CN1872828B

CN1872828B - Monocyclic polysubstitution cyclohexenol, and compound of ketones, and preparation method, and usage

Info

Publication number: CN1872828B
Application number: CN2005100748085A
Authority: CN
Inventors: 崔承彬; 李长伟; 蔡兵; 韩冰
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2005-06-03
Filing date: 2005-06-03
Publication date: 2010-06-02
Anticipated expiration: 2025-06-03
Also published as: CN1872828A

Abstract

This invention relates to a polysubstituted monocyclohexenol and ketone compound, extract containing the compound, and its preparation and application. The compound can be used as the cell cycle inhibitor, cell apoptosis inducer, cell proliferation inhibitor and antitumor drug.

Description

Monocyclic polysubstituted cyclohexenol and ketone compound and preparation method and application thereof

The technical field is as follows:

the invention relates to a monocyclic polysubstituted cyclohexenol and ketone compound and a preparation method and application thereof. In particular to monocyclic polysubstituted cyclohexenol and ketone compounds containing a long-chain hydrocarbon substituent, an extract containing the compounds, a method for extracting, separating and purifying the compounds and application of the extract or the compounds in preparing cell cycle inhibitors, apoptosis inducers, cell proliferation inhibitors, cancer cell killing agents and antitumor agents.

Background art:

monocyclic substituted cyclohexenol or ketone compounds refer to a series of compounds having a hydroxyl or carbonyl group and other various substituents attached to the carbon skeleton of cyclohexene. The compound has a single skeleton structure, and different compounds are formed mainly due to different substituent groups and different substituent positions. Some of the monocyclic polysubstituted cyclohexenone compounds containing a long-chain hydrocarbon substituent have been reported in the literature. Such as the documents S.R. Johns, et al, Campnosperma extracts, the optically effective long-chain 5-hydroxythex-2-enes and long-chain bicycles [3.3.1] none-3, 7-dienes: aust.j.chem., 40, 79-96, 1987, document e.f.queiroz, et al, New dihydroalkylhexeones from Lannea edition, j.nat.prod., 66, 578-cake 580, 2003, document s.j.coreia, et al, Alkyl phenol and derivatives from tapiria obtusese: phytochem, 56, 781-784, 2001 and A.Groweiss, et al, Novel cytoxic, alkylated hydroquinones from Lannea welwitschii, J.Nat.Prod., 60, 116-121, 1997 describe several monocyclic polysubstituted cyclohexenone compounds containing one long-chain hydrocarbon group (seventeen or nineteen carbons). The document S.J. Correia, et al, Alkyl phenols and derivitives from Tapiririraobtusie: phytochem.56, 781-784, 2001 also reported 4 cyclohexenol derivatives containing a hexadecyl hydrocarbon group, but the other end of the hexadecyl hydrocarbon group in these 4 compounds is connected with a benzene ring. To date, monocyclic polysubstituted cyclohexenols or ketones having the same type of long-chain hydrocarbon substituent as the compounds of the present invention have not been reported in the literature.

Choerospondias axillaris (Roxb.) Burtt et Hill is a plant of Choerospondias genus of Anacardiaceae family, the bark and fruit of which are used as medicines mainly for treating pyocutaneous disease, injury from hot water, eczema of scrotum, indigestion, abdominal pain due to dyspepsia, sobering up and detoxication of fruit stone, and sore or abscess due to wind-toxicity keloid (edited by Jiangsu New institute of medicine, dictionary of Chinese medicine, Shanghai people's publishing house, 1977, page 397-. The dried fruit has been used as Chinese medicine "Guangzao" and has been imported into pharmacopoeia of 2000 th edition of China (national pharmacopoeia Committee, pharmacopoeia of people's republic of China, one division, Beijing, chemical industry Press, 2000 th year, page 32). Various chemical components such as flavone, phenolic acid, organic acid, amino acid, steroid and the like in choerospondias axillaris have been reported, and various activities such as antibiosis, platelet aggregation inhibition and the like of a choerospondias axillaris crude extract, total flavone or a monomer compound have also been reported (such as bear winterization and the like; the research overview and the application prospect of choerospondias axillaris plants in the aspect of medicines are shown in Guangdong pharmacy, 10 th period and 5 th page in 2000, and 8-10 th page). However, the antineoplastic activity and the active ingredients of the choerospondias axillaris, in particular to the monocyclic polysubstituted cyclohexenol or ketone active ingredients related to the invention, have not been reported in research so far.

