CN106011193B - A kind of preparation method of noval chemical compound spherical cavity enamine - Google Patents
A kind of preparation method of noval chemical compound spherical cavity enamine Download PDFInfo
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Abstract
The present invention provides a kind of preparation method of noval chemical compound spherical cavity enamine, comprising the following steps: 1) obtains ZJLQ129 fermentation liquid；2) spherical cavity rhzomorph is obtained；3) spherical cavity enamine is obtained.
Description
Technical field
The present invention relates to chemical field, specifically a kind of preparation method of noval chemical compound spherical cavity enamine, name He Shi ball
Chamber bacteria strain ZJLQ129 can produce metabolite dibenzofurans class compound spherical cavity rhzomorph (-) mycousunine, the chemical combination
Object, which can derive, obtains spherical cavity enamine (-) mycousunine amide, and spherical cavity enamine (-) mycousunine amide has immune
Inhibitory activity.
Background technique
Plant endogenesis epiphyte (Plantendophyticfungi) refers to those in the certain phase or whole of its history of life
Stage moves in the fungi inside the various tissues and organ of health plant, infected host plant (at least temporarily) is no
External symptom is shown, it can the separation or from plant tissue through Histological method or from the plant tissue of stringent surface sterilization
The interior method for directly amplifying microbial DNA is raw in it to prove.It is the endogenetic fungus in phytomicroorganism system, is deposited extensively
It is in plant tissue, it is free from the influence of the external environment.It is the natural constituent in phytomicroorganism system, it is evolving
Harmonious symbiosis, secondary metabolite very abundant are established with plant host in the process.Plant endogenesis epiphyte is almost
It is present in all plants studied at present, distribution is wide, and type is more.Studies have shown that the plant host of infection endophyte is past
Toward having the advantages such as fast growing, degeneration-resistant border, disease resistance, anti-animal injury, struggle for existence power is had more than being uninfected by plant.Inside
In very raw bacterium-plant host-herbivore ecosystem, plant endogenesis epiphyte plays strange important ecological work
With.The material base for playing these effects is the rich and varied secondary metabolite of endogenetic fungus generation, they have a variety of
Bioactivity has important application potential in agricultural and (or) pharmaceutical sector.In recent years, with American scientist
A.Stierle has found the producing strains of antitumor drug paclitaxel on the short leaf China fir in the Pacific Ocean, in medicinal plant and endangered plants
Raw fungi and its active matter Quality Research become the important directions of drug development.China's plant resources very abundant, in it
The research of raw fungi is beneficial to the exploitation of microbial medicine and the protection of rare plant resource.
Common immunosuppressor mainly has five classes: (1) glucocorticoids, such as cortisone and prednisone；(2) microorganism
Metabolite, such as cyclosporin and fujimycin 506；(3) antimetabolite, such as imuran and Ismipur；(4) polyclonal
With monoclonal antilymphocyte antibody, such as antilymphocyte globulin (ALG) and OKT3；(5) alkylating agents, such as cyclophosphamide.Its
In, microorganism glycolysis has multiple product, such as cyclosporin CsA class, tacrolimus Tacrolimus as primary synthetic methods
(FK506), rapamycin Rapamycin (RPM) and its derivative SDZ RAD, Mizoribine (MZ) etc..It is with cyclosporin
Example, the Borel of the seventies later period Switzerland have found the one kind extracted in a kind of glycolysis product from mould containing only 11 amino acid
Circular polypeptides, be named as cyclosporine (CsA), effectively specificity can inhibit lymphocyte reaction and hyperplasia.To T cell,
Especially TH cell has preferable selective inhibitory, and then relatively weak to the inhibiting effect of other immunocytes, because
This achieves good curative effect in anti-organ-graft refection；It is also used for the treatment of autoimmunity disease, therefore is a kind of with very
The immunosuppressor of high clinical use value.Confirm its anti-rejection effect compared with other through clinical test application study in 10 years
Drug is strong and Small side effects it is more.Therefore it puts goods on the market application in the late nineteen eighties official register that goes through.CsA nearly 20 years face
Bed application shows magical effect so that in addition to small intestine transplantation, liver, kidney, the heart and the heart/lung, pancreas transplanting patient/graft one
Annual survival rate reaches 70-85%, and only 30-50% before this.The incidence of CsA associated neurological signs of toxicity is about
10-28% is a kind of more important factor for influencing patient's prognosis.It is slightly more with headache, limb tremor, sensory disturbance etc.
See, moderate is based on vision disorder.The severe performance incidence of CsA related neural toxicity is extremely low.
Plant endogenesis epiphyte is as the fungi with plant symbiosis, during with plant symbiosis, development possess with it is general
The different natural bioactive substances of logical pathogenic bacteria.New endogenetic fungus present in separating plant, the secondary metabolism for exploring the bacterium produce
Object activity, is a new developing way.
Summary of the invention
The present invention provides a kind of preparation method of noval chemical compound spherical cavity enamine: the following steps are included:
1) ZJLQ129 fermentation liquid is prepared: the ZJLQ129 bacteria cake in totally 10 access 500ml PDB for being 0.5 centimetre by diameter
Mycelia liquid is obtained in cultivating 10 days on 25 DEG C, 200 turns of shaking tables, mycelia liquid aseptically crushes；Bacterium is accessed in 500ml PDB
Silk liquid 25ml, stationary culture filters after 2 weeks at room temperature obtains fermentation liquid and mycelium respectively.
