CN107445933A - 3 cumarin formic acid compounds and as the application for preparing antibacterial agents for pathogenic bacteria - Google Patents
3 cumarin formic acid compounds and as the application for preparing antibacterial agents for pathogenic bacteria Download PDFInfo
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- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
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Abstract
Simple in construction there is the compound that suppresses phytopathogen growth activity the present invention relates to a kind of, and in particular to 3 cumarin formic acid compounds and its as the application for preparing antibacterial agents for pathogenic bacteria.3 cumarin formic acid compounds are as the application for preparing antibacterial agents for pathogenic bacteria.The described cumarin formic acid compound of two class 3 has following molecular characterization：Wherein, R is hydrogen, methyl, methoxyl group, hydroxyl, halogen, nitro.Its use Colony morphology method determine synthesis 28 target compounds, the 4 kind plant pathogenic fungi common to apple decay pathogen, ring rot of apple opportunistic pathogen, Curvularia pathogen, dry rot of potato opportunistic pathogen etc. external inhibitory activity, find all compounds to being respectively provided with different degrees of inhibitory action for examination bacterium.
Description
First, technical field：
The present invention relates to a kind of compound with suppression phytopathogen growth activity simple in construction, and in particular to 3-
Cumarin formic acid compound and its as the application for preparing antibacterial agents for pathogenic bacteria.
2nd, background technology：
China is agricultural and populous nation, and agriculture stable development is advantageous to the sustainable development of national economy, however, plant
Disease not only reduces the yield and quality of agricultural product, and huge loss is brought to agricultural production, and therefore, it is carried out
Effective prevention and control are one of essential measures for ensureing agricultural high-effiency production and stable development, and current main means of prevention is agricultural chemicals
Apply extensively.As can be seen here, novel pesticide of the research and development with China independent intellectual property right is for China's agricultural or even national economy
Development has particularly important strategic importance.The research and development of the antibacterial agent of high yield simple in construction are always that people exhaust
Try hard to pursue what is asked.
It is documented that the fungi of nature has kind more than 100,000, wherein thus harmful disease fungus causes up to kind more than 8000
Disease not only occupy the 80% of plant disease total amount, it is and very harmful.Some fungies can make agricultural product, timber, food etc.
Moldy metamorphism, some parasitize in people and animals' body and cause skin disease, and some even produces the noxious material of aflatoxins etc, meeting
Jeopardize the health of humans and animals.In addition fungus breeding speed is fast, and transmission capacity is strong, it is difficult to be removed.In recent years, China is in plant
Although pathogen study on prevention in terms of achieve larger development, there is also it is certain the problem of.Traditional agricultural is prevented
Control --- the straightforward procedure such as crop rotation is not implemented very well so that these disease getting worses；Current main means of prevention
It is still to use chemical pesticide, but long term frequent causes disease Yuan bacterium to produce the stronger resistance to the action of a drug using chemical antiseptic, therefore, how
Efficient, green, environmentally friendly method is taken to prevent and treat the key that disease fungus is peasant's increasing both production and income and ecological environmental protection.
3rd, the content of the invention
It is an object of the invention to provide 3- cumarins formic acid compound and its as preparing phytopathogen antibacterial
The application of agent, its use Colony morphology method determine 28 target compounds of synthesis to apple decay pathogen,
The external suppression of 4 kinds of common plant pathogenic fungis such as ring rot of apple opportunistic pathogen, Curvularia pathogen, dry rot of potato opportunistic pathogen
System activity, find all compounds to being respectively provided with different degrees of inhibitory action for examination bacterium.Compound A-45, B3, B4, B5, B6 couple
The inhibitory activity of apple decay disease fungus is obviously higher than positive control medicine --- kresoxim-methyl, this 5 compounds have into
One step research and development value, is expected to develop into antibacterial agent.
To achieve the above object, the technical solution adopted by the present invention is：
3- cumarins formic acid compound is as the application for preparing antibacterial agents for pathogenic bacteria.
The 3- cumarin formic acid compounds of synthesis, there is following molecular characterization：
Wherein, R is hydrogen, methyl, methoxyl group, hydroxyl, halogen, nitro.
The synthetic route of described 3- cumarin formic acid compounds is specific as follows：
Compared with prior art, the invention has the advantages that and effect：
(1) such compound structure is simple, and molecular weight is small, and activity is preferably；
(2) establish the synthetic method of such compound and its structure is identified；This method step is brief, operation
Convenient, post processing is simple；
(3) antibacterial activity in vitro experiment proves：Such compound has different degrees of inhibitory activity to all for examination bacterium,
The bacteriostatic activity for wherein having 5 compounds is significantly higher than positive drug, particularly suppressions of the compound B5 to apple decay pathogen
Make activity (EC50=13.4 μ g/mL) and compound B3 to the inhibitory activity (EC of Curvularia disease fungus50=46.8 μ g/
ML) it is respectively positive control kresoxim-methyl activity (EC50=84.7 and 89.6 μ g/mL) 6.3 times and 1.9 times.
