CN106420729A - Application of composition of derivative of Schiglautone A in antibacterial agents - Google Patents
Application of composition of derivative of Schiglautone A in antibacterial agents Download PDFInfo
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- CN106420729A CN106420729A CN201610609443.XA CN201610609443A CN106420729A CN 106420729 A CN106420729 A CN 106420729A CN 201610609443 A CN201610609443 A CN 201610609443A CN 106420729 A CN106420729 A CN 106420729A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4192—1,2,3-Triazoles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
- C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
- C07C323/25—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the field of organic synthesis and pharmaceutical chemistry, in particular to compositions, preparing methods and applications of the compositions in preparing antibacterial agents. The invention discloses a composition and a preparing method thereof. Pharmacology experiments indicate that the composition has an antibacterial effect and has a value in the development of antibacterial agents.
Description
Technical field
The present invention relates to organic synthesiss and medicinal chemistry art are and in particular to compositionss, preparation method and its usage.
Background technology
The enhancing of the diffusion of pathogenic bacterium and its drug resistance seriously threatens the health and lives of the mankind, antibacterials conduct
Routine administration is widely used in acquired immune deficiency syndrome (AIDS), the controlling of organ transplantation and Chronic consumptionss (as cancer, diabetes, uremia etc.)
Treat although clinically use at present antimicrobial agent (as Ketoconazole, amikacin, gentamycin, voriconazole, Itraconazole,
Terbinafine, amphotericin, fluconazol etc.) preferable to the curative effect of skin and superficial place infection, but the storage of these antibacterials
Long-pending toxicity is stronger, usually causes lesions of liver and kidney, digestive tract stimulation, dizziness, allergy etc., so finding the new of mechanism of action uniqueness
Antibacterials become one of focus of current medicament research and development.
Helicobacter pylori (Helicobacter pylori, Hp) is a kind of Gram-negative spiral bacteria.Research is aobvious
Show, helicobacter pylori is the primary pathogenic event of acute and chronic gastritis and taste-blindness rate, and may be with gastric cancer stomach function regulating
The morbidity of mucosa-associated lymphoid tissue (MALT) malignant lymphoma is relevant.Recently, World Health Organization (WHO) that Hp is classified as I class is carcinogenic
Thing, it plays a leading role in stomach cancer development.The scheme of currently a popular treatment Hp infection is to take proton pump inhibitor simultaneously
(PPI) add the triple therapy of two kinds of antibiotic (clarithromycin, amoxicillin, tetracycline, metronidazole etc. select two kinds).Affect three
The main factor of therapy is considered as the drug resistance to antibacterial for the Hp；Another serious problems are that proton pump inhibitor can induce and disappears
Change bad, a large amount of antibacterial then lead to the serious destruction of flora in digestive tract.Therefore, find efficient, safe anti-Hp activity one
Kind new medicine thing becomes an important and urgent task.
Global in recent years morbidity lungy, in increasing trend, is estimated according to World Health Organization (WHO) (WHO), the whole world is tied at present
The population that core mycobacteria (Mycobacterium tuberculosis, MTB) infects accounts for 1/3rd of world population, wherein
5～10% the infected becomes tuberculosis patient.China active tuberculosiss patient 1,300,000, wherein infectiousness every year
Pulmonary tuberculosis about 600,000, wherein infectiousness pulmonary tuberculosis about 600,000, are one of global tuberculosis high burden countries.
Come out one after another from antituberculotics, make treatment lungy play epoch-making change.Yet with tuberculosis sufferer
The Case management of person still not ten sectional specification, irregular chemotherapy, abuse antituberculotics, make drug resistance of tuberculosis situation increasingly serious,
And the change of drug resistance tends to multi-medicament drug resistance simultaneously, this causes extreme difficulties to preventing and controlling lungy.Therefore
Find new antituberculotics, the antituberculotics of especially anti-multidrug resistance, to protection people's health, have important
Meaning.
Find compound or lead compound and carry out structural modification and obtain its derivant, thus obtaining from natural product
The potential drug of high-efficiency low-toxicity has important value most.
Compound I according to the present invention be one deliver within 2011 (Fan-Yu Meng et al.,
2011.Schiglautone A,a New Tricyclic Triterpenoid with a Unique 6/7/9-Fused
Skeleton from the Stems of Schisandra glaucescens.Organic Letters 13(2011)
1502 1505) compound, we have carried out structural modification to compound I, and obtaining two new derivants is compound
III and compound IV, and be prepared for compositionss with compound III and compound IV and said composition antibacterial activity is commented
Valency, it has antibacterial activity.
