CN110041246A - The synthesis and application of novel Kessazulen sesquiterpene alkaloids - Google Patents
The synthesis and application of novel Kessazulen sesquiterpene alkaloids Download PDFInfo
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Abstract
The invention belongs to synthetic pharmacochemistry fields.More particularly to the chemical synthesis and application of a kind of new skeleton of Kessazulen sesquiterpene alkaloids from gorgonian source.The present invention synthesizes Muriceidine A using Kessazulen and nipecotic acid as raw material, using chemical method successful design.For the versatility for verifying synthetic method, Azulene aldehyde is connect with azacyclo-s segments such as piperidines, methyl piperidine, pyrroles, piperidine carbinols, 4- hydroxy piperidines, it is screened through anti tumor activity in vitro, present invention discover that the IC of structure optimization product 2 and 3 pair 231, MCF-7, K562, HCT-116, Hela, A549, H1975, HUVEC, MGC-803, SH-SY5Y, HO8910, Siha, PC-3 and BEL7402 this 14 kinds of tumor cell lines50Value is below 10 μM, therefore can be used for the research and development of anti-tumor drug.
Description
Technical field
The invention belongs to the synthesis of natural new molecule of the skeleton, structure optimization and its medical value fields, specifically relate to
And a kind of isolated Kessazulen sequiterpene is given birth to from the polo-neck class point gorgonian Muriceides collaris of South China Sea
The chemical synthesis and application of the new skeleton of alkaloids.
Background technique
This seminar to pick up from the polo-neck class point gorgonian Muriceides collaris in China, Weizhou Island sea area, the South Sea into
The chemical component and bioactivity research of system are gone, therefrom 5 isolated new molecule of the skeleton, are Kessazulen sesquialter
Terpenoid alkaloid is respectively designated as Muriceidine A/B/C.Preliminary biological activity test shows that such compound has preferably
Antitumor, anti-attachment isoreactivity.Anti tumor activity in vitro is the study found that wherein Muriceidine A and C are to human leukemia cell
The IC of K562 and HL-6050Value is respectively less than 10 μM.Structurally, two segments for constituting such compound scaffold are respectively more to create
The wooden Azulene and △1Nipecotic acid, wherein Kessazulen is isolated sequiterpene from a kind of yellow chamomile, can be original with guaiol
Material boiled through acid, dehydroaromatizationof and etc. conversion obtain.As a kind of typical polycyclic non-benzene aromatic compound, Kessazulen because
The distribution of its electronics has anti-inflammatory, antiallergy, antipepsin isoreactivity with external appearance characteristic and with distinctive physicochemical property,
Field of medicinal chemistry has certain researching value.△1Nipecotic acid, abbreviation P2C are a kind of special six-membered cyclic azomethines,
It is the important Metabolic Intermediate and multiple biological activities molecule of lysine in vivo, as rapamycin, Buddhist nun gram are mould
Element, Ropivacaine etc. must synthesize one of precursor.Therefore two segments for constituting skeleton all have potential medicinal study value.So
And the high cost with low efficiency of the developable finiteness of marine resources and natural product extraction limits the further medicine of such compound
With value research and exploitation.Therefore, this project, which is attempted to utilize, is chemically synthesized such new bone of Kessazulen sesquiterpene alkaloids
Frame compound, to carry out further pharmacology activity research.Currently, having no synthetic method in relation to such compound and anti-swollen
The research of the bioactivity such as tumor is reported.
Summary of the invention
The object of the present invention is to provide synthesis, structure optimization and the applications of a kind of new skeleton Azulene Alkaloid.
This study group is isolated from the polo-neck class point gorgonian Muriceides collaris for pick up from South China Sea
Product 2 and 3 after a kind of new natural product Muriceidine A (1) and its structure of modification, structure is as follows:
The present invention is to be implemented by following technical solution:
1. retrosynthetic analysis determines the reaction raw materials and route of New skeleton compound 1 in conjunction with document.
