CN107827752B

CN107827752B - Macrocyclic diterpenoid compound in euphorbia foliosa fruits, preparation method and multi-drug resistance reversing application

Info

Publication number: CN107827752B
Application number: CN201711064657.4A
Authority: CN
Inventors: 阿吉艾克拜尔·艾萨; 胡蕊
Original assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Current assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Priority date: 2017-11-02
Filing date: 2017-11-02
Publication date: 2020-07-28
Anticipated expiration: 2037-11-02
Also published as: CN107827752A

Abstract

The invention relates to a macrocyclic diterpenoid compound in euphorbia foliosa fruits, a preparation method and application of multidrug resistance reversal. The hypophyllum folicum (A) and (B)Euphorbia sororiaSchrenk) to obtain three new macrocyclic diterpenoid compounds, and the results of the multi-drug resistance reversal activity assay of the new macrocyclic diterpenoid compounds show that: the macrocyclic diterpenoid compounds (I) - (III) all have multidrug resistance reversing activity of different degrees, can reverse the drug resistance of drug-resistant cells to antitumor drugs of different degrees when being combined with antitumor drugs, and can be used for preparing multidrug resistance reversing drugs.

Description

Macrocyclic diterpenoid compound in euphorbia foliosa fruits, preparation method and multi-drug resistance reversing application

Technical Field

The invention relates to the technical field of medicines, in particular to three novel macrocyclic diterpenoid compounds and application thereof in preparing multidrug resistance reversal medicines or medicines combined with antitumor medicines.

Background

Euphorbia sororii a. schrenk is an annual herbaceous plant of the Euphorbia (Euphorbiaceae) genus, which is native to the middle asia and is now mainly distributed in the middle asia and the areas of the Xinjiang and Tian. At present, 57 compounds are isolated from euphorbia pekinensis, including flavones and their glycosides, sterols, coumarins, macrocyclic diterpenes, triterpenes, pyrimidines, adenosines, sphingolipids, ellagic acid, and the like. At present, 17 macrocyclic diterpenoids separated from euphorbia pekinensis are all pseudoolivil macrocyclic diterpenoids, and some of the macrocyclic diterpenoids have certain tumor multidrug resistance reversal activity.

Macrocyclic diterpenoid compounds, i.e., diterpenoid compounds containing more than six-membered ring structures in molecules, have rich and variable skeleton structures and various biological activities, and have the maximum quantity of the pseudoelemene (Jatrophane) diterpene, and the structural characteristics of the pseudoelemene diterpene are that the skeleton is formed by fusing a five-membered ring and a twelve-membered ring, namely 5/12 ring systems. The early-stage research of the subject group finds that the pseudo-elemene diterpene is usually in a high oxidation state and generally has a larger molecular structure with the molecular weight of more than 700, six compounds in the 'macrocyclic diterpene compound in euphorbia foliata fruits and a preparation method and application thereof' in the patent of the subject group application in 2013 are respectively completely different from substituents of carbons at 2,3, 5, 7, 8,9, 14 and 15 positions on the skeleton of three novel pseudo-elemene macrocyclic diterpenes in the research, wherein the substituents comprise acetoxyl, propionyloxy, benzoyloxy, isobutyryloxy and the like, and the positions, the numbers and the configurations of the substituents on the skeleton are different, so that the steric hindrance of the compounds is particularly changed, the structures of the compounds are completely different, and the abundant polytropy of the compounds has a larger influence on the biological activity. The literature research finds that part of the pseudoolivine diterpenoid shows stronger multidrug resistance reversal activity due to the determined substituent on the framework carbon, so that the biological activity of the new compound can not be presumed through the existing activity research results.

Tumor multidrug resistance (MDR), which is a phenomenon that after a drug acts on a tumor to cause drug resistance, the tumor has cross-resistance to multiple antineoplastic drugs which are never contacted, unrelated in structure, different in target and different in mechanism. MDR has multiple formation mechanisms, one of the most important mechanisms is the overexpression of ABC family transporter (the P-glycoprotein, P-gp, coded by ABCB1 gene, which is the most widely and deeply studied at present) to increase drug efflux and form drug resistance. In a patent applied to the subject group in 2013, "macrocyclic diterpenoid compounds in euphorbia foliata fruits and a preparation method and application thereof" determination of drug resistance reversal activity "is carried out, the activity aims at investigating the drug resistance index of tumor drug-resistant cells to the compounds indicated in the patent and evaluating drug resistance, the research detects the drug resistance reversal activity of a new compound on the basis of the prior period and aims at investigating whether the drug resistance reversal activity of the new compound is combined with a traditional antitumor drug in a competitive way or not and is used as a substrate of the drug resistance cell by transport proteins on drug-resistant cell membranes to achieve the aim of reversing drug resistance of the drug-resistant cells, so that the traditional antitumor drug can play a role of killing tumor cells in the cells and is used for evaluating the effectiveness of the new compound.

