CN105232513A - Composition and application thereof to antibacterial drugs - Google Patents

Abstract

The invention relates to the fields of organic synthesis and pharmaceutical chemistry, in particular to a composition, a preparing method and application thereof to preparation of antibacterial drugs. The invention discloses the composition and the preparing method thereof. Pharmacology experiments show that the composition has an antibacterial effect and has the value of developing the antibacterial drugs.

Description

A kind of compositions and the application in antibacterials thereof

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 in the diffusion of pathogenic bacterium and the enhancing serious threat of drug resistance thereof, antibacterials are widely used in acquired immune deficiency syndrome (AIDS) as routine administration, organ transplantation and Chronic consumptions are (as cancer, diabetes, uremia etc.) treatment, although the antimicrobial agent used clinically is at present (as ketoconazole, amikacin, gentamycin, voriconazole, itraconazole, terbinafine, amphotericin, fluconazol etc.) better to the curative effect of skin and superficial place infection, but the cumulative toxicity of these antibacterials is stronger, usually cause lesions of liver and kidney, digestive tract stimulates, dizzy, irritated etc., so the novel antibacterial medicine finding mechanism of action uniqueness becomes one of focus of current medicament research and development.

Helicobacter pylori (Helicobacterpylori, Hp) is a kind of Gram-negative spiral bacteria.Research display, helicobacter pylori is the primary pathogenic event of acute and chronic gastritis and taste-blindness rate, and may fall ill relevant with gastric cancer stomach function regulating mucosa-associated lymphoid tissue (MALT) malignant lymphoma.Recently, Hp is classified as I class carcinogen by World Health Organization (WHO), and it plays a leading role in stomach cancer development.Simultaneously the scheme that popular at present treatment Hp infects takes the triple therapy that proton pump inhibitor (PPI) adds two kinds of antibiotic (clarithromycin, amoxicillin, tetracycline, metronidazole etc. select two kinds).The main factor affecting triple therapy is considered to the drug resistance of Hp to antibacterial; Another serious problems are that proton pump inhibitor can bring out dyspepsia, and a large amount of antibacterial then causes the serious destruction of flora in digestive tract.Therefore, find the active kind new medicine thing of efficient, safe anti-Hp and become an important and urgent task.

Global morbidity lungy is in increasing trend in recent years, estimate according to World Health Organization (WHO) (WHO), the current whole world is by mycobacterium tuberculosis (Mycobacteriumtuberculosis, MTB) population infected accounts for 1/3rd of world population, and wherein the infected of 5 ~ 10% becomes tuberculosis patient.There is active tuberculosis patient 1,300,000 example in China, wherein infectiousness pulmonary tuberculosis about 600,000 example every year, wherein infectiousness pulmonary tuberculosis about 600,000 example, is one of global tuberculosis high burden country.

Come out one after another from antituberculotics, make treatment lungy play epoch-making change.But due to the Case management still not very specification of tuberculosis patient, irregular chemotherapy, abuse antituberculotics, makes drug resistance of tuberculosis situation day by day serious, and the change of drug resistance more trends towards 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 is to protection people's health, significant.

From natural product, find compound or lead compound and carry out structural modification and obtain its derivant, thus the potential drug obtaining high-efficiency low-toxicity there is important value most.

The Compound I that the present invention relates to is one and delivers (Fan-YuMengetal. in 2011, 2011.SchiglautoneA, aNewTricyclicTriterpenoidwithaUnique6/7/9-FusedSkeletonf romtheStemsofSchisandraglaucescens.OrganicLetters13 (2011) 1502 – 1505) compound, we have carried out structural modification to Compound I, obtain two new derivants and compound III and compound IV, and prepared compositions by compound III and compound IV and said composition antibacterial activity is evaluated, 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, and in said composition, the mass percent of compound III and compound IV is respectively 75% and 25%.

Compositions disclosed by the invention can make pharmaceutically acceptable salt or pharmaceutically acceptable carrier.

Pharmacodynamic experiment shows, compositions of the present invention has good antibacterial action.Pharmaceutically acceptable salt of the present invention has same drug effect.

Further, the experiment in vitro of compositions shows, compositions has very strong human body fungi activity, and therefore compositions of the present invention is expected to be used to prepare novel anti-human fungi medicine.