The invention content is as follows:

the invention aims to provide a monocyclic polysubstituted cyclohexenol or ketone compound containing a long-chain hydrocarbon substituent, which has the antitumor activities of cell cycle inhibition, apoptosis induction, direct killing of cancer cells and the like.

The inventor discovers for the first time that the crude extract of choerospondias axillaris has good antitumor activities such as killing cytotoxicity, apoptosis induction, cell cycle inhibition, inhibition on cancer cell proliferation and the like on cancer cells, researches the active ingredients of the crude extract, and discovers that the monocyclic polysubstituted cyclohexenol or ketone compound shown in the following formula I in the crude extract of choerospondias axillaris has the antitumor activity:

formula I

Wherein R is hydrogen and hydroxy (forming cyclohexenol), or oxygen (forming cyclohexenone); r₁、R₂、R₃、R₄、R₅、R₆Is hydrogen, hydroxy, amino, methoxy or saturated or unsaturated straight chain or branched chain C optionally substituted by hydroxy, amino and the like_15-20A hydrocarbon group, and, R₁-R₆At least one of them is said saturated or unsaturated straight chain or branched C optionally substituted with a substituent such as hydroxyl, amino or the like₁₅-C₂₀A hydrocarbyl group.

Accordingly, in a first aspect, the present invention relates to an extract characterized by comprising at least one monocyclic polysubstituted cyclohexenol and one compound represented by formula I:

formula I

Wherein R is hydrogen and hydroxy, or oxygen; r₁、R₂、R₃、R₄、R₅、R₆Is hydrogen, hydroxy, amino, methoxy or saturated or unsaturated straight chain or branched chain C optionally substituted by hydroxy, amino and the like_15-20A hydrocarbon group, and, R₁-R₆At least one of them is said saturated or unsaturated straight chain or branched C optionally substituted with a substituent such as hydroxyl, amino or the like₁₅-C₂₀A hydrocarbyl group.

The second aspect of the present invention relates to monocyclic polysubstituted cyclohexenol and ketone compounds represented by formula I:

formula I

A third aspect of the present invention relates to a process for preparing the above extract, which comprises extracting the relevant plant material such as Choerospondias axillaris with alcohol or aqueous alcohol to obtain a crude extract, i.e., the extract

A fourth aspect of the invention relates to a process for the isolation and purification of a compound of formula I from said extract, said process comprising extracting the relevant plant material, such as Choerospondias axillaris, with an alcohol or aqueous alcohol, followed by isolation and purification to obtain the compound of formula I.

A fifth aspect of the invention relates to pharmaceutical compositions comprising as active ingredient a monocyclic polysubstituted cyclohexanol and ketone compound of formula I together with one or more pharmaceutically acceptable carriers or excipients.

The sixth aspect of the present invention relates to the use of monocyclic polysubstituted cyclohexenol and ketone compounds of formula I for the preparation of cell cycle inhibitors, apoptosis inducers, cell proliferation inhibitors, cancer cell killers and antitumor agents.

In one embodiment of the present invention, the present invention relates to monocyclic polysubstituted cyclohexenol and ketone compounds represented by formula I:

formula I

In a preferred embodiment of the present invention, the present invention relates to monocyclic polysubstituted cyclohexenol and ketone compounds of formula I:

formula I

Wherein R is hydrogen and hydroxy, or oxygen; r₁、R₂、R₃And R₄Is hydroxy or optionally by hydroxy(Z) -14-alkenylnonadecyl group substituted with a substituent such as a group or an amino group, and R₂Or R₃At least one is (Z) -14-alkenylnonadecyl optionally substituted with a substituent such as hydroxy, amino or the like; r₅、R₆Is hydroxyl or hydrogen.

