CN105920014A - Application of composition of tetrahydropyrrolyl and morpholinyl derivatives of Virosaine A in antibacterial medicines - Google Patents
Application of composition of tetrahydropyrrolyl and morpholinyl derivatives of Virosaine A in antibacterial medicines Download PDFInfo
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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
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings
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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 discloses an application of a composition of tetrahydropyrrolyl and morpholinyl derivatives of Virosaine A in antibacterial medicines. The invention relates to the fields of organic synthesis and medicinal chemistry, and in particular to the composition, a preparation method and the application of the composition in preparing the antibacterial medicines. The invention discloses the composition and the preparation method thereof. Pharmacological experiments show that the composition disclosed by the invention has an antibacterial effect, and the composition has a value of developing the antibacterial medicines.
Description
Technical field
The present invention relates to organic synthesis and medicinal chemistry art, be specifically related to compositions, preparation method and its usage.
Background technology
The health and lives of the mankind, antibacterials conduct in the diffusion of pathogenic bacterium and the serious threat that strengthens of drug resistance thereof
Routine administration is widely used in acquired immune deficiency syndrome (AIDS), the controlling of organ transplantation and Chronic consumptions (such as cancer, diabetes, uremia etc.)
Treat, although the antimicrobial agent that uses clinically at present (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 relatively strong, usually causes the stimulation of lesions of liver and kidney, digestive tract, dizziness, allergy etc., so finding the novel 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
Showing, 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 that currently a popular treatment Hp infects is to take proton pump inhibitor simultaneously
(PPI) triple therapy of two kinds of antibiotic (clarithromycin, amoxicillin, tetracycline, metronidazole etc. select two kinds) is added.Affect three
The main factor of therapy is considered as the Hp drug resistance to antibacterial;Another serious problems are that proton pump inhibitor can induce and disappears
Changing bad, in a large amount of antibacterial then cause digestive tract, the serious of flora destroys.Therefore, efficient, safe anti-Hp activity one is found
Kind new medicine thing becomes an important and urgent task.
The most global morbidity lungy, in increasing trend, is estimated according to World Health Organization (WHO) (WHO), and 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 become tuberculosis patient.China occurs active tuberculosis patient 1,300,000 example, wherein infectiousness every year
Pulmonary tuberculosis about 600,000 example, wherein infectiousness pulmonary tuberculosis about 600,000 example, be one of whole world tuberculosis high burden country.
Come out one after another from antituberculotics, make treatment lungy play epoch-making change.Yet with tuberculosis sufferer
The Case management of person the most not ten sectional specification, irregular chemotherapy, abuse antituberculotics, make drug resistance of tuberculosis situation day by day serious,
And the change of drug resistance tends to multi-medicament drug resistance simultaneously, this causes extreme difficulties to preventing and controlling lungy.Therefore
Finding new antituberculotics, the antituberculotics of the most anti-multidrug resistance, to protection people's health, has important
Meaning.
From natural product, find compound or lead compound and carry out structural modification and obtain its derivant, thus obtaining
The potential drug of high-efficiency low-toxicity most has important value.
The compound I that the present invention relates to be one within 2012, deliver (Bing-Xin Zhao et al.,
2012.Virosaines A and B,Two New Birdcage-Shaped Securinega Alkaloids with an
Unprecedented Skeleton from Flueggea virosa.Organic Letters 14(2012)3096–
3099) compound, we have carried out structural modification to compound I, it is thus achieved that two new derivants i.e. compound III and changes
Compound IV, and be prepared for compositions with compound III and compound IV and said composition antibacterial activity is evaluated, its tool
There is antibacterial activity.
Summary of the invention
The invention discloses a new compositions, 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 45% and 55%.
Compositions disclosed by the invention can make pharmaceutically acceptable salt or pharmaceutically acceptable carrier.
Pharmacodynamic experiment shows, the compositions of the present invention has preferable antibacterial action.The present invention's is pharmaceutically acceptable
Salt there is same drug effect.