Content of the invention
The invention discloses new compositionss, said composition is made up of compound III and compound IV, said composition
The mass percent of middle compound III and compound IV is respectively 80% and 20%.
Compositionss disclosed by the invention can make pharmaceutically acceptable salt or pharmaceutically acceptable carrier.
Pharmacodynamic experiment shows, the compositionss of the present invention have preferable antibacterial action.The present invention's is pharmaceutically acceptable
Salt there is same drug effect.
Further, the experiment in vitro of compositionss shows, compositionss have very strong human body fungi activity, therefore this
Bright compositionss are expected to be used for preparing new anti-human fungi medicine.
Further, the experiment in vitro of compositionss shows, compositionss have very strong anti Helicobacter pylori activity, explanation
For the diseases such as the acute and chronic gastritis closely related with helicobacter pylori, duodenal ulcer, compositionss are one
The compound of individual great exploitation potential.It can be directly used for the treatment of corresponding disease and the preparation of related drugs.
Further, the experiment in vitro of compositionss shows, compositionss have very strong suppression escherichia coli, fluorescence vacation monospore
Bacterium, Staphylococcus aureus, Bacillus proteuss, the effect of neogenesis cryptococcus, so compositionss can be used as the change with antibacterial actions
Compound, and be expected to be applied in preparing anti-bacterial drug.
Further, the result according to preliminary test, the present invention with solid medium Dilution compositionss to card
The minimum suppression of Jie's Seedling, mycobacterium tuberculosis type strain H37Rv strain and substance of medicines-resistant branched tubercle bacillus (MDR MTB) three kinds of tulases
Bacteria concentration, experimental result confirms that compositionss have very strong anti-tubercle bacillus and anti-drug resistance tulase activity, can be used as treatment knot
The lead compound of solani infection disease is it can also be used to tubercular drugs are treated in preparation.
The present invention is further detailed explanation by the following examples, but protection scope of the present invention is not subject to concrete reality
Apply any restriction of example, but be defined in the claims.
Specific embodiment
The preparation of embodiment 1 compound Schiglautone A
Document (the Fan-Yu that the preparation method of compound Schiglautone A (I) is delivered with reference to Fan-Yu Meng et al.
Meng et al.,2011.Schiglautone A,a New Tricyclic Triterpenoid with a Unique 6/
7/9-Fused Skeleton from the Stems of Schisandra glaucescens.Organic Letters
13 (2011) 1,502 1505) method.
The synthesis of O- bromoethyl derivant (II) of embodiment 2 Schiglautone A
Compound I (502mg, 1.00mmol) is dissolved in 15mL benzene, adds tetrabutyl ammonium bromide (TBAB) in solution
(0.08g), 50% sodium hydroxide solution of glycol dibromide (7.520g, 40.00mmol) and 6mL.Mixture is Celsius 35
Degree stirring 8h.After 8h, reactant liquor is poured in frozen water, be extracted twice with dichloromethane immediately, merge organic phase solution.Then
Successively water and saturated common salt water washing 3 times are used to organic phase solution, then uses anhydrous sodium sulfate drying, last concentrating under reduced pressure removal is molten
Agent obtains product crude product.(mobile phase is product crude product silica gel column chromatography purification：Petroleum ether/acetone=100:1.0, v/v), receive
Collection brown is concentrated elution band and is flung to the brown ceramic powder (508mg, 71%) that solvent obtains compound II.
1H NMR(500MHz,DMSO-d6)δ13.40(s,1H),6.10(s,1H),5.63(s,1H),5.53(s,1H),
3.85 (d, J=11.2Hz, 4H), 3.52 (d, J=10.8Hz, 4H), 2.96 (s, 1H), 2.20 (s, 1H), 2.16 (s, 2H),
2.00 (s, 1H), 1.84 (d, J=13.9Hz, 4H), 1.69 (s, 1H), 1.58 (dd, J=22.2,8.5Hz, 4H), 1.51 (s,
1H), 1.47 (s, 1H), 1.26 (dd, J=9.1,4.4Hz, 4H), 1.21 (s, 1H), 1.08 0.98 (m, 4H), 0.96-0.94
(m,9H),0.94-0.85(m,6H).
13C NMR(125MHz,DMSO-d6)δ211.46(s),209.14(s),170.06(s),161.12(s),143.51
(s),132.01(s),127.77(s),85.96(s),82.40(s),70.19(s),69.10(s),57.14(s),52.73
(s),51.90(s),45.77(s),40.67(s),38.57(s),38.32(s),35.04(s),33.55(s),33.27(s),
29.85(s),28.98(s),26.71(s),25.50(s),24.05(s),22.31(s),21.06(s),20.56(s),20.00
(s),18.69(s),18.11(s),15.07(s).