4th, illustrate：
Fig. 1 is the regression curve that compound A-45 suppresses apple decay disease fungus；
Fig. 2 is the regression curve that compound A-45 suppresses ring rot of apple fungal pathogenses；
Fig. 3 is the regression curve that compound B3 suppresses apple decay disease fungus；
Fig. 4 is the regression curve that compound B3 suppresses Curvularia disease fungus；
Fig. 5 is the regression curve that compound B4 suppresses apple decay disease fungus；
Fig. 6 is the regression curve that compound B5 suppresses apple decay disease fungus；
Fig. 7 is the regression curve that compound B5 suppresses ring rot of apple fungal pathogenses；
Fig. 8 is the regression curve of compound B5 inhibition of potato dry rot disease funguses；
Fig. 9 is the regression curve that compound B-26 suppresses apple decay disease fungus.
5th, embodiment
In the present invention, the 3- cumarin formic acid compound general structures designed and synthesized are as follows：
Wherein, R is hydrogen, halogen, methyl, methoxyl group, hydroxyl, nitro etc..
3- cumarins formic acid compound is as the application for preparing antibacterial agents for pathogenic bacteria.
The chemical constitution and physical property of target compound are as shown in table 1.
The structure and physical property of the 3- cumarin formic acid compounds of table 1
The synthetic route for 3- cumarin formic acid compounds that what the present invention established prepare is as follows：
(1) synthesis of 3- cumarins Ethyl formate class compound
Using substituted salicylic aldehydes as raw material, under piperidines catalysis, make its flow back in absolute ethyl alcohol 2 with diethyl malonate~
5h, that is, Knoevnagal condensation reactions occur, are prepared for 14 3- cumarin Ethyl formate class compounds (A1-A14).
(2) synthesis of 3- cumarins formic acid compound
3- cumarin Ethyl formate class compounds hydrolyze in the basic conditions, then are acidified to obtain corresponding 3- cumarins formic acid
Class compound (B1-B14).
The concrete operation step for synthesizing different type 3- cumarin formic acid compounds is as follows:
1. compound A1 --- the synthesis of 3- cumarin Ethyl formates
Salicylide (4.9g, 4.3mL, 40mmol), diethyl malonate are sequentially added into dry round-bottomed flask
(7.25g, 6.8mL, 45mmol), 25mL absolute ethyl alcohols, 0.5mL piperidines and 1~2 drop glacial acetic acid.Load onto condenser pipe and fill nothing
After the drying tube of water calcium chloride, it is stirred vigorously, is heated to flowing back；TLC tracing detections, react after 2h complete.Question response liquid cools down
To room temperature, it is transferred in conical flask, 3~5mL water is added into bottle, ice-water bath cooling makes product separate out completely；Decompression filters,
Product washs 2 times (3~5mL every time) with 50% ethanol of cooling, drains, obtains white crystal.Weigh, obtain 7.3g products, produce
Rate is 83.4%.2. compound A-45 --- the synthesis of 7- methoxyl group-3- cumarin Ethyl formates
2- hydroxyls -4-methoxybenzaldehyde (6.0g, 40mmol), malonic acid two are sequentially added into dry round-bottomed flask
Ethyl ester (7.25g, 6.8mL, 45mmol), 25mL absolute ethyl alcohols, 0.5mL piperidines and 1~2 drop glacial acetic acid.Load onto condenser pipe and Sheng
After having the drying tube of anhydrous calcium chloride, it is stirred vigorously, is heated to flowing back；TLC tracing detections, react after 2.5h complete.Question response
Liquid is transferred in conical flask after being cooled to room temperature, adds 3~5mL water thereto, and ice-water bath cooling makes product separate out completely；Decompression is taken out
Filter, product wash 2 times (3~5mL every time) with 50% ethanol of cooling, drain, obtain white crystal.Weigh, obtain 8.9g products,
Yield is 89.8%.
3. compound B-11 --- the synthesis of 3- cumarin formic acid compounds
3- cumarins Ethyl formate (4.0g, 18.3mmol), sodium hydroxide are sequentially added into 100mL round-bottomed flask
(3g, 75mmol), 20mL 95% ethanol, 10mL water, condenser pipe is loaded onto, after heating makes ester and sodium hydroxide whole dissolving, is returned
Stream stirring, TLC tracing detections stop reaction to reacting complete.Mixed liquor is poured into while hot and mixed by 15mL concentrated hydrochloric acids and 50mL water
Close and be acidified in the watery hydrochloric acid formed, there are a large amount of white crystals to separate out, ice-water bath cooling makes crystal separate out completely；Decompression is taken out
Filter, product are washed 2 times with a small amount of frozen water, drain to obtain crude product.Weighed after drying, obtain 3.0g products, yield 86.2%.