In a further preferred embodiment of the invention, R in formula I is oxygen and R is₁、R₂、R₆Is hydrogen, R₃Is 2-hydroxy- (Z) -14-enenonalkyl, R₄、R₅Is hydroxy (absolute configuration 4R5S 8R).

In another further preferred embodiment of the invention, R in formula I is β -hydroxy and α -hydrogen, R₁And R₅Is hydroxy, R₂Is (Z) -14-enenonalkyl, R₃、R₄、R₆Is hydrogen (absolute configuration 1R4S 6S).

Extracts and pure compounds of formula I containing the above compounds of formula I can be prepared by the following method: extracting plant material such as Choerospondias axillaris with alcohol or aqueous alcohol to obtain crude extract, and separating and purifying to obtain compound of formula I.

According to the invention, the alcohol employed in the above process is ethanol; the aqueous alcohol is 60-95% aqueous ethanol; the separation and purification includes conventional methods and means for separation and purification of natural products, such as liquid-liquid extraction, column chromatography, thin layer chromatography, high performance liquid chromatography, recrystallization and the like, which are well known to those skilled in the art. Wherein column chromatography, high performance liquid chromatography and recrystallization refining can be repeatedly carried out for many times.

The invention adopts a method for detecting cell morphological characteristics under a microscope by combining a Lissamine rhodamine B (SRB) method and a flow cytometry, and tests the effects of the compound shown in the formula I on cell proliferation inhibition, cell cycle inhibition, cell apoptosis induction, cancer cell direct killing and the like of mouse breast cancer tsFT210 cells, human colorectal cancer HCT-15 cells, human cervical cancer HeLa cells, human breast cancer MCF-7 cells and human ovarian cancer A2780 cells. Experiments prove that the compound shown in the formula I can show the biological activity of inhibiting the proliferation of tumor cells on the tumor cells in the modes of inhibiting cell cycle turnover, inducing cancer cell apoptosis or directly killing and the like, thereby having the anti-tumor effect.

The compound of the formula I can be prepared into antitumor drugs by being compatible with various pharmaceutically acceptable carriers, excipients or auxiliary materials, and is used for treating tumors.

The term "pharmaceutically acceptable salt" in the present invention may be a pharmaceutically acceptable inorganic or organic salt. The compounds having a basic group in formula I of the present invention may form pharmaceutically acceptable salts with inorganic acids, such as sulfate, hydrochloride, hydrobromide, phosphate; pharmaceutically acceptable salts can also be formed with organic acids such as acetates, oxalates, citrates, gluconates, succinates, tartrates, p-toluenesulfonates, methanesulfonates, benzoates, lactates, maleates, and the like. The compounds having an acidic group in formula I of the present invention may form pharmaceutically acceptable salts with alkali metals or alkaline earth metals, preferably but not limited to sodium, potassium, magnesium or calcium salts.

The compounds of the present invention may be administered alone or in the form of pharmaceutical compositions. The route of administration may be oral, parenteral or topical. The pharmaceutical composition can be formulated into various suitable dosage forms according to the administration route.

Pharmaceutical compositions of the compounds of the present invention may be administered in any of the following ways: oral, aerosol inhalation, rectal, nasal, buccal, topical, parenteral, e.g. subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or via an external reservoir. Among them, oral, intraperitoneal or intravenous administration is preferable.

When administered orally, the compounds of the present invention may be formulated in any orally acceptable dosage form, including but not limited to tablets, capsules, aqueous solutions or suspensions. Among these, carriers for tablets generally include lactose and corn starch, and additionally, lubricating agents such as magnesium stearate may be added. Diluents used in capsule formulations generally include lactose and dried corn starch. Aqueous suspension formulations are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents. Optionally, some sweetener, aromatic or colorant may be added into the above oral preparation.

When applied topically to the skin, the compounds of the present invention may be formulated in a suitable ointment, lotion, or cream formulation wherein the active ingredient is suspended or dissolved in one or more carriers. Carriers that may be used in ointment formulations include, but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; carriers that can be used in lotions or creams include, but are not limited to: mineral oil, sorbitan monostearate, tween 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The compounds of the present invention may also be administered in the form of sterile injectable preparations, including sterile injectable aqueous or oleaginous suspensions or solutions. Among the carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

It is further noted that the dosage and method of administration of the compounds of the present invention will depend upon a variety of factors including the age, weight, sex, physical condition, nutritional status, the activity level of the compound, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The preferred dosage is between 0.01-100mg/kg body weight/day.

