silicon is gel column chromatography eluting, the chloroform-methanol of the elutriant of purification by silica gel column chromatography to be gradient be 60:1 to 10:1 (v/v), obtains purification of target compound 1 and 2.
3., by the preparation method of sulfo-diketopiperazine compound according to claim 1, it is characterized in that: the component that described collection chloroform-methanol 30:1 (v/v) wash-out obtains is compound 2; The component that collection chloroform-methanol 10:1 (v/v) wash-out obtains is compound 1.
4., by the application of sulfo-diketopiperazine compound according to claim 1, it is characterized in that: the compound shown in described formula I is preparing the application in inhibition of cell proliferation or tumor cytotoxicity agent.
5., by the application of sulfo-diketopiperazine compound according to claim 1, it is characterized in that: the compound shown in described formula I is preparing the application in inhibition tumor cell hyperproliferation agent.
6., by the application of sulfo-diketopiperazine compound according to claim 1, it is characterized in that: the compound shown in described formula I is preparing the application in antitumor drug.
The present invention relates to and prepare the method for sulfo-diketopiperazine compound with mould (Penicillium brocae) and this compounds is preparing the purposes in inhibition of cell proliferation or antineoplastic agent.
The present invention adopts mtt assay test compounds 1 and 2 to supply examination cell strain to 8 strains: the anti-tumor activity of human glioma cells (U251), human hepatoma cell strain (HepG2), human pancreas cancer cell strain (SW1990), human cervical carcinoma cell lines (HeLa), human prostate cancer cell line (DU145), Non-small cell lung carcinoma cell strain (NCI-H460), Breast cancer lines (MCF-7) and human stomach cancer cell line (SGC-7901), test proves, compound 1 and 2 pairs of selective inhibited proliferations of tumour cell.
Therefore sulfo-diketopiperazine compound 1 and 2 of the present invention can be used as inhibition of cell proliferation or tumor cytotoxicity agent.Meanwhile, compound 1 and 2 can make the treatment of antitumor drug for tumour.The aqueous solution that compound 1 and 2 dissolves in methyl-sulphoxide or methyl-sulphoxide is used.
The advantage that the present invention has:
The present invention is newly full of to be separated mangrove forest Avicennia marina stem obtains a strain Penicillium fungi Penicillium brocae from picking up from Hainan, this bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on January 17th, 2014, preserving number is: CGMCC8736, fermentation culture is carried out to it, chemical composition is separated, Structural Identification and bioactivity research, find that the sulfo-diketopiperazine compound of two novel structures has good anti-tumor activity, have not yet to see the report of chemical structure to this compound and cell inhibitory effect activity, therefore market also there is not yet medicine related to this.
The compound 1 and 2 that the present invention relates to by fermentable cultivate then from fermented product separation and purification obtain.Specifically to be newly full of to be separated in mangrove forest Avicennia marina stem to obtain a strain endogenetic fungus mould (Penicillium brocae) from picking up from Hainan, can be cultivated by artificial fermentation and obtain compound 1 and 2 through separation and purification.Our experiments show that compound 1 pair of human glioma cells (U251), human pancreas cancer cell strain (SW1990), human cervical carcinoma cell and human hepatoma cell strain (HepG2) have stronger inhibit activities, its half-inhibition concentration IC50 is respectively 6.13 ± 1.82,2.11 ± 0.79,4.34 ± 1.72,5.58 ± 1.18 μMs.And the IC50 of corresponding positive control is respectively 10.78 ± 2.15,2.21 ± 0.39,4.98 ± 1.57,5.08 ± 1.54 μMs.Compound 2 pairs of human glioma cells (U251), Non-small cell lung carcinoma cell strain (H460) and human hepatoma cell strain (HepG2) have strong inhibit activities, and its IC50 is respectively 5.35 ± 0.47,0.89 ± 0.45,2.89 ± 0.38 μMs.And the IC50 of corresponding positive control is respectively 10.78 ± 2.15,7.65 ± 1.76,5.08 ± 1.54.Therefore, compound 1 and 2 can be used as inhibition of cell proliferation or tumor cytotoxicity agent.
