CN106674163B - A kind of anti-mycobacterium tuberculosis compound and the preparation method and application thereof - Google Patents
A kind of anti-mycobacterium tuberculosis compound and the preparation method and application thereof Download PDFInfo
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- CN106674163B CN106674163B CN201710007440.3A CN201710007440A CN106674163B CN 106674163 B CN106674163 B CN 106674163B CN 201710007440 A CN201710007440 A CN 201710007440A CN 106674163 B CN106674163 B CN 106674163B
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 201000008827 tuberculosis Diseases 0.000 title abstract description 16
- 230000001355 anti-mycobacterial effect Effects 0.000 title abstract description 8
- 239000000126 substance Substances 0.000 claims abstract description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 153
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 88
- 239000003480 eluent Substances 0.000 claims description 59
- 238000010828 elution Methods 0.000 claims description 48
- 229940093499 ethyl acetate Drugs 0.000 claims description 47
- 235000019439 ethyl acetate Nutrition 0.000 claims description 47
- 239000003208 petroleum Substances 0.000 claims description 46
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 27
- 239000000741 silica gel Substances 0.000 claims description 27
- 229910002027 silica gel Inorganic materials 0.000 claims description 27
- 229960001866 silicon dioxide Drugs 0.000 claims description 27
- 238000010898 silica gel chromatography Methods 0.000 claims description 22
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
- 239000000284 extract Substances 0.000 claims description 20
- 238000004809 thin layer chromatography Methods 0.000 claims description 19
- 241000219112 Cucumis Species 0.000 claims description 18
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 claims description 18
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 claims description 18
- 239000003205 fragrance Substances 0.000 claims description 18
- 239000002023 wood Substances 0.000 claims description 17
- 239000013067 intermediate product Substances 0.000 claims description 14
- 239000002904 solvent Substances 0.000 claims description 14
- 239000000401 methanolic extract Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 230000005526 G1 to G0 transition Effects 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 239000012074 organic phase Substances 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000003463 adsorbent Substances 0.000 claims description 2
- NZCRJKRKKOLAOJ-XRCRFVBUSA-N rifaximin Chemical compound OC1=C(C(O)=C2C)C3=C4N=C5C=C(C)C=CN5C4=C1NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@H](C)[C@@H](OC)\C=C\O[C@@]1(C)OC2=C3C1=O NZCRJKRKKOLAOJ-XRCRFVBUSA-N 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims 1
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 6
- 241000894006 Bacteria Species 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 3
- 230000002365 anti-tubercular Effects 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 241000193830 Bacillus <bacterium> Species 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 238000000605 extraction Methods 0.000 description 9
- 239000012071 phase Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 5
- 241001189830 Fissistigma Species 0.000 description 4
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 239000004599 antimicrobial Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 239000007900 aqueous suspension Substances 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000000469 ethanolic extract Substances 0.000 description 2
- 239000012259 ether extract Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 201000009671 multidrug-resistant tuberculosis Diseases 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical group [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052805 deuterium Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 208000033065 inborn errors of immunity Diseases 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229960003350 isoniazid Drugs 0.000 description 1
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical group NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000013642 negative control Chemical group 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D307/40—Radicals substituted by oxygen atoms
- C07D307/46—Doubly bound oxygen atoms, or two oxygen atoms singly bound to the same carbon atom
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of anti-mycobacterium tuberculosis compound and the preparation method and application thereof, a kind of compound, structural formulas are as follows:The invention further relates to preparation methods and application, it is demonstrated experimentally that compound provided by the present invention has preferable anti-mycobacterium tuberculosis bacterium activity, are suitable for the research of antibacterial guide's chemical combination and prepare antituberculotic.
Description
Technical field
The present invention relates to a kind of anti-mycobacterium tuberculosis compounds and the preparation method and application thereof.
Background technique
Tuberculosis (Tuberculosis, TB) be by mycobacterium tuberculosis (MycobacteriumTuberculosis,
MTB a kind of chronic lethal disease caused by) is to endanger human health and lead to the great communicable disease of human death,
Lethality is only second to AIDS (Ac-quired Immunodeficiency syndrome, AIDS).
