CN110327328B - Application of 25 beta-sec butenyl-23 beta-isobutyryloxymilbemycin derivative in resisting tumors - Google Patents
Application of 25 beta-sec butenyl-23 beta-isobutyryloxymilbemycin derivative in resisting tumors Download PDFInfo
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Abstract
The scheme discloses an application of 25 beta-sec-butenyl-23 beta-isobutyryloxy milbemycin derivatives in the technical field of milbemycin derivatives in tumor resistance, in particular to an application of the 25 beta-sec-butenyl-23 beta-isobutyryloxy milbemycin derivatives in preparing medicines for resisting liver cancer, lung cancer, colon cancer, cervical cancer and nasopharyngeal cancer. The activity test proves that the 25 beta-sec-butenyl-23 beta-isobutanoyloxy milbemycin derivative has good anti-tumor effect.
Description
Technical Field
The invention belongs to the technical field of milbemycin derivatives, and particularly relates to an application of a 25 beta-sec-butenyl-23 beta-isobutyryloxy milbemycin derivative in tumor resistance.
Background
Milbemycins are a series of sixteen-membered ring macrolides antibiotics which are very similar in structure, can be produced by several streptomyces and have strong biological activities of killing insects, mites and the like. Since the discovery of the first milbemycins in 1967, a large number of other structurally similar compounds have been reported. There are several milbemycins that have been commercialized internationally and are widely used as veterinary drugs or crop protection pesticides.
According to whether the milbemycins have a hydrogenated benzofuran structure or not, the milbemycins can be simply divided into an alpha-type structure and a beta-type structure, wherein the activity of the alpha-type structure is far higher than that of the beta-type structure, so that people mainly focus on the research on the alpha-type structure, and the application research on the beta-type milbemycins is very rare. In addition, because of the strong biological activity of killing insects, mites and insects in the milbemycins, the research and application of the milbemycins are mainly focused on pesticides or veterinary drugs for resisting insects at present. The application of the milbemycins in the aspect of tumor resistance is reported.
Disclosure of Invention
The present invention is intended to provide an application of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives in antitumor to expand the use of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives.
The activity test proves that the 25 beta-sec-butenyl-23 beta-isobutanoyloxy milbemycin derivative has good anti-tumor effect. Therefore, the invention provides the application of the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative in preparing anti-tumor medicines or compositions.
Further, the 25 beta-sec butenyl-23 beta-isobutyryloxy milbemycin derivative is applied to the preparation of medicines or compositions for resisting liver cancer, lung cancer, colon cancer, cervical cancer and nasopharyngeal carcinoma.
The activity test proves that the 25 beta-sec-butenyl-23 beta-isobutanoyloxy milbemycin derivative has good anti-tumor effect, and particularly has high activity on liver cancer, lung cancer, colon cancer, cervical cancer and nasopharyngeal carcinoma.
Further, the structural formula of the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative is as follows:
drawings
FIG. 1 is a primary screening chart of cancer cell lines in the examples of the present invention;
FIG. 2 is a graph of the half inhibitory concentration of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative against Hep 1;
FIG. 3 is a graph of the half inhibitory concentration of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative against MHCC 97H;
FIG. 4 is a graph of the half inhibitory concentration of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative against A549;
FIG. 5 is a graph of the half inhibitory concentration of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives against LOVO;
FIG. 6 is a graph of the half inhibitory concentration of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative against CNE 1;
FIG. 7 is a graph showing the effect of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives on A549 cells;
FIG. 8 is a graph showing the effect of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives on LOVO cells;
FIG. 9 is a graph showing the effect of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives on MHCC97H cells;
FIG. 10 is a graph showing the effect of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives on Hep1 cells;
FIG. 11 is a graph showing the effect of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives on CNE1 cells.
Detailed Description
The following is further detailed by way of specific embodiments:
a method for the isolation of a 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative comprising the steps of:
step one, using ethyl acetate to extract and preserve the preservation number as follows: secondary metabolite of Streptomyces FJS 31-2 of CGMCC 4.7321 to obtain ethyl acetate extract;
the name of the preservation unit of the Streptomyces FJS 31-2 is that of the China general microbiological culture Collection center, the preservation date is 2016, 6 and 2 days, the classification name is Streptomyces sp, and the address of the preservation unit is No. 3 of No. 1 Homex of Beijing Korean district, Beicheng Xilu.