HRMS(ESI)m/z[M-H]-calcd for C34H51Br2O6:715.2032；found 715.2027.
The synthesis of O- (triazolyl) ethyl derivative (III) of embodiment 3 Schiglautone A
Compound II (358mg, 0.5mmol) is dissolved in the middle of 10mL acetonitrile, be added thereto to Anhydrous potassium carbonate (345mg,
2.5mmol), potassium iodide (84mg, 0.5mmol) and 1,2,3- triazoles (2760mg, 40mmol), mixture is heated to reflux 3h.
Reaction after terminating is poured reactant liquor in frozen water into, is extracted 2 times with equivalent dichloromethane, merges organic faciess.Use water and saturation successively
Brine It merge after organic faciess, then use anhydrous sodium sulfate drying, concentrating under reduced pressure removal solvent obtain product crude product.Produce
(mobile phase is thing crude product silica gel column chromatography purification：Petroleum ether/acetone=100:1.0, v/v), collect faint yellow concentration eluting
Band obtains the yellow gummy solid (245.7mg, 71%) of compound III.
1H NMR (500MHz, DMSO-d6) δ 18.00 (s, 1H), 7.96 (d, J=15.5Hz, 2H), 7.67 (d, J=
20.5Hz, 2H), 6.10 (s, 1H), 5.63 (s, 1H), 5.33 (s, 1H), 4.29 (d, J=19.8Hz, 2H), 4.08 (d, J=
4.0Hz, 2H), 3.83 (d, J=4.8Hz, 4H), 3.47 (s, 1H), 2.39 (d, J=2.9Hz, 3H), 2.16 (s, 1H), 1.84
(d, J=0.5Hz, 4H), 1.68 1.61 (m, 3H), 1.61 1.50 (m, 3H), 1.46 (s, 1H), 1.39 (d, J=11.0Hz,
2H),1.30–1.21(m,2H),1.21(s,1H),1.05–0.70(m,19H).
13C NMR(125MHz,DMSO-d6)δ211.62(s),209.42(s),170.24(s),161.39(s),143.68
(s),137.15(s),132.19(s),128.04(s),120.80(s),86.24(s),82.58(s),66.14(s),65.60
(s),57.43(s),52.89(s),52.18(s),46.47(s),46.06(s),40.83(s),38.85(s),38.50(s),
35.31(s),33.72(s),30.12(s),29.15(s),27.00(s),25.66(s),24.33(s),22.49(s),21.33
(s),20.73(s),20.29(s),18.85(s),18.39(s),15.25(s).
HRMS(ESI):m/z[M+H]+calcd for C38H57N6O6:693.4340；found:693.4338.
The synthesis of O- (two (2-methylmercaptoethyl) amido) ethyl derivative of embodiment 4 Schiglautone A
1st, the synthesis of O- (two hydroxyethylamines) ethyl derivative of Schiglautone A
Compound II (358mg, 0.5mmol) is dissolved in 15mL acetonitrile, adds Anhydrous potassium carbonate (0.345g, 2.5mmol),
Potassium iodide (0.084g, 0.5mmol) and diethanolamine (1.0514g, 10mmol), mixture is heated to reflux 9h.After reaction terminates
Reactant liquor is poured in cold water, is extracted three times with dichloromethane, merge organic faciess, use water and saturated common salt water washing successively, no
Aqueous sodium persulfate is dried, and concentrating under reduced pressure removes solvent.Product silica gel column chromatography purifies (petroleum ether/acetone 100:1, v/v), obtain
The faint yellow solid (0.199g, 52%) of O- (two hydroxyethylamines) ethyl derivative of Schiglautone A.
1H NMR(500MHz,DMSO-d6)δ17.77(s,1H),6.00(s,1H),5.40(s,1H),5.20(s,1H),
3.37 (d, J=17.8Hz, 4H), 3.28 (s, 8H), 2.87 (s, 1H), 2.51 (d, J=6.5Hz, 4H), 2.43 (s, 8H),
(2.24 s, 2H), 2.06 (d, J=12.7Hz, 2H), 1.76 1.68 (m, 5H), 1.57 (s, 1H), 1.53 1.39 (m, 8H),
1.36(s,1H),1.24(s,1H),1.20–1.14(m,3H),1.11(s,1H),0.94–0.82(m,10H),0.80(s,3H),
0.76 (s, 3H), 0.72 (d, J=20.0Hz, 3H).
