CN103122366A - Application of benzothiophene compound as mycobacterium tuberculosis inhibitor - Google Patents
Application of benzothiophene compound as mycobacterium tuberculosis inhibitor Download PDFInfo
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- CN103122366A CN103122366A CN2011103738273A CN201110373827A CN103122366A CN 103122366 A CN103122366 A CN 103122366A CN 2011103738273 A CN2011103738273 A CN 2011103738273A CN 201110373827 A CN201110373827 A CN 201110373827A CN 103122366 A CN103122366 A CN 103122366A
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- mycobacterium tuberculosis
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Abstract
The invention belongs to the technical field of drug targets and in particular relates to application of a small molecular lead compound with inhibiting effect on mycobacterium tuberculosis. The small molecular compound is a benzothiophene compound, with a chemical name being ethyl 2-[(3,5-dibromo-2,4-dihydroxybenzyl)amino]-4,5,6-7-tetrahydro-1-benzothiophene-3-carboxylate. The compound has the advantages of small molecular weight, simple structure, good permeability and the like and can effectively inhibit growth of mycobacterium tuberculosis. Mycobacterium tuberculosis is a pathogenic bacterium of tuberculosis, so the small molecular compound has the potential of treating tuberculosis and is expected to be developed into a new antituberculosis drug. Therefore, the small molecular compound can be developed as a new antituberculosis drug and provides a new approach for treatment and cure of tuberculosis. The invention also relates to application of preparations containing the small molecular inhibitor to preparation of antituberculosis drugs.
Description
Technical field
The invention belongs to the triage techniques field of antibacterial medicines.The micromolecular compound that is specifically related to a kind of special inhibition mycobacterium tuberculosis growth is that benzothiophenes is as the application of Killing Mycobacterium Tuberculosis inhibitor.
Background technology
Mycobacterium tuberculosis is a kind of pathogenic bacteria of serious harm human health, and mycobacterium tuberculosis can exist several months or several years in human body after infecting human body.According to World Health Organization statistics, annual nearly three million peoples in the whole world die from tuberculosis, and the whole world approximately has 1/3rd population to infect the mycobacterium tuberculosis (WHO, 2009) that is in latent state.At present, the tuberculosis chemotherapy has the mixture of a line and Second line Drug to consist of.Complicated and the long-term treatment plan that actual tuberculotherapy needs 4-5 kind medicine to surpass 6-9 month is eradicated this disease, has caused serious toxicity and resistance.In fact, owing to having uncommon cell walls, mycobacterium has resistance to most of common antibiotics natively.In addition, the genetics change also makes it obtain resistance.Due to the mycobacterium tuberculosis bacterial strain to more and more being used for the treatment of multiple drug resistance tuberculosis (multidrug-resistant tuberculosis, MDR-TB) Second line Drug produces resistance, therefore it has become the threat (Gandhi et al, 2010) of world wide publilc health.80% mortality ratio causes this disease to become serious global health problem for the mycobacterium tuberculosis of MDR.The drug resistance bacterial strain that occurs in recent years and with the coinfection problem of HIV, make this form severeer.Tuberculosis is not only the problem that is present in developing country.Along with whole world travelling and immigrant's increase, tuberculosis becomes a global disease of serious threat.Therefore, seek new drug targets and research and develop new antitubercular agent and diagnostic tool extremely urgent (Ginsberg and Spigelman, 2007).Especially, we need to have the activated novel drugs of antagonism medicated strain tool of novel mechanism.
Summary of the invention
The object of the invention is to benzothiophenes as the application of Killing Mycobacterium Tuberculosis inhibitor, described compound has the characteristic that suppresses the mycobacterium tuberculosis growth and finally kill mycobacterium tuberculosis.