Step two, mixing the ethyl acetate extract with 80-100 meshes of silica gel, filling the mixture into a column by using 200-300 meshes of silica gel through a petroleum ether wet method, and performing gradient elution by using petroleum ether-acetone (15: 1-3: 1) to obtain components Fr.1-Fr.6 with different polarity sections; performing ODS-A column chromatography (methanol-water 70: 30-90: 10) gradient elution on the Fr.4 to obtain 3 components Fr.4-1, Fr.4-2 and Fr.4-3; separating the fraction Fr.4-2 by Sephadex LH-20 column chromatography (chloroform-methanol 1:1), performing semi-preparative HPLC with methanol-water (72:28, v/v) as mobile phase, collecting fraction with retention time of 17.990min, and recovering solvent to obtain white amorphous powder compound: the structural formula of the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative and the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative is as follows:
TLC detection shows that the color of the color developing agent is peach-red after being detected by ultraviolet of 254nm and the color developing agent is 10% sulfuric acid ethanol, and the Rf value is 0.5 after being developed by using chloroform-acetone 10:1 or petroleum ether-acetone 3:1 as a developing agent.
25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives antitumor assays
The experimental steps are as follows:
(1) cell recovery: taking out the cancer cell strain frozen at-80 ℃, and quickly putting the cancer cell strain into water bath at 37 ℃ for unfreezing; the cell suspension was transferred to a 15mL centrifuge tube containing 2mL of medium, centrifuged at 800rpm for 3 min. The precipitated cells were resuspended in 1mL of medium, transferred to a cell culture flask containing 3mL of medium, blown down evenly, and placed in CO at 37 ℃2Culturing in an incubator.
(2) Cell passage: discarding the culture medium in the culture flask, washing with PBS solution for 2 times, adding 1mL of 0.25% trypsin, digesting, observing cell morphology under inverted microscope, and using when shrinking and roundingDigestion was stopped with 3mL of complete medium. Gently blow off adherent cells in the cell culture flask, transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 800rpm for 3 min. Taking 1mL of culture medium to re-suspend the precipitated cells, transferring to a cell culture bottle containing 3mL of culture medium, blowing off uniformly, placing at 37 deg.C and 5% CO2Culturing in an incubator.
(3) Cell administration: the passaged cells were removed, the flask was discarded of medium, washed 2 times with PBS, digested by the addition of 1mL of 0.25% trypsin, and the digestion was stopped with 3mL of medium when the cells had shrunk and become round. And (3) slightly blowing and beating the cells to ensure that adherent cells in the cell culture bottle fall off fully, collecting cell suspension, transferring the cell suspension to a 15mL centrifuge tube, and centrifuging at 800rpm for 3 min. The cell concentration of the medium for cell precipitation was adjusted to 5X 104Taking 100 mu L of cell suspension, adding the cell suspension into a 96-well plate culture well respectively, and placing the cell suspension in CO at 37 DEG C2The incubator is used for 24 h. When the cells grow to 80% of the culture wells, the drug wells are sequentially diluted to 8 concentrations of 2. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 32. mu.M, 64. mu.M, 128. mu.M, 256. mu.M, etc. as test concentrations, each drug group is plated in 6 duplicate wells at 37 ℃ with 5% CO2The incubator acts for 24 hours and then the culture is finished.
(4) And (3) measuring absorbance: the liquid in the 96-well plate was discarded, each plate was washed with PBS until no compound remained, 90. mu.L of the medium and 10. mu.L of cck8 reagent were added, incubation was continued at 37 ℃ for 4 hours, and then the incubation was terminated, and the OD value was measured at 490nm with a microplate reader.
(5) Experimental analysis:
according to each group OD490The mean and standard deviation of the values, the inhibition rate of cell growth can be calculated according to the following formula:
IC was calculated using SPSS18.0 and GraphPadprism6.0 statistical software50。
IC50(half maximum inhibition concentration) refers to the half inhibitory concentration of a drug. It can indicate that a drug or substance (inhibitor) is inhibiting a certain biological process,e.g. concentration or dose at half-time of cell death, IC50The value can be used to measure the ability of a drug to induce apoptosis, i.e., the stronger the inducing ability, the lower the value, and can also be used to reverse the degree of tolerance of a cell to a drug.
The cancer cell strain is liver cancer cell (MHCC97H, high metastasis), liver cancer cell (HEP1, low metastasis), lung cancer cell (A549), colon cancer cell (LOVO), cervical cancer cell (Hela) or nasopharyngeal cancer cell (CNE 1). The culture process and the test steps of the liver cancer cell, the lung cancer cell, the colon cancer cell, the cervical cancer cell and the nasopharyngeal carcinoma cell are the same, and the difference is only that in the culture process: DMEM basic medium for MHCC97H, MEM basic medium for Hep1, and 1640 basic medium for the rest; the culture medium is prepared by adding 10% of serum and 1% of double antibody into a basic culture medium. The half inhibitory concentration of 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives against each cancer cell is shown in FIGS. 2 to 6.
