CN110526925B - Separation method and application of 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative - Google Patents

Abstract

Translated fromChinese

本方案公开了米尔贝霉素衍生物技术领域的一种25β‑仲丁烯基‑23β‑异丁酰氧基米尔贝霉素衍生物的分离方法，步骤为：一、使用乙酸乙酯抽提链霉菌FJS 31‑2的次级代谢产物，得到乙酸乙酯提取物浸膏；二、对乙酸乙酯提取物浸膏进行拌样，然后用硅胶以石油醚湿法装柱，用石油醚－丙酮梯度洗脱，得到不同极性段的组分Fr.1～Fr.6；Fr.4经ODS‑A柱层析梯度洗脱，得到3个组分Fr.4‑1、Fr.4‑2和Fr.4‑3；再将组分Fr.4‑2通过葡聚糖凝胶Sephadex LH–20柱层析分离，再进一步通过半制备型高效液相色谱仪，以乙腈–水为流动相，收集保留时间为17.990min的组分，回收溶剂得25β‑仲丁烯基‑23β‑异丁酰氧基米尔贝霉素衍生物。本方案制备的25β‑仲丁烯基‑23β‑异丁酰氧基米尔贝霉素衍生物可应用于制备抗黑色素瘤药物。

This scheme discloses a method for separating 25β-sec-butenyl-23β-isobutyryloxy mirbemycin derivatives in the technical field of mirbemycin derivatives. The steps are: 1. Extraction with ethyl acetate The secondary metabolites of Streptomyces FJS 31-2 were obtained as ethyl acetate extracts; 2. The ethyl acetate extracts were mixed with samples, and then packed with silica gel with petroleum ether wet method, and petroleum ether- Acetone gradient elution to obtain components Fr.1-Fr.6 of different polar segments; Fr.4 is subjected to ODS‑A column chromatography gradient elution to obtain 3 components Fr.4‑1, Fr.4‑ 2 and Fr.4-3; the component Fr.4-2 was separated by Sephadex LH-20 column chromatography, and then further passed through a semi-preparative high performance liquid chromatograph with acetonitrile-water as the flow phase, the components with retention time of 17.990min were collected, and the solvent was recovered to obtain 25β-sec-butenyl-23β-isobutyryloxy milbemycin derivative. The 25β-sec-butenyl-23β-isobutyryloxy milbemycin derivatives prepared in this scheme can be used for the preparation of anti-melanoma drugs.

Description

Separation method and application of 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative

Technical Field

The invention belongs to the technical field of milbemycin derivatives, and particularly relates to a separation method and application of a 25 beta-sec-butenyl-23 beta-isobutyryloxy milbemycin derivative.

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.

The milbemycins can be simply divided into alpha-type structures and beta-type structures if the milbemycins have a hydrogenated benzofuran structure, wherein the activity of the alpha-type structures is far higher than that of the beta-type structures, so that people mainly focus on the alpha-type structures for research on the milbemycins. In addition, the milbemycins have strong biological activities of killing insects, mites and insects, so that the current research and application of the milbemycins are mainly focused on anti-insect pesticides or veterinary drugs. The application of the milbemycins in the aspect of tumor resistance is rarely reported.

Disclosure of Invention

The invention aims to provide a separation method of a 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative, which is used for extracting and separating the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative from streptomyces FJS 31-2 and researching the antitumor application of the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative.

According to the scheme, the separation method of the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative is characterized in that the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative is separated from a secondary metabolite of streptomyces FJS 31-2; the Streptomyces FJS 31-2 has the preservation number as follows: CGMCC 4.7321.

The name of the preservation unit of the Streptomyces FJS 31-2 is that of the China general microbiological culture Collection center with the preservation date of 2016, 6 and 2 days, the classification and the naming are that of the Streptomyces sp with the preservation unit address of No. 3 Xilu-1 Beichen of the Chaoyang district, Beijing.