The compound of formula I of the invention can also be used as a low molecular biological probe for inhibiting cell cycle or inducing apoptosis in life science research. When the compound of formula I is used as cell cycle inhibitor or apoptosis inducer in life science research, it can be dissolved in methanol or aqueous methanol, or dissolved in aqueous solution of dimethyl sulfoxide for application.

Description of the drawings:

FIG. 1 is a UV absorption spectrum of Compound I in methanol solution (40. mu.g/ml);

FIG. 2 is an infrared absorption spectrum (KBr) of Compound I;

FIG. 3 is of Compound I in deuterated chloroform¹H nuclear magnetic resonance spectrum;

FIG. 4 is of Compound I in deuterated chloroform¹³C nuclear magnetic resonance spectrum;

FIG. 5 is a Circular Dichroism (CD) spectrum of Compound I in methanol solution (0.1 mg/ml);

FIG. 6 is a UV absorption spectrum of Compound II in methanol solution (40. mu.g/ml);

FIG. 7 is an infrared absorption spectrum (KBr) of Compound II;

FIG. 8 is of Compound II in deuterated chloroform¹H nuclear magnetic resonance spectrum;

FIG. 9 is of Compound II in deuterated chloroform¹³C nuclear magnetic resonance spectrum;

FIG. 10 is a Circular Dichroism (CD) spectrum of Compound II in methanol solution (0.1 mg/ml);

FIG. 11 is a flow cytometric histogram of mouse breast cancer tsFT210 cells measured 17 hours after treatment with Compound I;

figure 12 is a flow cytometric histogram of mouse breast cancer tsFT210 cells measured 17 hours after treatment with compound II.

The specific implementation mode is as follows:

the following examples further illustrate the invention but are not intended to limit the invention thereto.

In the following examples, compounds of the present invention, hereinafter referred to as compound I, were identified by nuclear magnetic resonance or the like to have the following structures: formula IWherein R is oxygen, R₁、R₂、R₆Is hydrogen, R₃Is 2-hydroxy- (Z) -14-enenonalkyl, R₄、R₅Is hydroxy (absolute configuration 4R5S 8R); the compound of the present invention, hereinafter referred to as compound II, has the following structure as identified by nuclear magnetic resonance or the like: compounds of formula I wherein R is beta-hydroxy and alpha-hydrogen, R₁And R₅Is hydroxy, R₂Is (Z) -14-enenonalkyl, R₃、R₄、R₆Is hydrogen (absolute configuration 1R4S 6S):

formula I

Arabic numerals on carbocyclic rings and long chains indicate the index positions of the corresponding carbon atoms

In the structural study of the compound, the melting point is measured by an X-4 type precision micro melting point tester of Beijing Tian Di Yu science and technology Limited liability company, and the temperature is not corrected. The specific rotation was measured by a JSASCO P-1020 polarimeter. Positive and negative ion TOF-MS and HR-TOF-MS were measured with an API 3000 liquid chromatography-mass spectrometer (ABI, USA) and an LCT mass spectrometer (Micromass, UK), respectively, an ultraviolet spectrum was measured with a Shimadzu UV2501PC ultraviolet spectrophotometer, and an infrared spectrum was measured with a Nicolet Magn a-IR spectrometer^TMDetermination by 550 type Infrared spectrometer, nuclear magnetic resonance spectroscopy using JEOLECIlips-600 type superconducting nuclear magnetic resonance spectrometer (600 MHz)¹H-NMR，150MHz ¹³C-NMR). Circular Dichroism (CD) spectra were measured using JASCO J-810 Spectropolarimeter.