The discovery of the lead compound with biological activity is the precondition of innovative drug research and is also a decisive factor for influencing the period of the innovative drug. The source of the lead compound has a plurality of ways, and finding active compounds from natural products as leads to create new drugs is one of the accepted effective ways of the world pharmacy workers. The research aims at further researching and exploring macrocyclic diterpenoid components in euphorbia foliorum to discover macrocyclic diterpenoid compounds with novel structures and outstanding multidrug resistance reversal activities, and a compound library which takes natural products as templates is constructed on the basis of abundant molecular structures of the natural products, so that a large amount of compounds with abundant structural diversity are provided for high-throughput drug screening. Based on a large amount of structure-activity relationship information provided by a compound library, researchers can research the pharmacological activity and pharmacokinetic properties of the drug, modify or optimize the structure and provide a simple, convenient and effective synthesis method, so that a drug lead compound and even the drug are obtained, and a reference is provided for the development of a multi-drug resistance reversal drug for tumors.

Disclosure of Invention

The invention aims to provide macrocyclic diterpenoid compounds in euphorbia foliosa fruits, a preparation method and application of multidrug resistance reversal. Three novel macrocyclic diterpenoids were isolated from the fruits of Euphorbia heterophylla (Euphorbia sororia a. schrenk) and tested for multidrug resistance reversal activity, and the results showed that: the macrocyclic diterpenoid compounds (I) - (III) all have multidrug resistance reversing activity of different degrees, can reverse the drug resistance of drug-resistant cells to antitumor drugs of different degrees when being combined with antitumor drugs, and can be used for preparing multidrug resistance reversing drugs.

The macrocyclic diterpenoid compound in euphorbia pekinensis is represented by the following structural formula:

wherein: the compound of formula (I) is (2S)^*,3S^*,4R^*,5R^*,13R^*,15R^*) -5-benzoyloxy-3-hydroxy-15-acetoxypseudoolivine-6 (17), 11E-diene-9, 14-dione;

the compound of formula (II) is (2R)^*,3R^*,4S^*,5R^*,7S^*,8S^*,9S^*,13S^*,14S^*,15R^*) -5-benzoyloxy-14, 15-dihydroxy-2, 7-diisobutyloxy-3, 8, 9-triacetoxy pseudoolivine-6 (17), 11E-diene;

the compound of formula (III) is (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2' -methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy-pseudoolivine-6 (17), 11E-diene.

The preparation method of the macrocyclic diterpenoid compounds in the euphorbia foliosa fruits comprises the following steps:

a. pulverizing Euphorbiae Pekinensis fruit, percolating or cold-soaking with 5-10 times of 50-99% ethanol water solution, anhydrous ethanol, pure acetone, 50-99% methanol water solution, and anhydrous methanol at room temperature, or heating under reflux, and concentrating to obtain crude extract;

b. dispersing the crude extract obtained in the step a by using ethanol, adding petroleum ether, n-hexane or cyclohexane for extraction, or dispersing the crude extract by using petroleum ether, n-hexane or cyclohexane, adding ethanol for extraction for 3-5 times, and concentrating the ethanol extract to obtain an ethanol extract;

c. separating the ethanol extract obtained in step b by two or three of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, and Sephadex L H-20 gel column chromatography to obtain (2S) of formula (I)^*,3S^*,4R^*,5R^*,13R^*,15R^*) -5-benzoyloxy-3-hydroxy-15-acetoxypseudoolivine-6 (17), 11E-diene-9, 14-dione; the compound of formula (II) is (2R)^*,3R^*,4S^*,5R^*,7S^*,8S^*,9S^*,13S^*,14S^*,15R^*) -5-benzoyloxy-14, 15-dihydroxy-2, 7-diisobutyloxy-3, 8, 9-triacetoxy pseudoolivine-6 (17), 11E-diene; the compound of formula (III) is (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2' -methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy-pseudoolivine-6 (17), 11E-diene.

The normal phase silica gel column chromatography in step c is normal pressure or pressurized column chromatography, the filler is silica gel, and the eluant is a mixture of at least two solvents of petroleum ether, cyclohexane, normal hexane, acetone, chloroform, dichloromethane, ethyl acetate and methanol, and isocratic elution or gradient elution is adopted.

The reverse phase silica gel column chromatography in step c is normal pressure or pressure column chromatography, the eluent is 60-99% methanol water solution or 40-99% acetonitrile water solution, and isocratic elution or gradient elution is adopted.

And c, performing normal-pressure column chromatography on the Sephadex L H-20 gel column chromatography used in the step c, wherein an eluent is methanol, dichloromethane or a mixture of at least two solvents of the methanol and the dichloromethane, and performing isocratic elution or gradient elution.

