CN110179802A - The new application of pleuromulins compound - Google Patents
The new application of pleuromulins compound Download PDFInfo
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- CN110179802A CN110179802A CN201910348865.XA CN201910348865A CN110179802A CN 110179802 A CN110179802 A CN 110179802A CN 201910348865 A CN201910348865 A CN 201910348865A CN 110179802 A CN110179802 A CN 110179802A
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- amino
- pyrimidine
- staphylococcus aureus
- alpha hemolysin
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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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Abstract
The invention discloses pleuromulins compounds to inhibit the application in bacterium alpha hemolysin drug in preparation.Pleuromulins compound 14-O- [(4; 6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods to MRSA43300 have preferable bacteriostatic activity; inhibiting effect is generated to the hemolytic activity of alpha hemolysin in subinhibitory concentration, therefore can be used for treating the disease as caused by infection of staphylococcus aureus.
Description
Technical field
The invention belongs to chemicals technical fields, and in particular to the new application of pleuromulins compound.
Background technique
Staphylococcus aureus (Staphylococcus aureus) is that one kind is widely present in human body and animal is intracorporal
Essential condition pathogenic bacteria can cause many infectious diseases of human and animal.Studies have shown that caused by staphylococcus aureus
Infectious diseases is only second to escherichia coli (Escherichia coli), generates exotoxin and enzyme is major virulent factor
(Biosci.Biotechnol.Biochem.2004,68,981；Front.Cell.Infect.Mi.2012,2,2235).α-is molten
Sanguinin (HIa) is not only one of most important virulence factor of staphylococcus aureus, also participates in exempting from when the germ infects body
Epidemic disease escapes and induces the other pairs of unfavorable pathogenicity factors of body (Clin.Infect.Dis.2010,50,69).Therefore reduce or
Control staphylococcus aureus secretion alpha hemolysin has great importance to the germ infectious diseases is treated.
Methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus,
It MRSA) is a kind of drug-fast bacteria as caused by abuse or use beta-lactam antibiotic lack of standardization.In recent years, due to clinic
On non-beta-lactam antibiotic treatment MRSA infectious diseases is widely used, bringing certain pressure to the existence of MRSA makes
It gradually obtains new resistance mechanism, causes to clinically (Chinese Clinical pharmacology is miscellaneous for majority common antibiotics generation drug resistances
Will, 2014,30,251).Therefore, research does not carry out pressure to the living zone of MRSA, and by improving or inhibiting its virulence factor
The drug of subinhibitory concentration is used to treat or combination therapy MRSA infectious diseases has important theory significance and more practical value.
14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods (chemical structural formula is shown in formula 1) be by cut
Short pleurin C-14 side chains are modified, bacteriostatic test, acute toxicity test, preliminary stability test through inside and outside etc.
A series of researchs, a kind of pleuromulins drug candidate filtered out.The compound is to causing the most of thin of skin infection
Bacterium or drug-fast bacteria, as staphylococcus aureus, MRSA, streptococcus pyogenes (Streptococcus pyogenes,
S.pyogenes), mycoplasma pneumoniae (Mycoplasma pneumonia, M.Pneumonia), streptococcus pyogenes
(Pyogeniccoccus) etc. there is good bacteriostatic activity (Eur.J.Med.Chem.2017,126,687；
Microb.Pathogenesis,2019,127,202).In addition, the toxicity of compound is low, stability is good, and there is no rhizoma corydalis
Intense irritation possessed by sour Thailand's second rhzomorph.
14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods according to the present invention is in subinhibitory concentration
When, by inhibiting the alpha hemolysin encoding gene HIa of staphylococcus aureus and the table of its upstream regulator control system agrA gene
The secretion for inhibiting alpha hemolysin up to level, can be used for infectious diseases caused by clinical treatment staphylococcus aureus, has
High clinical development value and application value.
Summary of the invention
The purpose of the invention is to provide the new application of pleuromulins compound.
The purpose of the present invention is achieved by the following technical scheme:
Pleuromulins compound inhibits the application in bacterium alpha hemolysin drug in preparation.
Further, the pleuromulins compound is 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group]
Nurse body woods.
Further, the bacterium includes staphylococcus aureus.
The important virulence factor that alpha hemolysin causes a disease as staphylococcus aureus can be used as and treat germ infection
Important drugs target.14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods can significantly inhibit alpha hemolysin pair
The haemocylolysis of rabbit erythrocyte, and be in apparent drug concentration dependence.Although some compounds are living to the haemolysis of alpha hemolysin
Property there is certain inhibiting effect, but it is poor to the bacteriostatic activity of staphylococcus aureus, therefore these drugs are for clinic
Effect is poor when treating germ infection.Present invention discover that 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods
There is preferable bacteriostatic activity to MRSA43300, while the hemolytic activity of alpha hemolysin can be generated again in subinhibitory concentration
Inhibiting effect, therefore can be used for treating the disease as caused by infection of staphylococcus aureus.