The present invention is achieved through the following technical solutions:
The applicant obtains a kind of benzothiophenes by screening, and this compound has structure as described below:
This benzothiophenes is a kind of known compound, and its Chinese name is called ethyl 2-[(3,5-two bromo-2,4-dihydroxy benzyl) amino]-4,5,6,7-tetrahydrochysene-1-thionaphthene-3-carboxylic acid; English name is ethyl 2-[(3,5-dibromo-2,4-dihydroxybenzylidene) amino]-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate.
Above-claimed cpd is 690246 at ZINC database ID; Its molecular formula is C
18H
17Br
2NO
4S； Molecular weight is 503.21.In order to narrate conveniently, in this manual the applicant to be called for short this compound be 14# compound or inhibitor 14#.This compound can be bought from commercial channels and obtain, and this compound used in the present invention is to buy from Dutch Specs company (http://www.specs.net) to obtain.
The present invention has measured the restraining effect of this benzothiophenes to mycobacterium tuberculosis, in an embodiment of the present invention, the applicant describes involved some mycobacteriums of participating in the experiment in detail, for example: (public of M. smegmatics bacterial strain obtains the source: Chinese medicine bacterium preservation administrative center, bacterium numbering is the M. smegmatics bacterial strain: 93202.Network address: http://www.cmccb.org.cn/), (public of mycobacterium bovis BCG bacterial strain obtains the source: Chinese medicine bacterium preservation administrative center, bacterium numbering is the mycobacterium bovis BCG bacterial strain: 93006.Network address: http://www.cmccb.org.cn/), (public of mycobacterium tuberculosis bacterial strain obtains the source: Chinese medicine bacterium preservation administrative center, bacterium numbering is the mycobacterium tuberculosis bacterial strain: 93004.Network address: http://www.cmccb.org.cn/).Candidate inhibitor with 50 μ g/ml in the present invention adds in the culture of mycobacterium, then will add the mycobacterium of above-mentioned candidate inhibitor in 37 ℃ of cultivations 7～8 days, by reading each OD value and comparing to judge with contrast whether the growth of mycobacterium has been subject to inhibition.Contrast 1 is the treatment group of inhibitor 14#, and contrast 2 is that the antiphthisic first-line drug of use is Rifampin (RFP) treatment group.Data in experimentation are relatively to estimate with two control groups the inhibition of utilizing candidate inhibitor to obtain, seek the compound with inhibition.Adopting uses the same method has also measured ethyl 2-[(3,5-two bromo-2,4-dihydroxy benzyl) amino]-4,5,6,7-tetrahydrochysene-1-thionaphthene-3-carboxylic acid is to mycobacterium tuberculosis, the minimum inhibitory concentration of mycobacterium bovis BCG and M. smegmatics.
Advantage of the present invention is as follows
1, the micromolecular compound molecular weight of the present invention's acquisition is little, and structure is relatively simple, is soluble in multi-solvents.
2, the micromolecular compound specificity of the present invention's acquisition is good, can effectively kill mycobacterium tuberculosis, and to homology Pseudomonas M. smegmatics DeGrain.
3, the effect of the micromolecular compound inhibition mycobacterium tuberculosis of the present invention's acquisition can be near a line antitubercular agent Rifampin.More detailed technical scheme sees that " embodiment " is described.
Description of drawings
Fig. 1. the antibacterial result of candidate inhibitor 14# to each mycobacterium strain.
Fig. 2. solid is observed the survival condition of respectively processing sample on the inclined-plane, is from left to right the control group that does not add inhibitor 14# successively, adds the treatment group of inhibitor 14#, adds the control group of Rifampin.
Fig. 3. the growing state of mycobacterium tuberculosis bacterial strain under each inhibitor concentration in the minimal inhibitory concentration determination experiment.From left to right benzothiophenes is that the concentration of inhibitor 14# is respectively: 0.031,0.062,0.125,0.25,0.50,1.00,2.00,4.00, and 8.00,16.00,32.00 μ g/ml.