Two monomers of Azulene aldehyde and P2C can be obtained by disconnecting the carbon-carbon double bond among molecule.It reads up the literature it is found that Azulene aldehyde can be by guaiaci lignum
Azulene reacts to obtain through Vilsmeier-Haack, and Kessazulen is a kind of marketable material cheap and easy to get；C=N in P2C ring is satisfied
With change to obtain nipecotic acid, nipecotic acid is also a kind of marketable material cheap and easy to get；The condensation of Azulene aldehyde and P2C, bibliography
(Zoltewicz J A,Prokai-Tatrai K,Bloom L et al.Heterocycles,1993,35(1):171-
180.) method of condensing of benzaldehyde and anabasine reported, the synthetic schemes of target molecule has finally been determined.
2. being determined by adjusting factors such as reaction time, reaction temperature, charging sequence, acid/base ratio, material concentrations basic
The optimum reaction conditions of molecule of the skeleton 1, details are as shown in table 1.
The reaction condition of 1 compound 1 of table is explored
3. carrying out structure of modification to basic framework 1 by changing N- heterocyclic fragments ring substituents and ring size.By anti-
The structure-activity relationship of such compound is analyzed in tumor promotion screening, and attempts to look for the lead compound for having potential anti-tumor activity.
Present invention finds the compound of two potential broad-spectrum anti-tumor activities of tool, i.e. compound 2 and 3, the suppressions of initial in vitro tumour cell
Activity Results processed show that the two has significant inhibitory activity, details such as 2 institute of table for 14 kinds of tumour cells in screening range
Show.
The IC of 2 compound 2 and 3 of table50Test result
Detailed description of the invention
The 1H NMR (CDCl3) of Fig. 1 Kessazulen aldehyde
The 1H NMR (CD3OD) of Fig. 2 piperidines acetoacetic ester
The 13C NMR (CD3OD) of Fig. 3 piperidines acetoacetic ester
The 1H NMR (CDCl3) of Fig. 4 compound 1
The 13C NMR (CDCl3) of Fig. 5 compound 1
The mass spectrum of Fig. 6 compound 2
The 1H NMR (CDCl3) of Fig. 7 compound 2
The 13C NMR (CDCl3) of Fig. 8 compound 2
The DEPT-1 (CDCl3) of Fig. 9 compound 2
The DEPT-2 (CDCl3) of Figure 10 compound 2
The HSQC (CDCl3) of Figure 11 compound 2
The HMBC (CDCl3) of Figure 12 compound 2
The mass spectrum of Figure 13 compound 3
The 1H NMR (CDCl3) of Figure 14 compound 3
The 13C NMR (CDCl3) of Figure 15 compound 3
The DEPT-1 (CDCl3) of Figure 16 compound 3
The DEPT-2 (CDCl3) of Figure 17 compound 3
The HSQC (CDCl3) of Figure 18 compound 3
The HMBC (CDCl3) of Figure 19 compound 3
The mass spectrum of Figure 20 compound 4
The 1H NMR (CDCl3) of Figure 21 compound 4
The 13C NMR (CDCl3) of Figure 22 compound 4
The DEPT-1 (CDCl3) of Figure 23 compound 4
The DEPT-2 (CDCl3) of Figure 24 compound 4
The HMBC (CDCl3) of Figure 25 compound 4
The HSQC (CDCl3) of Figure 26 compound 4
The NOESY (CDCl3) of Figure 27 compound 4
The mass spectrum of Figure 28 compound 5
The 1H NMR (CDCl3) of Figure 29 compound 5
The 13C NMR (CDCl3) of Figure 30 compound 5
The DEPT-1 (CDCl3) of Figure 31 compound 5
The DEPT-2 (CDCl3) of Figure 32 compound 5
The HSQC (CDCl3) of Figure 33 compound 5
The HMBC (CDCl3) of Figure 34 compound 5
The mass spectrum of Figure 35 compound 6
The 1H NMR (CDCl3) of Figure 36 compound 6
The 13C NMR (CDCl3) of Figure 37 compound 6
The DEPT-1 (CDCl3) of Figure 38 compound 6
The DEPT-2 (CDCl3) of Figure 39 compound 6
The HSQC (CDCl3) of Figure 40 compound 6
The HMBC (CDCl3) of Figure 41 compound 6
The NOESY (CDCl3) of Figure 42 compound 6
Specific embodiment
Below by specific embodiment, the present invention will be described in detail.