Embodiment
Following specific embodiment is used for further illustrating the present invention, but the present invention is limited to absolutely not these examples.
In following embodiment, the chemical structure of the compound 1 and 2 of indication is (mark of the carbon atom in the Arabic numerals formula chemical structure in structural formula) respectively:
Embodiment 1
The fermentative production of compound 1 and 2 and separation and purification:
1) fermentative production
Produce the fermentation culture of bacterium:
Spawn culture: mould (Penicillium brocae) bacterial classification with agar-malt extract substratum (agar-malt extract substratum is malt extract 15g/L, agar 20g/L, full sea water 1000mL, pH7.4-7.8), 4 DEG C of preservations.Getting mould (Penicillium brocae) is inoculated on PDA flat board, cultivates 4 days in 28 DEG C of incubators.
It is 4cm that flat board is got size
2mycelium to put into the capacity that sterilizing fills 300ml liquid nutrient medium (liquid nutrient medium is potato 200g/L, glucose 20g/L, peptone 5g/L, yeast extract paste 3g/L, water 1000mL, pH6.5) be in the Erlenmeyer flask of 1000mL, leave standstill incubated at room temperature 30 days.
2) acquisition of medicinal extract
With gauze by mycelium and separation of fermentative broth, fermented liquid is extracted with ethyl acetate the underpressure distillation of 3 combined ethyl acetate extraction liquids and obtains crude extract.Mycelium acetone-water (acetone and volume of water are 80:20) ultrasonication extracts 3 times, and revolve after united extraction liquid and steam removing acetone, be extracted with ethyl acetate 3 times after revolving steaming, combining extraction liquid concentrates again, obtains mycelium crude extract; Above-mentioned medicinal extract and mycelium crude extract are merged.
3) separation and purification of compound
The material (45g) of above-mentioned merging is carried out decompression silicagel column (200-300 order) chromatography, and be the petroleum ether-ethyl acetate of 20:1 to 1:1 (v/v) and gradient by gradient is that 20:1 to 1:1 (v/v) chloroform-methanol carries out gradient elution (flow velocity 200ml/min) successively as solvent.
Collect the elution fraction that chloroform-methanol (10:1 (v/v)) obtains, carry out reversed phase column chromatography (using methanol-water as elutriant, flow velocity 1.5ml/min), collect the component that methanol-water 40:60 (v/v) wash-out obtains, recycle silicon is gel column chromatography eluting, with the chloroform-methanol wash-out of 60:1 to 10:1 (v/v) (flow velocity 3ml/min), namely the component that collection chloroform-methanol 30:1 (v/v) wash-out obtains obtains purification of target compound 2(59.1mg), namely the component that collection chloroform-methanol 10:1 (v/v) wash-out obtains obtains purification of target compound 1(54.1mg).
Its Structural Identification for such as shown in (I),
This compound has following physics and chemistry and spectral characteristic:
Compound 1, colorless needle crystals, specific rotatory power
-265.3 (c1.21, CH
3oH); UV (CH
3oH) λ
max(log ε) 201 (5.25) nm; Proton nmr spectra and carbon spectrum are as Table I; ESI mass spectrum m/z425 [M+H]
+, high resolution ESI mass spectrum m/z425.0836 [M+H]
+, C
18h
21o
6n
2s
2 +calculated value is 425.0836.
Compound 2, white powder, specific rotatory power
-210.3 (c1.35, CH
3oH); UV (CH
3oH) λ
max(log ε) 202 (4.46) nm; Proton nmr spectra and carbon spectrum are as Table I; ESI mass spectrum m/z423 [M+H]
+, high resolution ESI mass spectrum m/z423.0676 [M+H]
+, C
18h
19n
2o
6s
2 +calculated value is 423.0679.