According to the macrocyclic diterpenoid compound separated from the euphorbia pekinensis fruit, the three novel macrocyclic diterpenoid compounds are subjected to in vitro cytotoxicity activity determination and drug resistance reversal activity determination, and experimental results show that: the novel macrocyclic diterpenoid compound has weaker cytotoxic activity on a human breast cancer cell strain MCF-7 and a human breast cancer adriamycin drug-resistant cell strain MCF-7/ADR, and the compounds (I) to (III) have multidrug resistance reversal activity of different degrees.

The macrocyclic diterpene compound in the euphorbia pekinensis fruit can be obtained by separation and purification from plants or can be synthesized by chemical modification methods well known to those skilled in the art.

The structure of the macrocyclic diterpenoid compounds in the euphorbia foliosa fruits is determined by modern spectrum means such as high-resolution mass spectrum, one-dimensional and two-dimensional nuclear magnetic resonance spectrum and the like, and the structure identification process is as follows:

the compound of formula (I) is a colorless oil,

UV(MeOH)λ_max(log)201(3.21), 228(2.99), 281(1.94) nm; ECD (MeOH)206(Δ -2.79), 221(Δ +2.62), 237(Δ -1.47), 261(Δ -0.80), 301(Δ -4.96) nm; HRESI (+) MS gave the excimer peak M/z 519.2362[ M + Na ]]⁺(calculated value is C)₂₉H₃₆O₇Na 519.2359), and determining its molecular formula as C₂₉H₃₆O₇(ii) a According to¹H，¹³Determining the structure by C NMR and two-dimensional nuclear magnetic resonance data, wherein the skeleton type is pseudo-elemene (Jatrophane) type and is named as (2S)^*,3S^*,4R^*,5R^*,13R^*,15R^*) -5-benzoyloxy-3-hydroxy-15-acetoxypseudoolivine-6 (17), 11E-diene-9, 14-dione, which¹H and¹³c NMR data are shown in Table 1[400MHz ] (¹H)，100 MHz(¹³C) The solvent: CDCl₃]。

The compound of formula (II) is a colorless oil, [ α ]]_D ²⁰-27.7(c 0.1,MeOH)；UV(MeOH)λ_max(log)200(3.14), 229(2.78), 282(1.67) nm; ECD (MeOH)204(Δ -2.09), 209(Δ -1.72),224(Δ -2.58),281(Δ +0.11) nm. According to HRESI (+) MS (M/z 795.3568[ M + Na)]⁺Calculated value is C₄₁H₅₆O₁₄Na795.3568) is determined to be C₄₁H₅₆O₁₄(ii) a According to¹H，¹³Determining the structure by C NMR and two-dimensional nuclear magnetic resonance data, wherein the skeleton type is pseudo-elemene (Jatrophane) type and is named as (2R)^*,3R^*,4S^*,5R^*,7S^*,8S^*,9S^*,13S^*,14S^*,15R^*) -5-benzoyloxy-14, 15-dihydroxy-2, 7-diIsobutyryloxy-3, 8, 9-triacetoxy pseudoolivine-6 (17), 11E-diene. It is composed of¹H and¹³c NMR data are shown in Table 1[400MHz ] (¹H)，100MHz(¹³C) The solvent: CDCl₃]。

The compound of formula (III) is a white amorphous powder,

UV(MeOH)λ_max(log)201(3.27), 229(3.03), 282(1.70) nm; ecd (meoh)216(Δ -1.88), 239(Δ +6.31) nm; according to which¹³C NMR and HRESI (+) MS (M/z 851.3834[ M + Na ]]⁺Theoretical value C₄₄H₆₀O₁₅Na 851.3830) data to determine its molecular formula as C₄₄H₆₀O₁₅(ii) a According to¹H，¹³The structure is determined by C NMR and two-dimensional nuclear magnetic resonance data, the framework type is pseudo-elemene (Jatrophane) type, and the name is (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2' -methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy pseudo-elemene-6 (17), 11E-diene. It is composed of¹H and¹³c NMR data are shown in Table 1[400MHz ] (¹H)，100MHz(¹³C) The solvent: CDCl₃]；

TABLE 1 preparation of compounds of formula (I), formula (II) and formula (III)¹H and¹³c NMR data [ (ppm), J (Hz)]

ND is that the signal was not detected.

Drawings

FIG. 1 shows the preparation of the compounds of formula (I) according to the invention¹H NMR(400MHz,CDCl₃) A spectrogram;

FIG. 2 shows the preparation of the compounds of formula (I) according to the invention¹³C NMR(100MHz,CDCl₃) A spectrogram;

FIG. 3Of the formula (II) according to the invention¹H NMR(400MHz,CDCl₃) A spectrogram;

FIG. 4 shows the preparation of a compound of formula (II) according to the invention¹³C NMR(100MHz,CDCl₃) A spectrogram;

FIG. 5 shows the preparation of a compound of formula (III) according to the invention¹H NMR(400MHz,CDCl₃) A spectrogram;

FIG. 6 shows the preparation of a compound of formula (III) according to the invention¹³C NMR(100MHz,CDCl₃) Spectra.