The method for separating the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative specifically comprises the following steps:

step one, extracting a secondary metabolite of streptomycete FJS 31-2 by using ethyl acetate to obtain an ethyl acetate extract;

step two, mixing the ethyl acetate extract with 80-100 meshes of silica gel, filling the mixture into a column by using a 200-300 meshes of silica gel through a petroleum ether wet method, and performing gradient elution by using petroleum ether-acetone to obtain components Fr.1-Fr.6 of 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), further separating by semi-preparative high performance liquid chromatograph with acetonitrile-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: 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives.

The beneficial effect of this scheme: the method is simple to operate, the trace components are not easy to lose, and the controllability and the reproducibility are good.

Activity tests prove that the 25 beta-sec-butenyl-23 beta-isobutanoyloxy milbemycin derivative obtained by separation has a good anti-melanoma effect. Therefore, the invention provides an application of a 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative in preparing a medicament or a composition for resisting melanoma.

Further, the structural formula of the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative is as follows:

drawings

FIG. 1 shows 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives of the present inventionIs/are as follows¹H-NMR nuclear magnetic spectrum;

FIG. 2 shows a 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative in the present invention¹³C-NMR nuclear magnetic spectrum;

FIG. 3 is a 13C-NMR and DEPT135 and DEPT90 comparison spectra of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives according to the present invention;

FIG. 4 is a mass spectrum of a 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative according to the present invention;

FIG. 5 is a high resolution mass spectrum of a 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative of the present invention;

FIG. 6 is a primary screening chart of melanoma cancer cell lines;

FIG. 7 is a graph of the half maximal inhibitory concentration (IC50) of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives against melanoma;

FIG. 8 is a graph showing the effect of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives on melanoma cancer cells.

Detailed Description

The following is further detailed by way of specific embodiments:

separating and extracting

A method for isolating 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;

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 (1:0,15:1,10:1,8:1,5:1,3:1) to obtain components Fr.1-Fr.6 with different polarity sections; fr.4 is subjected to ODS-A column chromatography and gradient elution with methanol-water (70:30,80:20,90:10) 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), further separating by semi-preparative high performance liquid chromatograph with acetonitrile-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: 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives. TLC detection shows that the color of the product is peach-red after being developed by 10% sulfuric acid ethanol developer under 254nm ultraviolet, and Rf value is 0.5 after being developed by chloroform-acetone 10:1 or petroleum ether-acetone 3:1 as developing agent.

And (3) structural identification:

the structural formula of the 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivative is as follows:

white amorphous powder, ESI-MS (positive) gave M/z 693[ M + Na ]]⁺The molecular weight of the compound is suggested to be 670. The HR-ESI-MS spectrum gives M/z 693.3623[ M + Na [ ]]⁺(calculated value 693.3609), the molecular formula of the compound was determined to be C₃₈H₅₄O₁₀The unsaturation was calculated to be 12.

¹H-NMR(CDCl₃500MHz) spectrum, one can see 5 sets of doublet methyl hydrogen signals, of which 4 sets are methyl hydrogen δ 0.69(3H, d, J ═ 6.6Hz), δ 1.00(3H, d, J ═ 6.7Hz), δ 1.197(3H, d, J ═ 6.9Hz), δ 1.20(3H, d, J ═ 6.9 Hz);group 1 is methyl δ 1.66 attached to the terminal olefinic carbon (3H, d, J ═ 6.7 Hz); 3 groups of unimodal methyl hydrogen signals delta 1.53(3H, s), delta 1.59(3H, s), delta 1.87(3H, br s) connecting unsaturated carbons;group 1 oxygen-linked methylene hydrogen signal δ 4.68(2H, m).