The proton nmr spectra of Table I compound 1 and 2 and carbon spectrum (500MHz, in DMSO) data
A) this table signals assignment based on DEPT,
1h-
1h COSY, HSQC and HMBC spectrum analysis result, the multiplicity of carbon signal utilizes DEPT method to determine
Embodiment 2: anti-tumor activity test
Select following 8 strains for examination cell strain: human glioma cells (U251), human hepatoma cell strain (HepG2), human pancreas cancer cell strain (SW1990), human cervical carcinoma cell lines (HeLa), human prostate cancer cell line (DU145), Non-small cell lung carcinoma cell strain (NCI-H460), Breast cancer lines (MCF-7) and human stomach cancer cell line (SGC-7901) carry out anti-tumor activity test.
1) preparation of laboratory sample:
The preparation of sample solution: test sample is the sterling compound 1 and the compound 2 that are separated acquisition in above-described embodiment.Accurately take appropriate amount of sample, be mixed with the solution of desired concn by dimethyl sulfoxide (DMSO) (DMSO), for active testing.
3) growth of tumour cell inhibit activities test (mtt assay):
The present invention adopts tetrazolium staining (mtt assay) to test, have rated the growth inhibitory activity of tested sample to tumour cell.Tetrazolium MTT is that one can accept H
+yellow dyes.In viable cell mitochondrial respiratory chain, succinodehydrogenase and cytochrome C can make the tetrazole ring opening of MTT, generate hepatic formazan crystallization, and not containing this desaturase in dead cell.The growing amount of Formazan crystallization is directly proportional to viable count, and the DMSO solution of this crystallization has maximum absorption band in 570 nanometers, so the amount by detecting formazan crystallization evaluates the impact of medicine on cell proliferation.
The tumour cell of taking the logarithm vegetative period, is adjusted to 2 × 10 by cell density
5individual cells/ml, is inoculated in 96 porocyte culture plates by every hole 200 microlitre, passes in the incubator of 5% carbonic acid gas cultivate 4 hours in 37 DEG C.Sample sets 5 concentration gradients 10 respectively
-8, 10
-7, 10
-6, 10
-5with 10
-4mol/L, each concentration establishes three Duplicate Samples, and every hole adds sample liquid or each 2 microlitres of blank solution, cultivates 48 hours, and then every hole adds MTT liquid 10 microlitre that concentration is 5 mg/ml, continues cultivation 4 hours, removing supernatant liquor.Every hole adds each 100 microlitres of DMSO, and micro-oscillator shakes 10 minutes, and after dissolving completely to crystallization, microplate reader measures the light absorption value (OD value) of every hole 570 nanometers.
Get three hole mean OD value, by IR%=(OD
blank-OD
sample)/OD
blankthe inhibiting rate (IR%) of × 100% formula calculation sample on cell proliferation.
Experimental result is in table 2
Anti-tumor activity (the IC of table 2. compound 1, compound 2
50, μM)
From table 2, compound 1 pair of human glioma cells (U251), human pancreas cancer cell strain (SW1990), human cervical carcinoma cell (Hela) and human hepatoma cell strain (HepG2) have stronger inhibit activities.Compound 2 pairs of human glioma cells (U251), Non-small cell lung carcinoma cell strain (H460) and human hepatoma cell strain (HepG2) have strong inhibit activities, and its IC50 is less than positive control.
Above-mentioned the results show compound involved in the present invention has growth inhibitory activity to different tumor cell lines, they can be used as anti-tumor agent, and are expected to any acceptable salt or add clinical acceptable auxiliary material or carrier such as the form of vehicle, thinner and be applied preparing in related drugs.
Claims (6)
1. a sulfo-diketopiperazine compound, is characterized in that: sulfo-diketopiperazine compound is the compound 1 shown in formula I or compound 2
Formula I.