It is listed in following embodiments of the invention to have Guaiazulene (the 1,4- dimethyl-compared with high bioactivity
7- isopropyl Azulene, Guaiazulene) and nipecotic acid, methyl piperidine, piperidine carbinols be raw material, it is raw through three-step reaction synthesis Azulene class
The example of the new skeleton Muriceidine A of alkaloids and its structure optimization product.Reaction route is as follows:
Embodiment 1: the synthesis of Azulene aldehyde, bibliography (Takekuma S, Matsuoka H, Minematsu T et
Al.Tetrahedron, 2010,66 (16): 3004-3015.) it proceeds as follows:
Dry 3mL DMF is added into the 50mL three neck round bottom flask of dried and clean, is dripped under condition of ice bath by constant pressure
Phosphorus oxychloride is slowly added dropwise in liquid funnel, is stirred to react 35min.Then it is added dropwise from the other side of there-necked flask by dropping funel
The DMF solution of Kessazulen is finished and is transferred in 35 DEG C of water-baths.After reacting 10h, reaction solution is poured into enough ice water, is stirred
Uniformly.Reaction solution pH to 8 or so is adjusted with the aqueous solution of potassium hydroxide.Several minutes of stirring, is heated to 90 DEG C, drives reaction out of and generates
Dimethylamine gas.Then three times with methylene chloride extraction, combining extraction liquid is evaporated, and concentration gained crude product is through purification on normal-phase silica gel
Column separating purification, with petroleum ether/acetone system elution.Yield is up to 90%.
Embodiment 2: the synthesis of piperidines acetoacetic ester, bibliography (McFarlane A K, Thomas G, Whiting
A.Journal of the Chemical Society,Perkin Transactions 1:Organic and Bio-
Organic Chemistry, 1995, (21): 2803-2808.) it proceeds as follows:
1.46g (10.0mmol) lysine is added in 100mL round-bottomed flask, 50mL methanol is added.At 0 DEG C while stirring
It is added dropwise thionyl chloride (1.5mL, 20.6mmol), after reacting 3h, sampling TLC detection, discovery lysine raw material does not react completely, this
When reaction system be still suspension.Reaction solution is transferred in 70 DEG C of oil baths, after back flow reaction 4h, sampling TLC detection, lysine
Raw material disappears.Reaction was completed, and reaction solution is poured into 50mL ice water, adjusts pH to 6~7 with sodium hydroxide solution (10mol/L), steams
Do to obtain white solid.By products therefrom with the dissolution of 10mL anhydrous methanol, it is filtered to remove insoluble matter, filtrate is evaporated to get lysine
Methyl esters.
The synthesis of embodiment 3:P2C
1. existing t-butyl hypochlorate processed: sequentially adding 5mL liquor natrii hypochloritis's (active chlorine content in 25mL round-bottomed flask
>=5.2%, effective chlorine >=8.0mmol), 1mL (52.4mmol) tert-butyl alcohol be added, be placed in 0 DEG C of cryogenic thermostat stirring reactive bath
In, 1mL (17.2mmol) glacial acetic acid is added dropwise into flask, is protected from light 5min or so.Reaction flask is stood after completion of the reaction
2min draws reaction flask yellow oily liquid (i.e. t-butyl hypochlorate) at the middle and upper levels, be placed in it is another be protected from light it is standby in vial
With.
2. N- chlorination: 500mg (2.6mmol) nipecotic acid carbethoxy hydrochloride being added in 50mL round-bottomed flask, is added
30mL anhydrous ether, ultrasonic dissolution (largely feed intake insoluble), pump air in bottle later, seal bottleneck with rubber stopper rapidly.
The injector syringe of balloon and 5mL are connected, after being filled with nitrogen, is inserted into rubber stopper and gives in bottle with nitrogen protection.This is anti-
System is answered to be fixed in the cryogenic thermostat stirring reactive bath that temperature setting is 0 DEG C.(following all reactions and operation are being protected from light item
Carried out under part) in the t-butyl hypochlorate injection round-bottomed flask in 1mL syringe absorption penicillin bottle, persistently it is stirred to react
2~3h.Monitoring reaction stops reaction after all disappearing to nipecotic acid carbethoxy hydrochloride raw material.