Embodiment
The cultivation of embodiment 1 bacterial strain and the preparation of liquid inhibitor
The preparation of strains tested:
The bacterial strain of participating in the experiment of the present embodiment is the bacterial strain of open report: M. smegmatics bacterial strain (strain number: 93202; Administrative center obtains from the preservation of Chinese medicine bacterium, network address: http://www.cmccb.org.cn/, (lower same)), mycobacterium bovis BCG bacterial strain (strain number: 93006, administrative center obtains from the preservation of Chinese medicine bacterium), the mycobacterium tuberculosis bacterial strain (strain number: 93004, administrative center obtains from the preservation of Chinese medicine bacterium).
The component of screening culture medium and preparation:
The component of liquid screening substratum and compound method thereof: (this 7H9 liquid nutrient medium is available from U.S. company BD for the 7H9 liquid nutrient medium that adds 90ml to be purchased in the liquid screening substratum of 100ml, 271310) and 10ml OADC nutritive medium (5% bovine serum albumin article No.:, 0.2% glucose, 0.06% olein, 140mmol/l NaCl; These commodity OADC nutritive medium is available from U.S. company BD, and article No.: 211886), this liquid screening substratum is the typical liquid substratum for mycobacterium strain.
The component of solid screening culture medium and compound method thereof: add 7H10 solid medium that 90ml is purchased (available from U.S. company BD in the solid screening culture medium of 100ml, 262710) and 10ml OADC nutritive medium (5% bovine serum albumin article No.:, 0.2% glucose, 0.06% olein, 140mmol/l NaCl, these commodity OADC nutritive medium is available from U.S. company BD, and article No.: 211886), this solid medium is the typical solid substratum for mycobacterium strain.
The preparation of candidate inhibitor:
The inhibitor of the present embodiment (ethyl 2-[(3,5-two bromo-2,4-dihydroxy benzyl) amino]-4,5,6,7-tetrahydrochysene-1-thionaphthene-3-carboxylic acid) use with solution or suspension.Adopt dimethyl sulfoxide (DMSO) (DMSO) dissolution inhibitor pulvis so that subsequent experimental is used.concrete compound method: the inhibitor concentrated solution that first is mixed with original concentration and is 50mg/ml (0.1M) is stored standby, be ethyl 2-[(3 measuring inhibitor, 5-two bromo-2, the 4-dihydroxy benzyl) amino]-4, 5, 6, 7-tetrahydrochysene-1-thionaphthene-3-carboxylic acid is to mycobacterium tuberculosis, during the minimum inhibitory concentration of mycobacterium bovis BCG and M. smegmatics, carry out the concentration gradient experiment with the above-mentioned inhibitor concentrated solution for preparing, its concentration gradient is designed to respectively: 0.031, 0.062, 0.125, 0.25, 0.50, 1.00, 2.00, 4.00, 8.00, 16.00, 32.00 μ g/ml.
Inoculation and cultural method:
Above-mentioned three kinds of mycobacteriums are inoculated in respectively in the above-mentioned liquid screening substratum for preparing, in 37 ℃ of cultivations, wherein: M. smegmatics strain culturing 3 days, mycobacterium bovis BCG bacterial strain, the mycobacterium tuberculosis bacterial strain was all cultivated 21 days, to mycobacterium density be 1 * 10
8～2 * 10
8Or optical density value OD
600Be 0.8～1.Then each mycobacterium bacterium liquid is diluted that (final concentration is 1～2 * 10
4) and minute install in 96 orifice plates.Carry out under the whole aseptic technique program that operates in Biohazard Safety Equipment commonly used.
