CN106176726A - The application in antibacterials of the Atropurpuran derivative composition - Google Patents

The application in antibacterials of the Atropurpuran derivative composition Download PDF

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CN106176726A
CN106176726A CN201610619144.4A CN201610619144A CN106176726A CN 106176726 A CN106176726 A CN 106176726A CN 201610619144 A CN201610619144 A CN 201610619144A CN 106176726 A CN106176726 A CN 106176726A
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compositions
antibacterials
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concentration
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王卓婷
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Nanjing Fuhai Aosai Medicine Science & Technology Co Ltd
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Nanjing Fuhai Aosai Medicine Science & Technology Co Ltd
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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
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D257/04—Five-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • 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 present invention relates to organic synthesis and medicinal chemistry art, be specifically related to compositions, preparation method and the purposes in preparation antibacterials thereof.The invention discloses a kind of compositions and preparation method thereof.Pharmacological experiment shows, the compositions of the present invention has antibacterial action, has the value of exploitation antibacterials.

Description

The application in antibacterials of the Atropurpuran derivative composition
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 2009, deliver (Pei Tang et al., 2009.Atropurpuran,a novel diterpene with an unprecedented pentacyclic cage skeleton,from Aconitum hemsleyanum var.atropurpureum.Tetrahedron Letters 50 (2009) 460 462) compound, we have carried out structural modification to compound I, it is thus achieved that two i.e. chemical combination of new derivant Thing III and compound IV, and be prepared for compositions with compound III and compound IV and said composition antibacterial activity is carried out Evaluating, it has 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 70% and 30%.
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 Atropurpuran
Document (the Pei Tang et that the preparation method of compound Atropurpuran (I) is delivered with reference to Pei Tang et al. al.,2009.Atropurpuran,a novel diterpene with an unprecedented pentacyclic cage skeleton,from Aconitum hemsleyanum var.atropurpureum.Tetrahedron Letters 50 (2009) 460 462) method.
The synthesis of O-bromoethyl derivant (II) of embodiment 2 Atropurpuran
Compound I (312mg, 1.00mmol) is dissolved in 10mL benzene, in solution, adds tetrabutyl ammonium bromide (TBAB) (0.08g), glycol dibromide (3.760g, 20.00mmol) and 50% sodium hydroxide solution of 6mL.Mixture is Celsius 35 Degree stirring 6h.After 6h, 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 yellow powder (309mg, 74%) of compound II.
1H NMR(500MHz,DMSO-d6)δ9.86(s,1H),5.23(s,1H),4.87(s,1H),4.83(s,1H), 4.39 (s, 1H), 4.00 (s, 2H), 3.91 (s, 1H), 3.58 (s, 2H), 3.01 (s, 1H), 2.27 (s, 1H), 2.15 (d, J= 6.3Hz, 2H), 2.09 2.00 (m, 5H), 1.78 (dd, J=35.2,19.2Hz, 2H), 1.71 1.65 (m, 1H), 1.41 (s, 2H),0.99(s,3H).
13C NMR(125MHz,DMSO-d6)δ208.55(s),203.16(s),150.94(s),125.16(s),111.66 (s),78.63(s),71.39(s),57.77(s),47.73(s),40.79(s),34.58(s),33.01(s),32.69(s), 29.25(s),29.34(s),26.52(s),24.23(s),22.30(s),20.64(s).
HRMS(ESI)m/z[M+H]+calcd for C22H28BrO3:419.1222；found 419.1226.
The synthesis of the O-(nafoxidine base) ethyl derivative (III) of embodiment 3 Atropurpuran
Compound II (209mg, 0.5mmol) is dissolved in the middle of 18mL 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 2h.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=100:1.0, v/v) with silica gel column chromatography, collect brown and concentrate elution band, concentrate i.e. Obtain the brown solid (125mg, 61%) of compound III.