Further, the experiment in vitro of compositions shows, compositions has a strongest human body fungi activity, therefore this
Bright compositions is expected to be used for preparing novel anti-human fungi medicine.
Further, the experiment in vitro of compositions shows, compositions has the strongest anti Helicobacter pylori activity, explanation
From the point of view of the diseases such as acute and chronic gastritis closely-related with helicobacter pylori, duodenal ulcer, compositions is one
The compound of individual great exploitation potential.It can be directly used for treatment and the preparation of related drugs of corresponding disease.
Further, the experiment in vitro of compositions shows, compositions has the strongest suppression escherichia coli, fluorescence vacation monospore
Bacterium, Staphylococcus aureus, Bacillus proteus, the effect of neogenesis cryptococcus, so compositions can be as the change with antibacterial actions
Compound, and be expected to be applied in preparing anti-bacterial drug.
Further, according to the result of preliminary test, present invention solid medium dilution method determines compositions to card
The minimum of Jie's Seedling, mycobacterium tuberculosis type strain H37Rv strain and substance of medicines-resistant branched tubercle bacillus (MDR MTB) three kinds of tulases presses down
Bacteria concentration, experimental result confirms that compositions has the strongest anti-tubercle bacillus and anti-drug resistance tulase activity, can be as treatment knot
The lead compound of solani infection disease is it can also be used to tubercular drugs is treated in preparation.
The present invention is further detailed explanation by the following examples, but protection scope of the present invention is not by concrete real
Execute any restriction of example, but be defined in the claims.
Detailed description of the invention
The preparation of embodiment 1 compound Virosaine A
Document (the Bing-that the preparation method of compound Virosaine A (I) is delivered with reference to Bing-Xin Zhao et al.
Xin Zhao et al.,2012.Virosaines A and B,Two New Birdcage-Shaped Securinega
Alkaloids with an Unprecedented Skeleton from Flueggea virosa.Organic Letters
14 (2012) 3,096 3099) method.
The synthesis of O-bromoethyl derivant (II) of embodiment 2 Virosaine A
Compound I (235mg, 1.00mmol) is dissolved in 20mL benzene, in solution, adds tetrabutyl ammonium bromide (TBAB)
(0.08g), glycol dibromide (3.760g, 20.00mmol) and 50% sodium hydroxide solution of 5mL.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
Washing organic phase solution 3 times with water and saturated aqueous common salt successively, then be dried with anhydrous sodium sulfate, last concentrating under reduced pressure is removed molten
Agent obtains product crude product.Product crude product silica gel column chromatography purification (flowing is mutually: petroleum ether/acetone=100:1.0, v/v), receives
Collection brown is concentrated elution band and flings to solvent and i.e. obtain the brown ceramic powder (261mg, 78%) of compound II.
1H NMR(500MHz,DMSO-d6)δ5.91(s,1H),4.07(s,1H),3.81(s,2H),3.59(s,1H),
(3.43 s, 2H), 3.33 (s, 1H), 2.26 (s, 1H), 1.58 (d, J=9.8Hz, 3H), 1.41 (s, 2H).
13C NMR(125MHz,DMSO-d6)δ171.81(s),106.71(s),79.95(s),74.33(s),69.81
(s),66.68(s),44.21(s),35.56(s),33.28(s),25.85(s),21.88(s).
HRMS(ESI)m/z[M+H]+calcd for C14H17BrNO4:342.0341;found 342.0343.