EXAMPLE 1 extraction and isolation of Compound I

The first step is as follows: preparation of extract containing Compound I

Pulverizing dried bark (3.2kg) of Choerospondias axillaris (collected from Mengla region of Yunnan in 8 months 1999), extracting with ethanol at 25L room temperature for 4 times, each time soaking for 7 days. Mixing extractive solutions, concentrating, and drying to obtain 750g ethanol extract. 750g of the ethanol extract was suspended in 3L of water, and the mixture was extracted 4 times with chloroform (3L), ethyl acetate (3L) and n-butanol (3L) in this order to obtain 60g of the chloroform extract, 310g of the ethyl acetate extract, 300g of the n-butanol extract and 80g of the water layer residue, respectively. Wherein the chloroform extract is an extract containing compound I.

The second step is as follows: preparation of a chromatographic crude fraction containing Compound I

The chloroform extract (60g) was dried on a reduced pressure silica gel column (bed 7.5 cm. times.18.5 cm) and eluted with petroleum ether (P) -acetone (A) and methanol solvent system. The polarity of the elution solvent is increased by increasing the amount of acetone (A) in petroleum ether (P) to increase the polarity of the elution solvent in a gradient manner or by changing the amount of acetone (A) to methanol. Fractions were pooled according to thin layer assay and activity assay results to give 6 fractions Fr-1(3.2g, V)_P∶V_A100: 1 elution fraction), Fr-2(2.3g, V)_P∶V_A50: 1 elution fraction), Fr-3(2.0g, V_P∶V_A10: 1 elution fraction), Fr-4(10.8g, V)_P∶V_A5: 1 elution fraction), Fr-5(13.6g, V)_P∶V_A2: 1 elution fraction) and Fr-6(25g, methanol elution fraction). Fr-5(13.6g) was dried on a reduced pressure silica gel column (Becton Dickinson: 5.0 cm. times.22 cm), eluted with chloroform (C) -ethyl acetate (Ac), acetone, methanol system, and fractions were combined according to the results of thin layer assay and activity test to give 6 fractions Fr-5-1(4.1g, V)_C∶V_Ac8: 1 elution fraction), Fr-5-2(980mg, V)_C∶V_Ac4: 1 elution fraction), Fr-5-3(3.7g, V_C∶V_Ac2: 1 elution fraction), Fr-5-4(1.1g, ethyl acetate elution fraction), Fr-5-5(1.8g, acetone elution fraction), Fr-5-6(0.8g, methanol elution fraction). Wherein Fr-5-3 is a chromatography crude component containing the compound I.

The third step: purification and purification of Compound I

Fr-5-3(3.7g) was applied to Sephadex LH-20 column (bed 3.8 cm. times.36 cm) and eluted with chloroform-methanol (1: 1) to give a chromatography fraction mainly containing Compound I. This fraction was subjected to RP-18 reverse phase analytical high performance liquid chromatography (203nm detection) and when eluted with methanol gave an eluted fraction containing Compound I at a retention time of 8 minutes. Separating by RP-18 reversed-phase preparative high performance liquid chromatography (203nm detection) under the same conditions, eluting with methanol, and collecting corresponding eluate to obtain crude product of compound I. This crude compound I gave a single elution peak of compound I at retention time of 58 minutes in RP-18 reversed phase analytical HPLC (203nm detection) eluting with 80% methanol. Separating by RP-18 reversed phase preparative high performance liquid chromatography (203nm detection) under the same conditions, eluting with 80% methanol, collecting corresponding eluate, concentrating, and recrystallizing in chloroform-methanol to obtain 98mg of pure compound I.

Compound I white crystal powder with melting point 66-68 deg.C, [ alpha ]]_D ³¹-64.9(c 1.0，CHCl₃) Molecular formula C₂₅H₄₄O₄Positive ion TOF-MS m/z: 431[ M + Na ]]⁺，409[M+H]⁺，391[M+H-H₂O]⁺，373[M+H-2H₂O]⁺(ii) a Negative ion TOF-MS m/z: 453[ M-H + HCOOH ]]^-，443[M+Cl]^-，407[M-H]^-，389[M-H-H₂O]^-(ii) a Positive ion HR-TOF-MS m/z: found 409.3329[ M + H]⁺Calculated value 409.3318 (C)₂₅H₄₅O₄[M+H]⁺)。UVλ_max nm(logε)in MeOH：215(3.82)。IR(KBr)v_max cm^-1：3429(OH)，3004(＝CH-)，2925，2852(CH₃&CH₂) 1683 (conjugated CO), 1635, 1460(C ═ C), 1456, 1340, 1271, 1083, 1020, 1047(C-O), 920, 848, 721. CD lambda_max nm(mdeg)in MeOH at 0.1mg/ml：311(+1.92777)，237.6(-52.6351)，208.7(+59.1803)。¹H and¹³the C NMR data are shown in Table 1.