Further, the experiment in vitro of compositions shows, compositions has very strong anti Helicobacter pylori activity, illustrate for the diseases such as the closely-related acute and chronic gastritis of helicobacter pylori, duodenal ulcer, compositions is the compound of a great exploitation potential for its.It can be directly used in the treatment of corresponding disease and the preparation of related drugs.

Further, the experiment in vitro of compositions shows, compositions has the effect of very strong suppression escherichia coli, fluorescent pseudomonas, Staphylococcus aureus, Bacillus proteus, neogenesis cryptococcus, so compositions can be used as the compound with antibacterial actions, and be expected to be applied preparing in anti-bacterial drug.

Further, according to the result of preliminary test, the present invention with solid medium By Dilution compositions to the minimal inhibitory concentration of bacillus calmette-guerin vaccine, the H37Rv strain of mycobacterium tuberculosis type strain and substance of medicines-resistant branched tubercle bacillus (MDRMTB) three kinds of tulases, experimental result confirms that compositions has very strong anti-tubercle bacillus and anti-drug resistance tulase is active, can be used as the lead compound for the treatment of tubercle bacillus affection disease, also can be used for preparation treatment tubercular drugs.

The present invention is further detailed explanation by the following examples, but protection scope of the present invention is not by any restriction of specific embodiment, but be limited by claim.

Detailed description of the invention

The preparation of embodiment 1 compound S chiglautoneA

Document (the Fan-YuMengetal. that the people such as the preparation method reference Fan-YuMeng of compound S chiglautoneA (I) deliver, 2011.SchiglautoneA, aNewTricyclicTriterpenoidwithaUnique6/7/9-FusedSkeletonf romtheStemsofSchisandraglaucescens.OrganicLetters13 (2011) 1502 – 1505) method.

The synthesis of the O-bromoethyl derivant (II) of embodiment 2SchiglautoneA

By Compound I (502mg, 1.00mmol) be dissolved in 15mL benzene, add in solution tetrabutyl ammonium bromide (TBAB) (0.08g), 1,50% sodium hydroxide solution of 2-Bromofume (7.520g, 40.00mmol) and 6mL.Mixture stirs 8h at 35 degrees Celsius.After 8h, reactant liquor is poured in frozen water, use dichloromethane extraction twice immediately, merge organic phase solution.Then use water and saturated common salt water washing 3 times successively to organic phase solution, then use anhydrous sodium sulfate drying, last concentrating under reduced pressure is removed solvent and is obtained product crude product.Product crude product purification by silica gel column chromatography (mobile phase is: petroleum ether/acetone=100:1.0, v/v), collects brown concentrated elution band and flings to the brown ceramic powder (508mg, 71%) that namely solvent obtains Compound II per.

¹HNMR(500MHz,DMSO-d ₆)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).

¹³CNMR(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] ^-calcdforC ₃₄H ₅₁Br ₂O ₆:715.2032；found715.2027.

The synthesis of O-(diethylin) ethyl derivative (III) of embodiment 3SchiglautoneA

Compound II per (358mg, 0.5mmol) is dissolved in the middle of 10mL acetonitrile, adds Anhydrous potassium carbonate (690mg wherein, 5.0mmol), potassium iodide (168mg, 1.0mmol) and diethylamine (2920mg, 40mmol), mixture reflux 8h.After reaction terminates, reactant liquor is poured in frozen water, with equivalent dichloromethane extraction 2 times, merge organic facies.Organic facies after merging with water and saturated common salt water washing successively, then use anhydrous sodium sulfate drying, concentrating under reduced pressure is removed solvent and is obtained product crude product.Product crude product purification by silica gel column chromatography (mobile phase is: petroleum ether/acetone=100:1.0, v/v), collects brown concentrated elution band and flings to the brown ceramic powder (231.2mg, 66%) that namely solvent obtains compound III.

