CN108159039A - A kind of compound that can be used for preparing anti-tuberculosis drugs - Google Patents
A kind of compound that can be used for preparing anti-tuberculosis drugs Download PDFInfo
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- CN108159039A CN108159039A CN201810168892.4A CN201810168892A CN108159039A CN 108159039 A CN108159039 A CN 108159039A CN 201810168892 A CN201810168892 A CN 201810168892A CN 108159039 A CN108159039 A CN 108159039A
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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
- A61K31/28—Compounds containing heavy metals
- A61K31/305—Mercury compounds
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Abstract
The invention discloses a kind of compound that can be used for preparing anti-tuberculosis drugs, the compound is phenylmercuric acetate, pharmaceutically active substance can be used as to develop anti-mycobacterium tuberculosis drug.
Description
Technical field
The present invention relates to tuberculosis technical field, especially a kind of compound that can be used for preparing anti-tuberculosis drugs.
Background technology
Tuberculosis (tuberculosis, TB) be by mycobacterium tuberculosis (mycobacterium tuberculosis,
MTB a kind of great communicable disease for endangering human health and leading to human death caused by).A large amount of uses of antituberculotic
Mutation with mycobacterium tuberculosis treating tuberculosis action site causes resistant to many drug tuberculosis to continuously emerge.In addition tuberculosis and Chinese mugwort
It grows the concurrent incidence of disease to sharply increase, the appearance of these problems all carrys out huge challenge to treatment zone lungy.
Since treatment cycle lungy is long, and drug is not easy through cell wall, so clinically passing through frequently with drug
Associated with method treatment tuberculosis.Drug currently on the market is broadly divided into a line anti-tubercular drug and two wires anti-tubercular drug.One line
Anti-tubercular drug mainly includes isoniazid (isoniazid), rifampin (rifampin), pyrazinamide (pyrazinamide), second
Amine butanol (ethambutol) and fixed dosage compound formulation (fixed-dose combination formulations,
FDCs)；Advantage is that therapeutic effect is good, Small side effects, long half time, can kill the quick branch bar bred and bred at a slow speed simultaneously
Bacterium；Shortcoming is that mycobacterium tuberculosis is easily made to generate drug resistance.Two wires anti-tubercular drug has quinolones, macrolides, amino sugar
Glycoside, isonicotinic acid derivatives class, phenothiazines (Clofazimine), cyclic polypeptide class etc.；Advantage is not easy by mycobacteria drug resistance, but
It is that toxic side effect is bigger.
It is insufficient that the advantages of having both existing line anti-tubercular drug and two wires anti-tubercular drug therefore, it is necessary to one kind can avoid the two again
Drug.
Invention content
The purpose of the present invention is to provide a kind of compounds that can be used for preparing anti-tuberculosis drugs.
To achieve the above object, the present invention uses the following contents：
A kind of compound that can be used for preparing anti-tuberculosis drugs, the compound is phenylmercuric acetate, and structural formula is：
Further, the compound, which can be used for preparing, inhibits mycobacterium tuberculosis drug.
Further, the mycobacterium tuberculosis is H37Rv.
Phenylmercuric acetate for contraceptive, also acts as surgical operation disinfection earliest；Agriculturally it is being used for handling seed, to prevent
Control various diseases of agricultural plants.It is used for treatment lungy by the present inventor for the first time.By a series of tests, find for shame dirt
Mycobacteria and BCG, the minimal inhibitory concentration (MIC, Minimum inhibitory concentration) of phenylmercuric acetate
It is far smaller than rifampin；For standard strain, the MIC of phenylmercuric acetate is slightly above rifampin.In addition, by prolonged training
It supports, also no the phenomenon that finding drug resistance.
Specific embodiment
Embodiment
First, a series of drugs have been screened using the closer mycobacterium smegmatis of branch tubercle bacillus affinity, has obtained vinegar
Sour benzene mercury.
1 phenylmercuric acetate-mycobacterium smegmatis MIC experiments
1.1 experiment reagent
Phenylmercuric acetate；Rifampin；100 μ g/mL are dissolved into 10%DMSO.
1.2 cell strain
Mycobacterium smegmatis (MSMEG, mycobacterium smegmatis)【ATCC-70084】.