The In Vitro Bacteriostatic test of embodiment 2 inhibitor
According to the aseptic technique program of embodiment 1, (bacterium numbering is: 93004) be inoculated into the In Vitro Bacteriostatic of determining inhibitor 14# in the screening culture medium that is added with inhibitor 14# with the mycobacterium tuberculosis bacterium liquid of embodiment 1.In the present embodiment, the inhibitor 14# of 50 μ g/ml is added in the bacterial cultures of mycobacterium tuberculosis in 37 ℃ and cultivated 7～8 days, by reading each OD value and comparing to judge that with contrast (containing contrast 1 and contrast 2) mycobacterium tuberculosis is grown whether is suppressed.Contrast 1 is not for adding inhibitor 14# group, contrast 2 is for adding Rifampin 50 μ g/ml groups, data in experimentation are to compare to estimate inhibitor 14# to the mycobacterium tuberculosis inhibition with two, (bacterium numbering is the mycobacterium bovis BCG bacterial strain: 93006), (bacterium numbering is: above-mentioned same method is adopted in In Vitro Bacteriostasis experiment 93202) to the M. smegmatics bacterial strain, and result as shown in Figure 1.Can find out obviously that from Fig. 1 the candidate inhibitor benzothiophenes is that inhibitor 14# can effectively suppress the growth of mycobacterium tuberculosis and mycobacterium bovis BCG, and M. smegmatics is not produced inhibition.
Embodiment 3 determines the In Vitro Bacteriostatic of benzothiophenes
(bacterium numbering is: 93004) bacterium liquid is inoculated into the In Vitro Bacteriostatic of determining benzothiophenes in the liquid screening substratum that is added with benzothiophenes with mycobacterium tuberculosis.Concrete grammar is: the inhibitor 14# that is obtained by embodiment 2 screening of 50 μ g/ml is added in the bacterial cultures of mycobacterium tuberculosis, (bacterial concentration is 1～2 * 10
7) in 37 ℃ of cultivations, after processing through the inhibitor 14# of 8 days, 10 μ l mycobacterium tuberculosis bacterium liquid are coated onto on the solid screening culture medium in 37 ℃ cultivated one month, observe the growing state of each sample.Result shows: two samples that are added with inhibitor 14# group and Rifampin group all do not have single colony growth, and the mycobacterium tuberculosis of organizing without inhibitor 14# is well-grown, the results are shown in Figure 2.
Embodiment 4 determines the minimum inhibitory concentration (MIC) of candidate inhibitor
In antibacterial tests, the prior appraisal of the percentage ratio of inhibition is undertaken by measuring minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC).MIC is defined as the minimum concentration that antibacterials can suppress bacterial growth in substratum.According to the design of embodiment 1, the inhibitor 14# of different concns is carried out gradient dilution, and (its concentration gradient is respectively: 0.031,0.062,0.125,0.25,0.50,1.00,2.00,4.00,8.00,16.00,32.00 μ g/ml), adding to cultivate has in the liquid screening substratum of mycobacterium tuberculosis, cultivated 7～8 days for 37 ℃, judge that by reading each OD value whether the mycobacterium tuberculosis growth is suppressed, and the results are shown in shown in Figure 3.The M. smegmatics bacterial strain, the same mycobacterium tuberculosis of the inoculation of mycobacterium bovis BCG bacterial strain and culture condition, its MIC measurement result is as shown in table 1.
The minimal inhibitory concentration of table 1. inhibitor 14# to each Mycobacterium
Reference:
1.World?Health?Organization.Global?Tuberculosis?Control?2009：Epidemiology，Strategy，Financing.Nonserial?Publication.WHO，2009.
2.Gandhi?NR，Nunn?P，Dheda?K，Schaaf?HS，Zignol?M，van?Soolingen?D，Jensen?P，Bayona?J.Multidrug-resistant?and?extensively?drug-resistant?tuberculosis：a?threat?to?global?control?of?tuberculosis.Lancet.2010?May?22；375(9728)：1830-43.
3.Ginsberg?AM，Spigelman?M.Challenges?in?tuberculosis?drug?research?and?development.Nat?Med.2007?Mar；13(3)：290-4.