¹HNMR(500MHz,DMSO-d6)δ20.07(s,1H),6.13(s,1H),6.06(s,1H),5.55(s,1H),3.52(d,J＝5.1Hz,4H),3.01(s,1H),2.89(s,8H),2.72(s,2H),2.64(s,2H),2.40(s,2H),2.22(s,1H),2.10(s,1H),1.89(d,J＝18.6Hz,4H),1.72(d,J＝8.4Hz,2H),1.62(d,J＝11.5Hz,2H),1.55(s,1H),1.50(s,1H),1.40(d,J＝9.3Hz,2H),1.33–1.27(m,3H),1.25(s,1H),1.14(s,12H),1.08–0.60(m,19H).

¹³CNMR(125MHz,DMSO-d6)δ211.64(s),209.43(s),170.46(s),161.60(s),144.10(s),132.71(s),127.95(s),86.25(s),82.80(s),67.46(s),66.70(s),57.84(s),52.91(s),52.75(s),52.29(s),48.15(s),46.37(s),41.36(s),38.76(s),38.62(s),35.42(s),34.04(s),30.43(s),29.67(s),26.91(s),25.78(s),24.44(s),22.81(s),21.64(s),21.25(s),20.20(s),18.97(s),18.50(s),15.57(s),12.85(s).

HRMS(ESI):m/z[M+H] ⁺calcdforC ₄₂H ₇₃N ₂O ₆:701.5469；found:701.5465。

The synthesis of the O-(two hydroxyethylamines) ethyl derivative (IV) of embodiment 4SchiglautoneA

Compound II per (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 reflux 9h.Pour in cold water by reactant liquor after reaction terminates, with dichloromethane extraction three times, merge organic facies, use water and saturated common salt water washing successively, anhydrous sodium sulfate drying, concentrating under reduced pressure removes solvent.Product, with purification by silica gel column chromatography (petroleum ether/acetone 100:1, v/v), obtains the faint yellow solid (0.199g, 52%) of compound IV.

¹HNMR(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).

¹³CNMR(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] ⁺calcdforC ₄₂H ₇₃N ₂O ₁₀:765.5265；found:765.5261。

Embodiment 5 compositions human body fungi activity

The preparation of compositions: loaded by the powder of 25mg compound IV crossing 200 order nets after crossing the powder of the 75mg compound III of 200 order nets and grinding after grinding to mix in tubule with cover and with turbine stirring instrument and namely obtain 100mg compositions, obtains 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 adopting concentration dilution, and in triplicate, the pathogen of test has trichophyton, microsporum canis and trichophyton tonsurans to each mensuration, and bacterial concentration is 10 ⁵individual/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 liquid of equivalent volumes and test sample Mixed culture (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) in 96 orifice plates, Human Fungi cultivation temperature is respectively 28 DEG C, observe after incubation time 24h, if find, in the disposal hole not having bacterium colony to be formed, that minimum concentration is sample lowest concentration of antimicrobial, i.e. MIC value.This experiment positive control is ketoconazole, and 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 very strong human body fungi activity, therefore compositions of the present invention is expected to be used to prepare novel anti-human fungi medicine.Compound III and compound IV nonreactive Human Fungi activity, therefore can not for the preparation of novel anti-human fungi medicine.

Embodiment 6 compositions anti-helicobactor pylori activity

1) strains tested

Helicobacter pylori (Helicobacterpylori, Hp) reference culture ATCC43504 is purchased from U.S.'s Culture Collection (AmericanTypeCultureCollection, ATCC).13 strain Hp clinical strains are picked up from Nanjing drum tower hospital Dndoscope Laboratory and are accepted gastroscopic patient; To the patient of peptic ulcer, duodenal bulbar inflammation or gastritis verrucosa in continuous gastroscopy, first be defined as Hp positive through RUT experiment, get antral gastric mucosa 1-2 block again, be inoculated in containing 8% horse serum, trimethoprim (trimethropin after chopping, in the Columbia selectivity agar culture medium of TMP) 1.25g/L, polymyxin (polymyxin) B2500U/L, vancomycin (vancomycin) 10mg/L, in 37 DEG C of (5%O under micro-oxygen environment ₂, 10%CO ₂and 85%N ₂) cultivate 72 hours.Collect antibacterial, through smear Gram’s staining, after oxidase, catalase and urease are accredited as the positive, pure culture of going down to posterity, obtained strains is as experimental strain.

