CN105037397A - Sulfur-bridge-containing complex-ring-series alkaloids compound, preparation method and application thereof - Google Patents

Sulfur-bridge-containing complex-ring-series alkaloids compound, preparation method and application thereof Download PDF

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Publication number
CN105037397A
CN105037397A CN201510063692.9A CN201510063692A CN105037397A CN 105037397 A CN105037397 A CN 105037397A CN 201510063692 A CN201510063692 A CN 201510063692A CN 105037397 A CN105037397 A CN 105037397A
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compound
preparation
bridge
ring
series
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CN105037397B (en
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李德海
朱美林
李静
顾谦群
朱天骄
车茜
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Ocean University of China
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Ocean University of China
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms

Abstract

The invention relates to a sulfur-bridge-containing complex-ring-series alkaloids compound, a preparation method and an application thereof, wherein the novel sulfur-bridge-containing complex-ring-series alkaloids compound is produced from Penicillium sp.HDN13-309. A test proves that the compound can be used as a cell proliferation inhibitor or an anti-tumor agent.

Description

A kind of containing complicated ring system alkaloid compound of sulphur bridge and its production and use
Technical field:
The present invention relates to a strain mould Penicilliumsp.HDN13-309 (depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms centre address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, 100101 deposit numbers: CGMCC9429 preservation date: on July 9th, 2014) produce a kind of method containing the complicated ring system alkaloid compound of sulphur bridge; The invention still further relates to the purposes of this compound at anti-tumor aspect.
background technology:
The present inventor isolates a kind of containing the complicated ring system alkaloid compound of sulphur bridge from bacterial strain mould Penicilliumsp.HDN13-309 (deposit number is: CGMCC9429) liquid fermentation production.Shown in research finds, compound has anti-tumor activity, has not yet to see the chemical structure of this compound and the report of cell inhibitory effect activity, therefore market also there is not yet medicine relevant therewith.
summary of the invention:
The present invention aims to provide a kind of new compound with anti-tumor activity of novel structure.Its structural formula is
Formula I
embodiment:
Its constitutional features is: the complex structure that formula I is made up of a rarer nitrogen oxa-loop coupling six-ring and diketopiperazine ring and the structure of novelty, in addition, also containing a disulphide bridges and chlorine atom, has enriched the complexity of formula I.
Formula I of the present invention cultivates the fermented product obtained containing this compounds by fermentable, then from fermented product, adopt SephadexLH20 gel filtration chromatography, middle pressure MPLC, the method separation and purification such as half preparative HPLC obtain.The example utilizing mould Penicilliumsp.HDN13-309 (deposit number is: CGMCC9429) to prepare formula I is listed in following embodiment of the present invention.
In following embodiment, the chemical structure (Arabic numerals in structural formula are marks of carbon atom in chemical structure) of the Compound I of indication is:
Formula I
Compound I
The fermentative production of embodiment 1 Compound I and separation and purification
1 fermentative production
Produce the fermentation culture of bacterium: by the ordinary method of culturing micro-organisms, get bacterial strain mould Penicilliumsp.HDN13-309 (deposit number is: CGMCC9429) appropriate, be inoculated on PDA solid slant culture base, cultivate 5 days in 28 degrees Celsius of incubators.
Get the slant culture bacterial strain mould Penicilliumsp.HDN13-309 of 5 days appropriate, be inoculated into and 300mL substratum [substratum composition (grams per liter): maltose 20.0, glucose 10.0 is housed, N.F,USP MANNITOL 20.0, monosodium glutamate 10.0, yeast extract paste 3.0, corn steep liquor 1.0, KH 2pO 40.5, MgSO 47H 2o0.3, pH regulator to 6.5] 1000mL Erlenmeyer flask in, quiescent culture 30 days under 28 degrees celsius, obtains tunning.
The acquisition of 2 medicinal extract
With gauze, fermented liquid is separated with mycelium.By mycelium acetone extraction three times, be evaporated to without acetone, gained aqueous phase equivalent extraction into ethyl acetate three times.Fermented liquid directly uses equivalent extraction into ethyl acetate three times, combined ethyl acetate phase, and concentrating under reduced pressure, obtains crude extract, totally 8 grams.
The separation and purification of 3 compounds
Medicinal extract (8 grams), with after dissolve with methanol, is separated for eluent carries out purification on normal-phase silica gel with methyl alcohol with methylene dichloride, is divided into 6 components, and then component 4 is that solvent carries out LH20 column chromatography with methyl alcohol, is divided into 5 to flow part.Component 5, first with C-18ODS medium pressure column chromatography, is that solvent carries out gradient elution with methanol-water, is divided into again 10 components, and last component 8 is through anti-phase half preparative high-performance liquid chromatographic (methyl alcohol: water=55: 45) obtain Compound I (20mg).
Compound I white solid, molecular formula C 21h 21n 2o 8clS 2, HR-ESI-MSm/z:551.0335 [M+Na] +, calculated value 551.0326.IR(KBr)υ max3510，1660，1625，1606，1445，1384，1098，1030，709，670，652cm -1。 1h and 13c-NMR data are in table 1.
Table 1 Compound I (ind 6-DMSO) 1h and 13cNMR data (500 and 125MHz) a
A) this table signals assignment is based on DEPT, HMQC and HMBC spectrum analysis result.The multiplicity of carbon signal utilizes DEPT method to determine and represents with s (singlet), d (doublet), t (triplet) and q (quartet) respectively.
The anti-tumor activity test of embodiment 2 compound
1 laboratory sample and experimental technique
The preparation test sample of sample solution is the Compound I sterling of separation and purification in above-described embodiment 1.Precision takes appropriate amount of sample, is mixed with the solution of desired concn with DMSO, active for survey.
Srb assay: it is every milliliter 2 × 10 that Hela, HCT116, HO-8910 and MGC803 cell in vegetative period of taking the logarithm is mixed with density with fresh RPMI-1640 substratum 5the cell suspension of individual cell, is inoculated in 96 orifice plates by every hole 200 microlitre, and every hole adds sample or the blank solution of 2 μ L different concns, cultivates 72 hours at 37 DEG C.Cell after cultivating under getting drug effect, observes the morphological change that drug treating causes first under an optical microscope, determines whether Cyclin-dependent kinase, the morphological feature of apoptosis or necrocytosis.Add 80% Tricholroacetic Acid 50 μ L in every porocyte, be placed in 4 DEG C and fix 1 hour, rinse 5 times and dry air with water.Every hole adds the acetum 50 μ L of 0.4%SRB and leaves standstill 30 minutes in room temperature.Clean 4 times with 1% acetic acid water, remove unconjugated free SRB dyestuff.Every hole adds 150 μ LTris damping fluid (10mmol/L, pH10.5) soluble protein combination dyes and utilizes MD company product SPECTRAMAXPlus type microplate reader to measure optical density(OD) (OD) value of every hole at 520nm place.In same 96 orifice plate, each concentration of sample all arranges three holes, separately establishes three hole blanks and acellular zeroing hole (if medicine has color will do the acellular zeroing of relative medicine concentration).Each hole OD value first does corresponding acellular zeroing, then gets three hole mean OD value by IR%=(OD blank-OD sample)/OD blank× 100% formula calculates the cell proliferation inhibition rate (IR%) under each concentration, and adopts bliss method to calculate half inhibiting rate IC 50.
Mtt assay: HL-60 and the K562 tumour cell in vegetative period of taking the logarithm, is adjusted to every milliliter 2 × 10 by cell density 5individual cell, is added in 96 porocyte culture plates by every hole 200 μ L, passes into 5%CO in 37 DEG C 2incubator in cultivate 4h.Sample sets 5 concentration gradients respectively, and each concentration establishes 3 Duplicate Samples, establishes the positive, negative control simultaneously, every hole adds sample liquid or each 2 μ L of blank solution, cultivates 72h, and then every hole adds MTT liquid 10 μ L, continue cultivation 4 hours, 37 DEG C, 2000 revs/min centrifugal 8 minutes, suck supernatant.Every hole adds each 100 μ L of DMSO, and micro oscillator vibrates 15 minutes, and after dissolving completely to crystallization, microplate reader measures the light absorption value (OD value) at 570nm place, every hole.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, and adopt bliss method to calculate half inhibiting rate IC 50.
2 experimental results
Table 2 Compound I is to the in vitro cytotoxic effect of kinds of tumor cells
3 conclusions
Compound I has good anti-tumor activity to HCT116, and all has inhibit activities to kinds of tumor cells model, and this provides compound basis for development of new anticarcinogen.