13C NMR(125MHz,DMSO-d6)δ211.39(s),208.98(s),169.80(s),160.73(s),143.02
(s),131.42(s),127.05(s),85.91(s),82.25(s),66.70(s),65.63(s),58.62(s),56.55
(s),56.61(s),53.30(s),52.45(s),51.52(s),45.29(s),40.06(s),38.52(s),38.17(s),
34.73(s),33.17(s),29.35(s),28.38(s),26.34(s),25.00(s),23.45(s),22.05(s),20.67
(s),20.07(s),19.31(s),18.41(s),17.73(s),14.59(s).
HRMS(ESI):m/z[M+H]+calcd for C42H73N2O10:765.5265；found:765.5261.
O- (two hydroxyethylamines) ethyl derivative of Schiglautone A
2nd, the synthesis of O- (bischloroethylamines base) ethyl derivative of Schiglautone A
Prepare O- (two hydroxyethylamines) ethyl derivative of the Schiglautone A of 1g according to above-mentioned steps, take
The O- (two hydroxyethylamines) ethyl derivative (382mg, 0.5mmol) of Schiglautone A is dissolved in 8mL chloroform, is added dropwise over chlorine
Change sulfoxide (0.238g, 2mmol), reactant is heated to reflux 2h.Reactant is cooled to room temperature, the excessive chlorine of Deca Methanol Decomposition
Change sulfoxide, concentrating under reduced pressure removes solvent.Product is through silica gel column chromatography purification (petroleum ether/acetone 100:1, v/v), obtain
The faint yellow colloidal solid (0.297g, 71%) of O- (bischloroethylamines base) ethyl derivative of Schiglautone A.
1H NMR (500MHz, DMSO-d6) δ 13.87 (s, 1H), 6.01 (s, 1H), 5.50 (d, J=8.4Hz, 2H),
3.61 (s, 4H), 3.55 (s, 4H), 3.45 (s, 2H), 3.40 (s, 2H), 2.98 (s, 1H), 2.73 (d, J=17.4Hz, 4H),
2.67–2.57(m,6H),2.54(s,2H),2.40(s,1H),2.10(s,1H),1.99(s,2H),1.78(s,1H),1.73
(d, J=16.9Hz, 4H), 1.58 (d, J=3.0Hz, 2H), 1.46 (d, J=3.0Hz, 2H), 1.43 (s, 1H), 1.37 (s,
1H), 1.22 (s, 1H), 1.17 (dd, J=18.9,11.1Hz, 4H), 0.95 0.83 (m, 10H), 0.80 (d, J=20.0Hz,
3H), 0.78 (d, J=20.0Hz, 3H), 0.73 (d, J=20.0Hz, 3H).
13C NMR(125MHz,DMSO-d6)δ211.19(s),208.87(s),169.79(s),160.85(s),143.24
(s),131.74(s),127.50(s),85.69(s),82.13(s),66.71(s),65.84(s),56.87(s),55.80
(s),53.19(s),52.44(s),51.64(s),45.51(s),40.38(s),39.22(s),38.30(s),38.05(s),
34.77(s),33.28(s),29.56(s),28.72(s),26.45(s),25.21(s),23.79(s),22.05(s),20.77
(s),20.30(s),19.76(s),18.41(s),17.85(s),14.81(s).
HRMS(ESI):m/z[M+H]+calcd for C42H69Cl4N2O6:839.3880；found:839.3881.
O- (bischloroethylamines base) ethyl derivative of Schiglautone A
3rd, the synthesis of O- (two (2-methylmercaptoethyl) amido) ethyl derivative (IV) of Schiglautone A
Prepare O- (bischloroethylamines base) ethyl derivative of the Schiglautone A of 1g according to above-mentioned steps, will
The O- (bischloroethylamines base) ethyl derivative (0.419g, 0.5mmol) of Schiglautone A is dissolved in 15mL ethanol, adds under room temperature
Enter sodium methyl mercaptide (0.21g, 3mmol), reactant is heated to reflux 3h.Concentrating under reduced pressure removes solvent, products therefrom silica gel column layer
Analysis carries out purification (petroleum ether/acetone 100:0.2, v/v), obtain yellow solid, i.e. compound IV (0.296g, 67%).
1H NMR(500MHz,Chloroform-d1)δ13.21(s,1H),6.02(s,1H),5.48(s,1H),5.44
(s,1H),3.45(s,2H),3.38(s,2H),3.00(s,1H),2.57–2.48(m,20H),2.16–2.09(m,4H),1.96
(s, 12H), 1.73 (d, J=15.4Hz, 4H), 1.55 (s, 1H), 1.44 (dd, J=7.8,2.8Hz, 4H), 1.35 (s, 1H),
1.29–1.14(m,6H),0.97–0.49(m,19H).