4. compound B3 --- the synthesis of 7- methylcoumarin-3- formic acid
As described in compound B-11, after charging reaction, condenser pipe is loaded onto, after heating makes ester and sodium hydroxide whole dissolving, is returned
Stream stirring, TLC tracing detections stop reaction to reacting complete.Mixed liquor is poured into while hot and mixed by 15mL concentrated hydrochloric acids and 50mL water
Close and be acidified in the watery hydrochloric acid formed, there are a large amount of white crystals to separate out, ice-water bath cooling makes crystal separate out completely；Decompression is taken out
Filter, product are washed 2 times with a small amount of frozen water, drain to obtain crude product.Weighed after drying, obtain 2.8g products, yield 79.2%.
5. compound B4 --- the synthesis of 6-Methylcoumarin-3- formic acid
As described in compound B-11, after charging reaction, condenser pipe is loaded onto, after heating makes ester and sodium hydroxide whole dissolving, is returned
Stream stirring, TLC tracing detections stop reaction to reacting complete.Mixed liquor is poured into while hot and mixed by 15mL concentrated hydrochloric acids and 50mL water
Close and be acidified in the watery hydrochloric acid formed, there are a large amount of white crystals to separate out, ice-water bath cooling makes crystal separate out completely；Decompression is taken out
Filter, product are washed 2 times with a small amount of frozen water, drain to obtain crude product.Weighed after drying, obtain 2.9g products, yield 82.5%.
6. compound B5 --- the synthesis of ayapanin-3- formic acid
As described in compound B-11, after charging reaction, condenser pipe is loaded onto, after heating makes ester and sodium hydroxide whole dissolving, is returned
Stream stirring, TLC tracing detections stop reaction to reacting complete.Mixed liquor is poured into while hot and mixed by 15mL concentrated hydrochloric acids and 50mL water
Close and be acidified in the watery hydrochloric acid formed, there are a large amount of white crystals to separate out, ice-water bath cooling makes crystal separate out completely；Decompression is taken out
Filter, product are washed 2 times with a small amount of frozen water, drain to obtain crude product.Weighed after drying, obtain 2.8g products, yield 80.7%.
7. compound B-26 --- the synthesis of 6- methoxy coumarin-3- formic acid
As described in compound B-11, after charging reaction, condenser pipe is loaded onto, after heating makes ester and sodium hydroxide whole dissolving, is returned
Stream stirring, TLC tracing detections stop reaction to reacting complete.Mixed liquor is poured into while hot and mixed by 15mL concentrated hydrochloric acids and 50mL water
Close and be acidified in the watery hydrochloric acid formed, there are a large amount of white crystals to separate out, ice-water bath cooling makes crystal separate out completely；Decompression is taken out
Filter, product are washed 2 times with a small amount of frozen water, drain to obtain crude product.Weighed after drying, obtain 3.0g products, yield 83.2%.
(3) spectral data of part of compounds
Applicant using spectroscopic technique (1H NMR and13C NMR) structural characterization has been carried out to all compounds, it is below 7
The data of the nmr spectrum of kind representation compound.
3- cumarins Ethyl formate (A1):1H NMR(500MHz,CDCl3)δ:8.54(1H,s,H-4),7.64(2H,q,J
=8.4Hz), 7.35 (2H, q, J=4.1Hz), 4.42 (2H, q, J=7.1Hz ,-CH 2-CH3), 1.41 (3H, t, J=7.1Hz ,-
CH2-CH 3)；13C NMR(125MHz,CDCl3)δ:163.1,156.8,155.2,148.6,134.4,129.5,124.9,
118.3,117.9,116.8,62.0,14.3。
7- methoxyl group -3- cumarins Ethyl formates (A5):1H NMR(500MHz,CDCl3)δ:8.51(1H,s,H-4),
7.51 (1H, d, J=8.7Hz), 6.90 (1H, q, J=3.7Hz), 6.82 (1H, d, J=2.0Hz), 4.40 (2H, q, J=
7.1Hz,-CH 2-CH3),3.91(3H,s,-OCH 3), 1.41 (3H, t, J=7.1Hz ,-CH2-CH 3)；13C NMR(125MHz,
CDCl3)δ:165.2,163.5,157.6,157.2,149.0,130.8,114.1,113.7,111.6,100.4,61.7,
56.1,14.3。
3- cumarins formic acid (B1):1H NMR(500MHz,DMSO-d6)δ:12.25(1H,s,-OH),8.96(1H,s,H-