¹³C-NMR(CDCl₃125MHz) spectrum, including, in combination with DEPT90 and 135 spectra: 8 quaternary carbon signals, of which 2 are the ester carbonyl carbon signal (δ 178.0,173.7), and 4 are the alkene carbon signal (δ 139.6,137.9,137.5,133.2), 2 are the carbon signals to oxygen (δ 100.3, 80.3); 17 methine carbon signals, 6 alkene carbon signals (delta 142.9,125.0,123.5,120.5,120.4,118.2), 7 oxygen linked carbon signals (delta 80.3,79.3,76.1, 75.5,68.6,68.3,67.8),4Carbon signals associated with fatty chains (Δ 45.8,37.8,36.1, 34.4); 5 methylene carbon signals, 5 methylene carbon signals linked to the aliphatic carbon chain (δ 48.6,36.3,36.2,34.7), 1 methylene carbon signal linked to oxygen (δ 68.5); 8 methyl carbon signals (δ 22.4,20.0,19.3,19.1,15.7,13.3,13.1, 10.9).

Of the compound¹H-NMR and¹³C-NMR nuclear magnetic data were compared with that of compound VM48130 (Haxell et al 1992; Baker et al 1996), and the chemical shift values and coupling constant values were found to be substantially identical, so that the compound was identified as VM 48130.

The detailed data are shown in the following table:

process for preparing 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives¹H and¹³C-NMR data (500: (¹H)and 125(¹³C)MHz,Chloroform–d)

C₃₈H₅₄O₁₀White amorphous powder. Positive ESI-MS M/z 693[ M + Na ]]⁺；¹H NMR(CDCl₃,500 MHz)δ_H:5.76(m,1H),5.75(dd,J＝14.4,11.2Hz,1H),5.45(dq,J＝6.7,1.2Hz,1H),5.42(m, 1H),5.31(m,1H),5.30(m,1H),4.97(m,1H),4.93(dd,J＝10.6,9.5Hz,1H),4.68(m,2H),4.29 (d,J＝6.0Hz,1H),3.95(d,J＝6.2Hz,1H),3.89(s,1H),3.61(m,1H),3.58(d,J＝10.4Hz,1H), 3.27(q,J＝2.5Hz,1H),3.21(d,J＝9.6Hz,1H),2.61(heptet,J＝6.9Hz,1H),2.42(m,1H),2.32 (br d,J＝7.9Hz,1H),2.25(m,2H),2.20(m,1H),1.90(m,1H),1.87(br s,3H),1.85(m,1H), 1.82(m,1H),1.80(m,2H),1.66(d,J＝6.7Hz,3H),1.59(s,3H),1.53(s,3H),1.20(d,J＝6.9Hz, 3H),1.19(d,J＝6.9Hz,3H),1.19(d,J＝8.6Hz,1H),1.00(d,J＝6.7Hz,3H),0.90(q,J＝12.4 Hz,1H),0.69(d,J＝6.6Hz,3H).The¹³C NMR(CDCl₃,125MHz)δ_C:178.0(s,C-35),173.7(s, C-1),142.9(d,C-11),139.6(s,C-8),137.9(s,C-4),137.5(s,C-14),133.2(s,C-31),125.0(d, C-32),123.5(d,C-10),120.5(d,C-15),120.4(d,C-9),118.2(d,C-3),100.3(s,C-21),80.3(s, C-7),80.3(d,C-25),79.3(d,C-6),76.1(d,C-23),75.5(d,C-22),68.6(d,C-19),68.5(t,C-27), 68.3(d,C-17),67.8(d,C-5),48.6(t,C-13),45.8(d,C-2),37.8(d,C-24),36.3(t,C-18),36.2(t, C-20),36.1(d,C-12),34.7(t,C-16),34.4(d,C-36),22.4(q,C-28),20.0(q,C-26),19.3(q,C-37), 19.1(q,C-38),15.7(q,C-29),13.3(q,C-33),13.1(q,C-30),10.9(q,C-34)。

25 beta-sec butenyl-23 beta-isobutyryloxymilbemycin derivatives anti-melanoma tests

The experimental steps are as follows:

(1) cell recovery: taking out melanoma (B16) cancer cell strain frozen at-80 deg.C, and rapidly thawing in water bath at 37 deg.C; the cell suspension was transferred to a 15mL centrifuge tube containing 2mL of medium, centrifuged at 800rpm for 3 min. Resuspending the pelleted cells in 1mL of medium, transferring to a cell culture flask containing 3mL of medium, sparging uniformly, and placing in CO at 37 deg.C₂Culturing in an incubator.