TABLE 1 600MHz of Compound I in deuterated chloroform¹H and 400MHz¹³C nuclear magnetic resonance data^a)

a) The method comprises the following steps Signals in the table are according to DEPT, PFG¹H-¹And attributing the analysis results of the difference spectra of H COSY, PFG HMQC, PFG HMBC and NOE. b) The method comprises the following steps The symbols in this column are represented in PFG¹H-¹The hydrogen nuclei in the H COSY spectrum, which are in the same line position with the hydrogen nuclei in the same line position, give coupling-related signals. c) The method comprises the following steps The symbols in this column are represented in the PFG HMBC spectra (A), (B), (C)^IrJ_CHEither 8.3 or 4Hz) to the hydrogen nucleus in the same row index position gives the carbon atom of the HMBC coupling related signal. d) E), f): the signal cannot be attributed exactly because it overlaps with other signals. g) The method comprises the following steps The signal assignments between two signals with the same superscript are interchangeable.

NOE difference spectrum test analysis of compound I: NOE was observed on H-3 when H-2 was irradiated; NOE were observed on H-2 and H-4, respectively, when H-3 was irradiated; when H-4 is irradiated, NOE is observed separately on H-3, Ha-6, Ha-7, Hb-7 and H-8; NOE was observed on H-4, He-6 when Ha-6 was irradiated; when He-6 was irradiated, NOE signals were observed on Ha-6 and H-8.

EXAMPLE 2 extraction and isolation of Compound II

The first step is as follows: preparation of extract containing Compound II

Referring to the first step of example 1, the same procedure was followed to obtain 110 g of chloroform extract, the main extract containing compound II, from 6 kg of choerospondias axillaris raw material.

The second step is as follows: separation and purification of Compound II from chloroform extract

60g of chloroform extract containing compound II was applied to a vacuum silica gel column (column bed 7.5 cm. times.18.5 cm) by dry method, and eluted with petroleum ether (P) -acetone (A) and methanol solvent system. The polarity of the elution solvent is increased by increasing the amount of acetone (A) in petroleum ether (P) to increase the polarity of the elution solvent in a gradient manner or by changing the amount of acetone (A) to methanol. Combining the fractions according to the results of thin layer detection and activity test to obtain 6 groupsFr-1(3.3g, V)_P∶V_A100: 1 elution fraction), Fr-2(2.1g, V)_P∶V_A50: 1 elution fraction), Fr-3(2.2g, V_P∶V_A10: 1 elution fraction), Fr-4(11.0g, V_P∶V_A5: 1 elution fraction), Fr-5(14.0g, V_P∶V_A2: 1 elution fraction) and Fr-6(24.5g, methanol elution fraction). Fr-5(14.0g) was dried on a reduced pressure silica gel column (bed 5.0 cm. times.22 cm), eluted with chloroform (C) -ethyl acetate (Ac), acetone, methanol system, and fractions were combined according to the results of thin layer assay and activity test to give 6 fractions Fr-5-1(4.3g, V)_C∶V_Ac8: 1 elution fraction), Fr-5-2(1.0g, V)_C∶V_Ac4: 1 elution fraction), Fr-5-3(3.8g, V_C∶V_Ac2: 1 elution fraction), Fr-5-4(1.0g, ethyl acetate elution fraction), Fr-5-5(1.9g, acetone elution fraction), Fr-5-6(0.9g, methanol elution fraction). Wherein Fr-5-4 is a chromatography crude component containing a compound II.

Fr-5-4 was separated by repeated preparative thin layer chromatography (chloroform-methanol 7: 1 development), and recrystallized from chloroform-methanol to give 87mg of pure compound II.