Detailed Description

Example 1

a. Pulverizing Euphorbiae radix 10kg, cold soaking in 50% ethanol-water solution of 50L at room temperature, and evaporating solvent under reduced pressure to obtain crude extract;

b. dispersing the crude extract obtained in the step a by using ethanol, adding petroleum ether for extraction, combining ethanol layers, and evaporating to dryness under reduced pressure to obtain an ethanol extract;

c. separating the ethanol extract obtained in step B with normal phase silica gel column, performing gradient elution with petroleum ether-ethyl acetate at volume ratio of 10:1-0:1, analyzing the fraction by silica gel thin layer chromatography (T L C), combining the same fractions to obtain 6 components (F1-F6), performing normal phase silica gel column separation on component F4, performing gradient elution with chloroform-acetone at volume ratio of 100:0-0:100 to obtain components F4A-F4D, separating component F4B with RP-18 reverse phase column, performing gradient elution with 65% -100% methanol-water solution, collecting 65% methanol-water solution (F4B1) and 80% methanol-water solution (F4B3), evaporating to dryness under reduced pressure, separating F4B1 with normal phase silica gel column, performing gradient elution with n-hexane-acetone at volume ratio of 20:1-0:1 to obtain components F4B1A-F4B1K, and preparing F4B1B with reverse phase column (B C)₁₈5 μm10 × 250mm) and isocratic elution with 68% strength methanol-water solution to give (2S) as the compound of formula (I)^*,3S^*,4R^*,5R^*,13R^*,15R^*) -5-benzoyloxy-3-hydroxy-15-acetoxypseudoolivine-6 (17), 11E-diene-9, 14-dione; section F4B1G adopts preparation of reverse phase column (C)₁₈5 μm10 × 250mm) and isocratic with 60% strength acetonitrile-water solutionEluting to obtain the compound (2R) of the formula (II)^*,3R^*,4S^*,5R^*,7S^*,8S^*,9S^*,13S^*,14S^*,15R^*) -5-benzoyloxy-14, 15-dihydroxy-2, 7-diisobutyloxy-3, 8, 9-triacetoxy pseudoolivine-6 (17), 11E-diene; section F4B3 is prepared by reverse phase column preparation (C)₁₈5 μm10 × 250mm) and isocratic elution with 80% acetonitrile-water solution to obtain the compound of formula (III) which is (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2' -methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy pseudoolivine-6 (17), 11E-diene.

Example 2

a. Pulverizing Euphorbiae radix 10kg, extracting with 60L ethanol-water solution of 99% concentration at 80 deg.C under reflux, and evaporating solvent under reduced pressure to obtain crude extract;

b. dispersing the crude extract obtained in the step a by using ethanol, adding n-hexane for extraction, combining ethanol layers, and evaporating to dryness under reduced pressure to obtain an ethanol extract;

c. separating the ethanol extract obtained in step B with normal phase silica gel column, gradient eluting with n-hexane-ethyl acetate at volume ratio of 10:1-0:1, analyzing the fraction by silica gel thin layer chromatography (T L C), mixing the same fractions to obtain 6 components (F1-F6), separating component F4 with normal phase silica gel column, gradient eluting with chloroform-methanol at volume ratio of 100:0-0:100 to obtain component F4A-F4D, separating component F4B with RP-18 reverse phase column, gradient eluting with acetonitrile-water solution at concentration of 80% -100%, collecting 80% acetonitrile-water solution (F4B1) and 90% acetonitrile-water solution (F4B3), evaporating to dryness under reduced pressure, separating F4B1 with normal phase silica gel column, gradient eluting with n-hexane-ethyl acetate at volume ratio of 20:1-0:1 to obtain components F4B1A-F4B1K, preparing F4B1B with reverse phase column (C B)₁₈5 μm10 × 250mm) and isocratic elution with 48% strength acetonitrile-water solution to give (2S) as the compound of formula (I)^*,3S^*,4R^*,5R^*,13R^*,15R^*) -5-benzoyloxy-3-hydroxy-15-acetoxypseudoolivine-6 (17), 11E-diene-9, 14-dione(ii) a Section F4B1G adopts preparation of reverse phase column (C)₁₈5 μm10 × 250mm) and isocratic eluted with 85% strength methanol-water solution to give (2R) compound of formula (II)^*,3R^*,4S^*,5R^*,7S^*,8S^*,9S^*,13S^*,14S^*,15R^*) The section F4B3 is separated by a Sephadex L H-20 gel column and eluted by absolute methanol to obtain the compound shown as the formula (III), namely (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2 '-methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy-pseudoolivine-6 (17), 11E-diene, wherein the compound is (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2' -methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy-pseudoolivine-6 (17).