1H NMR (500MHz, DMSO-d6) δ 9.78 (s, 1H), 5.15 (s, 1H), 4.61 (d, J=10.5Hz, 2H), 4.45(s,1H),3.83(s,1H),3.53(s,2H),2.94(s,1H),2.62(s,2H),2.46(s,4H),2.20(s,1H), 2.08 (d, J=6.0Hz, 2H), 2.00 1.91 (m, 3H), 1.75 (d, J=2.1Hz, 2H), 1.69 (s, 2H), 1.62 (d, J= 5.5Hz,5H),1.30(s,2H),0.91(s,3H).
13C NMR(125MHz,DMSO-d6)δ208.65(s),203.36(s),151.04(s),125.36(s),111.76 (s), 78.83 (s), 67.20 (s), 57.97 (s), 54.33 (d, J=17.1Hz), 47.82 (s), 41.00 (s), 34.67 (s), 32.79(s),29.45(s),29.22(s),26.61(s),25.14(s),24.43(s),22.40(s),20.84(s).
HRMS(ESI):m/z[M+H]+calcd for C26H36NO3:410.2695；found:410.2699.
The synthesis of O-(the 1H-tetrazole base) ethyl derivative of embodiment 4 Atropurpuran
Compound II (209mg, 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 1H-tetrazole (1401mg, 20mmol), mixture is heated to reflux 4h.Reaction Reactant liquor is poured in 20mL frozen water after end, extract 3 times with equivalent dichloromethane, merge organic facies.Successively with water and saturated Organic facies after brine It merging, then be dried with anhydrous sodium sulfate, concentrating under reduced pressure is removed solvent and is obtained product crude product.Cause For tautomerization, 1H-tetrazole base and two kinds of substitution products of 2H-tetrazole base can be generated at reaction conditions.Product is thick Product silica gel column chromatography purification (flowing is mutually: petroleum ether/acetone=100:0.8, v/v), collects yellow and concentrates elution band, then will Elution band concentrates, and purifies (flowing is mutually: petroleum ether/acetone=100:0.4, v/v) with silica gel column chromatography, and collection two is light successively The elution band of yellow, concentrates front 1 elution band and i.e. obtains the yellow powder (83.6mg, 41%) of compound IV.
1H NMR(500MHz,DMSO-d6)δ10.51(s,1H),9.74(s,1H),5.05(s,1H),4.61–4.48(m, 3H),4.19(s,1H),4.12(s,1H),3.80(s,2H),3.75(s,1H),2.86(s,1H),2.08(s,1H),1.97(d, J=2.9Hz, 2H), 1.90 1.81 (m, 3H), 1.69 1.57 (m, 4H), 1.54 (s, 1H), 1.25 (s, 2H), 0.80 (s, 3H).
13C NMR(125MHz,DMSO-d6)δ208.46(s),203.17(s),150.83(s),144.83(s),125.18 (s),111.56(s),78.65(s),66.57(s),57.79(s),47.63(s),46.37(s),40.80(s),34.49(s), 32.59(s),29.27(s),29.02(s),26.43(s),24.24(s),22.21(s),20.67(s).
HRMS(ESI):m/z[M+H]+calcd for C23H29N4O3:409.2240；found:409.2244.
Embodiment 5 compositions human body fungi activity
The preparation of compositions: the powder that will cross the 70mg compound III of 200 mesh nets after grinding crosses 200 after grinding The powder of the 30mg 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 6 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 35mm (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 1.5 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 7 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 8 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 is without anti-tubercle bacillus With anti-drug resistance tulase activity, it is impossible to as the lead compound for the treatment of tubercle bacillus affection disease, preparation can not be used for and control Treat tubercular drugs.
The preparation of embodiment 9 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 10 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 70% and 30%,
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 70% and 30%.
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.
CN201610619144.4A 2016-07-29 2016-07-29 The application in antibacterials of the Atropurpuran derivative composition Pending CN106176726A (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BUCHBAUER G等: "二萜的生物活性", 《国外医学 药学分册》 *
PEI TANG等: "Atropurpuran, a novel diterpene with an unprecedented pentacyclic cage skeleton, from Aconitum hemsleyanum var. atropurpureum", 《TETRAHEDRON LETTERS》 *

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