Claims (4)

1. compound structure is shown below
Compound I
2. the preparation method of compound described in claim 1, it is characterized in that fermentation culture mould Penicilliumsp.HDN13-309 (deposit number is: CGMCC9429) obtains the fermented product containing above-claimed cpd, then from fermented product, separation and purification goes out this compounds.
3. preparation method according to claim 2, wherein by described fermented product through SephadexLH20 gel filtration chromatography, methyl alcohol is solvent, then must this compounds through anti-phase middle pressure MPLC and anti-phase half preparative HPLC separation and purification.
4. compound described in claim 1 is in the purposes of anti-tumor aspect.
CN201510063692.9A 2015-02-05 2015-02-05 Sulfur-bridge-containing complex-ring-series alkaloids compound, preparation method and application thereof Active CN105037397B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109810055A (en) * 2019-01-17 2019-05-28 广东轻工职业技术学院 A kind of new bio alkali cpd in marine fungi source and its preparing the application in anti-lung-cancer medicament

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3883561A (en) * 1972-10-16 1975-05-13 Lilly Co Eli Antibiotic lactones
CN101812079A (en) * 2010-03-08 2010-08-25 中国海洋大学 Piprazine compound containing polysulfide bond and preparation method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3883561A (en) * 1972-10-16 1975-05-13 Lilly Co Eli Antibiotic lactones
CN101812079A (en) * 2010-03-08 2010-08-25 中国海洋大学 Piprazine compound containing polysulfide bond and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LING-HONG MENG ET AL.: ""Penicibrocazines A–E, Five New Sulfide Diketopiperazines from the Marine-Derived Endophytic Fungus Penicillium brocae"", 《MARINE DRUGS》 *
REN XIANG TAN ET AL.: ""Isolation and Structure Assignments of Rostratins A-D, Cytotoxic Disulfides Produced by the Marine-Derived Fungus Exserohilum rostratum"", 《JOURNAL OF NATURAL PRODUCTS》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109810055A (en) * 2019-01-17 2019-05-28 广东轻工职业技术学院 A kind of new bio alkali cpd in marine fungi source and its preparing the application in anti-lung-cancer medicament

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