Example 3

a. Pulverizing Euphorbiae radix fruit 10kg, cold soaking with 70L anhydrous ethanol at room temperature, and evaporating solvent under reduced pressure to obtain crude extract;

b. b, dispersing the crude extract obtained in the step a by using ethanol, adding cyclohexane for extraction, combining ethanol layers, and evaporating to dryness under reduced pressure to obtain an ethanol extract;

c. separating the ethanol extract obtained in step B with normal phase silica gel column, performing gradient elution with cyclohexane-ethyl acetate at volume ratio of 10:1-0:1, analyzing the fraction by silica gel thin layer chromatography (T L C), combining the same fractions to obtain 6 components (F1-F6), performing normal phase silica gel column separation on component F4, performing gradient elution with dichloromethane-acetone at volume ratio of 100:0-0:100 to obtain components F4A-F4D, separating component F4B with RP-18 reverse phase column, performing gradient elution with 65% -100% methanol-water solution, collecting 65% methanol-water solution (F4B1) and 80% methanol-water solution (F4B3), evaporating to dryness under reduced pressure, separating F4B1 with normal phase silica gel column, performing gradient elution with petroleum ether-ethyl acetate at volume ratio of 20:1-0:1 to obtain components F4B1A-F4B1K, preparing F4B1B with reverse phase column (C) at volume ratio of 20:1-0:1 to obtain components F4B B₁₈5 μm10 × 250mm) and isocratic elution with 60% strength methanol-water solution to give (2S) as the compound of formula (I)^*,3S^*,4R^*,5R^*,13R^*,15R^*) -5-benzoyloxy-3-hydroxy-15-acetoxyPseudoelemene-6 (17), 11E-diene-9, 14-dione; section F4B1G adopts preparation of reverse phase column (C)₁₈5 μm10 × 250mm) and isocratic elution with 60% strength acetonitrile-water solution to give the compound of formula (II) (2R)^*,3R^*,4S^*,5R^*,7S^*,8S^*,9S^*,13S^*,14S^*,15R^*) The section F4B3 is separated by a Sephadex L H-20 gel column, and is eluted by dichloromethane-methanol with the volume ratio of 1:1 to obtain the compound (III) which is (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2' -methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy-pseudoolivine-6 (17), 11E-diene.

Example 4

a. Taking 10kg of euphorbia pekinensis fruit, crushing, percolating and extracting with 80L pure acetone at room temperature, and evaporating the solvent under reduced pressure to obtain a crude extract of euphorbia pekinensis;

b. dispersing the crude extract obtained in the step a by using petroleum ether, adding ethanol for extraction for 3 times, combining ethanol layers, and evaporating to dryness under reduced pressure to obtain an ethanol extract;

c. separating the ethanol extract obtained in step B with normal phase silica gel column, performing gradient elution with petroleum ether-ethyl acetate at volume ratio of 10:1-0:1, analyzing the fraction by silica gel thin layer chromatography (T L C), combining the same fractions to obtain 6 components (F1-F6), performing normal phase silica gel column separation on component F4, performing gradient elution with chloroform-acetone at volume ratio of 100:0-0:100 to obtain components F4A-F4D, separating component F4B with RP-18 reverse phase column, performing gradient elution with acetonitrile-water solution at concentration of 80% -100%, collecting 80% acetonitrile-water solution (F4B1) and 90% acetonitrile-water solution (F4B3), evaporating to dryness under reduced pressure, separating F4B1 with normal phase silica gel column, performing gradient elution with n-hexane-acetone at volume ratio of 20:1-0:1 to obtain components F4B1A-F4B1K, and preparing F4B1B with reverse phase column (B C)₁₈5 μm10 × 250mm) and isocratic elution with 40% strength acetonitrile-water solution to give (2S) as the compound of formula (I)^*,3S^*,4R^*,5R^*,13R^*,15R^*) -5-benzoyloxy-3-hydroxy-15-acetoxypseudoolivine-6 (17), 11E-diene-9, 14-dione; section F4B1G adopts preparation of reverse phase column (C)₁₈5 μm10 × 250mm) and isocratic eluted with 85% strength methanol-water solution to give (2R) compound of formula (II)^*,3R^*,4S^*,5R^*,7S^*,8S^*,9S^*,13S^*,14S^*,15R^*) -5-benzoyloxy-14, 15-dihydroxy-2, 7-diisobutyloxy-3, 8, 9-triacetoxy pseudoolivine-6 (17), 11E-diene; section F4B3 is prepared by reverse phase column preparation (C)₁₈5 μm10 × 250mm) and isocratic elution with 80% acetonitrile-water solution to obtain the compound of formula (III) which is (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2' -methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy pseudoolivine-6 (17), 11E-diene.