2) strain culturing

We adopt micro-aerobic bag (purchased from Shanghai Medical Univ) to carry out the strain culturing of HP, and it produces the micro-aerobic environment required for Hp by chemical reaction.

3) biological activity determination

Paper disk method (Microbiologicalpapermethod) is adopted to measure the inhibitory action of medicine to helicobacter pylori, the minimum inhibitory concentration (minimalinhibitoryconcentration, MIC) of test sample is measured with agar dilution.

I. paper disk method experiment

(A) culture medium is prepared by the Columbia culture medium for preparing after high pressure steam sterilization, be cooled to 50-60 DEG C, add 8% horse serum or Sheep Blood, mixing is poured in the culture dish of sterilizing, every ware 7-10ml, culture medium thickness is 1.5mm (sterile working).

(B) experimental bacteria of transferring (being coated with bacterium) gets diluted 10 with microscale sampler ⁸cFU/ml (1OD ₆₆₀=10 ⁸cFU/ml) the bacteria suspension 0.1ml of Hp spreads upon suitable culture dish surface equably.Be inverted in 37 DEG C of drying bakers and take out after 15min, object makes agar surface dry, for subsequent use.

(C) paste sample scraps of paper microscale sampler to get 6 μ l testing samples (mass concentration 2mg/ml) and inject on the round filter paper of sterilizing.With aseptic nipper tweezer containing the scraps of paper of sample and the blank scraps of paper of contrast, by sterile working respectively the scraps of paper be close to containing bacterio-agar surface, paste a piece of paper sheet at a certain distance.Often kind of bacterium is cooked 3 wares, and acquired results asks its meansigma methods.

(D) cultivate each plate is placed in micro-aerobic bag, sealing, opens gas generator, then is placed in 37 DEG C of incubators and cultivates 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 the result of matched group, the result of testing sample sensitive experiment can be drawn.In triplicate.

II. agar dilution measures MIC

(A) first medicine dimethyl sulfoxide (DMSO) solution preparation of test is become the mother solution of 0.5mg/ml by the preparation of Drug plates, then with sterilized water dilution, is finally made into 100.0,80.0,60.0,45.0,40.0,35.0,30.0,25.0,20.0,15.0,10.0, the concentration series of 5.0 and 2.5 μ g/ml, DMSO concentration is in media as well less than 1%.The testing drug solution that 1ml is prepared be incubated 8ml Columbia medium in 50 DEG C, separately add the horse serum that 1ml is incubated in 50 DEG C and fully mix, cast in culture dish cool (final concentration of culture dish Chinese medicine is 10.0,8.0,6.0,4.5,4.0,3.5,3.0,2.5,2.0,1.5,1.0,0.5 and 0.25 μ g/ml, if there is bacteriostasis, then MIC value namely these worthwhile in one).

(B) experimental bacteria of transferring (being coated with bacterium) draws diluted 1 × 10 with microscale sampler ⁸the bacteria suspension 0.1ml of CFU/mlHp spreads upon culture dish surface equably, is inverted in 37 DEG C of drying bakers and takes out after 15min, and object makes agar surface dry, for subsequent use.

(C) determine that test dish (contains: 85%N at micro-aerobic bag by MIC ₂, 10%CO ₂and 5%O ₂) in, be incubated 37 DEG C and cultivate 72 hours, observe Hp growing state, contrast with blank group, there is no the sample least concentration of bacteria growing completely for minimum inhibitory concentration (minimalinhibitoryconcentration, MIC) value.Positive control is ampicillin (Ampicillin).

Experimental result

Experiment in vitro shows, compositions has very strong anti Helicobacter pylori activity, and it is 23mm (ATCC43504) that paper disk method shows its antibacterial circle diameter.With agar dilution display, it can suppress the growth of 5 random clinical strains and 1 reference culture (ATCC43504) completely, and minimal inhibitory concentration (MIC) is 3.0 μ g/ml.Make positive control with ampicillin, its Cmin (MIC) suppressed completely 6 strain test bacterium is 1.0 μ g/ml.Compound III and compound IV are without anti Helicobacter pylori activity.