2) prepare spherical cavity rhzomorph: extract liquor is concentrated to give extractum A, weight 1.72g, mycelia after fermentation liquid is extracted 3 times with ethyl acetate
Body is concentrated into small size with acetone soak after overnight, obtain medicinal extract B, weight 1.76g；Merge two parts and obtains medicinal extract C, weight 3.5g；It takes
Medicinal extract C 3.5g adds 15g silica gel mixed sample, and Rotary Evaporators are evaporated, 950 grams of dry column-packings of 200-300 silica gel, will mix the sample after sample
Column chromatography for separation is carried out after product loading, successively uses petroleum ether: ethyl acetate volume ratio 4:1, petroleum ether: ethyl acetate volume ratio 3:
2, petroleum ether: ethyl acetate volume ratio 3:7, ethyl acetate: methanol volume ratio 4:1 carries out gradient elution separation respectively, and silica gel is thin
Analysis and anisaldehyde chromogenic reagent layer by layer, merging Rf value is 0.46 and anisaldehyde colour developing is the component of blue；Component 1.70g is used
Gel filtration chromatography isolates and purifies, using chloroform: methanol volume ratio 1:1 is separated as eluent, silica gel thin-layer chromatography and anisaldehyde
Chromogenic reagent obtains spherical cavity rhzomorph sterling 300mg.
3) it prepares spherical cavity enamine: sterling spherical cavity rhzomorph 200mg being taken, to 0 DEG C in ice salt bath, to be passed through ammonia in three-necked bottle
Reaction 30 minutes, to doing, 7 grams of sample after taking concentration are dissolved sample concentration with methanol, add 14g silica gel mixed sample upper prop, addition is washed
De- agent is petroleum ether: chloroform: methanol=18: the separation of 4: 1,200-300 mesh silica gel column chromatography obtains noval chemical compound spherical cavity enamine
150mg。
Invention also provides above-mentioned name spherical cavity enamines to have immunosuppressive activity.Spherical cavity enamine is by name He Shi spherical cavity
The secondary metabolite spherical cavity rhzomorph ammonification that bacterium Mycosphaerella nawae ZJLQ129 fermentation generates is derived.Name and
Family name's spherical cavity bacterium Mycosphaerella nawae ZJLQ129 preservation title are as follows: Mycosphaerella nawae ZJLQ129,
Depositary institution: China typical culture collection center, preservation address: Wuhan, China Wuhan University；Preservation date: 2015 9
The moon 9, deposit number: CCTCC NO:M2015517.
Above-mentioned preferred, spherical cavity enamine has immunosuppressive activity.Preferably, under 2nM concentration spherical cavity enamine to sword bean
The immunosupress rate of the T cell proliferation of albumin A induction is 32.4 ± 6.2%.
Spherical cavity rhzomorph compound has immunosuppressive activity.Preferably, the immune suppression of 10 μM of concentration spherical cavity rhzomorph compounds
Rate processed is 51.28%.
Immunosupress: refer to the inhibiting effect for immune response, or perhaps have to the immune response of body and inhibit
Effect.Immunosuppressor can inhibit and be immunoreacted proliferation and function in relation to cell (macrophages such as T cell and B cell),
Antibody mediated immunity reaction can be reduced.
Detailed description of the invention
Fig. 1: spherical cavity rhzomorph (-) mycousunine structural formula figure
Fig. 2: spherical cavity enamine (-) mycousunine amide structural formula figure
Fig. 3: spherical cavity enamine (-) mycousunine amide absolute configuration figure
Fig. 4: spherical cavity enamine (-) mycousunine amide (compound 2) selective depression T cell proliferation.(A): sword bean
The T cell proliferation of albumin A (Con A) induction.(B): the B cell proliferation of lipopolysaccharides (LPS) induction.(C): PANC-1 and A549 are thin
Born of the same parents' proliferation.Ordinate be cell opposite proliferation rate, n=4, compared with control group (0nM group),bThe He of P < 0.01cP<0.001。
Fig. 5: (A and B): spherical cavity enamine (-) mycousunine amide (compound 2) inhibits CD3+T cell proliferation.It is vertical
Coordinate representation CD3+The ratio of T cell.N=3, compared with the control group,bThe He of P < 0.01cP<0.001.(C): the cell of compound 2
Toxicity.Ordinate expression cell survival rate, n=4, compared with control group (0nM group),aP<0.05,bThe He of P < 0.01cP<0.001。
Fig. 6: spherical cavity enamine (-) mycousunine amide (compound 2) inhibits point of cell factor in activating T cell
It secretes.The concentration of ordinate expression cell factor, n=3, compared with the control group,aP<0.05,bThe He of P < 0.01cP<0.001。
Specific embodiment
The preparation method of noval chemical compound spherical cavity enamine is mainly by name He Shi spherical cavity bacteria strain (Mycosphaerella
Nawae) the metabolite spherical cavity rhzomorph derivatization that ZJLQ129 fermentation generates obtains, with reference to the accompanying drawing to tool of the invention
Body embodiment is described in further detail.