3. N- chloro piperidines acetoacetic ester dehydrochlorination and ester hydrolysis reaction (all operations and reaction carry out at the place of being protected from light)
Experimental implementation: walking in reaction system directly up and 100mg (2.5mmol, excessive) sodium hydrate solid particle be added,
10mL methanol is added simultaneously.After 0 DEG C is protected from light 1h, it is transferred to back flow reaction 1h in 50 DEG C of oil bath pans.TLC detection is sampled, with
Nipecotic acid carbethoxy hydrochloride is control, with petroleum ether: methylene chloride 1:1 expansion.Ultra-violet analysis, in R under 365nm wavelengthfValue 0.9
Nearby there is an apparent phosphor dot, 254nm wavelength light shows a clearly purple blackening according to the lower position；Ninhydrin color developing agent
A yellow dots are presented in colour developing, the position.
Post-processing: reaction solution is evaporated, and petroleum ether dissolution is added, has more white solid insoluble, solution layer is glassy yellow.
I.e. after back flow reaction, reaction solution is stood into 2h, is evaporated under reduced pressure at 20 DEG C, steams the ether in reaction system.Residue
The as methanol solution of the acid containing unsaturated piperidines, filtering, filter cake are washed 3~5 times with methanol, and filtrate sealing is kept in dark place, and are directly made
For the raw material of next step reaction.
Embodiment 4: the synthesis of compound 1
Experimental implementation: in 930mg (17.2mmol) sodium methoxide is added in 50mL round-bottomed flask, 1mL is added with pipette
(17.2mmol) glacial acetic acid adds 5mL methanol and makes it dissolve in bottle.The methanol for the unsaturated piperidines acid for taking step to react is molten
The system is added in liquid, and 200mg (0.9mmol) Kessazulen aldehyde solid is added, and supplies 30mL methanol, 60 DEG C of oil bath heating stirrings.
Reaction is for 24 hours.After completion of the reaction, it is evaporated reaction solution, by obtained solid with the dissolution of 30mL distilled water, appropriate sodium bicarbonate is added and neutralizes
Acetic acid in reaction system, until being released there is no bubble.(30mL × 3) are extracted with ethyl acetate, then use extracting n-butyl alcohol
Once.Merge organic layer, is evaporated.With methylene chloride, methylene chloride: the mobile phase of methanol (20:1~10:1) carries out gradient and washes
It is de-.Target component is red, in methylene chloride: eluting when methanol 10:1.Merge eluent, is evaporated weighing.
Embodiment 5: the synthesis of compound 2
1. synthesis and its dehydrochlorination of N- chloromethyl piperidines: in addition 400mg (4.7mmol) in 25mL round-bottomed flask
Piperidines liquid, 15mL ether, which is added, is uniformly mixed it.Air pump pumps air in bottle, seals bottleneck with rubber stopper rapidly.Nitrogen
It protects and is operated with the chlorination of piperidines acetoacetic ester, reaction flask is placed in 0 DEG C of cryogenic thermostat stirring reactive bath.(following institute
Thering is reaction and operation to carry out under the conditions of being protected from light) syringe takes 1mL (excess) now t-butyl hypochlorate processed injects reaction system,
Persistently it is stirred to react 2~3h.Excessive sodium hydrate solid is directly added thereto, while 5mL methanol is added, low temperature is anti-
It is transferred in 50 DEG C of oil bath pans after answering 1h, back flow reaction 1h.Reaction was completed, stands 2h, and decompression steams ether, residue mistake at 20 DEG C
Filter, gained filtrate are protected from light sealing, in case reaction uses in next step.
2. the synthesis of compound 2: in 2.0g (14.3mmol) sodium acetate solid is added in 50mL round-bottomed flask, using pipette
1mL (17.2mmol) glacial acetic acid is added, adds 5mL methanol and is made it dissolve in bottle.The unsaturated piperidines acid for taking step to react
Methanol solution be added the system, 200mg (0.9mmol) Kessazulen aldehyde solid is added, supplies 30mL methanol, 60 DEG C of oil baths add
Thermal agitation.Reaction was completed after hour for 24 hours, evaporated under reduced pressure, and obtained solid is added in appropriate sodium bicarbonate with the dissolution of 30mL distilled water
With the acetic acid in reaction system, to there is no bubble release until.It is extracted with ethyl acetate (30mL × 3), merges organic layer, steam
It is dry.Normal phase column chromatography, with methylene chloride for initial eluent, then with methylene chloride: methanol 50:1,30:1,20:1 and 15:1
Gradient elution, red objects component is in methylene chloride: eluting when methanol 15:1.Merge eluent, is evaporated.