Due to a large amount of mutation using with treating tuberculosis action site of antituberculotic, lead to multi-drug resistance tuberculosis
(multidrug-resistant tuberculosis, MDR-TB) continuously emerges, along in recent years with AIDS population
The growth of quantity sends out tuberculosis and human immunodeficiency virus (human immunodeficiency virus, HIV) concurrently
Sick rate sharply increases, and the appearance of these problems all gives treatment zone lungy to carry out huge challenge, this has undoubtedly illustrated tuberculosis
Through the significant threat for becoming human health, it would be highly desirable to solve.
Summary of the invention
The object of the present invention is to provide a kind of antimicrobial compounds and the preparation method and application thereof.
Compound provided by the present invention is as shown in formula I.
The compound also belongs to protection scope of the present invention in the application prepared in anti-mycobacterium tuberculosis drug.
It is also another object of the present invention to provide a kind of antibacterials.
The active constituent of antibacterials provided by the present invention is I compound represented of above formula.
In the present invention, the antibacterials are anti-mycobacterium tuberculosis drug.
It is a further object to provide the preparation methods of the compound.
The preparation method of the compound provided by the present invention is to carry out methanol eddy to only mountain melon fragrance wood dry root to mention
It takes, only mountain melon fragrance wood methanol extract is obtained, then gained methanol extract is separated and purified, to obtain the compound.
Further, the methanol extract is prepared as follows: only mountain melon fragrance wood dry root is cut
Block is added the methanol of 2 times of weight, refluxing extraction 3 times, each 1.5h, removes methanol (being such as recovered under reduced pressure with Rotary Evaporators), water
Bath is evaporated, and obtains only mountain melon fragrance wood methanolic extract.
In the above-mentioned methods, the method for the separation and purifying specifically may include following steps:
(a) add water in Xiang Suoshu methanolic extract, extracted with the organic solvent that can dissolve the compound, collection has
Machine phase removes organic solvent, obtains the extract containing compound described in claim 1；
(b) extract is successively passed through to the silica gel column chromatography A, silica gel column chromatography B, prepares thin-layer chromatography, is separated
Obtain I compound represented of above formula.
In step (a), the solvent that can dissolve the compound can be ethyl acetate.
In step (b), when carrying out the silica gel column chromatography A, for 200-300 mesh, elution volume is the silica gel granularity used
5500mL, the eluent that the 1-500mL of elution volume is used by volume ratio for the petroleum ether of 10:1 and ethyl acetate mixing and
At, the eluent that the 501-1000mL of elution volume is used by volume ratio for the petroleum ether of 10:2 and ethyl acetate mixing and
At, the eluent that the 1001-1500mL of elution volume is used by volume ratio for the petroleum ether of 10:3 and ethyl acetate mixing and
At, the eluent that the 1501-2000mL of elution volume is used is mixed by volume ratio for the petroleum ether of 10:4 and ethyl acetate,
The eluent that the 2001-2500mL of elution volume is used is mixed by volume ratio for the petroleum ether of 10:5 and ethyl acetate, is washed
The eluent that the 2501-3000mL of lift-off product is used is mixed by volume ratio for the petroleum ether of 10:6 and ethyl acetate, is eluted
The eluent that the 3011-3500mL of volume is used is mixed by volume ratio for the petroleum ether of 10:7 and ethyl acetate, elution
The eluent that the 3501-4000mL of volume is used is mixed by volume ratio for the petroleum ether of 10:8 and ethyl acetate, elution
The eluent that the 4001-4500mL of volume is used is mixed by volume ratio for the petroleum ether of 10:9 and ethyl acetate, elution
The eluent that the 4501-5000mL of volume is used is mixed by volume ratio for the petroleum ether of 10:10 and ethyl acetate, elution
The eluent ethylacetate that the 5001-5500mL of volume is used elutes.