13C NMR(125MHz,DMSO-d6)δ211.72(s),209.42(s),170.36(s),161.38(s),143.79
(s),132.31(s),128.03(s),86.24(s),82.70(s),67.24(s),66.39(s),57.44(s),53.73
(s),53.00(s),52.69(s),52.17(s),46.06(s),40.95(s),38.84(s),38.61(s),35.32(s),
33.82(s),32.41(s),30.13(s),29.25(s),27.00(s),25.77(s),24.34(s),22.59(s),21.33
(s),20.85(s),20.28(s),18.96(s),18.40(s),15.35(s),15.03(s).
HRMS(ESI):m/z[M+H]+calcd for C46H81N2O6S4 +:885.4977；found:885.4979.
Embodiment 5 compositionss human body fungi activity
The preparation of compositionss：Cross the powder of the 80mg compound III of 200 mesh nets after grinding and cross 200 after grinding
The powder of the 20mg compound IV of mesh net loads in tubule with cover and obtains 100mg compositionss with the mixing of turbine stirring instrument,
Obtain the solution of compositionss with the compositionss of this 100mg of water dissolution during use.
The experiment of human body fungi activity is the method using concentration dilution, measures in triplicate every time, the pathogen of test
There are trichophyton, microsporum canis and trichophyton tonsurans, bacterial concentration is 105Individual/mL.The initial concentration of medicine is
50.00 μ g/mL (5% dimethyl sulfoxide DMSO), gradient dilution to 0.10 μ g/mL, mix by the bacterium solution of equivalent volumes and test sample
(ultimate density of formation has in 96 orifice plates to close culture：25.00、12.50、6.25、3.13、1.56、0.78、0.39、0.20、
0.10 and 0.05 μ g/mL), Human Fungi cultivation temperature is respectively 28 DEG C, observes after incubation time 24h, if finding do not have bacterium colony
In the disposal hole being formed, that minimum concentration is sample lowest concentration of antimicrobial, i.e. MIC value.This experiment positive control is ketone health
Azoles, compositionss human body funguses the results are shown in Table 1.
Table 1 compositionss human body funguses MIC value (μ g/mL)
Conclusion：Compositionss have very strong human body fungi activity, and the compositionss of the therefore present invention are expected to be used for preparing
New anti-human fungi medicine.Compound III and compound IV nonreactive Human Fungi activity, therefore cannot be used for preparing new resisting
Human Fungi medicine.
Embodiment 6 compositionss anti-helicobactor pylori activity
1) strains tested
Helicobacter pylori (Helicobacter pylori, Hp) reference culture ATCC 43504 is purchased from U.S.'s fungi preservation
Center (American Type Culture Collection, ATCC).13 plants of Hp clinical strains pick up from Nanjing drum tower hospital
Dndoscope Laboratory accepts gastroscopic patient；To peptic ulcer, duodenal bulbar inflammation or gastritis verrucosa in continuous gastroscopy
Patient, be first defined as Hp positive through RUT experiment, then take antral gastric mucosa 1-2 block, be inoculated in after chopping containing 8% horse
Serum, trimethoprim (trimethropin, TMP) 1.25g/L, polymyxin (polymyxin) B2500U/L, through the ages
In the Columbia selectivity agar culture medium of mycin (vancomycin) 10mg/L, in 37 DEG C of (5%O under micro- oxygen environment2,
10%CO2And 85%N2) cultivate 72 hours.Collect antibacterial, through smear Gram’s staining, oxidase, catalase and urease are accredited as
After the positive, pass on pure culture, obtained strains are as experimental strain.
2) strain culturing
We carry out the strain culturing of HP using micro- aerobic bag (purchased from Shanghai Medical Univ), and it can be produced by chemical reaction
Micro- aerobic environment required for raw Hp.
3) biological activity determination
Measure medicine using paper disk method (Microbiological paper method) suppression of helicobacter pylori is made
With, measured with agar dilution test sample minimum inhibitory concentration (minimal inhibitory concentration,
MIC).
I. paper disk method experiment
(A) preparation culture medium, by the Columbia preparing culture medium after high pressure steam sterilization, is cooled to 50-60 DEG C,
Plus 8% horse serum or Sheep Blood, mix and be poured in sterilized culture dish, every ware 7-10ml, culture medium thickness are 1.5mm
(sterile working).