The synthesis of the O-(nafoxidine base) ethyl derivative (III) of embodiment 3 Virosaine A
Compound II (171mg, 0.5mmol) is dissolved in the middle of 15mL acetonitrile, be added thereto to Anhydrous potassium carbonate (345mg,
2.5mmol), potassium iodide (84mg, 0.5mmol) and pyrrolidine (1420mg, 20mmol), mixture is heated to reflux 1h.Reaction knot
After bundle, reactant liquor is poured in frozen water, extract 2 times with equivalent dichloromethane, merge organic facies.Successively with water and saturated aqueous common salt
Organic facies after washing merging, then be dried with anhydrous sodium sulfate, concentrating under reduced pressure is removed solvent and is obtained product crude product.Product crude product
Purify (flowing is mutually: petroleum ether/acetone=100:1.5, v/v) with silica gel column chromatography, collect faint yellow concentration elution band and get final product
Faint yellow colloidal solid (111.6mg, 67%) to the O-(nafoxidine base) ethyl derivative (III) of Virosaine A.
1H NMR(500MHz,DMSO-d6)δ5.92(s,1H),4.11(s,1H),3.66(s,1H),3.53(s,2H),
(3.45 d, J=66.9Hz, 1H), 2.64 (s, 2H), 2.52 (s, 4H), 2.29 (s, 1H), 1.71 (s, 4H), 1.64 (s, 2H),
1.61(s,1H),1.50(s,2H).
13C NMR(125MHz,DMSO-d6)δ171.83(s),106.73(s),79.97(s),74.35(s),66.72(d,
J=9.2Hz), 54.42 (d, J=17.1Hz), 44.23 (s), 35.58 (s), 25.86 (s), 25.23 (s), 21.90 (s).
HRMS(ESI):m/z[M+H]+calcd for C18H25N2O4:333.1814;found:333.1811.
The synthesis of O-(morpholinyl) ethyl derivative of embodiment 4 Virosaine A
Compound II (171mg, 0.5mmol) is dissolved in the middle of 15mL acetonitrile, be added thereto to Anhydrous potassium carbonate (345mg,
2.5mmol), potassium iodide (84mg, 0.5mmol) and morpholine (1742mg, 20mmol), mixture is heated to reflux 1h.Reaction terminates
After reactant liquor is poured in 20mL frozen water, with equivalent dichloromethane extract three times, merge organic facies.Successively with water and saturated common salt
Organic facies after water washing merging, then be dried with anhydrous sodium sulfate, concentrating under reduced pressure is removed solvent and is obtained product crude product.Product is thick
Product silica gel column chromatography purification (flowing is mutually: petroleum ether/acetone=100:1.0, v/v), collects yellow and concentrates elution band and get final product
Yellow solid (IV) (125.3mg, 72%) to O-(morpholinyl) ethyl derivative of Virosaine A.
1H NMR (500MHz, DMSO-d6) δ 5.95 (s, 1H), 4.12 (s, 1H), 3.68 (s, 1H), 3.63 (d, J=
8.4Hz,1H),3.61(s,4H),3.57–3.26(m,3H),2.61(s,2H),2.56(s,2H),2.51–2.03(m,5H),
1.70 (d, J=3.1Hz, 1H), 1.65 (d, J=25.6Hz, 1H), 1.52 (s, 2H).
13C NMR(125MHz,DMSO-d6)δ171.85(s),106.73(s),79.95(s),74.34(s),66.75(t,
J=4.6Hz), 54.46 (s), 52.93 (s), 44.22 (s), 35.55 (s), 25.88 (s), 21.89 (s).
HRMS(ESI):m/z[M+H]+calcd for C18H25N2O5:349.1763;found:349.1761.
Embodiment 5 compositions human body fungi activity
The preparation of compositions: the powder that will cross the 45mg compound III of 200 mesh nets after grinding crosses 200 after grinding
The powder of the 55mg compound IV of mesh net loads in tubule with cover and i.e. obtains 100mg compositions with the mixing of turbine stirring instrument,
Obtain the solution of compositions by the compositions of this 100mg of water dissolution during use.
The experiment of human body fungi activity is the method using concentration dilution, measures in triplicate, the pathogen of test every time
Having 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, the bacterium solution of equivalent volumes and test sample mixes
Close and cultivate in 96 orifice plates (ultimate density of formation has: 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 formed, that minimum concentration is sample lowest concentration of antimicrobial, i.e. MIC value.This experiment positive control is ketone health
Azoles, compositions human body fungus the results are shown in Table 1.