Key instrument in the present invention includes: 3K-30 high speed tabletop centrifuge, German Sigma；RE-6000A rotary evaporation
Device, Shanghai are sub- flourish；Glass chromatography column；LCQ-Advantage type mass spectrograph, U.S. Finnigan；DRX-500 type nuclear magnetic resonance
Instrument, German Bruker company.MCO-15AC type cell incubator, SANYO GS company；SpX-250B-Z biochemical cultivation case, Shanghai
Bo Xun Industrial Co., Ltd.；1700 type electronic balances, German Sartorius company；SW-CJ-1F type superclean bench, Suzhou peace
Safe air technique Co., Ltd；LD5-2A type centrifuge, Beijing Medical Centrifugal Machine Factory；1-13 type centrifuge, German SIGMA are public
Department；MH-1 type micro oscillator, its woods Bell's instrument manufacturing Co., Ltd, Jiangsu Haimen City；680 type enzyme-linked immunosorbent assay instruments, beauty
Bio-Rad company, state；Synergy-2SLFPA all-wave length multi-function microplate reader, BioTek company, the U.S.；XDS-1B type biology falls
Set microscope, Chongqing optical instrument factory；IX73 research grade inverted fluorescence microscope, Japanese Olympus company；5804R type high speed
Freeze desk centrifuge, German Eppendorf company；PTC-200PCR instrument, Bio-Rad company, the U.S.；ABI
7900 fluorescence quantitative PCR instruments, Applied biosystems；ND-1000 all-wave length ultraviolet/visible light is swept
Retouch spectrophotometer: Sai Mofei company, the U.S.；FACS Calibur type flow cytometer: Becton Dickson company, the U.S..
Main agents in the present invention include: column chromatography silica gel (200-300 mesh), Haiyang Chemical Plant, Qingdao；Column chromatography is solidifying
Glue (Sephadex LH-20), Pharmacia company.Concanavalin A (Con A), lipopolysaccharides LPS, dexamethasone acetate, 3-
[4,5-dimethylthylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide (MTT), platform phenol is blue,
Dimethyl sulphoxide solution (DMSO) is purchased from Sigma company；Cyclosporin A (CsA) is presented by Huadong Medicine Co., Ltd.；
RPMI1640 cell culture fluid and DMEM culture solution are purchased from Thermo Fisher company；Newborn bovine serum is purchased from Hangzhou Chinese holly
Bioengineering Co., Ltd；100 × mycillin solution and Hank ' s liquid are purchased from the green skies Bioengineering Research Institute in Jiangsu；Contain
10% fire extinguishing fetal calf serum and the RPMI1640 cell culture fluid of 1 × mycillin solution are complete culture solution；Between 40 μm of holes
Cell strainer is purchased from Biologix company；Antibody FITC-anti-CD3, PE-anti-CD4, PE-anti-CD8, PE-anti-
CD69 and PE-anti-CD25 is purchased from eBioscience company；Mouse cytokine (IL-2 and IFN-γ) Elisa detection reagent
Box is purchased from Wuhan doctor moral bio-engineering corporation；Trizol is purchased from Invitrogen company；MMLV reverse transcriptase is purchased from
Fermentas company；SYBR Green PCR kit is purchased from TIANGEN company；PCR primer and other PCR reagents are purchased from upper
Hai Shenggong biotechnology Co., Ltd；Remaining reagent is that domestic analysis is pure.
The acquisition of embodiment one, name He Shi spherical cavity bacteria strain (Mycosphaerella nawae) ZJLQ129
The Isolation and identification of name He Shi spherical cavity bacteria strain (Mycosphaerella nawae) ZJLQ129:
It is separately cultured in the lab by following condition and step, can be obtained name He Shi spherical cavity bacterium bacterium of the present invention
Strain ZJLQ129:
1) it is acquired from China Longquan, Zhejiang Province nature reserve area, by the chinaroot greenbier rhizome leaf of the acquisition secondary chlorine of concentration 0.5% (w/v)
Sour sodium carries out surface sterilization 10min, and then rinsed with sterile water, which is placed on aseptic filter paper, blots water.Chinaroot greenbrier, also referred to as Smilax china L, hundred
Conjunction section smilax, perennial liana fallen leaves climber.It is born in hillside hayashishita, is distributed in drought various regions, climbs up by holding on to filling
Wood, leaf Bao Gezhi or hard papery, usual red, brown or age of recent antiquity coppery after doing, round, oval or other shapes are 3-10 centimetres long,
Wide 1.5-6 (- 10) centimetre, below usual light green, less pale asphyxia；Petiole is 5-15 millimeters long, accounts for about the 1/2-2/3 tool of overall length
The sheath of 0.5-1 millimeters wide (side) nearly all has tendril, rare exception, and separation point is located at tendril, and rhizomes is slightly thick,
It is hard, be irregular bulk, thick 2-3 centimetres.1-3 meters of stem length, a small number of up to 5 meters, sparsely grow is pierced.