Conclusion: it is active that compositions has stronger suppression helicobacter pylori, illustrate for the disease such as the closely-related acute and chronic gastritis of helicobacter pylori, duodenal ulcer, compositions is the medicine of a great exploitation potential for its.It can be directly used in the treatment of corresponding disease and the preparation of related drugs.Compound III and compound IV, without anti Helicobacter pylori activity, cannot be used for the treatment of corresponding disease and the preparation of related drugs.

Embodiment 7 compositions antibacterial activity

Antibacterial activity test is the method adopting concentration dilution, and in triplicate, test pathogen has escherichia coli, fluorescent pseudomonas, Staphylococcus aureus, Bacillus proteus, neogenesis cryptococcus to each mensuration, and bacterial concentration is 10 ⁵individual/mL.Medicine initial concentration is 50.00 μ g/mL (5% dimethyl sulfoxide DMSO), gradient dilution to 0.10 μ g/mL, the bacterium liquid of equivalent volumes and test sample Mixed culture (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) in 96 orifice plates, antibacterial culturing temperature is respectively 37 DEG C, observe after incubation time 24h, if find, in the disposal hole not having bacterium colony to be formed, that minimum concentration is sample lowest concentration of antimicrobial, i.e. MIC value.Compositions the anti-bacterial result is in table 2.

Table 2 compositions antibacterium MIC value (μ g/mL)

Conclusion: compositions has very strong antibacterial activity, therefore compositions of the present invention is expected to be used to prepare novel anti-bacterial drug.Compound III and compound IV, without antibacterial activity, can not be used to prepare novel anti-bacterial drug.

Embodiment 8 compositions anti-tubercle bacillus is active

(1) the anti-bacillus calmette-guerin vaccine of solid medium By Dilution compositions (BCG) absolute concentration

Scraping BCG cultures from inclined-plane, join in 3mlMiddlebrook7H9 broth bouillon, add a small amount of bead, screw test tube cap, high vibration grinding in vortex oscillator, with standard Maxwell opacity tube (MacFarlandNo.1) than turbid, be namely made into bacillus calmette-guerin vaccine (BCG) bacteria suspension of 1mg/ml.

Medicine DMSO is made into the stock solution of high concentration, by the tween 80 aseptic ultra-pure water dilution stock solution containing 5% to desired concn, the medicine diluted is joined 4mlMiddlebrook7H11 agar culture medium (this culture medium 121 DEG C of high pressure steam sterilizations 15 minutes, be cooled to 50 ~ 55 DEG C) by required dosage, 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, the isocyatic slant medium of 0.75ug/ml, 0.5ug/ml.

Be that bacillus calmette-guerin vaccine (BCG) the bacteria suspension inoculating loop of 1mg/ml dips ring of numbers by concentration, be inoculated in containing in the culture medium of each medicine series concentration and blank medium slant respectively, be placed in 37 DEG C to cultivate 4 ~ 8 weeks, observation experiment result, result is as shown in table 3.

In the present embodiment, Middlebrook7H9 broth bouillon used and Middlebrook7H11 agar culture medium are the conventional culture medium that those skilled in the art carry out when tulase is cultivated, and its formula adopts conventional formulation.

(2) solid medium By Dilution compositions Killing Mycobacterium Tuberculosis type strain H37Rv strain absolute concentration

Scraping mycobacterium tuberculosis type strain H37Rv strain culture from inclined-plane, join in 3mlMiddlebrook7H9 broth bouillon, add a small amount of bead, screw test tube cap, high vibration grinding in vortex oscillator, with standard Maxwell opacity tube (MacFarlandNo.1) than turbid, be namely made into the H37Rv strain bacteria suspension of 1mg/ml.

Medicine is made into respectively the stock solution of high concentration with DMSO, by the tween 80 aseptic ultra-pure water dilution stock solution containing 5% to desired concn, the medicine diluted is joined 4mlMiddlebrook7H11 agar culture medium (this culture medium 121 DEG C of high pressure steam sterilizations 15 minutes, be cooled to 50 ~ 55 DEG C) by required dosage, 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, the isocyatic slant medium of 0.75ug/ml, 0.5ug/ml.

Be that the H37Rv strain bacteria suspension inoculating loop of 1mg/ml dips ring of numbers by concentration, in the culture medium being inoculated in drug containing series concentration respectively and blank medium slant, be placed in 37 DEG C and cultivate 4 ~ 8 weeks, observation experiment result, result is as shown in table 3.