In step (b), when carrying out the silica gel column chromatography B, the silica gel granularity used can be 200-300 mesh, eluent
It is extremely mixed by petroleum ether and acetic acid second that volume ratio is 10:9, elution volume 1000mL.It carries out described preparing thin-layer chromatography
When, the adsorbent used for silica gel, silica gel granularity can be 200-400 mesh, solvent by volume ratio be 24:1 methylene chloride and
Methanol mixes.
In one embodiment of the invention, carry out described when preparing thin-layer chromatography, the silica gel used is GF254 model
Silica gel plate (is purchased from Qingdao Haiyang chemical industry).
In the above-mentioned methods, step (a) can are as follows: adds water in Xiang Suoshu methanolic extract, is extracted with petroleum ether, is collected
Water phase；The water phase is extracted with ethyl acetate again, collects organic phase, is i.e. " the extraction containing the compound described in acquisition
Take liquid ".In one embodiment of the invention, step (a) is more specific are as follows: 1 times of weight is added in Xiang Suoshu methanolic extract
Water, stir to get aqueous suspensions；Isometric petroleum ether is added into the aqueous suspensions again, extracts 30 minutes, extraction three times, is received
Collect water phase；Isometric ethyl acetate is added into the water phase again, extracts 30 minutes, extraction three times, collects organic phase, removal
Organic solvent is evaporated described in obtaining " extract containing the compound ".
In the above-mentioned methods, step (b) is concretely: the extract being carried out the silica gel column chromatography A, is collected intermediate
Product Fra.I, the intermediate product Fra.I using volume ratio for 10:9 petroleum ether and ethyl acetate mixtures as expansion
Agent, using silica gel that granularity is 100-200 mesh as in the thin-layer chromatography (27 DEG C of operation temperature) of stationary phase, Rf value (Rf) is
0.30-0.42；The intermediate product Fra.I is subjected to the silica gel column chromatography B again, collects intermediate product Subfra.I3, it is described
Intermediate product Subfra.I3 is being the petroleum ether of 10:9 and the mixed liquor of ethyl acetate as solvent using volume ratio, with granularity
For 200-300 mesh silica gel as in the thin-layer chromatography (27 DEG C of operation temperature) of stationary phase, Rf value (Rf) is 0.28-0.35；
Again by the intermediate product Subfra.I3 carry out it is described prepare thin-layer chromatography, collect the sample that Rf value (Rf) is 0.27, obtain
I compound represented of above formula.
Wherein, the intermediate product Fra.I is when carrying out the silica gel column chromatography A, is the petroleum of 10:9 with volume ratio
Obtained by ether and ethyl acetate mixtures are eluted as eluent, the 4001-4500mL of corresponding elution volume.The intermediate production
Object Subfra.I3 is when carrying out the silica gel column chromatography B, is the mixing of the petroleum ether and ethyl acetate of 10:9 with volume ratio
Obtained by liquid is eluted as eluent, the 401-600mL of corresponding elution volume.
More specifically, in step (b), when carrying out the silica gel column chromatography A, the specification of the chromatographic column used is 60X
900mm, column temperature are 27 DEG C, used silica gel 1.8kg, and applied sample amount is extract described in 36g, and flow velocity is * ml/min, are carrying out institute
When stating silica gel column chromatography B, the specification of the chromatographic column used is 20X 900mm, and column temperature is 27 DEG C, used silica gel 900g, loading
Amount is intermediate product Fra.I described in 1.5g, and flow velocity is * ml/min.Carry out it is described prepare thin-layer chromatography when, the lamellae of use
Specification be 20X 20cm, 27 DEG C of operation temperature；Applied sample amount 0.5ml；Duration of run 1h.
It is demonstrated experimentally that compound structure provided by the invention is completely new, there is preferable tubercle bacillus resistant activity, be suitable for resisting
The research of bacterium lead compound or preparation antibacterials.Natural product active ingredient preparation method used in the present invention has selected to produce
Raw only mountain melon fragrance with excellent antibacterial activity compound is wooden (Fissistigma cavaleriei), and extracting method is mature, work
Skill is easy, and products therefrom yield is high, and through magnetic resonance detection, structure is correct.