2) the chinaroot greenbrier leaf of surface sterilization is cut into sterile razor blade the tissue of 1cm or so size, tissue is moved
It connects on 2% (w/v) water agar plate containing 100 μ g/ml ampicillins and streptomysin, is cultivated in illumination box, cultivated
Condition: 25 DEG C, illumination in 16 hours, 8 hours dark；
3) it after mycelia grows, will be cultivated in single bacterium silk top transposing to PDA solid medium, 25 DEG C of cultures are continuous pure
Change 3 times, obtains pure culture.Name He Shi spherical cavity bacteria strain ZJLQ129 (i.e. Mycosphaerella nawae of the invention
ZJLQ129 CCTCC NO:M2015517) have the property that slow growth in PDA culture medium, 20-25d colony diameter are 2-
3cm, 60-66d colony diameter are 3-4cm, and growth rate reduces.In 25 DEG C in PDA culture medium, 12 hours dark alternate cultures,
Bacterium colony is in irregular shape, and no aerial hyphae, mycelia initial stage is grey, and the later period becomes grey black, and there are fold, bacterium in the back side and front
It is irregular to fall edge, non-pigment secretion；Ascostome is black, does not generate spore, and mycelia has diaphragm.Name He Shi spherical cavity bacteria strain
ZJLQ129 (i.e. Mycosphaerella nawae ZJLQ129 CCTCC NO:M2015517), it belongs to mycota
(Fungi), double-core suberathem (Dikarya), Ascomycota (Ascomycota), cup fungi subphylum (Pezizomycotina), ascus
Gammaproteobacteria (Ascomycetes), Pezizale (Pezizales), cup fungi section (Pezizaceae), mycosphaerella
(Mycosphaerella)。
4) strain idenfication: being carried out beta-tubulin gene sequencing for ZJLQ129, and be compared using blast program,
Phylogenetic Analysis is carried out using PAUP program.
Bacterial strain mycelia is ground into a powder in liquid nitrogen, takes 100mg powder to be loaded in 1.5mL centrifuge tube, using DNeasy
Plant Mini Kits (QIAGEN) presses the Program extraction genomic DNA of manufacturer.Using universal primer BT1 (5'-
AACATGCGTGAGATTGTAAGT-3') and BT22 (5'-TCTGGATGTTGTTGGGAATCC-3') expands beta-tubulin
Gene, PCR system: 5.0 μ L, 10 × PCR buffer (MgCl containing 25mmol/L of DNA profiling (100ng/ μ L)2) 5.0 μ L,
1.0 μ L of dNTPs (5mmol/L), primer (50 μ g/mL) 1 μ L of each 0.5 μ L, Taq archaeal dna polymerase (2U/ μ L), adds water to supply 50 μ
L carries out PCR reaction, PCR reaction condition: 94 DEG C of 3min in the PCR instrument of BIORAD company S1000 model after mixing；94℃
30s, 53 DEG C of 50s, 72 DEG C of 90s, 35 circulations；72℃10min.
Pcr amplification product is purified with Axygen pillar DNA plastic recovery kit.The ITS rDNA PCR amplification of purifying
Product is respectively sequenced ITS1 and ITS4 with amplimer, the beta-tubulin amplified production sequencing of purifying.Sequencing is by Shanghai
Raw work bioengineering Services Co., Ltd is completed.The sequence measured carries out blast search on GenBank.It is tied based on BLAST
Fruit is compared the sequence of bacterial strain ZJLQ129 and the correlated series of mycosphaerella on GenBank with software Clustal X 1.81
It is right, then carry out Phylogenetic Analysis.The above result shows that bacterial strain ZJLQ129 can name He Shi spherical cavity bacterium of running after fame
Mycosphaerella nawae。
Embodiment two, the preparation of spherical cavity enamine precursor spherical cavity rhzomorph (-) mycousunine and Structural Identification
1) name He Shi spherical cavity bacteria strain (Mycosphaerella nawae) ZJLQ129 fermented and cultured successively carries out following
Step:
It will be trained in ZJLQ129 bacteria cake (diameter is 0.5 centimetre) totally 10 access 500ml PDB on 25 DEG C, 200 turns of shaking tables
It supports 10 days, culture solution aseptically crushes, and is added in 500ml PDB and cultivates, every bottle of access mycelia liquid 25ml connects 40 bottles altogether
That is 20L, stationary culture filters after 2 weeks at room temperature obtains fermentation liquid and mycelium respectively.Wherein fermentation liquid extracts 3 with ethyl acetate
Extract liquor is concentrated to give medicinal extract 1.72g (part A) after secondary, and mycelium is concentrated into small size with acetone soak after overnight, obtains medicinal extract
1.76 (part Bs).Merge two parts and obtains medicinal extract 3.5g (C portion).
Wherein the ingredient of above-mentioned culture medium or culture solution is as follows:
PDA culture medium: i.e. potato dextrose agar (potato dextrose agar) peeled potatoes 200g is cut into
Fritter adds boiling to boil 20min, filters off solid residue with gauze, and filtrate adds water to complement to 1000mL, and the dissolution of 20g glucose is added,
The agar powder that 15g dissolves is added, is dispensed, 121 DEG C of sterilizing 20min are spare.
PDB culture solution: i.e. potato glucose (potato dextrose broth): peeled potatoes 200g is cut into small
Block adds boiling to boil 20min, filters off solid residue with gauze, and filtrate adds water to complement to 1000mL, and the dissolution of 20g glucose is added, point
Dress, 121 DEG C of sterilizing 20min are spare.