Example 5

a. Pulverizing Euphorbiae radix 10kg, extracting with 90L methanol-water solution with concentration of 50% at 80 deg.C under reflux, and evaporating solvent under reduced pressure to obtain crude extract;

b. dispersing the crude extract obtained in the step a by using normal hexane, adding ethanol for extraction for 4 times, combining ethanol layers, and evaporating to dryness under reduced pressure to obtain an ethanol extract;

c. separating the ethanol extract obtained in step B with normal phase silica gel column, gradient eluting with n-hexane-ethyl acetate at volume ratio of 10:1-0:1, analyzing the fraction by silica gel thin layer chromatography (T L C), combining the same fractions to obtain 6 components (F1-F6), separating component F4 with normal phase silica gel column, gradient eluting with chloroform-methanol at volume ratio of 100:0-0:100 to obtain components F4A-F4D, separating component F4B with RP-18 reverse phase column, gradient eluting with 65% -100% methanol-water solution, collecting 65% methanol-water solution (F4B1) and 80% methanol-water solution (F4B3), evaporating under reduced pressure, separating F4B1 with normal phase silica gel column, gradient eluting with n-hexane-ethyl acetate at volume ratio of 20:1-0:1 to obtain components F4B 1-F4B K, preparing F4B B with reverse phase column 361C (B C)₁₈5 μm10 × 250mm) and isocratic with 68% strength methanol-water solutionEluting to obtain the compound (2S) of the formula (I)^*,3S^*,4R^*,5R^*,13R^*,15R^*) -5-benzoyloxy-3-hydroxy-15-acetoxypseudoolivine-6 (17), 11E-diene-9, 14-dione; section F4B1G adopts preparation of reverse phase column (C)₁₈5 μm10 × 250mm) and isocratic elution with 60% strength acetonitrile-water solution to give the compound of formula (II) (2R)^*,3R^*,4S^*,5R^*,7S^*,8S^*,9S^*,13S^*,14S^*,15R^*) The section F4B3 is separated by a Sephadex L H-20 gel column and eluted by absolute methanol to obtain the compound shown as the formula (III), namely (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2 '-methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy-pseudoolivine-6 (17), 11E-diene, wherein the compound is (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2' -methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy-pseudoolivine-6 (17).

Example 6

a. Pulverizing Euphorbiae radix fruit 10kg, percolating with 100L anhydrous methanol at room temperature, and evaporating solvent under reduced pressure to obtain crude extract;

b. b, dispersing the crude extract obtained in the step a by using cyclohexane, adding ethanol for extraction for 5 times, combining ethanol layers, and evaporating to dryness under reduced pressure to obtain an ethanol extract;

c. separating the ethanol extract obtained in step B with normal phase silica gel column, performing gradient elution with cyclohexane-ethyl acetate at volume ratio of 10:1-0:1, analyzing the fraction with silica gel thin layer chromatography (T L C), combining the same fractions to obtain 6 components (F1-F6), performing normal phase silica gel column separation on component F4, performing gradient elution with dichloromethane-acetone at volume ratio of 100:0-0:100 to obtain components F4A-F4D, separating component F4B with RP-18 reverse phase column, performing gradient elution with acetonitrile-water solution at concentration of 80% -100%, collecting 80% acetonitrile-water solution (F4B1) and 90% acetonitrile-water solution (F4B3), evaporating to dryness under reduced pressure, separating F4B1 with normal phase silica gel column, performing gradient elution with petroleum ether-ethyl acetate at volume ratio of 20:1-0:1 to obtain components F4B1A-F4B1K, preparing F4B1B with reverse phase column (C) at volume ratio of 20:1-0:1₁₈5 μm10 × 250mm) separationIsocratically eluting with 48% acetonitrile-water solution to obtain (2S) as compound of formula (I)^*,3S^*,4R^*,5R^*,13R^*,15R^*) -5-benzoyloxy-3-hydroxy-15-acetoxypseudoolivine-6 (17), 11E-diene-9, 14-dione; section F4B1G adopts preparation of reverse phase column (C)₁₈5 μm10 × 250mm) and isocratic elution with 60% strength acetonitrile-water solution to give the compound of formula (II) (2R)^*,3R^*,4S^*,5R^*,7S^*,8S^*,9S^*,13S^*,14S^*,15R^*) The section F4B3 is separated by a Sephadex L H-20 gel column, and is eluted by dichloromethane-methanol with the volume ratio of 1:1 to obtain the compound (III) which is (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2' -methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy-pseudoolivine-6 (17), 11E-diene.