The invention has the following advantages:
Pleuromulins compound 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse provided by the invention
Body woods has preferable bacteriostatic activity to MRSA43300, generates in subinhibitory concentration to the hemolytic activity of alpha hemolysin and inhibits to make
With, therefore can be used for treating the disease as caused by infection of staphylococcus aureus.
Detailed description of the invention
Fig. 1 is to train after 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods co-cultures with MRSA43300
Support object supernatant hemolytic activity (1:Normal, 2:1/2MIC, 3:1/4MIC, 4:1/8MIC, 5:1/16MIC, 6:1/32MIC, 7:
Control) * P < 0.05 indicates significant difference compared to the blank group；* P < 0.01 indicates that difference is extremely significant compared to the blank group.
Fig. 2 is α-after 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods and MRSA43300 co-cultivation
The immunoblotting assay of hemolysin.
Fig. 3 is 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods to MRSA43300HIa and agrA base
Because of the influence of transcriptional level.
Specific embodiment
Hereinafter, preferred embodiments of the present invention will be described, it should be understood that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
The preparation method of 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods is detailed in reference paper
“Eur.J.Med.Chem.2017,126,687”。
1 14-O- of embodiment [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods subinhibitory concentration inhibits
Hemolytic activity of the MRSA43300 culture supernatant to rabbit erythrocyte
- 70 DEG C of 43300 strain inoculated of MRSA will be stored on blood plate, repeatedly after rejuvenation three times, picking individual colonies
It is inoculated into after cultivating 12-16h on TSB culture medium again, obtains the bacterium of logarithmic growth phase.It is carried out using MHA culture plate thin
Bacterium counts, and the bacterium after counting is adjusted using PBS to 3.2 × 106ML is added in 6 porocyte culture plates, then in 6 hole cells
14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods is added in culture plate, making the final concentration of drug is respectively 1/
2MIC, 1/4MIC, 1/8MIC, 1/16MIC, 1/32MIC, while being equipped with blank control group and (bacterium solution being not added, only adds androgynous with drug
Long-pending PBS, i.e. Normal in attached drawing) and positive group (drug is not added, only plus the PBS of bacterium solution and same volume, i.e., in attached drawing
Control), the 6 orifice plates handled well are placed in 37 DEG C of incubators after cultivating 16h and take out culture plate, collected bacterium solution in each hole and set
In 2mL centrifuge tube, the sample gathered is centrifuged 5min with 4 DEG C of 9000g, collects supernatant of bacteria solution.By 200 μ L of supernatant of bacteria solution with
200 μ L of PBS is added in 1.5mL centrifuge tube simultaneously, mixes, the sterile de- fiber rabbit erythrocyte of 50 μ L is then added to supernatant of bacteria solution
It is statically placed in room temperature 2h in PBS centrifuge tube, after mixing, the sample handled well is put in centrifuge and is centrifuged with 4 DEG C of 10000g
After 8min, collects supernatant and be added in 96 orifice plates, use absorbance (the haemolysis OD value of microplate reader test sample on 543nm wavelength
Sample beyond detection range is diluted using PBS).Calculation formula is as follows: n=(A1-A2)/(A3-A2), and in formula: n is hemolysis rate
(%)；A1 is test sample group absorbance；A2 is negative control group absorbance；A3 is positive controls absorbance.
The result shows that the subinhibitory concentration of 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods can press down
MRSA43300 processed is to the hemolytic activity (such as Fig. 1) of rabbit erythrocyte, and rejection ability enhances with the raising of drug concentration.With it is right
It is compared according to group, 1/2MIC, 1/4MIC, 1/8MIC and 1/16MIC DPTM can significantly inhibit MRSA43300 supernatant of bacteria solution to rabbit
The haemocylolysis (Fig. 1-A) of red blood cell, to the inhibiting rate of hemolytic be respectively (97.24 ± 1.15) %, (96.4 ± 2.1) %,
(73.93.74) % and (31.5 ± 7.86) % (Fig. 1-B).
2 Western Blot of embodiment detects 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods and makees
With the expression of lower alpha hemolysin
Bacterial supernatant total protein concentration in " embodiment 1 " is measured using BCA determination of protein concentration kit,
It measures detailed step to carry out by kit specification, using the minimum value of measured sample concentration as standard, other samples is tuned into
Consistent concentration carries out the expression of WesternBlot detection alpha hemolysin.Concrete operation method are as follows: take the supernatant for having adjusted concentration
20 μ L and 5 × Loading Buffer of liquid, 5 μ L is mixed, and after boiling 10min using water-bath, carries out SDS-PAGE electrophoresis, electrophoresis
After, PAGE gel is taken out, the protein after separation is transferred on pvdf membrane using wet turn of method, by pvdf membrane
It is split by albumen Marker band, the band divided is placed in no protein blocking liquid and closes 4h, uses anti-alpha hemolysin
Polyclonal antibody is incubated overnight the closed band of institute in 4 DEG C of refrigerators, after incubation, is carried out using TBST solution to it
Washing 3 times, each 10min after washing, is incubated for 70min using the secondary antibody of HRP label, takes out pvdf membrane, wash to it
It washs 3 times, each 10min, finally detects its band in Alpha chemiluminescence gel imaging system using ECL luminescent solution.