4), 7.79 (2H, q, J=7.9Hz, Ph-H), 7.48-7.52 (m, 2H, J=4.8Hz, Ph-H)；13C NMR(125MHz,DMSO-
d6)δ:164.1,162.4,154.6,151.5,135.8,130.5,126.5,118.5,117.3,114.9。
7- methyl -3- cumarins formic acid (B3):1H NMR(500MHz,DMSO-d6)δ:13.03(1H,s,-OH),8.71
(1H, s, H-4), 7.79 (1H, d, J=7.3Hz), 7.28 (1H, s, H-8), 7.26 (1H, d, J=8.0Hz), 2.47 (3H, s,
7-CH3)；13C NMR(125MHz,DMSO-d6)δ:164.4,157.5,155.1,148.8,146.3,130.4,126.5,
117.5,116.6,116.1,21.9。
6- methyl -3- cumarins formic acid (B4):1H NMR(500MHz,CDCl3)δ:13.03(1H,s,-OH)8.65(1H,
S, H-4), 7.67 (1H, s, H-5), 7.54 (1H, dd, J=8.5,1.9Hz), 7.33 (1H, d, J=8.5Hz), 2.3748 (3H,
s,Ph-CH 3)；13C NMR(125MHz,CDCl3)δ:165.1,158.0,153.7,149.3,149.1,136.2,135.2,
130.7,130.6,119.3,118.7,116.9,21.2。
7- methoxyl group -3- cumarins formic acid (B5):1H NMR(500MHz,CDCl3)δ:8.75(1H,s,H-4),7.66
(1H, d, J=8.7Hz), 7.00 (1H, d, J=8.7Hz), 6.92 (1H, s, Ph-H), 4.24 (1H, s ,-OH),3.94(3H,s,
Ph-CH 3)；13C NMR(125MHz,CDCl3)δ:166.2,164.2,162.2,157.3,150.8,131.6,114.5,
112.2,111.6,100.6,56.0。
6- methoxyl group -3- cumarins formic acid (B6):1H NMR(500MHz,DMSO-d6)δ:13.22(1H,s,-OH),8.70
(1H, s, H-4), 7.48 (1H, d, J=3.0Hz), 7.00 (1H, d, J=8.7Hz), 7.40 (1H, d, J=9.1Hz), 7.34
(1H, dd, J=9.1,3.0Hz), 3.84 (3H, s, Ph-OCH3)；13C NMR(125MHz,DMSO-d6)δ:164.5,157.5,
156.2,149.4,148.6,122.5,119.0,118.9,117.7,112.3,56.3。
(3) antibacterial activity of 3- cumarins formic acid compound
1. for examination bacterium
Apple decay disease fungus (Valsa mali, VM)
Ring rot of apple fungal pathogenses (Botryosphaeria dothidea, BD)
Curvularia disease fungus (Curvularia lunata, CL)
Dry rot of potato fungal pathogenses (Fusarium solani, FS)
2.PDA culture mediums are prepared
According to the total amount of required culture medium, according to adding 200g potatoes, 15g agar, 20g glucose in every 1000mL water
Amount weighs raw material and prepares culture medium.Comprise the following steps that：Appropriate distilled water is added in stainless-steel pan, is heated to boiling；
The potato wash clean that will be weighed up, peeling, stripping and slicing, added when boiling water in pot, firing rate is adjusted after being boiled by fire, kept micro-
Boil 30min；With the above-mentioned mixed liquor of filtered through gauze to required volume, pH to 7 is adjusted, adds load weighted agar and grape in advance
Sugar, is stirred well to well mixed, is divided in conical flask after accurately weighing aequum with graduated cylinder, is sealed with special sealing membrane,
It is stand-by after autoclaving.
3. test sample solution is prepared
Precise testing sample, is loaded into 20mL bottle, and precalculated appropriate two are added with liquid-transfering gun
Methyl sulfoxide (DMSO), ultrasonication is completely dissolved it, covers bottle cap, is put into superclean bench；Then open ultraviolet
Lamp, irradiation is equipped with the bottle for treating sample measuring liquid, and sterilize 30min；It is 1 according to the volume ratio of DMSO and sterilized water:19 amount, with advance
The quantitative sterile distilled water of the pipette, extract that sterilized added in sample bottle, and testing sample is configured into 5% supplies test liquid,
It is stand-by after it is fully mixed.Negative control is made with isometric 5%DMSO aqueous solution, positive drug compound method and treats test sample
Condition is same.
4. the example of preliminary examination target compound antibacterial activity
In Vitro Bacteriostatic of the compound A-45 of example 1 to apple decay disease fungus
5mg compound A-45s are accurately weighed with 20mL bottles, 0.5mL DMSO and 9.5mL sterilized waters is injected thereto, will treat
Test sample product are made into 5% DMSO solution；Then add after sterilizing in the 90mL melted PDA culture medium, mix, be made into 100mL
Culture medium containing testing sample, is poured into sterile petri dish, and band medicine plating medium is made.It is 5% with equivalent solvent
DMSO is used as positive control as blank control using commercial antimicrobial medicine kresoxim-methyl.