(B) switching experimental bacteria (applying bacterium) takes 10 having diluted with microscale sampler8CFU/ml(1OD660=108CFU/ml)Hp
Bacteria suspension 0.1ml be equably applied to suitable culture dish surface.Take out after being inverted in 15min in 37 DEG C of drying bakers, purpose
Agar surface is made to be dried, standby.
(C) patch sample scraps of paper microscale sampler takes 6 μ l testing sample (mass concentration 2mg/ml) injections sterilized
On circle filter paper.Contain the scraps of paper and the comparison virgin paper sheet of sample with aseptic nipper tweezer, the scraps of paper are close to respectively by sterile working
Surface containing bacterio-agar, pastes a piece of paper piece at a certain distance.Every kind of bacterium is cooked 3 wares, and acquired results seek its meansigma methods.
(D) each plate is placed in micro- aerobic bag for culture, sealing, opens gas generator, then is placed in 37 DEG C of incubators
Culture 72h.
(E), after surveying antibacterial circle diameter taking-up flat board, measure the size of antibacterial circle diameter around each scraps of paper respectively.With reference to comparison
The result of group, can draw the result of testing sample sensitive experiment.In triplicate.
II. agar dilution measures MIC
(A) medicine dimethyl sulfoxide (DMSO) solution of test is configured to 0.5mg/ml by the preparation of Drug plates first
Mother solution, then with sterilized water dilution, be finally made into 100.0,80.0,60.0,45.0,40.0,35.0,30.0,25.0,20.0,
The concentration series of 15.0,10.0,5.0 and 2.5 μ g/ml, DMSO concentration in media as well is less than 1%.The test that 1ml is prepared
Drug solution and the 8ml Columbia medium being incubated in 50 DEG C, the horse serum that another plus 1ml is incubated in 50 DEG C fully mixes, and pours
Make in culture dish cooling (final concentration of the 10.0 of medicine in culture dish, 8.0,6.0,4.5,4.0,3.5,3.0,2.5,
2.0,1.5,1.0,0.5 and 0.25 μ g/ml, if there are bacteriostasis, then MIC value is that these are one of worthwhile).
(B) switching experimental bacteria (applying bacterium) draws, with microscale sampler, 1 × 10 having diluted8The bacteria suspension of CFU/ml Hp
0.1ml is equably applied to culture dish surface, takes out after being inverted in 15min in 37 DEG C of drying bakers, and purpose makes agar surface be dried,
Standby.
(C) determine that test dish (is contained by MIC in micro- aerobic bag：85%N2, 10%CO2And 5%O2) in, it is incubated 37 DEG C
Culture 72 hours, is observed Hp growing state, is contrasted with blank group, be minimum with the sample least concentration not having bacteria growing completely
Mlc (minimal inhibitory concentration, MIC) value.Positive control is ampicillin
(Ampicillin).
Experimental result
Experiment in vitro shows, compositionss have very strong anti Helicobacter pylori activity, and paper disk method shows that its inhibition zone is straight
Footpath is 21mm (ATCC43504).Show that it can completely inhibit 5 random clinical strains and 1 standard bacteria with agar dilution
The growth of strain (ATCC43504), minimal inhibitory concentration (MIC) is 2.0 μ g/ml.Make positive control with ampicillin, it is to 6
The Cmin (MIC) of strain test bacterium complete inhibition is 1.5 μ g/ml.Compound III and compound IV no helicobacter pylori resistant
Activity.
Conclusion：Compositionss have stronger suppression helicobacter pylori activity, illustrate for close with helicobacter pylori
For the diseases such as the acute and chronic gastritis of correlation, duodenal ulcer, compositionss are the medicines of a great exploitation potential.It
Can be directly used for the treatment of corresponding disease and the preparation of related drugs.Compound III and compound IV nonreactive H. pylori
Bacterium activity is it is not possible to be used for the treatment of corresponding disease and the preparation of related drugs.
Embodiment 7 compositionss antibacterial activity
Antibacterial activity test is the method using concentration dilution, measures in triplicate every time, and test pathogen has large intestine bar
Bacterium, fluorescent pseudomonas, Staphylococcus aureus, Bacillus proteuss, neogenesis cryptococcus, bacterial concentration is 105Individual/mL.Medicine initiates
Concentration is 50.00 μ g/mL (5% dimethyl sulfoxide DMSO), and gradient dilution is to 0.10 μ g/mL, the bacterium solution of equivalent volumes and test
In 96 orifice plates, (ultimate density of formation has for sample mix culture：25.00、12.50、6.25、3.13、1.56、0.78、0.39、
0.20th, 0.10 and 0.05 μ g/mL), antibacterial culturing temperature is respectively 37 DEG C, observes after incubation time 24h, if finding do not have bacterium colony
In the disposal hole being formed, that minimum concentration is sample lowest concentration of antimicrobial, i.e. MIC value.Compositionss the anti-bacterial result is shown in Table 2.