Table 1 compositions human body fungus MIC value (μ g/mL)
Conclusion: compositions has the strongest human body fungi activity, and therefore the compositions of the present invention is expected to be used for preparation
Novel anti-human fungi medicine.Compound III and compound IV nonreactive Human Fungi activity, therefore cannot be used for preparing novel resisting
Human Fungi medicine.
Embodiment 8 compositions 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 strain 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.Collecting antibacterial, through smear Gram’s staining, oxidase, catalase and urease are accredited as
After the positive, passing on pure culture, obtained strains is as experimental strain.
2) strain culturing
We use micro-aerobic bag (purchased from Shanghai Medical Univ) to carry out the strain culturing of HP, and it can be produced by chemical reaction
Micro-aerobic environment required for raw Hp.
3) biological activity determination
Use paper disk method (Microbiological paper method) to measure medicine the suppression of helicobacter pylori is made
With, with agar dilution measure test sample minimum inhibitory concentration (minimal inhibitory concentration,
MIC)。
I. paper disk method experiment
(A) prepare culture medium by the Columbia culture medium for preparing after high pressure steam sterilization, be cooled to 50-60 DEG C,
Adding 8% horse serum or Sheep Blood, mixing is poured in the most sterilized culture dish, and every ware 7-10ml, culture medium thickness is 1.5mm
(sterile working).
(B) switching experimental bacteria (being coated with bacterium) takes 10 diluted with microscale sampler8CFU/ml(1OD660=108CFU/ml)Hp
Bacteria suspension 0.1ml spread upon suitable culture dish surface equably.It is inverted in 37 DEG C of drying bakers and takes out after 15min, purpose
Agar surface is made to be dried, standby.
(C) patch sample scraps of paper microscale sampler take 6 μ l testing sample (mass concentration 2mg/ml) inject the most sterilized
On circle filter paper.With the blank scraps of paper of the aseptic nipper tweezer scraps of paper containing sample and comparison, it is close to by sterile working's scraps of paper respectively
Containing bacterio-agar surface, patch a piece of paper sheet at a certain distance.Every kind of bacterium is cooked 3 wares, and acquired results seeks its meansigma methods.
(D) each plate is placed in micro-aerobic bag by cultivation, and sealing is opened gas generator, then is placed in 37 DEG C of incubators
Cultivate 72h.
(E), after surveying antibacterial circle diameter taking-up flat board, the size of antibacterial circle diameter around each scraps of paper is measured 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) first medicine dimethyl sulfoxide (DMSO) solution of test is configured to 0.5mg/ml by the preparation of Drug plates
Mother solution, then with sterilized water dilute, 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 be incubated the 8ml Columbia medium in 50 DEG C, separately add the horse serum that 1ml is incubated in 50 DEG C and fully mix, water
Make in culture dish cooling (final concentration of the 10.0 of culture dish Chinese medicine, 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 being bacteriostasis, then during i.e. these are worthwhile one of MIC value).
(B) switching experimental bacteria (being coated with bacterium) draws, with microscale sampler, 1 × 10 diluted8The bacteria suspension of CFU/ml Hp
0.1ml spreads upon culture dish surface equably, is inverted in 37 DEG C of drying bakers and takes out after 15min, and purpose makes agar surface be dried,
Standby.
(C) determine that test dish (is contained: 85%N by MIC at micro-aerobic bag2, 10%CO2And 5%O2In), it is incubated 37 DEG C
Cultivate 72 hours, observe Hp growing state, contrast with blank group, be minimum with the sample least concentration entirely without bacteria growing
Mlc (minimal inhibitory concentration, MIC) value.Positive control is ampicillin
(Ampicillin)。
Experimental result
Experiment in vitro shows, compositions has the strongest anti Helicobacter pylori activity, and paper disk method shows that its inhibition zone is straight
Footpath is 32mm (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 2.5 μ g/ml.Making positive control with ampicillin, it is to 6
The Cmin (MIC) that strain test bacterium completely inhibits is 1.0 μ g/ml.Compound III and compound IV is without helicobacter pylori resistant
Activity.