The eugonic apple decay pathogen bacteria cake of colony edge is taken out with a diameter of 5mm card punch, uses transfer needle
Transfer them on above-mentioned plating medium, mycelia one is face-down, is attached on culture medium, triangular in shape to be positioned over per 3 pieces of ware
Centre, is placed in constant incubator after capping and cultivates, and temperature is 26 DEG C, and humidity 80%, illumination is set to 0%.After 72h, using ten
Word interior extrapolation method measures the square crossing diameter of bacterium colony with ruler, takes its average value.Often processing sets 3 repetitions.
Bacteriostasis rate is calculated as follows：
Mycelial growth inhibition rate (%)={ [blank control colony diameter-processing colony diameter] }/[blank colony diameter-
5]×100
Compound A-45 is to the average inhibition of apple decay disease fungus：58.1%.
In Vitro Bacteriostatics of the compound B3 of example 2 to Curvularia disease fungus
5mg compound B3 are accurately weighed with 20mL bottles, 0.5mL DMSO and 9.5mL sterilized waters is injected thereto, will treat
Test sample product are made into 5% DMSO solution；Then add after sterilizing in the 90mL melted PDA culture medium, mix, be made into 100mL
Culture medium containing testing sample, is poured into sterile petri dish, and band medicine plating medium is made.It is 5% with equivalent solvent
DMSO is used as positive control as blank control using commercial antimicrobial medicine kresoxim-methyl.
The eugonic Curvularia pathogen bacteria cake of colony edge is taken out with a diameter of 5mm card punch, uses transfer needle
Transfer them on above-mentioned plating medium, mycelia one is face-down, is attached on culture medium, triangular in shape to be positioned over per 3 pieces of ware
Centre, is placed in constant incubator after capping and cultivates, and temperature is 26 DEG C, and humidity 80%, illumination is set to 0%.After 72h, using ten
Word interior extrapolation method measures the square crossing diameter of bacterium colony with ruler, takes its average value.Often processing sets 3 repetitions.
Bacteriostasis rate is calculated as follows：
Mycelial growth inhibition rate (%)={ [blank control colony diameter-processing colony diameter] }/[blank colony diameter-
5]×100
Compound B3 is to the average inhibition of Curvularia disease fungus：49.8%.
In Vitro Bacteriostatics of the compound B5 of example 3 to ring rot of apple fungal pathogenses
5mg compound B5 are accurately weighed with 20mL bottles, 0.5mL DMSO and 9.5mL sterilized waters is injected thereto, will treat
Test sample product are made into 5% DMSO solution；Then add after sterilizing in the 90mL melted PDA culture medium, mix, be made into 100mL
Culture medium containing testing sample, is poured into sterile petri dish, and band medicine plating medium is made.It is 5% with equivalent solvent
DMSO is used as positive control as blank control using commercial antimicrobial medicine kresoxim-methyl.
The eugonic ring rot of apple opportunistic pathogen bacteria cake of colony edge is taken out with a diameter of 5mm card punch, uses transfer needle
Transfer them on above-mentioned plating medium, mycelia one is face-down, is attached on culture medium, triangular in shape to be positioned over per 3 pieces of ware
Centre, is placed in constant incubator after capping and cultivates, and temperature is 26 DEG C, and humidity 80%, illumination is set to 0%.After 72h, using ten
Word interior extrapolation method measures the square crossing diameter of bacterium colony with ruler, takes its average value.Often processing sets 3 repetitions.
Bacteriostasis rate is calculated as follows：
Mycelial growth inhibition rate (%)={ [blank control colony diameter-processing colony diameter] }/[blank colony diameter-
5]×100
Compound B5 is to the average inhibition of ring rot of apple fungal pathogenses：82.3%.
In Vitro Bacteriostatics of the compound B5 of example 4 to dry rot of potato fungal pathogenses
5mg compound B5 are accurately weighed with 20mL bottles, 0.5mL DMSO and 9.5mL sterilized waters is injected thereto, will treat
Test sample product are made into 5% DMSO solution；Then above-mentioned prepare liquid is added after sterilizing in the 90mL melted PDA culture medium, mixed
It is even, the culture medium that 100mL contains testing sample is made into, is poured into sterile petri dish, band medicine plating medium is made.It is molten with equivalent
Agent be 5% DMSO as blank control, positive control is used as using commercial antimicrobial medicine kresoxim-methyl.
The eugonic dry rot of potato opportunistic pathogen bacteria cake of colony edge is taken out with a diameter of 5mm card punch, with inoculation
Pin is transferred them on above-mentioned plating medium, and mycelia one is face-down, is attached on culture medium, triangular in shape to be positioned over per 3 pieces of ware
Center, it is placed in constant incubator and cultivates after capping, temperature is 26 DEG C, humidity 80%, illumination 0%.After 72h, using ten
Word interior extrapolation method measures the square crossing diameter of bacterium colony with ruler, takes its average value.Often processing sets 3 repetitions.