Table 2 compositionss antibacterium MIC value (μ g/mL)
Conclusion：Compositionss have very strong antibacterial activity, and the compositionss of the therefore present invention are expected to be used for prepare new
Anti-bacterial drug.Compound III and compound IV no antibacterial activity are it is impossible to be used for preparing new anti-bacterial drug.
Embodiment 7 compositionss anti-tubercle bacillus activity
(1) the anti-bacillus calmette-guerin vaccine of solid medium Dilution compositionss (BCG) absolute concentration
Scrape BCG cultures from inclined-plane, be added in 3ml Middlebrook7H9 broth bouillon, add few
Amount bead, screws test tube cap, and in vortex oscillator, high vibration grinds, with standard Maxwell opacity tube (MacFarland
No.1) ratio is turbid, that is, be made into bacillus calmette-guerin vaccine (BCG) bacteria suspension of 1mg/ml.
Medicine is made into the stock solution of high concentration with DMSO, extremely required with the tween 80 aseptic ultra-pure water dilution stock solution containing 5%
Concentration, the medicine having diluted is added to 4ml Middlebrook7H11 agar culture medium by required dosage, and (this culture medium is
121 DEG C of high pressure steam sterilizations 15 minutes, it is cooled to 50～55 DEG C), mix, make drug containing, concentration is respectively 6.0ug/ml,
The isocyatic inclined-plane of 4.0ug/ml, 3.0ug/ml, 2.0ug/ml, 1.5ug/ml, 1.0ug/ml, 0.75ug/ml, 0.5ug/ml is trained
Foster base.
Bacillus calmette-guerin vaccine (BCG) the bacteria suspension inoculating loop for 1mg/ml for the concentration is dipped ring of numbers, is inoculated in respectively containing each medicine
In the culture medium of thing series concentration and blank medium slant, it is placed in 37 DEG C and cultivates 4～8 weeks, observation experiment result, result
As shown in table 3.
In the present embodiment, Middlebrook7H9 broth bouillon used and Middlebrook7H11 agar culture medium are this
Skilled person carries out conventional culture medium during tulase culture, and its formula adopts conventional formulation.
(2) solid medium Dilution compositionss Killing Mycobacterium Tuberculosis type strain H37Rv strain absolute concentration
Scrape mycobacterium tuberculosis type strain H37Rv strain culture from inclined-plane, be added to 3ml Middlebrook7H9
In broth bouillon, add a small amount of bead, screw test tube cap, high vibration grinds in vortex oscillator, with standard Maxwell
Opacity tube (MacFarland No.1) ratio is turbid, that is, be made into the H37Rv strain bacteria suspension of 1mg/ml.
Medicine is made into respectively the stock solution of high concentration with DMSO, with the tween 80 aseptic ultra-pure water dilution stock solution containing 5% extremely
Desired concn, the medicine having diluted is added to 4ml Middlebrook7H11 agar culture medium (this culture medium by required dosage
121 DEG C of high pressure steam sterilizations 15 minutes, be cooled to 50～55 DEG C), mix, make drug containing, concentration is respectively 6.0ug/
Ml, 4.0ug/ml, 3.0ug/ml, 2.0ug/ml, 1.5ug/ml, 1.0ug/ml, 0.75ug/ml, 0.5ug/ml are isocyatic tiltedly
Face culture medium.
The H37Rv strain bacteria suspension inoculating loop for 1mg/ml for the concentration is dipped ring of numbers, is inoculated in drug containing series respectively dense
In the culture medium and blank medium slant of degree, it is placed in 37 DEG C and cultivates 4～8 weeks, observation experiment result, result such as table 3 institute
Show.
(3) solid medium Dilution medicine Ad tuberculosis are clinically separated the MTB of resistance to ISRE strain absolute concentration
From inclined-plane scraping mycobacterium tuberculosis be clinically separated the MTB of resistance to ISRE strain (resistance to isoniazid, streptomycin, rifampicin,
Ethambutol mycobacterium tuberculosis are clinically separated post) culture, it is added in 3ml Middlebrook7H9 broth bouillon, plus
Enter a small amount of bead, screw test tube cap, high vibration grinds in vortex oscillator, with standard Maxwell opacity tube
(MacFarland No.1) ratio is turbid, that is, be made into the bacteria suspension of 1mg/ml.