1.3 experimental method
Preparation LBGT culture mediums (tryptone 1%, yeast extract 0.5%, sodium chloride 1%, glucose 0.5%,
Tween 80 is 0.025%).MSMEG glycerol stocks are connected in 5mL LBGT culture mediums, 37 DEG C of cultures to OD6000.6-0.8, will
Bacterium is diluted to OD6000.1, then 20 times are diluted, until final concentration is about 105CFU/mL.Prepare to add good LBGT culture mediums and phase in advance
Answer 96 orifice plates of drug concentration (drug concentration is according to proportional diluted method).By the bacterium solution diluted according to 1：10 add in 96 holes
(bacterial concentration is 10 in final apertures4CFU/mL).Microplate reader OD is used after 37 DEG C of constant temperature incubation 20h600Detect the bacterium solution growth in each hole
Situation.
1 phenylmercuric acetate of table is to the In Vitro Bacteriostasis of MSMEG
2nd, phenylmercuric acetate is used on BCG vaccine and mycobacterium tuberculosis type strain H37Rv.
2nd, phenylmercuric acetate-BCG MIC are tested
2.1 experiment reagent
Phenylmercuric acetate；Rifampin；100 μ g/mL are dissolved into 10%DMSO.
2.2 cell strain
BCG vaccine (BCG, bacillus Calmette-Gu é rin).
2.3 experimental method
Using 7H9 solid medium activated b CG, after cultivating a period of time, when BCG bacterium colonies are agglomerating, in sterile super-clean bench
In by about 10mg bacterium colonies scrape in sterilizing homogenate tubule in, add in sterilizing magnetic bead and filtered TBST (0.01M pH7.4TBS,
0.025%Tween 80), upper homogenizer is fully homogenized, after reuse 8 μm of strainer filterings, until final concentration is about 105CFU/mL。
Prepare to add in 96 orifice plate of V bottoms of 7H9 (+ADC) culture medium and relative medicine, and add in the resazurin of debita spissitudo in advance.It will
The bacterium solution diluted is according to 1：(bacterial concentration is 10 in final apertures in 10 96 holes of addition4CFU/mL).37 DEG C of constant temperature incubations for 24 hours after
With fluorescence microplate reader bacterium solution upgrowth situation (resazurin being made to be irreversibly changed detection using the amount of oxygen of metabolic exhaustion).
2 phenylmercuric acetate of table is to the In Vitro Bacteriostasis of BCG
3rd, phenylmercuric acetate-H37Rv MIC are tested
3.1 experiment reagent
Phenylmercuric acetate；Rifampin；100 μ g/mL are dissolved into 10%DMSO.
3.2 cell strain
Mycobacterium tuberculosis type strain H37Rv.
3.3 experimental method
1) preparation of strain subject
Strain subject is transferred to fluid nutrient medium, after activated, is cultivated 2 weeks in 37 DEG C, absorption culture bacterium solution is a little, is placed in
In 4mL fluid nutrient mediums, diameter 2-3mm sterile glass beads 10-20 are added in, vibrate 20-30s, quiescent setting 10-20min, are inhaled
Bacteria suspension supernatant is taken, than turbid to 1 maxwell unit is adjusted with fluid nutrient medium, is equivalent to 1 × 107CFU/mL is spare.
2) preparation of test-compound
Compound is dissolved to 100 μ g/mL, 0.22 μm of filter filtering with appropriate DMSO.Institute is diluted to fluid nutrient medium again
Need experimental concentration (2 × final concentration).The setting of test-compound final concentration is as follows：0.0039μg/mL、0.0078μg/mL、0.0156
μg/mL、0.03125μg/mL、0.0625μg/mL、0.125μg/mL、0.25μg/mL、0.5μg/mL、1μg/mL、2μg/mL、4μ
G/mL, totally 11 concentration gradients.
The setting of control drug rifampin is a concentration of：0.0625 μ g/mL, through 1:1 dilution, sets 11 concentration gradients.