Bacteriostasis rate is calculated as follows：
Mycelial growth inhibition rate (%)={ [blank control colony diameter-processing colony diameter] }/[blank colony diameter-
5]×100
Compound B5 is to the average inhibition of dry rot of potato fungal pathogenses：57.0%.
In Vitro Bacteriostatic of the compound B-26 of example 5 to apple decay disease fungus
5mg compound B-26s are accurately weighed with 20mL bottles, 0.5mL DMSO and 9.5mL sterilized waters is injected thereto, will treat
Test sample product are made into 5% DMSO solution；Then add after sterilizing in the 90mL melted PDA culture medium, mix, be made into 100mL
Culture medium containing testing sample, is poured into sterile petri dish, and band medicine plating medium is made.It is 5% with equivalent solvent
DMSO is used as positive control as blank control using commercial antimicrobial medicine kresoxim-methyl.
The eugonic apple decay pathogen bacteria cake of colony edge is taken out with a diameter of 5mm card punch, uses transfer needle
Transfer them on above-mentioned plating medium, mycelia one is face-down, is attached on culture medium, triangular in shape to be positioned over per 3 pieces of ware
Centre, is placed in constant incubator after capping and cultivates, and temperature is 26 DEG C, and humidity 80%, illumination is set to 0%.After 72h, using ten
Word interior extrapolation method measures the square crossing diameter of bacterium colony with ruler, takes its average value.Often processing sets 3 repetitions.
Bacteriostasis rate is calculated as follows：
Mycelial growth inhibition rate (%)={ [blank control colony diameter-processing colony diameter] }/[blank colony diameter-
5]×100
Compound B-26 is to the average inhibition of apple decay disease fungus：92.7%.
Suppression of the 23 kinds of compounds of synthesis to 4 kinds of frequently seen plants disease funguses is determined using above-mentioned mycelial growth rate method
System activity, preliminary experimental results are as shown in table 2.
The target compound antifungal activity preliminary survey result of table 2 (50 μ g/mL, 72h)
As shown in Table 2：28 3- cumarins formic acid compounds of synthesis for 4 kinds of plant pathogenic fungis of examination to being respectively provided with
Different degrees of bacteriostatic activity.As can be seen from Table 1, when mass concentration is 50 μ g/mL, 28 kinds of target compounds have difference
The bacteriostatic activity of degree.Wherein B5, B6 have reached more than 90% to the inhibiting rate of apple decay pathogen；B3 is to canker of apple fruit
The inhibiting rate of opportunistic pathogen has reached 80% or so；B5 has reached more than 80% to the inhibiting rate of ring rot of apple opportunistic pathogen.Other targets
Compound is relatively low to the bacteriostatic activity of 4 kinds of phytopathogens compared with positive control kresoxim-methyl, to such compound structure
Optimization and bacteriostatic activity need further to further investigate.
In general, for examination bacterium, (apple decay pathogen, ring rot of apple are former to 4 kinds by compound A-45, B3, B4, B5, B6
Bacterium, dry rot of potato opportunistic pathogen, Curvularia pathogen) inhibitory activity generally compared with the height of kresoxim-methyl, we will further survey
Measure the antibacterial virulence of this 5 compounds.
5. determine the EC that partial target compound suppresses 4 kinds of plant pathogenic fungis50The example of value
Example 1 determines the EC that compound A-45 suppresses apple decay disease fungus50Value is (referring to Fig. 1)
A certain amount of compound A-45 is weighed with 20mL bottle, 5mL medicaments are dissolved as with 5% DMSO solution, addition is gone out
In the 95mL melted after bacterium PDA culture medium, mix, be made into the culture medium that 100mL contains testing sample, be finally configured to concentration
For 150,125,100,75,50,25,12.5 μ g/mL culture mediums, pour into sterile petri dish, band medicine plating medium is made, adopts
Mycelial growth inhibition rate of the test compound to bacterium to be measured is determined with above-mentioned mycelial growth rate method.With goodness of fit method analog drug
Thing docs-effect S curve, the S type curve linearization(-sation)s of amount-result relation are made by Scatchard methods and Hill methods, obtain cutting edge aligned poison
Power regression equation y=kx+b, EC is calculated using the softwares of GraphPad Prism 5.050Value.
Example 2 determines the EC that compound A-45 suppresses ring rot of apple fungal pathogenses50Value is (referring to Fig. 2)
A certain amount of compound A-45 is weighed with 20mL bottle, 5mL medicaments are dissolved as with 5% DMSO solution, addition is gone out
In the 95mL melted after bacterium PDA culture medium, mix, be made into the culture medium that 100mL contains testing sample, be finally configured to concentration
For 150,125,100,75,50,25,12.5 μ g/mL culture mediums, pour into sterile petri dish, band medicine plating medium is made, adopts
Mycelial growth inhibition rate of the test compound to bacterium to be measured is determined with above-mentioned mycelial growth rate method.With goodness of fit method analog drug
Thing docs-effect S curve, the S type curve linearization(-sation)s of amount-result relation are made by Scatchard methods and Hill methods, obtain cutting edge aligned poison
Power regression equation y=kx+b, EC is calculated using the softwares of GraphPad Prism 5.050Value.