Embodiment 6: the synthesis of compound 3
1. synthesis and its dehydrochlorination of N- chloro piperidine carbinols: in addition 800mg (7.0mmol) in 25mL round-bottomed flask
Piperidine carbinols solid, 30mL ether, which is added, is uniformly mixed it.1. part of remaining operation with embodiment 5.Gained △4,5Piperidines
MeOH methanol solution is yellow.
2. the synthesis of compound 3: with the 2. part of embodiment 5.
Embodiment 7: the synthesis of compound 4
1. the synthesis of 3,4,5,6- tetrahydropyridines: in 25mL round-bottomed flask be added 400mg (4.7mmol) piperidines liquid,
15mL ether, which is added, is uniformly mixed it, adds 5mL methanol and makes it dissolve in bottle.Air pump pumps air in bottle, uses rapidly
Rubber stopper seals bottleneck.Nitrogen protection is operated with the chlorination of piperidines acetoacetic ester, and the cryogenic thermostat that reaction flask is placed in 0 DEG C is stirred
It mixes in reactive bath technique.(following all reactions and operation carry out under the conditions of being protected from light) syringe takes 1mL (excess) now hypochlorous acid processed
The tert-butyl ester injects reaction system, is persistently stirred to react 2-3h.Excessive sodium hydrate solid is directly added thereto, is added simultaneously
5mL methanol, low temperature are transferred in 50 DEG C of oil bath pans after reacting 1h, back flow reaction 1h.Reaction was completed, 2h is stood, at 20 DEG C
Decompression steams ether, and residue filtering, gained yellow filtrate is protected from light sealing, in case reaction uses in next step.
2. the synthesis of compound 4: in 1.61g (29.6mmol) sodium methoxide solid is added in 50mL round-bottomed flask, using liquid relief
2mL (34.4mmol) glacial acetic acid is added in pipe, adds 5mL methanol and makes it dissolve in bottle.The unsaturated piperidines for taking step to react
The system is added in the methanol solution of acid, and 200mg (0.9mmol) Kessazulen aldehyde solid is added, supplies 30mL methanol, 60 DEG C of oil baths
Heating stirring.TLC detection is sampled after 12h, is compared with Azulene aldehyde raw material, with methylene chloride: methanol 15:1 (v:v) expansion, in Rf
Occurs a pink color dot at 0.4 position of value, thus it is speculated that the point is target product.Reaction was completed after hour for 24 hours, evaporated under reduced pressure, institute
Solid is obtained with the dissolution of 30mL distilled water, the acetic acid in appropriate sodium bicarbonate neutralization reaction system is added, until there is no bubble releasings
Until.It is extracted with ethyl acetate (30mL x 3), merges organic layer, be evaporated.Normal phase column chromatography is initial elution with methylene chloride
Liquid, then with methylene chloride: methanol 50:1,30:1,20:1 and 15:1 gradient elution, red component is in methylene chloride: methanol 15:
It is eluted when 1.Merge eluent, is evaporated.After purification through HPLC analysis, nuclear magnetic resonance spectroscopy is done.
Embodiment 8: the synthesis of compound 5
1. the synthesis of 3,4,5,6- tetrahydropyridines: in 25mL round-bottomed flask be added 400mg (4.7mmol) piperidines liquid,
15mL ether, which is added, is uniformly mixed it, adds 5mL methanol and makes it dissolve in bottle.Air pump pumps air in bottle, uses rapidly
Rubber stopper seals bottleneck.Nitrogen protection is operated with the chlorination of piperidines acetoacetic ester, and the cryogenic thermostat that reaction flask is placed in 0 DEG C is stirred
It mixes in reactive bath technique.(following all reactions and operation carry out under the conditions of being protected from light) syringe takes 1mL (excess) now hypochlorous acid processed
The tert-butyl ester injects reaction system, is persistently stirred to react 2-3h.Excessive sodium hydrate solid is directly added thereto, is added simultaneously
5mL methanol, low temperature are transferred in 50 DEG C of oil bath pans after reacting 1h, back flow reaction 1h.Reaction was completed, 2h is stood, at 20 DEG C
Decompression steams ether, and residue filtering, gained yellow filtrate is protected from light sealing, in case reaction uses in next step.