2) preparation of compound:
Above-mentioned medicinal extract C 3.5g is taken to add 15g silica gel mixed sample, Rotary Evaporators are evaporated.In addition, with 950 grams of 200-300 silica gel
Dry column-packing carries out column chromatography for separation after mixing the sample loading after sample, successively uses petroleum ether: ethyl acetate 4:1 (v/v), stone
Oily ether: ethyl acetate 3:2 (v/v), petroleum ether: ethyl acetate 3:7 (v/v), ethyl acetate, ethyl acetate: methanol 4:1 (v/v)
Gradient elution separation is carried out respectively, and every 500ml receives 1 flow point, receives 31 flow points altogether, and it is thin that received sample is carried out silica gel
Analyse layer by layer, after anisaldehyde chromogenic reagent, by Rf value be 0.46 and anisaldehyde develop the color by spraying be blue flow point merge, obtain
This part of Fr8.The formula of anisaldehyde color developing agent is as follows: anisaldehyde (P-methoxybenzal-dehyde) 1: wide spectrum.Preparation method: 135
Ethyl alcohol+5mL the concentrated sulfuric acid+1.5mL of glacial acetic acid+3.7mL anisaldehyde, is vigorously stirred, makes to be uniformly mixed.Anisaldehyde is (to methoxyl group
Benzaldehyde) 2: terpenes, cineole (cineoles), withanolides, out phyllanthus emblica alkali (acronycine).Preparation method: fennel
Fragrant aldehyde: HClO4: acetone: water (1:10:20:80).Fr8 part (value=0.46 Rf) total 1.70g is separated pure using gel filtration chromatography
Change, with chloroform: methanol 1:1 (v/v) is that washing and dehydrating integrated machine elution separates repeatedly, and silica gel thin-layer chromatography detection is aobvious with anisaldehyde color developing agent
After color, merge the identical flow point of component, it is final to obtain sterling 300mg, it is denoted as compound (1).Compound (1) is from name He Shi ball
The metabolite of acquisition is separated in chamber bacterial strain ZJLQ129.
3) qualification process of compound (1) spherical cavity rhzomorph (-) mycousunine is as follows:
Compound (1) is shown in Fig. 1, yellow needles, mp 86-89 DEG C (MeOH)；HRESIMS m/z:376.1158M+
(calcd for C19H20O8 376.1158)1H NMR (400MHz, CDCl3), 3.17 (1H, d, J=18Hz, H-4a), 3.31
(1H, d, J=18Hz, H-4b), 1.63 (3H, s, H-10), 2.56 (3H, s, H-12), 2.59 (3H, s, H-14), 2.04 (3H,
S, H-15), 3.54 (3H, s, H-16)13C NMR (100MHz, CDCl3) 197.7 (s, 1-C), 110.7 (s, 2-C), 194.6
(s, 3-C), 37.9 (t, 4-C), 51.3 (q, 4a-C), 156.6 (s, 5a-C), 101.8 (s, 6-C), 163.4 (s, 7-C),
107.2 (s, 8-C), 159.1 (s, 9-C), 105.3 (s, 9a-C), 59.8 (s, 9b-C), 17.3 (q, 10-C), 202.4 (s, 11-
C), 27.4 (q, 12-C), 201.0 (s, 13-C), 31.2 (q, 14-C), 7.2 (q, 15-C), 51.3 (q, C-16).Compound (1)
Molecular weight it is much like with usnic acid with carbon modal data with nuclear magnetic resonance spectroscopy, illustrate the compound be dibenzofurans class chemical combination
Object.Compound 1 methoxyl group (δ C 51.4, δ H 3.56, s 3H) more than usnic acid, ABq methylene (δ C 37.9,
δ H 3.17d, J=18.0, δ H 3.31d, J=18.0), but lacked a phenyl ring tertiary carbon and corresponding Hydrogen Proton signal (δ C98.2,
δ H 5.98, s 1H), illustrate the position 4a of compound 1 by methoxy substitution, and No. 4 positions are methylene, compound (1)1H NMR
And13C NMR is consistent with the data of (-)-mycousnine in document, therefore compound (1) is accredited as (-) mycousnine,
Chinese name spherical cavity rhzomorph.
Embodiment three: the acquisition and name of compound (2) spherical cavity enamine (-) mycousunine amide
1, compound (2) is that compound (1) is obtained by aminating reaction, specific as follows:
The total 200mg of above-mentioned sterling compound (1) is taken, is placed in three-necked bottle, is placed in ice salt bath to 0 DEG C, it is anti-to lead to ammonia
It answers 30 minutes, by sample concentration to dry.Reaction front and back product is subjected to silica gel thin-layer chromatography comparison, discovery reaction is in Rf value
0.4 (solvent is petroleum ether: chloroform: methanol 3: 3: 1) generates a noval chemical compound, and 7 grams are dissolved with proper amount of methanol, add 14g silicon
Glue mixes sample, and Rotary Evaporators are evaporated.It is separated using 200-300 mesh silica gel column chromatography, eluant, eluent is petroleum ether: chloroform: methanol=
18: 4: 1, it carries out separation and obtains noval chemical compound 150mg, be denoted as compound (2).