Example 3 changes the EC that measure compound B3 suppresses apple decay disease fungus50Value is (referring to Fig. 3)
A certain amount of compound B3 is weighed with 20mL bottle, 5mL medicaments are dissolved as with 5% DMSO solution, addition is gone out
In the 95mL melted after bacterium PDA culture medium, mix, be made into the culture medium that 100mL contains testing sample, be finally configured to concentration
For 150,125,100,75,50,25,12.5 μ g/mL culture mediums, pour into sterile petri dish, band medicine plating medium is made, adopts
Mycelial growth inhibition rate of the test compound to bacterium to be measured is determined with above-mentioned mycelial growth rate method.With goodness of fit method analog drug
Thing docs-effect S curve, the S type curve linearization(-sation)s of amount-result relation are made by Scatchard methods and Hill methods, obtain cutting edge aligned poison
Power regression equation y=kx+b, EC is calculated using the softwares of GraphPad Prism 5.050Value.
Example 4 determines the EC that compound B3 suppresses Curvularia disease fungus50Value is (referring to Fig. 4)
A certain amount of compound B3 is weighed with 20mL bottle, 5mL medicaments are dissolved as with 5% DMSO solution, addition is gone out
In the 95mL melted after bacterium PDA culture medium, mix, be made into the culture medium that 100mL contains testing sample, be finally configured to concentration
For 150,125,100,75,50,25,12.5 μ g/mL culture mediums, pour into sterile petri dish, band medicine plating medium is made, adopts
Mycelial growth inhibition rate of the test compound to bacterium to be measured is determined with above-mentioned mycelial growth rate method.With goodness of fit method analog drug
Thing docs-effect S curve, the S type curve linearization(-sation)s of amount-result relation are made by Scatchard methods and Hill methods, obtain cutting edge aligned poison
Power regression equation y=kx+b, EC is calculated using the softwares of GraphPad Prism 5.050Value.
Example 5 determines the EC that compound B4 suppresses apple decay disease fungus50Value is (referring to Fig. 5)
Compound B4 is weighed to the sample of different quality in sample flasket, 5mL medicines are dissolved as with 5% DMSO solution
Agent, add after sterilizing in the 95mL melted PDA culture medium, mix, be made into the culture medium that 100mL contains testing sample, finally
It is 150,125,100,75,50,25,12.5 μ g/mL culture mediums to be configured to concentration, is poured into sterile petri dish, and band medicine is made and puts down
Face culture medium, mycelial growth inhibition rate of the test compound to bacterium to be measured is determined using above-mentioned mycelial growth rate method.With fitting
Goodness method aids drug docs-effect S curve, the S type curve straight lines of amount-result relation are made by Scatchard methods and Hill methods
Change, obtain cutting edge aligned virulence regression equation y=kx+b, EC is calculated using the softwares of GraphPad Prism 5.050Value.
Example 6 determines the EC that compound B5 suppresses apple decay fungal pathogenses50Value is (referring to Fig. 6)
A certain amount of compound B5 is weighed with 20mL bottle, 5mL medicaments are dissolved as with 5% DMSO solution, addition is gone out
In the 95mL melted after bacterium PDA culture medium, mix, be made into the culture medium that 100mL contains testing sample, be finally configured to concentration
For 150,125,100,75,50,25,12.5 μ g/mL culture mediums, pour into sterile petri dish, band medicine plating medium is made, adopts
Mycelial growth inhibition rate of the test compound to bacterium to be measured is determined with above-mentioned mycelial growth rate method.With goodness of fit method analog drug
Thing docs-effect S curve, the S type curve linearization(-sation)s of amount-result relation are made by Scatchard methods and Hill methods, obtain cutting edge aligned poison
Power regression equation y=kx+b, EC is calculated using the softwares of GraphPad Prism 5.050Value.
Example 7 determines the EC that compound B5 suppresses ring rot of apple fungal pathogenses50Value is (referring to Fig. 7)
A certain amount of compound B5 is weighed with 20mL bottle, 5mL medicaments are dissolved as with 5%DMSO solution, add sterilizing
In the 95mL melted afterwards PDA culture medium, mix, be made into the culture medium that 100mL contains testing sample, being finally configured to concentration is
150th, 125,100,75,50,25,12.5 μ g/mL culture mediums, are poured into sterile petri dish, and band medicine plating medium is made, uses
Mycelial growth inhibition rate of the above-mentioned mycelial growth rate method measure test compound to bacterium to be measured.With goodness of fit method aids drug
Docs-effect S curve, the S type curve linearization(-sation)s of amount-result relation are made by Scatchard methods and Hill methods, obtain cutting edge aligned virulence
Regression equation y=kx+b, EC is calculated using the softwares of GraphPad Prism 5.050Value.