3) operating procedure
During detection, 100 μ L of above compound solution are respectively taken, is added in 96 hole microwell plates, adds 104CFU/mL (by
107CFU/mL dilutions obtain) the 100 μ L of bacterium solution of concentration, compound concentration is made to reach the final concentration of 2) setting.37 DEG C of cultures, it is empty
White control group is not added with any drug, and is inoculated with 100%, 10%, 5% and 1% bacterium amount.Same diluted chemical compound degree sets two groups of groups
Parallel control.Minimum inhibitory concentration of the compound to mycobacterium tuberculosis is observed, observation method is to be positioned over 96 hole U-shaped boards certainly
Result is observed and recorded on the inversion magnifying glass of system.It is the positive that naked eyes visible white bacterial sediment, which occurs, in bottom hole, and no naked eyes can
It is feminine gender to see bacterial sediment, and MIC is defined as the lowest concentration of drug that bottom hole visible white bacterial sediment is less than 1% control wells.
3 phenylmercuric acetate of table is to the In Vitro Bacteriostasis of H37Rv
3rd, phenylmercuric acetate carries out cytotoxicity experiment
4th, phenylmercuric acetate-MDA-MB-231 cells (single the moon breast cancer cell) MTT cytoactive detections experiment
1) pancreatin digestion logarithmic phase cell, is collected by centrifugation after termination, cell suspension is made, cell count adjusts its concentration extremely
5-10×104/mL；
2) after cell suspension is prepared, gently mixing, 100 μ L is added in per hole, the density of cell to be measured in this way is 5000-
10000/hole (edge hole is filled with sterile PBS)；
3) tissue culture plate being inoculated with is put into incubator and cultivated, (96 holes are flat until cell monolayer is paved with bottom hole
Plate), add in the drug of concentration gradient；It in principle, can dosing after cell is adherent；
4) 5%CO2, 37 DEG C are incubated 72h, and the function and effect of drug are observed under inverted microscope；
5) 10 μ LMTT solution (5m μ g/mL, i.e. 0.5%MTT) are added in per hole, continue to cultivate 4h；If drug can with MTT
Reaction, can first centrifuge and discard culture solution afterwards, carefully rush 2-3 after with PBS, add the culture solution containing MTT；
6) culture is terminated, it is dissolving crystallized with DMSO；570nm surveys absorbance after all dissolvings to be crystallized；
7) IC50 is calculated.
4 phenylmercuric acetate of table is to the toxicity test of MDA-MB-231 cells
From above-mentioned MIC experiments and MTT experiment it is found that phenylmercuric acetate to the MIC of standard strain in 60ng/mL, to thin
The safe dose of born of the same parents is between 12.5-25ng/mL.Phenylmercuric acetate shows good anti-tubercular in vitro, without apparent
Cytotoxicity is expected to develop a kind of antituberculotic efficiently, less toxic.In addition, by prolonged culture, also do not find
The phenomenon that drug resistance, this may be because phenylmercuric acetate is not belonging to antibiotic.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention for those of ordinary skill in the art, may be used also on the basis of the above description
To make other variations or changes in different ways, all embodiments can not be exhaustive here, it is every to belong to this hair
The obvious changes or variations that bright technical solution is extended out are still in the row of protection scope of the present invention.
Claims (3)
1. a kind of can be used for preparing the compounds of anti-tuberculosis drugs, which is characterized in that the compound is phenylmercuric acetate, structure
Formula is：
2. compound according to claim 1, which is characterized in that the compound, which can be used for preparing, inhibits tuberculosis branch bar
Bacterium drug.
3. compound according to claim 2, which is characterized in that the mycobacterium tuberculosis is H37Rv.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810168892.4A CN108159039A (en) | 2018-02-28 | 2018-02-28 | A kind of compound that can be used for preparing anti-tuberculosis drugs |
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| CN201810168892.4A CN108159039A (en) | 2018-02-28 | 2018-02-28 | A kind of compound that can be used for preparing anti-tuberculosis drugs |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012032360A2 (en) * | 2010-09-10 | 2012-03-15 | Helperby Therapeutics Limited | Novel use |
| CN105560891A (en) * | 2016-01-13 | 2016-05-11 | 成都大学 | Traditional Chinese medicine eye drops containing borneol and preparation method thereof |
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2018
- 2018-02-28 CN CN201810168892.4A patent/CN108159039A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012032360A2 (en) * | 2010-09-10 | 2012-03-15 | Helperby Therapeutics Limited | Novel use |
| CN105560891A (en) * | 2016-01-13 | 2016-05-11 | 成都大学 | Traditional Chinese medicine eye drops containing borneol and preparation method thereof |
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Application publication date: 20180615 |
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