Conclusion: compositions has stronger suppression helicobacter pylori activity, illustrates for close with helicobacter pylori
From the point of view of the diseases such as relevant acute and chronic gastritis, duodenal ulcer, compositions is the medicine 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, it is not possible to for treatment and the preparation of related drugs of corresponding disease.
Embodiment 9 compositions 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 proteus, 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
Sample mix cultivate in 96 orifice plates (ultimate density of formation has: 25.00,12.50,6.25,3.13,1.56,0.78,0.39,
0.20,0.10 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 formed, that minimum concentration is sample lowest concentration of antimicrobial, i.e. MIC value.Compositions the anti-bacterial result is shown in Table 2.
Table 2 compositions antibacterium MIC value (μ g/mL)
Conclusion: compositions has a strongest antibacterial activity, therefore the compositions of the present invention is expected to be used for prepare novel
Anti-bacterial drug.Compound III and compound IV is without antibacterial activity, it is impossible to be used for preparing novel anti-bacterial drug.
Embodiment 10 compositions anti-tubercle bacillus activity
(1) solid medium dilution method measures compositions anti-bacillus calmette-guerin vaccine (BCG) absolute concentration
From inclined-plane, scrape BCG cultures, join 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, is i.e. made into bacillus calmette-guerin vaccine (BCG) bacteria suspension of 1mg/ml.
Medicine DMSO is made into the stock solution of high concentration, dilutes stock solution to required with the aseptic ultra-pure water of tween 80 containing 5%
Concentration, by required dosage, the medicine diluted is joined 4ml Middlebrook7H11 agar culture medium, and (this culture medium is
121 DEG C of high pressure steam sterilizations 15 minutes, it is cooled to 50~55 DEG C), mixing, 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
Support base.
Bacillus calmette-guerin vaccine (BCG) the bacteria suspension inoculating loop that concentration is 1mg/ml 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 when tulase is cultivated, and its formula uses conventional formulation.
(2) solid medium dilution method measures compositions Killing Mycobacterium Tuberculosis type strain H37Rv strain absolute concentration
From inclined-plane, scrape mycobacterium tuberculosis type strain H37Rv strain culture, join 3ml Middlebrook7H9
In broth bouillon, adding a small amount of bead, screw test tube cap, in vortex oscillator, high vibration grinds, with standard Maxwell
Opacity tube (MacFarland No.1) ratio is turbid, is i.e. made into the H37Rv strain bacteria suspension of 1mg/ml.
Medicine is made into DMSO respectively the stock solution of high concentration, dilutes stock solution extremely with the aseptic ultra-pure water of tween 80 containing 5%
Desired concn, joins 4ml Middlebrook7H11 agar culture medium (this culture medium by the medicine diluted by required dosage
121 DEG C of high pressure steam sterilizations 15 minutes, be cooled to 50~55 DEG C), mixing, 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 that concentration is 1mg/ml is dipped ring of numbers, is inoculated in drug containing series respectively dense
In the culture medium of degree and blank medium slant, 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 method measures medicine Ad tuberculosis and is clinically separated the MTB of resistance to ISRE strain absolute concentration
From inclined-plane scrape mycobacterium tuberculosis be clinically separated the MTB of resistance to ISRE strain (resistance to isoniazid, streptomycin, rifampicin,
Ethambutol mycobacterium tuberculosis is clinically separated post) culture, join in 3ml Middlebrook7H9 broth bouillon, add
Entering a small amount of bead, screw test tube cap, in vortex oscillator, high vibration grinds, with standard Maxwell opacity tube
(MacFarland No.1) ratio is turbid, is i.e. made into the bacteria suspension of 1mg/ml.