Detailed description of the invention
Fig. 1 is TLC of the present invention figure；
The position Fig. 2 active testing figure of the present invention；
Detailed description of the invention: Fig. 1 is from left to right followed successively by ethanol extract, petroleum ether extract, acetic acid ethyl ester extract and extraction
Take rear residue；Fig. 2 is from left to right successively are as follows: remains after ethanol extract, petroleum ether extract, acetic acid ethyl ester extract, extraction
Excess.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Only mountain melon fragrance wood dry root (Fissistigma cavaleriei): Guizhou Province's Qiandongnan Prefecture of Guizhou Province Dushan County is picked up from.Solely
It is big that mountain melon fragrance wood dry root (Fissistigma cavaleriei) voucher specimen has been preserved in Guizhou on 04 03rd, 2011
Learn pharmaceutical college.Certainly, only mountain melon fragrance wood dry root (Fissistigma cavaleriei) is also commercially bought.
Mycobacterium tuberculosis (MycobacteriumTuberculosis, MTB) is H37Rv type strain.
The preparation and identification of 1 antimicrobial compound of embodiment
Agents useful for same is to analyze pure (Tianjin Zhi Yuan chemical reagent Co., Ltd)；Silica gel column chromatography used silica gel 100-
200 mesh, 200-300 mesh are purchased from Haiyang Chemical Plant, Qingdao；Thin-layer chromatography used silica gel GF254 is prepared purchased from Qingdao City Qingdao
Marine chemical industry subsidiary factory.
One, the preparation of antimicrobial compound
1, only mountain melon fragrance wood dry root 3Kg, stripping and slicing, are added proper amount of methanol in batches, and heating and refluxing extraction 3 times, every time
Solvent is recovered under reduced pressure with Rotary Evaporators in 1.5h, to remove methanol, obtains only mountain melon fragrance wood methanolic extract.
The acquisition of only mountain melon fragrance wood dry root acetic acid ethyl ester extract
2, the water of 1 times of weight is added into the resulting only mountain melon fragrance wood root methanolic extract of step 1, stirring makes its suspension,
Isometric petroleum ether is added thereto again, extracts 0.3h, extraction three times, collects water phase；The bodies such as addition into the water phase again
Long-pending ethyl acetate, extracts 0.5h, and extraction three times, collects organic phase, solvent is recovered under reduced pressure with Rotary Evaporators, to remove second
Acetoacetic ester obtains only mountain melon fragrance wood root acetic acid ethyl ester extract.
3, the silica gel column chromatography of only mountain melon fragrance wood vinegar ethyl ester extract
Silica gel column chromatography condition is as follows
Silicagel column (normal pressure column) specification: 60X 900mm (Hua Kai experimental glass Instrument Ltd., Haimen City)；Column temperature is 27
℃；Used silica gel (200-300 mesh) amount is 1.8kg；The applied sample amount of only mountain melon fragrance wood root acetic acid ethyl ester extract is 36g；Elution
Liquid and elution program are as shown in table 1；Flow velocity is 2ml/min；Continuous to collect eluent, every 100mL collects a pipe.
The elution program of the silica gel column chromatography of the only mountain melon fragrance wood root acetic acid ethyl ester extract of table 1
| Eluent number | Eluent ingredient petroleum ether: ethyl acetate | Elution volume (ml) |
| Eluent 1 | Volume ratio 10:1 | 1-500 |
| Eluent 2 | Volume ratio 10:2 | 501-1000 |
| Eluent 3 | Volume ratio 10:3 | 1001-1500 |
| Eluent 4 | Volume ratio 10:4 | 1501-2000 |
| Eluent 5 | Volume ratio 10:5 | 2001-2500 |
| Eluent 6 | Volume ratio 10:6 | 2501-3000 |
| Eluent 7 | Volume ratio 10:7 | 3001-3500 |
| Eluent 8 | Volume ratio 10:8 | 3501-4000 |
| Eluent 9 | Volume ratio 10:9 | 4001-4500 |
| Eluent 10 | Volume ratio 10:10 | 4501-5000 |
| Eluent 11 | Volume ratio 0:1 | 5001-5500 |
Thin-layer chromatography (TLC) detection is carried out to collected each pipe eluent, silica gel column chromatography is carried out with each pipe eluent
The eluent of the respective components used when elution is used as stationary phase as solvent, using the silica gel that granularity is 100-200 mesh, operates
Temperature is 27 DEG C.Calculate the Rf value (Rf) of corresponding 10 fractions of 10 kinds of eluents.