2. the synthesis of compound 5: with the 2. part of embodiment 5.
Embodiment 9: the synthesis of compound 6
1. the synthesis of N, N- Dimethyl Hydan: in 50mL round-bottomed flask be added 1.21g (20.0mmol) glyoxal liquid,
Its pH to 9 or so is adjusted with triethylamine；1.80g (20.0mmol) 1,3- dimethyl urea separately is taken, after dissolving with 5mL distilled water dropwise
Above-mentioned glyoxal system, 35 DEG C of oil bath heating stirrings are added.After reacting 3h, it is dense to be slowly added to 0.4mL dropwise into reaction system
Reaction temperature is risen to 100 DEG C, back flow reaction 6h by sulfuric acid.It is down to room temperature after reaction, is concentrated under reduced pressure.Products therefrom is added
The dissolution of 20mL water adjusts pH value of solution to 7 or so with solid sodium carbonate, extracts (30mL x 3) with ethyl acetate, combining extraction liquid,
Sample TLC detection.Isolate and purify: normal phase column chromatography, with petroleum ether: ethyl acetate 3:1 elution, TLC monitoring are eluted in flow point
Until target product.Final gained target product light yellow oil.Weighing, calculated yield are sealed.
2. the synthesis of compound 6: reacting gained imidazoles two in step on 256mg (2.0mmol) is added in 50mL round-bottomed flask
Ketone, is added 276mg (3.6mmol) ammonium acetate and 5mL glacial acetic acid, 115 DEG C of oil bath heatings, after back flow reaction 10h, is down to room temperature,
It is concentrated under reduced pressure.The dissolution of 20mL water is added in gained coarse extract product, adjusts pH value of solution to 7 or so, with acetic acid second with solid sodium carbonate
Ester extracts (30mL x 3), combining extraction liquid, sampling TLC detection.
Reaction experiment operation is catalyzed with sodium ethoxide: in addition 150mg (1.2mmol) imidazoles two in 25mL round-bottomed flask
4mL ethyl alcohol is added in ketone, and 159mg (2.3mmol) sodium ethoxide solid is added after mixing；Separately take 120mg Kessazulen aldehyde with 6mL second
Alcohol dissolution, is added dropwise above-mentioned imidazole diketone system.50 DEG C of oil bath heatings are down to room temperature after reacting 6h, are concentrated under reduced pressure.Gained
The dissolution of 20mL water is added in product, extracts (30mL x 3) with ethyl acetate, combining extraction liquid.It isolates and purifies: normal phase silicagel column layer
Analysis, with petroleum ether: methylene chloride 5:1,2:1 and absolute dichloromethane elution are eluted with target product when dichloromethane eluent,
For yellow-green soln.
Embodiment 10: the test of in-vitro antibacterial, anti-tumor activity
1. experimental material
The preparation of sample solution: test sample is object sterling in above-described embodiment 4,5,6.Precision weighs in right amount
Sample is configured to the solution of required concentration with methanol, for surveying activity.
Cell strain source: 231 (human breast cancer cells), MCF-7 (human breast cancer cell), K562 (people with red blood leukemia are used
Cell), HCT-116 (human colon cancer cell), Hela (human cervical carcinoma cell), A549 (human lung carcinoma cell), H1975 (human lung cancer
Cell), HUVEC (Human umbilical vein endothelial cells), MGC-803 (gastric carcinoma cells), (human neuroblastoma is thin by SH-SY5Y
Born of the same parents), HO8910 (Proliferation of Human Ovarian Cell), Siha (human cervical carcinoma cell), PC-3 (Human Prostate Cancer Cells) and BEL7402 (people
Hepatoma cell strain) cell strain be Chinese Marine University's Medicine and pharmacy college molecule pharmacological room provide for oneself.
MTT and SRB is purchased from Sigma company, with physiological saline solution, is made into the working solution of 5mg/mL, -20 DEG C of preservations；Two
Methyl sulfoxide (DMSO), analysis is pure, produces for Xi'an chemical reagent factory.