2, the identification of compound (2) spherical cavity enamine (-) mycousunine amide:
Compound (2) is the aminating reaction product of compound (1), and structural formula is shown in Fig. 2, yellow needles, optical activity:(c=0.1, MeCN) mp HRESIMS m/z:376.1399 [M+H]+(calcd for C19H21O7N
376.1399).Fewer than the molecular weight of compound (1) 1.1H NMR (500MHz, CDCl3), 13C NMR (125MHz, CDCl3)
It is shown in Table 1.By comparing compound (1) and (2)1H and13C NMR data determines that its structure is (-) mycousnine amide.
In order to further verify its structure and absolute configuration, X-ray analysis is carried out to compound (2).It is final to confirm the exhausted of compound (2)
To being configured as (4aR, 9bS, E) -6-acetyl-2- (1-aminoethylidene) -7,9-dihydroxy-4a-methoxy-
8,9b-dimethyl-4,4a-dihydrodibenzo [b, d] furan-1,3 (2H, 9bH)-dione, Fig. 3 are compound (2)
Absolute configuration.
1 compound of table2's13C NMR with1H NMR data (CDCl3, δ ppm；Hz)
Embodiment three: the preparation method of noval chemical compound spherical cavity enamine
It is realized by the following method:
1) ZJLQ129 fermentation liquid is prepared: the ZJLQ129 bacteria cake in totally 10 access 500ml PDB for being 0.5 centimetre by diameter
Mycelia liquid is obtained in cultivating 10 days on 25 DEG C, 200 turns of shaking tables, mycelia liquid aseptically crushes；Bacterium is accessed in 500ml PDB
Silk liquid 25ml, stationary culture filters after 2 weeks at room temperature obtains fermentation liquid and mycelium respectively.
2) prepare spherical cavity rhzomorph: extract liquor is concentrated to give extractum A, weight 1.72g, mycelia after fermentation liquid is extracted 3 times with ethyl acetate
Body is concentrated into small size with acetone soak after overnight, obtain medicinal extract B, weight 1.76g；Merge two parts and obtains medicinal extract C, weight 3.5g；It takes
Medicinal extract C 3.5g adds 15g silica gel mixed sample, and Rotary Evaporators are evaporated, 950 grams of dry column-packings of 200-300 silica gel, will mix the sample after sample
Column chromatography for separation is carried out after product loading, successively uses petroleum ether: ethyl acetate volume ratio 4:1, petroleum ether: ethyl acetate volume ratio 3:
2, petroleum ether: ethyl acetate volume ratio 3:7, ethyl acetate: methanol volume ratio 4:1 carries out gradient elution separation respectively, and silica gel is thin
Analysis and anisaldehyde chromogenic reagent layer by layer, merging Rf value is 0.46 and anisaldehyde colour developing is the component of blue；Component 1.70g is used
Gel filtration chromatography isolates and purifies, using chloroform: methanol volume ratio 1:1 is separated as eluent, silica gel thin-layer chromatography and anisaldehyde
Chromogenic reagent obtains spherical cavity rhzomorph sterling 300mg.
3) it prepares spherical cavity enamine: sterling spherical cavity rhzomorph 200mg being taken, to 0 DEG C in ice salt bath, to be passed through ammonia in three-necked bottle
Reaction 30 minutes, to doing, 7 grams of sample after taking concentration are dissolved sample concentration with methanol, add 14g silica gel mixed sample upper prop, addition is washed
De- agent is petroleum ether: chloroform: methanol=18: the separation of 4: 1,200-300 mesh silica gel column chromatography obtains noval chemical compound spherical cavity enamine
150mg。
Example IV: the immunosuppressive activity measurement of compound (2) spherical cavity enamine (-) mycousunine amide:
Experimental method is as follows:
1, test sample compound (2) (-) mycousnine enamine is prepared: compound (2) (-) mycousnine
Enamine purity > 99%.Compound (2) is first dissolved as 50mM concentration, -20 DEG C of freezen protectives with DMSO.It uses again before use
RPMI1640 cell culture fluid is diluted to respective concentration.Han Liang≤0.2% of DMSO, does not have the bioactivity of cell in dilution
Have an impact.
2, cell strain and experimental animal source: PANC-1 and A549 cell is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.Culture
In containing 10% inactivated fetal bovine serum, the DMEM culture medium (Dulbecco's of 100IU/mL penicillin, 100 μ g/mL streptomysins
Modified eagle medium) in；37 DEG C are placed in, 5%CO2Cell incubator in cultivate.To cell growth 80% with
When upper fusion degree, with 0.25% pancreatin had digestive transfer culture.Female SPF C57BL/6 mouse, 6 week old, 18-22g are purchased from Shanghai
Western Poole-Bi Kai experimental animal Co., Ltd, experimental animal are raised one week after buying, and are tested again after adapting to environment.Experiment
The processing routine of animal is executed according to the regulation of Zhejiang Academy of Medical Sciences Laboratory Animal Welfare Ethics Committee.