As the result is shown: by 10 fractions collected by the step, it is denoted as Fra.A, Fra.B, Fra.C, Fra.D respectively,
Fra. E, Fra.F, Fra.G, Fra.H, Fra.I, Fra.J, Fra.K.Wherein, Fra.A is with eluent petroleum ether: acetic acid second
(1-500ml of corresponding elution volume) that ester (volume ratio 10:1) affords, Rf value (Rf) is 0.55-0.70；Fra.B
For with eluent petroleum ether: (501-1000ml of corresponding elution volume) that ethyl acetate (volume ratio 10:2) affords,
Rf value (Rf) is 0.50-0.67；Fra.C is with eluent petroleum ether: ethyl acetate (volume ratio 10:3) affords (right
Answer the 1001-1500ml of elution volume), Rf value (Rf) is 0.5-0.60；Fra.D is with eluent petroleum ether: acetic acid second
(1501-2000ml of corresponding elution volume) that ester (volume ratio 10:4) affords, Rf value (Rf) is 0.45-0.62；
Fra.E is with eluent petroleum ether: (the 2001- of corresponding elution volume that ethyl acetate (volume ratio 10:5) affords
2500ml), Rf value (Rf) is 0.40-0.58；Fra.F is with eluent petroleum ether: ethyl acetate (volume ratio 10:6) elutes
Obtained (2501-3000mL of corresponding elution volume), Rf value (Rf) is 0.37-0.55；Fra.G is with eluent petroleum
Ether: (3001-3500ml of corresponding elution volume) that ethyl acetate (volume ratio 10:7) affords, Rf value (Rf) is
0.35-0.55；Fra.H is with eluent petroleum ether: (the corresponding elution volume that ethyl acetate (volume ratio 10:8) affords
3501-4000ml), Rf value (Rf) is 0.33-0.53；Fra.I is with eluent petroleum ether: ethyl acetate (volume ratio
(4001-4500ml of corresponding elution volume) 10:9) afforded, Rf value (Rf) is 0.30-0.42；Fra.J is to use
Eluent petroleum ether: (4501-5000ml of corresponding elution volume) that ethyl acetate (volume ratio 10:10) affords, than
Shifting value (Rf) is 0.26-0.40.It is (corresponding volume 5001-5500ml) that ethyl acetate affords that Fra.K, which is with eluent,
Rf value 0.35-0.67.
4, the silica gel column chromatography of fraction Fra.I.
Silicagel column (normal pressure column) specification: 20X 900mm (Hua Kai experimental glass Instrument Ltd., Haimen City)；Column temperature is 27
℃；Used silica gel (200-300 mesh) amount is 900g；Applied sample amount is 1.5g (solid after 1.5g fraction Fra.I solvent evaporated)；It washes
De- liquid is petroleum ether: ethyl acetate (volume ratio 10:9)；Flow velocity is * ml/min；Elution volume is 1000ml.It is continuous to collect elution
Liquid, every 20ml collect a pipe.The 1-200mL of elution volume be the 1st fraction, elution volume 201-400ml be the 2nd
A fraction, elution volume 401-600ml be the 3rd fraction, elution volume 601-800ml be the 4th fraction, elution
The 801-1000ml of volume is the 5th fraction.
Thin-layer chromatography (TLC) detection is carried out to collected each pipe eluent, is used with when carrying out the above silica gel column chromatography
Eluent (volume ratio be 10:9 petroleum ether: ethyl acetate) be used as solvent, using granularity be 200-300 mesh silica gel as
Stationary phase, operation temperature are 27 DEG C.Calculate the Rf value (Rf) of 5 fractions.