Example 7

The application of the macrocyclic diterpenoid compound separated from the euphorbia pekinensis fruit in preparing multidrug resistance reversal medicines or preparing antitumor medicines by combining with the antitumor medicines takes a human breast cancer cell strain and an adriamycin resistance strain thereof as examples;

cytotoxicity and resistance index testing of Compounds of formula (I) -formula (III):

materials and reagents: RPMI 1640 medium was purchased from Hyclone; double antibody and fetal bovine serum were purchased from Hyclone; trypsin was purchased from Gibco; thiazole blue (MTT) was purchased from Biosharp; dimethyl sulfoxide (DMSO) was purchased from Amresco corporation; rhodamine 123 is available from Sigma; verapamil hydrochloride was purchased from Sigma; doxorubicin hcl was purchased from shanghai bio ltd;

cell lines: human breast cancer cell line MCF-7 (purchased from Shanghai cell bank of Chinese academy of sciences) and human breast cancer adriamycin-resistant cell line MCF-7/ADR (purchased from Shanghai Dou Biotech, Inc.);

cell culture: human breast cancer cell line MCF-7 and adriamycin resistant cell line MCF-7/ADR in RPMI 1640 complete medium (RPMI 1640 medium + 10% FBS + 1% double antibody); all cells were placed in CO₂Incubator (temperature 37 ℃, 5% CO)₂) Maintaining subculture, carrying out drug-resistant culture on the drug-resistant strain (MCF-7/ADR cells) in a complete culture medium with a final adriamycin concentration of 500ng/m L for one week, carrying out drug-resistant culture in a complete culture medium with a final adriamycin concentration of 1000ng/m L for one week, and then carrying out culture in a complete culture medium without an antitumor agent for two weeks for experiment;

the experimental method comprises the steps of adopting an MTT method, inoculating MCF-7 or MCF-7/ADR cells in a logarithmic growth phase into a 96-hole micro-culture plate at the density of 5000 cells/hole, incubating the micro-culture plate in an incubator at the temperature of 37 ℃ for 24 hours, adding a monomer compound to be tested, setting a concentration gradient of 100 mu L in each hole and 6 repeated holes, setting a cell-free zero-adjustment group, a solvent (DMSO) control group and a positive drug control group, setting tumor cells at the temperature of 37 ℃ and 5% CO in the tumor cells, and setting a concentration gradient of 6 repeated holes in each hole and setting a concentration gradient of 5 mu₂Culturing for 48 hr under the condition, discarding supernatant, adding MTT solution (5mg/m L, prepared with physiological saline, mixed with complete culture medium at a ratio of 1:9, 100 μ L per well), and culturing at 37 deg.C with 5% CO₂Culturing for 4h, discarding supernatant, adding 150 μ L DMSO into each well, dissolving formazan, detecting absorbance value (A) at 570nm of each well with microplate reader, and calculating inhibition rate (IC) of monomer compound to be tested on tumor cell growth according to the following formula₁₀Value and median suppression IC₅₀Values were calculated using GraphPad Prism 5.0 software) and drug resistance index: inhibition ratio (%) ═ a_{Control group}－A_{Administration set})/A_{Control group}× 100%, and adriamycin Resistance Index (RI) ═ IC as antineoplastic agent_50(MCF-7)/IC_{50(MCF-7/ADR)}；

The experimental results are as follows: half-growth inhibition of compounds of formula (i) -formula (iii) fractionated from euphorbia foliata fruits, see table 2:

TABLE 2 half-growth inhibition of the compounds of formula (I) -formula (III) in Euphorbia foliosa fruits on human breast cancer cell line MCF-7 and human breast cancer Adriamycin resistant cell line MCF-7/ADR

As is clear from Table 2, neither of the compounds of the formulae (I) to (III) exhibited cytotoxicity (IC) against the human breast cancer cell line MCF-7 nor against the human breast cancer doxorubicin-resistant cell line MCF-7/ADR₅₀Greater than 70 mu M), 39.83 is obtained by calculating the drug resistance index of the antitumor drug adriamycin to MCF-7/ADR cells;

the compound of formula (I) -formula (III) reverses the activity of tumor multidrug resistance test:

in the experiment, macrocyclic diterpenoid compounds (I) - (III) separated from euphorbia lobata fruits are combined with antitumor drug adriamycin (DOX), growth inhibition on drug-resistant cells before and after combination is detected, and multi-drug resistance reversal activity test is carried out.