3, single splenocyte suspension preparation: mouse is sterile to take spleen, after grinding, adds appropriate Hank ' s liquid to be suspended, with 40 μm of holes
Between cell strainer filter, 1500rpm be centrifuged 5 minutes, abandon supernatant, add Hank ' s liquid wash repeatedly 2 times.Splenocyte is collected, is added
Single splenocyte suspension is made in the suspension of RPMI1640 complete culture solution.The numeration of dye method is refused with 0.4% phenol indigo plant, viable count is many
In 95%.
4, the B cell proliferation measuring of the T cell proliferation of Con A induction and LPS induction: in 96 hole flat-bottomed plates
In, every hole adds 100 μ L splenocyte suspensions (5 × 106Cell/ml), and 50 μ l Con solution As (2 μ g/ml) of addition or LPS are molten
Liquid (2 μ g/ml) is eventually adding various concentration (0,2,10,50,250nM) for the 50 μ l of dilution of reagent object, and each concentration repeats
4 holes separately set normal cell controls hole.37 DEG C, 5%CO2After culture 44 hours, MTT solution (2mg/ml) 50 μ l is added in each hole, after
Continuous culture 4h.It is centrifuged (1800rpm, 5min), discards each hole supernatant, respectively Plus acidic 150 μ of DMSO solution (4%1mol/lHCl)
L, is protected from light oscillation 15min, measures OD490nm, is calculated as 100% with the proliferation rate of mitogen functional hole (drug concentration 0), calculates medicine
Relative cell proliferation rate after object effect.MTT is a kind of powdered chemical reagent, and full name is 3- (4,5)-
Dimethylthiahiazo (- z-y1) -3,5-di-phenytetrazoliumromide, the entitled 3- (4,5- bis- of Chinese chemistry
Methylthiazol -2) -2,5- diphenyltetrazolium bromide bromide, trade name: thiazolyl blue is a kind of dyestuff of yellow color.The detection of mtt assay
Principle is that the bluish violet that the succinate dehydrogenase in living cells mitochondria can make exogenous MTT be reduced to water-insoluble crystallizes first a ceremonial jade-ladle, used in libation
(Formazan) it and is deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell,
Its absorbance value is measured at 490nm wavelength with microplate reader, within the scope of certain cell number, MTT crystallizes the amount to be formed and cell
Number is directly proportional.According to the absorbance value (OD value) measured, to judge Living cells proliferation quantity, OD value is bigger, and cell activity is stronger,
Appreciation rate is higher.
5, PANC-1 and A549 cell proliferation experiment measures: the cell of logarithmic growth phase, adjustment concentration to 1 × 105A/
ML is inoculated in 100 hole μ L/ in 96 porocyte culture plates.After culture 24 hours, it is 200 that ZJLQ129 dilution to total volume, which is added,
μ L after continuing culture 48 hours, measures cell proliferation rate using above-mentioned mtt assay.
6, cytotoxicity assay: in 96 hole flat-bottomed plates, every hole adds 100 μ L splenocyte suspensions (5 × 106cell/
Ml), and 50 μ l various concentrations are added for the dilution of reagent object, add 50 μ l of RPMI1640 culture solution, multiple 4 holes, 37 DEG C,
5%CO2 is cultivated 48 hours, measures cell survival rate by aforementioned mtt assay.
7、CD3+T cell ratio measuring: in 24 hole flat-bottomed plates, every hole adds 1ml splenocyte suspension (5 × 106Cell/ml), and be added Con solution A (2 μ g/ml) and various concentration for trying each 0.5ml of drug solution, each concentration answers 3 holes.
37 DEG C are set, 5%CO2After cultivating 24 hours and 48 hours respectively, cell is collected, is marked with FITC-anti-CD3.PBS washing is gone
Except unbonded fluorescence antibody, the ratio of flow cytometer measurement fluorescence labeled cell is set.
8, the efficiency test of cell factor: in 24 hole flat-bottomed plates, every hole add 1ml splenocyte suspension (5 ×
106Cell/ml), and be added Con solution A (2 μ g/ml) and various concentration for trying each 0.5ml of drug solution, each concentration multiple 3
Hole.37 DEG C are set, 5%CO2After culture 24 and 48 hours, culture supernatant is collected, detects cell factor IL-2 by ELISA kit
With the content of IFN-γ.
It is above-mentioned the results showed that
Observe the influence of compound (2) to Con A inducing T cell proliferation.As a result as shown in Figure 4 A, as CsA, chemical combination
Object (2) can significantly inhibit the proliferation of T cell in extremely low concentration (2nM), spherical cavity enamine (-) mycousunine under 2nM concentration
Amide is 32.4 ± 6.2% to the immunosupress rate for the T cell proliferation that concanavalin A (Con A) induces.In tested concentration model
(2~250nM) is in preferable concentration-dependent relation in enclosing.The medium effective concentration (EC50) of compound (2) be 26.97 ±
The EC50 of 6.00nM, CsA are 18.27 ± 4.60nM.But in the concentration range, the B that compound (2) and CsA induce LPS is thin
Born of the same parents are proliferated but without any influence (Fig. 4 B).Even after improving 100 times of concentration (25 μM), compound (2) is to human pancreas cancer
The proliferation of cell PANC-1 and lung cell A549 is without any influence (Fig. 4 C).