2. experimental method
A.MTT method
Principle: MTT (3- (4,5- dimethylthiazole -2) -2,5- hexichol tetrazole bromide) is a kind of weld, can connect
By H+.In the mitochondrial respiratory chain of living cells, cromoci and succinate dehydrogenase can make the tetrazole ring-splitting of MTT, raw
It is crystallized at the formazan of bluish violet, this dehydrogenase is then free of in dead cell.Formazan crystallizes production quantity and living cells
Quantity is directly proportional, and the maximum absorption band of the DMSO solution of the crystallization is at 570nm, and therefore, the influence of drug cell proliferation can
It is evaluated by the amount of detection formazan crystallization.
Test method:
(1) tumour cell of logarithmic phase is taken, adjustment concentration of cell suspension is 200000/mL, and 96 holes are added in every 200 μ L of hole
In tissue culture plate；
(2) in incubator cultivate 4h (37 DEG C, 5%CO2)；
(3) sample to be tested sets 5 concentration gradients (3 Duplicate Samples of each concentration) respectively, while setting negative, positive control,
Every hole adds 2 μ L of blank solution or sample liquid, cultivates 72h；
(4) 10 μ L of MTT liquid is added in every hole, continues to cultivate 4h；
(5) culture is terminated, is centrifuged 8min (37 DEG C, 2000r/min), sucks culture solution in hole；
(6) each 100 μ L of DMSO is added in every hole, and 15min is vibrated in micro oscillator, until crystallization sufficiently dissolution；
(7) microplate reader measures light absorption value (i.e. OD value) of every hole at 570nm.The mean OD value for taking three holes, by formula
IR%=(ODBlank control-ODSample)/ODBlank control× 100% calculates the inhibiting rate (IR%) of sample cell proliferation.
B.SRB method
Principle: SRB is a kind of pink anionic dye, soluble easily in water, in acid condition can specifically with cell
The basic amino acid of interior constitutive protein matter combines；Absorption peak, light absorption value and the linear positive of cell concentration are generated under 520nm wavelength
It closes, therefore can be used as the quantitative detection of cell number.It can also be stablized the long period with the SRB that Tris-base solution dissolves.It is particularly suitable for
Large-scale Screening drug is disadvantageous in that and is only limitted to attached cell, and staining procedure is more, cumbersome, easily causes human error.
Measuring method:
(1) tumour cell for taking logarithmic phase is 2 × 10 with fresh RPMI-1640 culture medium adjustment concentration of cell suspension5
A/mL, every 200 μ L of hole are added in 96 orifice plates, and the 2 μ L of sample solution of various concentration is added in every hole；
(2) 37 DEG C of culture 17h；
(3) 3min (4 DEG C, 3000prm) are centrifuged, suck supernatant；
(4) the 80%TCA solution of 50 μ L pre-cooling is added in every hole, moves into 4 DEG C of refrigerators and fixes 1.5h, then is rushed with deionized water
It washes 5 times, room temperature is dried；
(5) 0.5%SRB dye liquor (1% TCA solution) 50 μ L are added in every hole, after being stored at room temperature 30min, with the 1% of pre-cooling
TCA aqueous solution rinses 4 times, removes unbonded dyestuff, room temperature is dried；
(6) non-buffered Tris solution (10mM, the pH=10.5) dissolution of 150 μ L and protein bound dyestuff is added in every hole, uses
Microplate reader measures every hole absorbance OD value at 520nm.Each sample concentration sets 3 holes in 96 orifice plates, separately sets blank control 3
Hole takes 3 hole average values, by formula IR (%)=(ODBlank-ODSample)/ODBlank× 100% calculates cell proliferation inhibition rate (IR%),
Bliss method is recycled to find out half-inhibitory concentration (IC by the IR of various concentration50) standard deviation and average value.
3. experimental result
The inhibiting rate of 3 three compound on tumor cell Hela of table
The inhibiting rate of 4 three compound on tumor cell H1975 of table
The inhibiting rate of 5 three compound on tumor cell A549 of table
The inhibiting rate of 6 three compound on tumor cell 231 of table
The inhibiting rate of 7 three compound on tumor cell HCT-116 of table
The inhibiting rate of 8 two compound on tumor cell HL-60 of table
The inhibiting rate of 9 three compound on tumor cell K562 of table
Test result shows that seven kinds of tumour cells of 1 pair of compound test do not show apparent inhibitory activity, and changes
It closes object Z/E-2 and Z/E-3 and significant inhibitory activity, further expansion cell is showed to all tumour cells within the scope of primary dcreening operation
Strain screening range tests the two compounds, as a result, it has been found that its IC to the 14 kinds of tumour cells surveyed50Value is below
10 μM, test result is as shown in table 2.