The results show that 5 fractions collected by the step are denoted as Subfra.Il, Subfra.I2, Subfra. respectively
I3, Subfra.I4, Subfra.I5.Wherein, Subfra.Il corresponds to the 1-200mL of elution volume, and Rf value (Rf) is
0.38-0. 50；Subfra.I2 corresponds to the 201-400mL of elution volume, and Rf value (Rf) is 0.35-0.47；Subfra.I3
The 401-600m of corresponding elution volume, Rf value (Rf) are 0.32-0.40；Subfra.I4 corresponds to the 601- of elution volume
800mL, Rf value 0.26-0.34, Subfra.I5 correspond to the 801-1000ml of elution volume, Rf value 0.20-
0.30。
The preparation chromatography of 5, fraction Subfra.I3
Lamellae specification: 20X 20cm (prepares thin-layer chromatography used silica gel GF254 to study purchased from Yantai City's chemical industry
Institute)；27 DEG C of operation temperature；Applied sample amount 0.5ml；Duration of run 1h；Solvent is the methylene chloride that volume ratio is 24:1: methanol.
Loading expansion finishes, and develops the color, the sample that will test scrapes simultaneously together with silica gel, uses eluent (volume ratio 24:1
Methylene chloride: methanol) component is eluted, eluent is evaporated.40mg type I compound is obtained, in thin layer color made above
Rf value (Rf) in spectrum is 0.5.
Two, the Structural Identification of antimicrobial compound formula I
The type I compound that step 1 obtains is identified
(1) appearance: type I compound is yellow or weak yellow liquid to solid.
(2) organic solvents such as ethyl acetate, methylene chloride, methanol dissolubility: are soluble in.
(3) nuclear magnetic resoance spectrum: Fig. 1 is type I compound1H-NMR spectrum, Fig. 2 are type I compounds13C-NMR spectrogram.
According to compound1H-NMR,13C-NMR, to the nuclear magnetic resoance spectrum of two compounds carried out research and it is right13C signal carries out
Ownership, is shown in Table 2.And finally determine that structure is as follows:
The ownership of type I compound hydrogen spectrum and carbon spectral peak,
ES-MS m/z 126[M]+；H-NMR(CDCl3) δ: 4.9 (- OH), 4.7 (6-H), 6.5 (4-H), 7.3 (3-H),
9.5(CHO)；13C-NMR (CDCl 3) δ:: 57.0 (6-C), 161.5 (5-C), 111.6 (4-C), 124.0 (3-C), 152.0
(2-C),178.1(CO)。
Note: solvent is methylene chloride,
The NMR test of type I compound uses Li Waan nuclear magnetic resonance apparatus (400 megahertzs), and the solvent of test is deuterium
For methylene chloride.
Embodiment 2, the tubercle bacillus resistant activity measurement of type I compound
One, experimental method
Tubercle bacillus resistant activity measurement
Tubercle bacillus well-grown on modified Russell medium grows the proliferation of bacterium colony by observing it on culture medium
Situation measures the antibacterial activity of type I compound, and concrete operations are as follows.
By tubercle bacillus H37Rv is inoculated into consolidating containing modified Russell medium (winning microorganism Science and Technology Ltd. in Shanghai)
On body inclined-plane, drug concentration is respectively 500 μ g/ml, 2500 μ g/ml, 5000 μ g/ml, and positive controls are 2 μ g/ml of isoniazid,
Negative control group is 5% dimethyl sulfoxide, is placed in 37 DEG C of constant incubator and cultivates 10 days, counts untested compound to tuberculosis
The minimum inhibitory concentration value (MIC) of bacillus, by observing the growing state of tubercle bacillus, growth is suppressed 80% or more chemical combination
Object concentration is minimum inhibitory concentration value of the compound to tubercle bacillus.
Experiment is repeated 3 times.
Two, experimental result
The results show that the type I compound that embodiment 1 is prepared resists tubercle bacillus in anti-mycobacterium tuberculosis experiment
Bacterium activity MIC is determined as 500 μ g/ml, and concrete outcome is as shown in table 3.