(2) Cell passage: the medium in the flask was discarded, washed 2 times with PBS, and 1mL of 0.25% trypsin was added to digest the cells under an inverted microscope, and digestion was stopped with 3mL of medium when they were rounded off. 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. Resuspending the precipitated cells in 1mL of medium, transferring to a cell culture flask containing 3mL of medium, blowing off, and standing at 37 deg.C with 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 to5X 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. The culture medium is prepared by adding 10% of serum and 1% of double antibody on the basis of 1640 basic culture medium.

(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 the concentration or dose, IC, of a drug or substance (inhibitor) at which it inhibits certain biological processes, such as half-cell death₅₀The value can be used to measure the ability of a drug to induce death, i.e., the more inducible, the lower the value, and can be used to reverse the degree of tolerance of a cell to the drug.

The half inhibitory concentration of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivative on melanoma cancer cells is shown in fig. 7.

The inhibitory rates of the various concentrations of 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives against melanoma cancer cells are shown in the following table

The experiment adopts 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives with different concentrations to carry out the experiment on melanoma cell strains through the steps, the melanoma cancer cells after the experiment are shown in figure 8, the grid jiugongge in figure 8 is from top to bottom, the first row shows from left to right that the concentrations of the used 25 beta-sec-butenyl-23 beta-isobutyryloxymilbemycin derivatives are respectively as follows: 0. mu.M, 2. mu.M, 4. mu.M; the second row, from left to right, shows the concentrations of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives used: 8. mu.M, 16. mu.M, 32. mu.M; the third row shows from left to right the concentrations of 25 β -sec-butenyl-23 β -isobutyryloxymilbemycin derivatives used: 64 μ M, 128 μ M and 256 μ M. In FIG. 8, 50. mu.M in white is a scale, and a graph showing the effect of the cells after being enlarged 200 times is shown.

As can be seen from FIG. 8, no significant difference was observed in cell morphology and number from the control group when the drug concentration was 2. mu.M, 4. mu.M, 8. mu.M; however, when the concentration of the drug is 16. mu.M, the cells are not only reduced in number but also changed in morphology, and part of the cells are disintegrated; when the concentration of the drug is increased to 32 mu M, the cells are disintegrated, and the number of the cells is sharply reduced; when the drug concentration was increased to 64 μ M, most of the cells floated and most of the cells died.

The effect of 25 beta-sec butenyl-23 beta-isobutyryloxymilbemycin on melanoma cancer cells is shown in figure 8.

The effect is obvious that the 25 beta-sec-butenyl-23 beta-isobutyryloxy milbemycin derivative has obvious inhibition effect on melanoma cancer cells; it can be widely used for preparing anti-melanoma drugs.

Claims (1)

Translated fromChinese

1.一种25β-仲丁烯基-23β-异丁酰氧基米尔贝霉素衍生物的应用，其特征在于：25β-仲丁烯基-23β-异丁酰氧基米尔贝霉素衍生物在制备抗黑色素瘤的药物或组合物中的应用，所述25β-仲丁烯基-23β-异丁酰氧基米尔贝霉素衍生物的结构式为：1. the application of a kind of 25β-sec-butenyl-23β-isobutyryloxy mirbemycin derivative, is characterized in that: 25β-sec-butenyl-23β-isobutyryloxy mirbemycin derivative The application of the compound in the preparation of anti-melanoma medicine or composition, the structural formula of the 25β-sec-butenyl-23β-isobutyryloxy mirbemycin derivative is:

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