Compound II white crystal powder with melting point 60-62 deg.C, [ alpha ]]_D ³¹+46.4(c 1.0，CHCl₃) Molecular formula C₂₅H₄₆O₃. Positive ion TOF-MS m/z: 433[ M + K ]]⁺，417[M+Na]⁺，412[M+NH₄]⁺，395[M+H]⁺，394[M]⁺，377[M+H-H₂O]^-，359[M+H-2H₂O]⁺(ii) a Negative ion TOF-MS m/z: 439[ M-H + HCOOH]^-，429[M+Cl]^-(ii) a Positive ion HR-TOF-MS m/z: found 395.3523[ M + H]⁺Calculated value 395.3525 (C)₂₅H₄₇O₃[M+H]⁺)。UVλ_maxnm (log ε) in MeOH: 200(3.77, terminal absorptions). IR (KBr) v_max cm^-1：3367(OH)，2923，2852(CH₃&CH₂)，1460，1457(C＝C)，1379(CH₃)，1091，1063，1028(C-O)，835，721。CDλ_max nm(mdeg)in MeOH at0.1mg/ml：201.1(+98.4664)，194.2(-49.1499)，191.1(+11.0594)。¹H and¹³the CNMR data are shown in Table 2.

TABLE 2 600MHz of Compound II in deuterated chloroform¹H and 400MHz¹³C nuclear magnetic resonance data^a)

a) The method comprises the following steps The signals in the table were assigned according to the results of differential spectrum analysis of DEPT, PEG1H-1HCOSY, PFG HMQC, PFG HMBC, and NOE. b) The method comprises the following steps The symbols in this column are represented in PFG¹H-¹The hydrogen nuclei in the H COSY spectrum, which are in the same line position with the hydrogen nuclei in the same line position, give coupling-related signals. c) The method comprises the following steps The symbols in this column are represented in the PFG HMBC spectra (A), (B), (C)^IrJ_CHEither 8.3 or 4Hz) to the hydrogen nucleus in the same row index position gives the carbon atom of the HMBC coupling related signal. d) E), f): the signal cannot be attributed exactly because it overlaps with other signals. g) H): the signal assignments between two signals with the same superscript are interchangeable.

NOE difference spectrum test analysis of compound II: when H-1 is irradiated, at 1-OHNOE was observed in H-2, Ha-5 and Ha-7; when H-4 is irradiated, at H-3, 4-OH、He-5、6-OHNOE was observed above, respectively; when Ha-5 is irradiated, H-1, He-5, H₂NOE observed at-7, respectively; when He-5 is irradiated, H-4, Ha-5, H₂NOE signals were observed at-7 and Ha-8.

Example 3 bioactivity test

Experimental sample and experimental method

Preparing a solution of a sample to be detected: the compound I separated and purified in example 1 and the compound II separated and purified in example 2 were weighed out precisely and mixed with methanol to give a solution of the desired concentration for measuring the activity.

Cell lines and cell culture: the activity test adopts a mouse breast cancer tsFT210 cell line, a human colorectal cancer HCT-15 cell line, a human cervical cancer HeLa cell line, a human breast cancer MCF-7 cell line and a human ovarian cancer A2780 cell line.

The cells were subcultured in RPMI-1640 medium containing 10% FBS at 32 deg.C (tsFT210 cells) or 37 deg.C (HCT-15, HeLa, MCF-7 and A2780 cells) in an incubator with 5% carbon dioxide.