2. a preparation method for sulfo-diketopiperazine compound according to claim 1, is characterized in that comprising the steps:
1) by mould Penicillium broca through fermentation culture, be then extracted with ethyl acetate 3-4 time by fermented liquid, united extraction liquid concentrates, and obtains fermentation broth coarse extract and mycelium;
Mycelium acetone-water (80-20) ultrasonication is extracted 3-4 time, and revolve after united extraction liquid and steam removing acetone, be then extracted with ethyl acetate 3-4 time, combining extraction liquid concentrates again, obtains mycelium crude extract;
Described mould (Penicillium brocae) is preserved in China General Microbiological culture presevation administrative center, preservation date: on January 17th, 2014, preserving number is: CGMCC8736;
2) after the fermentation broth coarse extract in step 1) and mycelium crude extract being merged, carry out decompression silica gel column chromatography, the petroleum ether-ethyl acetate of the elutriant of employing to be gradient be successively 20:1 to 1:1 (v/v) and gradient are the chloroform-methanol of 20:1 to 1:1 (v/v);
3) by step 2) in carry out reversed phase column chromatography using methanol-water (ratio of methanol-water) as elutriant with the component under chloroform-methanol 10:1 (v/v) gradient elution, collect the component that methanol-water 40:60 (v/v) wash-out obtains, recycle silicon is gel column chromatography eluting, the chloroform-methanol of the elutriant of purification by silica gel column chromatography to be gradient be 60:1 to 10:1 (v/v), obtains purification of target compound 1 and 2.
3., by the preparation method of sulfo-diketopiperazine compound according to claim 1, it is characterized in that: the component that described collection chloroform-methanol 30:1 (v/v) wash-out obtains is compound 2; The component that collection chloroform-methanol 10:1 (v/v) wash-out obtains is compound 1.
4., by the application of sulfo-diketopiperazine compound according to claim 1, it is characterized in that: the compound shown in described formula I is preparing the application in inhibition of cell proliferation or tumor cytotoxicity agent.
5., by the application of sulfo-diketopiperazine compound according to claim 1, it is characterized in that: the compound shown in described formula I is preparing the application in inhibition tumor cell hyperproliferation agent.
6., by the application of sulfo-diketopiperazine compound according to claim 1, it is characterized in that: the compound shown in described formula I is preparing the application in antitumor drug.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106397459A (en) * | 2016-08-31 | 2017-02-15 | 中国科学院海洋研究所 | Sulfo-diketopiperazine compound and application thereof |
| CN111087402A (en) * | 2018-10-24 | 2020-05-01 | 华东师范大学 | Method for asymmetrically synthesizing Epicocin G alkaloid of ETP natural product |
| CN111205308A (en) * | 2020-01-19 | 2020-05-29 | 中国科学院海洋研究所 | Sulfo-diketone piperazine compound and preparation method and application thereof |
-
2014
- 2014-01-29 CN CN201410042650.2A patent/CN104804020B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| JIA-MING WANG等: "Study on absolute configurations of a/a0 chiral carbons of thiodiketopiperazines by experimental and calculated circular dichroism spectra", 《TETRAHEDRON》 * |
| REN XIANG TAN等: ""Isolation and Structure Assignments of Rostratins A-D, Cytotoxic Disulfides Produced by the Marine-Derived Fungus Exserohilum rostratum"", 《J. NAT. PROD. 》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106397459A (en) * | 2016-08-31 | 2017-02-15 | 中国科学院海洋研究所 | Sulfo-diketopiperazine compound and application thereof |
| CN106397459B (en) * | 2016-08-31 | 2018-07-13 | 中国科学院海洋研究所 | Thio diketopiperazine compound and its application |
| CN111087402A (en) * | 2018-10-24 | 2020-05-01 | 华东师范大学 | Method for asymmetrically synthesizing Epicocin G alkaloid of ETP natural product |
| CN111087402B (en) * | 2018-10-24 | 2022-09-20 | 华东师范大学 | Method for asymmetrically synthesizing Epicocin G alkaloid of ETP natural product |
| CN111205308A (en) * | 2020-01-19 | 2020-05-29 | 中国科学院海洋研究所 | Sulfo-diketone piperazine compound and preparation method and application thereof |
| CN111205308B (en) * | 2020-01-19 | 2022-03-15 | 中国科学院海洋研究所 | Sulfo-diketone piperazine compound and preparation method and application thereof |
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