Compound (2) shows (Fig. 5 C) that the cytotoxicity assay of splenocyte, compound (2) starts to show at 6.25 μM
Faint cytotoxicity (cell survival inhibiting rate is 8.5%), half toxic concentration (TC50) it is 120.37 ± 17.97 μM；CsA
At 6.25 μM, cell survival inhibiting rate is 43.7%, TC50It is 16.17 ± 4.80 μM.According to the EC of above-mentioned acquisition50Value, successively
3, tri- 30,300nM concentration are set, FITC label are carried out to the particular surface antigens c D3 molecule of T cell, using fluidic cell
Technology further looks at the influence that compound (2) is proliferated T cell.As the result is shown (Fig. 5 A, B): compound (2) can obviously inhibit
The increase of T cell ratio in splenocyte caused by Con A, and preferable concentration-dependent relation and time-dependent relation is presented.
IL-2 and IFN-γ are the outstanding features of the major cytokine that T cell activation generates and T cell activation.Into
The cell secretion situation discovery of IL-2 and IFN-γ is determined in one pacing, and compound 2 can significantly be dropped to concentration dependent in 3nM or more
IL-2 and IFN-γ secretion caused by low Con A.
Result above sufficiently shows: compound 2 can selectively inhibit T cell to be proliferated and activate in extremely low concentration,
Activity is not that its cytotoxicity causes.Although the action intensity of T cell proliferation is inhibited to be not so good as CsA, cytotoxicity is markedly less than
CsA.Therapeutic index (the Therapeutic index, TI, TC of ZJLQ12950/EC50) (4463.5) be much larger than CsA (885.0).
Therefore, compound 2 has efficient, low toxicity, highly selective feature, has preferable research and development prospect.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (1)
1. a kind of preparation method of compound spherical cavity enamine, it is characterized in that: the following steps are included:
1) ZJLQ129 fermentation liquid is prepared: the ZJLQ129 bacteria cake in totally 10 access 500ml PDB in 25 for being 0.5 centimetre by diameter
It is cultivated 10 days on oC, 200r/min shaking table and obtains mycelia liquid, mycelia liquid aseptically crushes；Bacterium is accessed in 500ml PDB
Silk liquid 25ml, stationary culture filters after 2 weeks at room temperature obtains fermentation liquid and mycelium respectively, the deposit number of the ZJLQ129:
CCTCC NO:M2015517；
2) prepare spherical cavity rhzomorph: extract liquor is concentrated to give extractum A, weight 1.72g after fermentation liquid is extracted 3 times with ethyl acetate, and mycelium is used
Acetone soak is concentrated into small size after overnight, obtain medicinal extract B, weight 1.76g；Merge two parts and obtains medicinal extract C, weight 3.5g；Take medicinal extract C
3.5g adds 15g silica gel mixed sample, and Rotary Evaporators are evaporated, 950 grams of dry column-packings of 200-300 mesh silica gel, will mix on the sample after sample
Column chromatography for separation is carried out after sample, successively uses petroleum ether: ethyl acetate volume ratio 4:1, petroleum ether: ethyl acetate volume ratio 3:2, stone
Oily ether: ethyl acetate volume ratio 3:7, ethyl acetate: methanol volume ratio 4:1 carries out gradient elution separation, silica gel thin-layer layer respectively
Analysis and anisaldehyde chromogenic reagent merge RfValue is 0.46 and anisaldehyde colour developing is the component of blue；Component 1.70g gel
Column chromatographic isolation and purification, using chloroform: methanol volume ratio 1:1 is separated as eluent, silica gel thin-layer chromatography and anisaldehyde colour developing
Agent colour developing, obtains spherical cavity rhzomorph sterling 300mg；
3) it prepares spherical cavity enamine: taking sterling spherical cavity rhzomorph 200mg, in three-necked bottle, to 0 DEG C in ice salt bath, be passed through ammonia reaction
30 minutes, to doing, 7 grams of sample after taking concentration were dissolved sample concentration with methanol, add 14g silica gel mixed sample upper prop, eluant, eluent is added
For petroleum ether: chloroform: methanol=18:4:1, the separation of 200-300 mesh silica gel column chromatography obtain noval chemical compound spherical cavity enamine 150mg.
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| CN105330620A (en) * | 2015-12-07 | 2016-02-17 | 西宁意格知识产权咨询服务有限公司 | New enamine compound and preparation method and medical application thereof |
| CN105566130A (en) * | 2015-12-18 | 2016-05-11 | 姚天文 | Novel enamine compound and preparation method and medical application thereof |
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| CN105566130A (en) * | 2015-12-18 | 2016-05-11 | 姚天文 | Novel enamine compound and preparation method and medical application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Structures of (- )-Mycousnine, (+ )-Isomycousnine and (+ )-Oxymycousnine, New Usnic Acid Derivatives from Phytopathogenic Mycosphaerella nawae;Takeshi SASSA 等;《Agricultural and Biological Chemistry》;19901231;第54卷(第9期);第2231-2237页 * |
| Usnic Acid Derivatives with Cytotoxic and Antifungal Activities from the Lichen Usnea longissima;Xuelong Yu 等;《Journal of Natural Products》;20160517;第79卷(第5期);第1373-1380页 * |
| 植物响应病原真菌的代谢组学研究进展;张凡忠 等;《中国细胞生物学学报》;20160430;第38卷(第4期);第434-440页 * |
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