Medicine is made into respectively the stock solution of high concentration with DMSO, with the tween 80 aseptic ultra-pure water dilution stock solution containing 5% extremely
Desired concn, the medicine having diluted is added to 4ml Middlebrook7H11 agar culture medium (this culture medium by required dosage
121 DEG C of high pressure steam sterilizations 15 minutes, be cooled to 50～55 DEG C), mix, make drug containing, concentration is respectively 6.0ug/
Ml, 4.0ug/ml, 3.0ug/ml, 2.0ug/ml, 1.5ug/ml, 1.0ug/ml, 0.75ug/ml, 0.5ug/ml are isocyatic tiltedly
Face culture medium.
The mycobacterium tuberculosis for 1mg/ml for the concentration are clinically separated the MTB of resistance to ISRE strain bacteria suspension inoculating loop and dip number
Ring, is inoculated in the culture medium and blank medium slant containing each medicine series concentration respectively, be placed in 37 DEG C culture 4～
8 weeks, observation experiment result, result is as shown in table 3.
Table 3 solid medium Dilution compositionss anti-tubercle bacillus absolute concentration result
Table 4 solid medium Dilution compound III anti-tubercle bacillus absolute concentration result
Table 3 solid medium Dilution compound IV anti-tubercle bacillus absolute concentration result
Conclusion：Compositionss have very strong anti-tubercle bacillus and anti-drug resistance tulase activity, can be used as treatment tulase sense
The lead compound of dye disease is it can also be used to tubercular drugs are treated in preparation.Compound III and compound IV no anti-tubercle bacillus and
Anti-drug resistance tulase activity it is impossible to as treatment tubercle bacillus affection disease lead compound, nor for preparation treat
Tubercular drugs.
The preparation of embodiment 8 composition tablet involved in the present invention
Take 2 grams of compositionss, 18 grams of the customary adjuvant of tablet is prepared in addition, mix, conventional tablet presses make 100.
The preparation of embodiment 9 composition capsule involved in the present invention
Take 2 grams of compositionss, customary adjuvant such as 18 grams of the starch of capsule is prepared in addition, mix, encapsulated make 100.
Claims (10)
1. a kind of compositionss, it is characterized by said composition is made up of compound III and compound IV, compound in said composition
The mass percent of III and compound IV is respectively 80% and 20%,
2. the preparation method of compositionss as claimed in claim 1, it is characterized by：By the powder of compound III and compound IV
Powder be respectively according to mass percent and 80% and 20% be sufficiently mixed.
3. application in antibacterials for a kind of compositionss as claimed in claim 1.
4. application in antibacterials for the compositionss as claimed in claim 3, it is characterized by：Described bacterium is antibacterial.
5. application in antibacterials for the compositionss as claimed in claim 3, it is characterized by：Described bacterium is funguses.
6. application in antibacterials for the compositionss as claimed in claim 3, it is characterized by：Described bacterium is helicobacter pylori.
7. application in antibacterials for the compositionss as claimed in claim 3, it is characterized by：Described bacterium is tulase.
8. application in antibacterials for the compositionss as claimed in claim 5, it is characterized by：Described funguses are red hair tinea
Bacterium, microsporum canis or trichophyton tonsurans.
9. application in antibacterials for the compositionss as claimed in claim 4, it is characterized by：Described antibacterial be escherichia coli,
Fluorescent pseudomonas, Staphylococcus aureus, Bacillus proteuss or neogenesis cryptococcus.
10. application in antibacterials for the compositionss as claimed in claim 7, it is characterized by：Described tulase be bacillus calmette-guerin vaccine,
Mycobacterium tuberculosis type strain H37Rv strain and substance of medicines-resistant branched tubercle bacillus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610609443.XA CN106420729A (en) | 2016-07-28 | 2016-07-28 | Application of composition of derivative of Schiglautone A in antibacterial agents |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610609443.XA CN106420729A (en) | 2016-07-28 | 2016-07-28 | Application of composition of derivative of Schiglautone A in antibacterial agents |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106420729A true CN106420729A (en) | 2017-02-22 |
Family
ID=58184343
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610609443.XA Withdrawn CN106420729A (en) | 2016-07-28 | 2016-07-28 | Application of composition of derivative of Schiglautone A in antibacterial agents |
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| Country | Link |
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| CN (1) | CN106420729A (en) |
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2016
- 2016-07-28 CN CN201610609443.XA patent/CN106420729A/en not_active Withdrawn
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Application publication date: 20170222 |