The result shows that 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods of various concentration can be reduced training
The content of the alpha hemolysin in object supernatant is supported, and is in apparent concentration dependent, as a result as shown in Figure 2.
3 Real-time PCR of embodiment detects 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto acetyl group] nurse body woods and makees
With the lower influence to alpha hemolysin mRNA
According to the sequence HIa and 16rRNA of the expression alpha hemolysin of the Genbank staphylococcus aureus USA300 announced
(16S ribosomal RNA) sequence and the binary regulator control system agrA sequence of regulation alpha hemolysin expression carry out design of primers,
And compare primer specificity by blast program, pick out the good three pairs of primers of specificity send to Beijing Huada gene company into
Row synthesis (table 1).The thallus of " embodiment 1 " centrifuged deposit is taken, 1mL TRIzol reagent is added in every a sample, mixes well
Total serum IgE is extracted to MRSA43300 thallus according to specification.Total serum IgE after extraction is subjected to reverse transcription into cDNA, operating process
By specification carries out.Using BeyoFast SYBR Green qPCR Mix (2X, Low ROX) reagent, using cDNA as template into
The detection of row real-time fluorescence quantitative PCR, system and step are as follows: GreenReal-time PCR
Master Mix, 2 μ L DNA, 0.5 μ L forward and reverse primer and 7 μ L DEPC H2O, after eight unions have added reagent, with 95
DEG C initial denaturation 2min carries out 30s then with 95 DEG C of 15s at 59 DEG C, 45 circulations is repeated, each sample is repeated 3 times.
Result after detection analyzes relative expression's abundance of gene using 2- Δ Δ Ct method, and the specific formula for calculation of-Δ Δ t is as follows: Δ
Δ t=Δ Ct (experimental group) Δ Ct (control group), wherein the corresponding reference gene of Δ Ct value=target gene Average Ct values-is average
Ct value.
1 Real-time PCR primer list of table
The result shows that 1/2MIC, 1/4MIC, 14-O- [(4,6- di-amino-pyrimidine -2- base) mercapto of 1/8MIC concentration is added
Acetyl group] after nurse body woods, difference is extremely significant compared with the control group for the gene transcription level of HIa, agrA gene transcription level with it is right
Compare significant difference according to group (result is shown in Fig. 3).Although the drug effect of 1/16MIC concentration is after MRSA43300, the gene of HIa
Transcriptional level significant difference compared with the control group, but agrA gene transcription level is not influenced significantly；1/32MIC concentration
Drug effect HIa gene transcription level is neither influenced after MRSA43300, nor affect on the gene transcription level of agrA.
It is merely a preferred embodiment of the present invention, is not intended to restrict the invention, although referring to aforementioned implementation described in upper
Invention is explained in detail for example, for those skilled in the art, still can be to foregoing embodiments
Documented technical solution is modified or equivalent replacement of some of the technical features.It is all in spirit of the invention
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (3)
1. pleuromulins compound inhibits the application in bacterium alpha hemolysin drug in preparation.
2. application according to claim 1, the pleuromulins compound is 14-O- [(4,6- di-amino-pyrimidines-
2- yl) mercapto acetyl group] nurse body woods.
3. application according to claim 1, the bacterium includes staphylococcus aureus.
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| CN201910348865.XA CN110179802A (en) | 2019-04-28 | 2019-04-28 | The new application of pleuromulins compound |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112089715A (en) * | 2020-09-27 | 2020-12-18 | 山东齐发药业有限公司 | Composition for resisting respiratory positive bacteria infection for livestock and poultry and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104803926A (en) * | 2015-03-25 | 2015-07-29 | 中国农业科学院兰州畜牧与兽药研究所 | Pleuromutilin derivative with pyrimidine side chain and application of pleuromutilin derivative |
| CN105622524A (en) * | 2016-03-22 | 2016-06-01 | 中国农业科学院兰州畜牧与兽药研究所 | Pleuromutilin derivative and application thereof |
-
2019
- 2019-04-28 CN CN201910348865.XA patent/CN110179802A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104803926A (en) * | 2015-03-25 | 2015-07-29 | 中国农业科学院兰州畜牧与兽药研究所 | Pleuromutilin derivative with pyrimidine side chain and application of pleuromutilin derivative |
| CN105622524A (en) * | 2016-03-22 | 2016-06-01 | 中国农业科学院兰州畜牧与兽药研究所 | Pleuromutilin derivative and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| YUNPENG YI等: "Synthesis and antibacterial activities of novel pleuromutilin derivatives with a substituted pyrimidine moiety", 《EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY》 * |
| YUNPENG YI等: "Synthesis and Biological Activity Evaluation of Novel Heterocyclic Pleuromutilin Derivatives", 《MOLECULES》 * |
| 郑峰等: "金黄色葡萄球菌α-溶血素的研究进展", 《中华微生物学和免疫学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112089715A (en) * | 2020-09-27 | 2020-12-18 | 山东齐发药业有限公司 | Composition for resisting respiratory positive bacteria infection for livestock and poultry and application thereof |
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Application publication date: 20190830 |