The experimental method comprises inoculating human breast cancer adriamycin drug-resistant cell strain MCF-7/ADR cells in logarithmic growth phase into 96-well microplate (100 mu L per well) at a density of 5000 cells/well, incubating at 37 deg.C for 24 hr, adding adriamycin and test monomer compound or positive control drug verapamil, setting concentration gradient of 7 in each well at 100 mu L, setting 6 multiple wells, setting blank control group and solvent (DMSO) control group, and culturing at 37 deg.C with 5% CO₂Culturing for 48 hr under the condition, discarding supernatant, adding MTT solution (5mg/m L, prepared with physiological saline, mixed with complete culture medium at a ratio of 1:9, 100 μ L per well), and culturing at 37 deg.C with 5% CO₂Culturing for 4h under the condition, removing the supernatant, adding 150 mu L DMSO into each well, detecting the absorbance value (A) of 570nm of each well by using a microplate reader after the formazan is dissolved, and calculating the inhibition rate of the test monomer compound on the growth of the tumor cells according to the following formula, namely the inhibition rate (%) - (A)_{Control group}－A_{Administration set})/A_{Control group}× 100%, and adriamycin Resistance Index (RI) ═ IC as antineoplastic agent_50(MCF-7)/IC_{50(MCF-7/ADR)}. And calculating the reversion multiple according to the following formula;

inverse multiple (RF) IC_{50 (Adriamycin)}/IC_{50 (Adriamycin + Compound)}

The experimental results are as follows: the half-growth inhibition and fold-reversal of MCF-7/ADR cells by the combination of compounds of formula (I) -formula (III) and doxorubicin are shown in tables 3-5:

TABLE 3 half-growth inhibition of MCF-7/ADR cells by the combination of a Compound of formula (I) with Adriamycin

As shown in Table 3, the IC of the compound of formula (I) when used in combination with doxorubicin was compared to the effect of doxorubicin alone₅₀The value is reduced, and the reduction degree is expressed by reversal multiple, namely, the multi-drug resistance reversal activity is displayed to a certain degree;

TABLE 4 half-growth inhibition of MCF-7/ADR cells by the combination of a Compound of formula (II) with Adriamycin

As can be seen from Table 4, the IC of doxorubicin combined with the compound of formula (II) was compared to the effect of doxorubicin alone₅₀The value is obviously reduced, the reduction degree is expressed by a reversal multiple, the reversal multiple (14.27 times) of 5 mu M of the compound shown in the formula (II) is equivalent to 10 mu M of the positive control drug verapamil (10.35 times), namely, the compound shown in the formula (II) has stronger activity of reversing the multidrug resistance of the tumor;

TABLE 5 half-growth inhibition of MCF-7/ADR cells by combination of a Compound of formula (III) with Adriamycin

As can be seen from Table 5, the IC of doxorubicin combined with the compound of formula (III) was compared to the effect of doxorubicin alone₅₀The value is obviously reduced, the reduction degree is expressed by a reversal multiple, the reversal multiple (10.60 times) of the compound of the formula (III) with the concentration of 2.5 mu M is equivalent to (10.35 times) of the verapamil which is a positive control drug with the concentration of 10 mu M, and the compound of the formula (III) has stronger activity of reversing the multidrug resistance of the tumor.

Claims

1. Macrocyclic diterpenoid compounds in euphorbia pekinensis fruits are characterized in that the compound has a structural formula as follows:

2. The method for the preparation of macrocyclic diterpenoids in the fruit of Euphorbia peplus according to claim 1, characterized by the following steps:

c. separating the ethanol extract obtained in step b by two or three of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, and Sephadex L H-20 gel column chromatography, i.e. separatingTo obtain (2S) of the formula (I)^*,3S^*,4R^*,5R^*,13R^*,15R^*) -5-benzoyloxy-3-hydroxy-15-acetoxypseudoolivine-6 (17), 11E-diene-9, 14-dione; the compound of formula (II) is (2R)^*,3R^*,4S^*,5R^*,7S^*,8S^*,9S^*,13S^*,14S^*,15R^*) 5-benzoyloxy-14, 15-dihydroxy-2, 7-diisobutyloxy-3, 8, 9-triacetoxy pseudoolivine-6 (17), 11E-diene, wherein the compound of formula (III) is (2R,3R,4S,5R,7S,8S,9S,13S,14S,15R) -14-benzoyloxy-15-hydroxy-5- (2' -methyl-butyryloxy) -7-isobutyryloxy-2, 3,8, 9-tetraacetoxy pseudoolivine-6 (17), 11E-diene, wherein the normal phase silica gel column chromatography is normal pressure or pressure column chromatography, the nitrile filler is silica gel, the eluent is a mixture of at least two solvents selected from petroleum ether, cyclohexane, n-hexane, acetone, chloroform, dichloromethane, ethyl acetate and methanol, the eluent is isocratic or gradient elution, wherein the eluent for the volume ratio of 10:1-0:1 is cyclohexane-ethyl acetate, ethyl acetate or petroleum ether-ethyl acetate, the eluent for the dichloromethane to 100: 0: 1: 0: 10: 1: 0: 1: 0: 10: 1: 0: 10: 0: 1: 0: 25: 0: 25: 0: 25: 0: 25: 10.

3. Use of the macrocyclic diterpenoid compounds of the euphorbia pekinensis, compounds of formulae i-iii according to claim 1 for the preparation of a medicament with multidrug resistance reversing activity.