Conclusion:
Above-mentioned experimental configuration shows new natural product Kessazulen sesquiterpene alkaloids Muriceidine A's of the present invention
Structure optimization product-compound Z/E-2 and Z/E-3 has significant broad-spectrum anti-tumor activity, can be used for grinding for anti-tumor drug
Study carefully and develop, enriches antineoplastic new chemical entities library.
Claims (8)
1. the new skeleton Muriceidine A (1) of the Kessazulen sesquiterpene alkaloids in gorgonian source and its structure optimization product
(E/Z-2, E/Z-3, E/Z-4, E/Z-5, E/Z-6), structure is as follows:
2. the synthetic method of the new skeleton of Kessazulen sesquiterpene alkaloids and its derivative described in claim 1, including following step
Rapid: (1) Vilsmeier-Haack reaction prepares Azulene aldehyde: DMF and phosphorus oxychloride react formation under conditions of anhydrous and oxygen-free
Vilsmeier nucleopilic reagent, is then added Kessazulen, carries out in the position 1- of its electron rich plus carbon reacts；(2)△1Nipecotic acid
(P2C) and its one pot reaction the preparation method of derivative: nipecotic acid, piperidines, methyl piperidine, piperidine carbinols, isonipecotic acid, pyrrolidines
Equal N- heterocycle carries out N- chloro, then the dehydrochlorination shape under the effect of the highly basic such as KOH, NaOH under now t-butyl hypochlorate effect processed
At the azomethine segment containing C=N；(3) it the condensation of Azulene aldehyde and P2C and its derivative: is reacted using Knoevenagel, with methanol
Or ethyl alcohol is solvent, heating reaction 10-30 hours in acetic acid/sodium acetate or sodium methoxide or sodium ethoxide buffer solution system.
3. the synthetic method of the new skeleton of Kessazulen sesquiterpene alkaloids according to claim 2, it is characterised in that: the party
Reconstruction structure (change of N- heterocyclic fragments substituent group, the change of ring size or Azulene of the method to compound 1 described in claim 1
Ring is changed to other fragrant segments) use.
4. the synthetic method of P2C according to claim 2 and its derivative, it is characterised in that: in N- heterocycle raw molecule
If the group of isopolarity containing COOH causes it to be difficult to dissolve in ether, first raw material is esterified, then carries out chlorination, N- chlorine
It can be carried out simultaneously under highly basic effect for intermediate dehydrochlorination and ester hydrolysis.
5. the synthetic method of P2C according to claim 2 and its derivative, it is characterised in that: used in N- chlorination
Chlorinating agent includes NCS and existing t-butyl hypochlorate processed (tert-butyl alcohol is protected from light 5 minutes or so with 0 DEG C of sodium hypochlorite), but after
Person's reaction effect is more preferable, more easy to handle.
6. the synthetic method of P2C according to claim 4 and its derivative, it is characterised in that: due to N- chloro intermediate
And the product after dehydrochlorination is unstable, it is more sensitive to light, temperature, air etc., total overall reaction be both needed to low temperature, be protected from light,
It is carried out under nitrogen protection.It is purified without isolation after obtaining azomethine segment by one pot reaction method, but uses low-temperature reduced-pressure
The method of distillation is replaced it to methanol system by ether system, in case using in next step.
7. the condensation reaction of Azulene aldehyde according to claim 2 and P2C and its derivative, it is characterised in that: in reaction system
Weak bronsted lowry acids and bases bronsted lowry need to be contained simultaneously, wherein weak acid often uses acetic acid, and alkali can be sodium alkoxide, sodium acetate etc..By investigating reaction time, reaction
Influence of five factors such as temperature, charging sequence, acid/base ratio, material concentration to object yield, find different compounds its
Respective optimum reaction condition and yield have certain difference.
8. compound described in claim 1 or 3 is preparing the purposes in anti-tumor drug and inhibition of cell proliferation.
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| CN114957081B (en) * | 2022-05-27 | 2024-04-05 | 中国科学院上海有机化学研究所 | Azulene compounds, intermediates, preparation method and application thereof |
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