Example 8 determines the EC of compound B5 inhibition of potato dry rot disease funguses50Value is (referring to Fig. 8)
A certain amount of compound B5 is weighed with 20mL bottle, 5mL medicaments are dissolved as with 5% DMSO solution, addition is gone out
In the 95mL melted after bacterium PDA culture medium, mix, be made into the culture medium that 100mL contains testing sample, be finally configured to concentration
For 150,125,100,75,50,25,12.5 μ g/mL culture mediums, pour into sterile petri dish, band medicine plating medium is made, adopts
Mycelial growth inhibition rate of the test compound to bacterium to be measured is determined with above-mentioned mycelial growth rate method.With goodness of fit method analog drug
Thing docs-effect S curve, the S type curve linearization(-sation)s of amount-result relation are made by Scatchard methods and Hill methods, obtain cutting edge aligned poison
Power regression equation y=kx+b, EC is calculated using the softwares of GraphPad Prism 5.050Value.
Example 9 determines the EC that compound B-26 suppresses apple decay disease fungus50Value is (referring to Fig. 9)
A certain amount of compound B-26 is weighed with 20mL bottle, 5mL medicaments are dissolved as with 5% DMSO solution, addition is gone out
In the 95mL melted after bacterium PDA culture medium, mix, be made into the culture medium that 100mL contains testing sample, be finally configured to concentration
For 100,75,50,40,30,20,10 μ g/mL culture mediums, pour into sterile petri dish, band medicine plating medium is made, in use
State mycelial growth inhibition rate of the mycelial growth rate method measure test compound to bacterium to be measured.With goodness of fit method analog drug agent
Amount-effect S curve, the S type curve linearization(-sation)s of amount-result relation are made by Scatchard methods and Hill methods, obtain cutting edge aligned virulence and return
Return equation y=kx+b, EC is calculated using the softwares of GraphPad Prism 5.050Value.
As a result as shown in table 3,5 kinds of higher compounds of activity are to the virulence equation of fraction of pathogens bacterium and its antibacterial with kresoxim-methyl
The contrast of activity.
Table 3 determines virulence regression equation of 5 kinds of higher compounds of activity to Activities of Some Plants pathogen
The virulence curve of the bacteriostatic activity of 5 kinds of 3- cumarin formic acid compounds of the above is shown in Fig. 1-9.By table 3 and Fig. 1-9
Data are understood：5 compounds chosen show preferable inhibitory activity to test plant disease fungus, its EC50It is worth model
Enclose and be located at 11.6-73.1 μ g/mL；The EC of which part compound50Value is less than kresoxim-methyl.It is all to supply examination thing to apple decay cause of disease
The inhibitory activity of bacterium is above kresoxim-methyl (EC50=84.7 μ g/mL), the EC of wherein B5 and B6 to apple decay pathogen50Value point
Not Wei 13.4 and 16.7 μ g/mL, illustrate apple decay pathogen to for try thing sensitivity it is higher.Comparatively, B5 suppresses
Apple decay, potato dry rot, the active highest of ring rot of apple opportunistic pathogen, corresponding EC50 values are respectively 13.4,11.6 and
14.9μg/mL；And B3 suppresses the EC of Curvularia pathogen50It is worth and is also significantly greater than kresoxim-methyl for 46.8 μ g/mL, its activity
(EC50=89.6 μ g/mL).Therefore, compound B3 and B5 is expected to develop into new Coumarins antiseptic.
These results absolutely prove test sample --- 3- cumarin formic acid compounds have antibacterial effect.
The foregoing is only a preferred embodiment of the present invention, is not intended to limit the scope of the present invention.
Claims (3)
1.3- cumarins formic acid compound is as the application for preparing antibacterial agents for pathogenic bacteria.
2. two classes 3- cumarin formic acid compounds according to claim 1, it is characterised in that：With following molecular structure
Feature：
Wherein, R is hydrogen, methyl, methoxyl group, hydroxyl, halogen, nitro.
3. 3- cumarins formic acid compound according to claim 2, it is characterised in that：3- cumarin formic acid compounds
Synthetic route it is specific as follows：
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| BERNADETTE S. CREAVEN ET AL.: "Synthesis, characterization and antimicrobial activity of a series of substituted coumarin-3-carboxylatosilver(I) complexes", 《INORGANICA CHIMICA ACTA》 * |
| J.C.MCGOWAN ET AL.: "The fungistatic activity of ethylenic and acetylenic compounds", 《ANNALS OF APPLIED BIOLOGY》 * |
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| CN113519530A (en) * | 2021-07-30 | 2021-10-22 | 西北农林科技大学 | Application of coumarin compound in prevention and treatment of apple tree canker |
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