Claims (1)
1. a benzothiophenes as the application of inhibitor, is characterized in that in screening Killing Mycobacterium Tuberculosis medical compounds, and this compound is ethyl 2-[(3,5-two bromo-2, the 4-dihydroxy benzyl) amino]-4,5,6,7-tetrahydrochysene-1-thionaphthene-3-carboxylic acid, its molecular formula is C
18H
17Br
2NO
4S, molecular weight are 503.21, and its structural formula is as follows;
Its step is as follows:
(1) with M. smegmatics, mycobacterium tuberculosis var bovis, mycobacterium tuberculosis is the bacterium of participating in the experiment;
(2) preparation of liquid screening substratum: add 90ml 7H9 liquid nutrient medium and 10ml OADC nutritive medium in the liquid screening substratum of 100ml;
(3) the described three kinds of mycobacteriums of step (1) are inoculated in respectively in the described liquid screening substratum of step (2), in 37 ℃ of cultivations, wherein: M. smegmatics was cultivated 3 days, and mycobacterium tuberculosis var bovis and mycobacterium tuberculosis were cultivated 21 days, to mycobacterium density be 1 * 10
8～2 * 10
8Or optical density value OD
600Be 0.8～1, then each mycobacterium bacterium liquid diluted and divides and install in 96 orifice plates, the final concentration after dilution is 1～2 * 10
4
(4) with the ethyl 2-[(3 of 50 μ g/ml, 5-two bromo-2,4-dihydroxy benzyl) amino]-4,5,6, the cultivation that 7-tetrahydrochysene-1-thionaphthene-3-carboxylic acid adds step (3) to has in 96 orifice plates of mycobacterium bacterium liquid, by reading OD
600Value and with compare, judge whether the growth of mycobacterium has been subject to inhibition;
(5) method of repeating step (4) is measured ethyl 2-[(3,5-two bromo-2, the 4-dihydroxy benzyl) amino]-4,5,6,7-tetrahydrochysene-1-thionaphthene-3-carboxylic acid is to mycobacterium tuberculosis, the minimum inhibitory concentration of mycobacterium tuberculosis var bovis and M. smegmatics (MIC);
Wherein
The described OADC nutrient solution prescription of step (2) and proportioning are as follows: 5% bovine serum albumin, 0.2% glucose, 0.06% olein, 140mmol/l NaCl;
The described minimum inhibitory concentration of step (5) is as described below respectively:
Mycobacterium tuberculosis 2 μ g/ml;
Mycobacterium tuberculosis var bovis 2-4 μ g/ml.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040171603A1 (en) * | 2001-05-18 | 2004-09-02 | Janos Pato | Novel therapeutic targets for the treatment of mycobacterial infections and compounds useful therefor |
| CN1297670C (en) * | 2004-10-19 | 2007-01-31 | 中国人民解放军第三○九医院 | M.tuberculosis drug resistant gene detection reagent kit and process for preparation |
| CN101932579A (en) * | 2007-03-23 | 2010-12-29 | 拜欧蒂姆实验室公司 | New imidazolo-heteroaryl derivatives with antibacterial properties |
| CN102174638A (en) * | 2011-01-18 | 2011-09-07 | 贵州省中国科学院天然产物化学重点实验室 | Fast screening method of anti-bacilus tuberculosis medicine |
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2011
- 2011-11-21 CN CN2011103738273A patent/CN103122366A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040171603A1 (en) * | 2001-05-18 | 2004-09-02 | Janos Pato | Novel therapeutic targets for the treatment of mycobacterial infections and compounds useful therefor |
| CN1297670C (en) * | 2004-10-19 | 2007-01-31 | 中国人民解放军第三○九医院 | M.tuberculosis drug resistant gene detection reagent kit and process for preparation |
| CN101932579A (en) * | 2007-03-23 | 2010-12-29 | 拜欧蒂姆实验室公司 | New imidazolo-heteroaryl derivatives with antibacterial properties |
| CN102174638A (en) * | 2011-01-18 | 2011-09-07 | 贵州省中国科学院天然产物化学重点实验室 | Fast screening method of anti-bacilus tuberculosis medicine |
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Application publication date: 20130529 |