The inhibition rate of the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives against cancer cells was as follows.
25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives at various concentrations
The inhibition rate on liver cancer (HEP1) cells is shown in the following table
25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives at varying concentrations
The inhibition rate of liver cancer (MHCC97H) cells is shown in the following table
25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives at varying concentrations
The inhibition rate of lung cancer (A549) cells is shown in the table
The cytostatic rate of various concentrations of 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives against colon cancer (LOVO) is shown in the following table
Different concentrations of 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives against nasopharyngeal carcinoma
(CNE1) cell inhibitory rates are shown in the following Table
The experiment adopts the steps that 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives with different concentrations are used for carrying out the experiment on cancer cell strains, the action effect graphs of the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives on different cancer cells are shown in figures 7-11, 50 mu M in white parts in figures 7-11 is a scale and shows the effect graphs after the cells are amplified by 200 times; in FIGS. 7 to 8, from top to bottom, the top row and from left to right, the concentrations of the 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives used are as follows: 0. mu.M, 2. mu.M, 4. mu.M; the second row, from left to right, shows the concentrations of the 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative used: 8. mu.M, 16. mu.M, 32. mu.M; the third row, from left to right, shows the concentrations of the 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative used: 64 μ M, 128 μ M and 256 μ M. In FIGS. 9 to 11, the concentrations of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives used are shown from left to right: 0. mu.M, 40. mu.M.
As can be seen from FIGS. 7-8, the cell morphology and number were not significantly different from those of the control group when the drug concentration was 2. mu.M or 4. mu.M; however, when the concentration of the drug is 8 μ M, the cells are not only reduced in number but also changed in morphology, and a part of the cells are disintegrated; when the concentration of the medicine is increased to 16 mu M, the cell shape is contracted and rounded, the cell disintegration is more obvious, and the cell number is obviously reduced; with increasing dose, the number of cell deaths increased, and when the drug concentration increased to 32 μ M, most of the cells floated and most of the cells died.
As is apparent from FIGS. 9 to 11, when the concentration of the 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative reached 40 μ M, most of the cells floated and most of the cells died.
The effect is obvious that the 25 beta-sec-butenyl-23 beta-isobutyryloxy milbemycin derivative has obvious inhibition effect on liver cancer cells, lung cancer cells, colon cancer cells and nasopharyngeal carcinoma cells; it can be widely used for preparing anti-tumor drugs.
Claims (1)
- The application of 25 beta-sec butenyl-23 beta-isobutyryloxymilbemycin derivatives in preparing antitumor drugs is characterized in that: the tumor is liver cancer, lung cancer, colon cancer, cervical cancer or nasopharyngeal carcinoma, and the structural formula of the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative is as follows:
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999017760A2 (en) * | 1997-10-02 | 1999-04-15 | Microcide Pharmaceuticals, Inc. | Fungal or mammalian cell efflux pump inhibitors for enhancing susceptibility of the cell to a drug |
| WO2016076359A1 (en) * | 2014-11-11 | 2016-05-19 | 国立研究開発法人産業技術総合研究所 | Anticancer agent containing ivermectin or milbemycin d as active ingredient |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999017760A2 (en) * | 1997-10-02 | 1999-04-15 | Microcide Pharmaceuticals, Inc. | Fungal or mammalian cell efflux pump inhibitors for enhancing susceptibility of the cell to a drug |
| WO2016076359A1 (en) * | 2014-11-11 | 2016-05-19 | 国立研究開発法人産業技術総合研究所 | Anticancer agent containing ivermectin or milbemycin d as active ingredient |
Non-Patent Citations (2)
| Title |
|---|
| A milbemycin compound isolated from Streptomyces Sp. FJS31-2 with cytotoxicity and reversal of cisplatin resistance activity in A549/DDP cells;Xiao-Qian Li等;《Biomedicine & Pharmacotherapy》;20200831;第128卷;第110322页 * |
| Reversal of P-glycoprotein-mediated multidrug resistance in vitro by milbemycin compounds in adriamycin-resistant human breast carcinoma (MCF-7/adr) cells;Wensheng Xiang等;《Toxicology in Vitro》;20100723;第24卷;第1474-1481页 * |
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