Medicine is made into DMSO respectively the stock solution of high concentration, dilutes stock solution extremely with the aseptic ultra-pure water of tween 80 containing 5%
Desired concn, joins 4ml Middlebrook7H11 agar culture medium (this culture medium by the medicine diluted by required dosage
121 DEG C of high pressure steam sterilizations 15 minutes, be cooled to 50~55 DEG C), mixing, 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 that concentration is 1mg/ml is clinically separated the MTB of resistance to ISRE strain bacteria suspension inoculating loop and dips number
Ring, is inoculated in the culture medium containing each medicine series concentration and blank medium slant respectively, be placed in 37 DEG C cultivate 4~
8 weeks, observation experiment result, result was as shown in table 3.
Table 3 solid medium dilution method measures compositions anti-tubercle bacillus absolute concentration result
Table 4 solid medium dilution method measures compound III anti-tubercle bacillus absolute concentration result
Table 5 solid medium dilution method measures compound IV anti-tubercle bacillus absolute concentration result
Conclusion: compositions has the strongest anti-tubercle bacillus and anti-drug resistance tulase activity, can be as treatment tulase sense
The lead compound of dye disease is it can also be used to tubercular drugs is treated in preparation.Compound III and compound IV without anti-tubercle bacillus and
Anti-drug resistance tulase activity, it is impossible to as the lead compound for the treatment of tubercle bacillus affection disease, can not be used for preparing treatment
Tubercular drugs.
The preparation of embodiment 8 composition tablet involved in the present invention
Taking 2 grams of compositionss, the customary adjuvant 18 grams of tablet is prepared in addition, and mixing, conventional tablet presses makes 100.
The preparation of embodiment 9 composition capsule involved in the present invention
Taking 2 grams of compositionss, the customary adjuvant such as starch 18 grams of capsule is prepared in addition, mixing, encapsulated makes 100.
Claims (10)
1. a compositions, is characterized by that 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 45% and 55%,
2. the preparation method of compositions as claimed in claim 1, is characterized by: by powder and the compound IV of compound III
Powder be sufficiently mixed according to mass percent respectively 45% and 55%.
3. a compositions as claimed in claim 1 application in antibacterials.
4. compositions application in antibacterials as claimed in claim 3, is characterized by: described bacterium is antibacterial.
5. compositions application in antibacterials as claimed in claim 3, is characterized by: described bacterium is fungus.
6. compositions application in antibacterials as claimed in claim 3, is characterized by: described bacterium is helicobacter pylori.
7. compositions application in antibacterials as claimed in claim 3, is characterized by: described bacterium is tulase.
8. compositions application in antibacterials as claimed in claim 5, is characterized by: described fungus is red hair tinea
Bacterium, microsporum canis or trichophyton tonsurans.
9. compositions application in antibacterials as claimed in claim 4, is characterized by: described antibacterial be escherichia coli,
Fluorescent pseudomonas, Staphylococcus aureus, Bacillus proteus or neogenesis cryptococcus.
10. compositions application in antibacterials as claimed in claim 7, is characterized by: described tulase be bacillus calmette-guerin vaccine,
Mycobacterium tuberculosis type strain H37Rv strain and substance of medicines-resistant branched tubercle bacillus.
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CN201610278686.XA CN105920014A (en) | 2016-04-28 | 2016-04-28 | Application of composition of tetrahydropyrrolyl and morpholinyl derivatives of Virosaine A in antibacterial medicines |
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Citations (1)
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CN104193756A (en) * | 2014-09-04 | 2014-12-10 | 南京标科生物科技有限公司 | Method for extracting securinine from securinega |
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CN104193756A (en) * | 2014-09-04 | 2014-12-10 | 南京标科生物科技有限公司 | Method for extracting securinine from securinega |
Non-Patent Citations (1)
Title |
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BING-XIN ZHAO: "《Virosaines A and B,Two New Birdcage-Shaped Securinega Alkaloids with an Unprecedented Skeleton from Flueggea virosa》", 《ORGANIC LETTERS》 * |
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