Table 3, the antibacterial activity of type I compound
The test of 3 tubercle bacillus resistant activity of table
Claims (3)
1. a kind of preparation method of type I compound, it is characterised in that:
The preparation method of the compound is to carry out methanol to only mountain melon fragrance wood dry root to return
Stream extracts, and only mountain melon fragrance wood methanol extract is obtained, then gained methanol extract is separated and purified, to obtain the chemical combination
Object；The method of the separation and purifying includes the following steps:
(a) methanol aqueous solution for adding 5% in Xiang Suoshu methanol extract, is extracted with the organic solvent of dissolvable type I compound
Organic phase is collected, organic solvent is removed, obtains the extract containing type I compound；
(b) extract successively passed through into silica gel column chromatography A, silica gel column chromatography B, prepare TLC separation and obtain formula I and changes
Close object；In step (a), the solvent that can dissolve type I compound is ethyl acetate.
2. according to the method described in claim 1, it is characterized by:
In step (b), when carrying out the silica gel column chromatography A, for 200-300 mesh, elution volume is the silica gel granularity used
5500mL, the eluent that the 1-500mL of elution volume is used by volume ratio for the petroleum ether of 10:1 and ethyl acetate mixing and
At, the eluent that the 501-1000mL of elution volume is used by volume ratio for the petroleum ether of 10:2 and ethyl acetate mixing and
At, the eluent that the 1001-1500mL of elution volume is used by volume ratio for the petroleum ether of 10:3 and ethyl acetate mixing and
At, the eluent that the 1501-2000mL of elution volume is used is mixed by volume ratio for the petroleum ether of 10:4 and ethyl acetate,
The eluent that the 2001-2500mL of elution volume is used is mixed by volume ratio for the petroleum ether of 10:5 and ethyl acetate, is washed
The eluent that the 2501-3000mL of lift-off product is used is mixed by volume ratio for the petroleum ether of 10:6 and ethyl acetate, is eluted
The eluent that the 3001-3500mL of volume is used is mixed by volume ratio for the petroleum ether of 10:7 and ethyl acetate, elution
The eluent that the 3501-4000mL of volume is used is mixed by volume ratio for the petroleum ether of 10:8 and ethyl acetate, elution
The eluent that the 4001-4500mL of volume is used is mixed by volume ratio for the petroleum ether of 10:9 and ethyl acetate, elution
The eluent that the 4501-5000mL of volume is used is mixed by volume ratio for the petroleum ether of 10:10 and ethyl acetate, elution
The eluent ethylacetate that the 5001-5500mL of volume is used elutes；In step (b), the silica gel column chromatography B is carried out
When, for 200-300 mesh, eluent is mixed the silica gel granularity used by the petroleum ether that volume ratio is 10:9 and ethyl acetate；
In step (b), carry out it is described for the adsorbent used for silica gel, silica gel granularity is 200-400 mesh when preparing thin-layer chromatography, be unfolded
Agent is mixed by the methylene chloride that volume ratio is 24:1 and methanol.
3. according to the method described in claim 2, it is characterized by: the extract is carried out the silicagel column in step (b)
A is chromatographed, collects intermediate product Fra.I, the intermediate product Fra.I is mixed for the petroleum ether and ethyl acetate of 10:9 with volume ratio
Close liquid and be used as solvent, using granularity for 100-200 mesh silica gel as in the thin-layer chromatography of stationary phase, Rf value (Rf) is
0.52-0.79；The intermediate product Fra.F is subjected to the silica gel column chromatography B again, collects intermediate product Subfra.I3, it is described
Intermediate product Subfra.I3 is being the petroleum ether of 10:9 and the mixed liquor of ethyl acetate as solvent using volume ratio, with granularity
For 200-300 mesh silica gel as in the thin-layer chromatography of stationary phase, Rf value 0.28-0.35；Again by the intermediate product
Subfra.I3 progress is described to prepare thin-layer chromatography, collects the sample that Rf value is 0.5, obtains shown in claim 1 Chinese style I
Compound.
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