(3) the clinical separation of solid medium By Dilution medicine Ad tuberculosis resistance to ISREMTB strain absolute concentration

The clinical separation of scraping mycobacterium tuberculosis resistance to ISREMTB strain (resistance to isoniazid, streptomycin, rifampicin, the clinical detached dowel of ethambutol mycobacterium tuberculosis) culture from inclined-plane, join in 3mlMiddlebrook7H9 broth bouillon, add a small amount of bead, screw test tube cap, high vibration grinding in vortex oscillator, with standard Maxwell opacity tube (MacFarlandNo.1) than turbid, be namely made into the bacteria suspension of 1mg/ml.

Medicine is made into respectively the stock solution of high concentration with DMSO, by the tween 80 aseptic ultra-pure water dilution stock solution containing 5% to desired concn, the medicine diluted is joined 4mlMiddlebrook7H11 agar culture medium (this culture medium 121 DEG C of high pressure steam sterilizations 15 minutes, be cooled to 50 ~ 55 DEG C) by required dosage, 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, the isocyatic slant medium of 0.75ug/ml, 0.5ug/ml.

Be that the clinical separation of the mycobacterium tuberculosis resistance to ISREMTB strain bacteria suspension inoculating loop of 1mg/ml dips ring of numbers by concentration, be inoculated in containing in the culture medium of each medicine series concentration and blank medium slant respectively, be placed in 37 DEG C to cultivate 4 ~ 8 weeks, observation experiment result, result is as shown in table 3.

Table 3 solid medium By Dilution compositions anti-tubercle bacillus absolute concentration result

Table 4 solid medium By Dilution compound III anti-tubercle bacillus absolute concentration result

Table 3 solid medium By Dilution compound IV anti-tubercle bacillus absolute concentration result

Conclusion: compositions has very strong anti-tubercle bacillus and anti-drug resistance tulase is active, can be used as the lead compound for the treatment of tubercle bacillus affection disease, also can be used for preparation treatment tubercular drugs.Compound III and compound IV without anti-tubercle bacillus and anti-drug resistance tulase active, can not as the lead compound for the treatment of tubercle bacillus affection disease, can not for the preparation for the treatment of tubercular drugs.

The preparation of embodiment 9 composition tablet involved in the present invention

Get 2 grams of compositionss, add the customary adjuvant 18 grams preparing tablet, mixing, conventional tablet presses makes 100.

The preparation of embodiment 10 composition capsule involved in the present invention

Get 2 grams of compositionss, add prepare capsule customary adjuvant as starch 18 grams, mixing, encapsulatedly makes 100.

Claims (10)

1. a compositions, it is characterized by said composition and be made up of compound III and compound IV, in said composition, the mass percent of compound III and compound IV is respectively 75% and 25%,

2. the preparation method of compositions as claimed in claim 1, is characterized by: the powder of compound III and the powder of compound IV are respectively 75% and 25% according to mass percent and fully mix.

3. the application of compositions as claimed in claim 1 in antibacterials.

4. the application of compositions in antibacterials as claimed in claim 3, is characterized by: described bacterium is antibacterial.

5. the application of compositions in antibacterials as claimed in claim 3, is characterized by: described bacterium is fungus.

6. the application of compositions in antibacterials as claimed in claim 3, is characterized by: described bacterium is helicobacter pylori.

7. the application of compositions in antibacterials as claimed in claim 3, is characterized by: described bacterium is tulase.

8. the application of compositions in antibacterials as claimed in claim 5, is characterized by: described fungus is trichophyton, microsporum canis or trichophyton tonsurans.

9. the application of compositions in antibacterials as claimed in claim 4, is characterized by: described antibacterial is escherichia coli, fluorescent pseudomonas, Staphylococcus aureus, Bacillus proteus or neogenesis cryptococcus.

10. the application of compositions in antibacterials as claimed in claim 7, is characterized by: described tulase is bacillus calmette-guerin vaccine, the H37Rv strain of mycobacterium tuberculosis type strain and substance of medicines-resistant branched tubercle bacillus.