Cell proliferation inhibitory activity test method (lissamine rhodamine B method, SRB method): preparing human colorectal cancer HCT-15 cells, human cervical cancer HeLa cells, human breast cancer MCF-7 cells and human ovarian cancer A2780 cells in logarithmic growth phase into 2 x 10/ml cells by using fresh RPMI-1640 culture medium⁵The cell suspension of each cell was inoculated into a 96-well plate at 200. mu.l per well, and 2. mu.l of each sample or blank solution at different concentrations was added thereto, followed by incubation at 37 ℃ for 24 hours. The cells cultured under the action of the medicine are taken, and the morphological change caused by the medicine treatment is observed under an optical microscope to judge whether the morphological characteristics of cell cycle inhibition, cell apoptosis or cell necrosis exist. The supernatant was then aspirated and 50. mu.l of 20% trichloroacetic acid was added to each well of cells, fixed at 4 ℃ for 1 hour, rinsed 5 times with water and air dried. 50 microliters of 0.4% SRB in acetic acid was added to each well and allowed to stand at room temperature for 30 minutes. Unbound free SRB dye was removed by washing 4 times with 1% acetic acid water. 150 μ L of Tris buffer (10mmol/L, pH 10.5) was added to each well to dissolve the protein-binding dye and the Optical Density (OD) at 520nm of each well was measured using a SPECTRA MAX Plus type microplate reader manufactured by MD. Three wells were placed for each concentration of sample in the same 96 well plate, three additional well blanks were placed, and the mean OD values in IR% (OD)_{Blank control}-OD_{Sample (I)})/OD_{Blank control}X 100% formula the inhibition of cell proliferation (IR%) was calculated at each concentration.

Flow cytometry test methods: taking tsFT210 cells in logarithmic growth phase, preparing the cells into a density of 2 x 10 per ml by using fresh RPMI-1640 medium⁵Individual cell ofThe suspension was inoculated into 24-well plates at 0.5 ml per well, and 5. mu.l of each sample solution was added thereto at different concentrations, followed by incubation at 32 ℃ for 17 hours. The cells cultured under the action of the drug are taken, the morphological change caused by the drug treatment is observed under an optical microscope, the morphological characteristics of the cells such as cell cycle inhibition, cell apoptosis or cell necrosis are judged, and if necessary, the cells are photographed. Then, the cells were transferred from 24-well plates to 1.5 ml Eppendorf centrifuge tubes, centrifuged at 3000 rpm at 4 ℃ for 3 minutes, the supernatant was aspirated, washed once with shaking by adding 0.5 ml Phosphate Buffer Solution (PBS), centrifuged under the same conditions to collect the cells, added with 150. mu.l of an aqueous solution of Propidium Iodide (PI) (5 mg of PI, 100mg of sodium citrate, and 200 mg of NP-40 in 100 ml of water), stained at 4 ℃ for 30 minutes, diluted with 150. mu.l of PBS, and analyzed by flow cytometry to determine the DNA content distribution in the cells.

Results of the experiment

Inhibitory Activity of Compound I and Compound II on human cancer cell proliferation

In the SRB method test, the compound I and the compound II both show certain inhibitory activity to the cell proliferation of human colorectal cancer HCT-15 cells, and the test results of the inhibitory activity of different concentrations of the compound I and the compound II to the cell proliferation of human colorectal cancer HCT-15 cells are shown in Table 3.

TABLE 3 SRB assay results of compound I and compound II for inhibition of HCT-15 cell proliferation in human colorectal cancer

In the SRB method test, the compound I also shows inhibitory activity of different degrees on the proliferation of human cervical cancer HeLa cells, human ovarian cancer A2780 cells and human breast cancer MCF-7 cells, and the test results of the inhibitory activity of different concentrations of the compound I on the proliferation of the human cancer cells are shown in Table 4.

TABLE 4 SRB assay results for inhibition of human cancer cell proliferation by Compound I

The results in tables 3 and 4 show that both compound I and compound II show varying degrees of anticancer activity against the cancer cells tested in the SRB assay.

Flow cytometry results

Flow cytometry detection analysis results: tsFT210 cells were treated with different concentrations of compound I and compound II and assayed by flow cytometry. The results show that: compound I predominantly exhibits cell cycle inhibitory activity at concentrations ranging from 0.19 micrograms per ml to 0.78 micrograms per ml, inhibiting the cell cycle of tsFT210 cells at stage G0/G1; and the apoptosis induction activity is presented at the same time from the concentration of 0.78 microgram per milliliter, obvious apoptosis peaks are detected in a sub-G0/G1 region, the apoptosis peaks are more obvious along with the increase of the concentration, and the apoptosis induction activity is mainly presented from 0.78 microgram per milliliter; necrotic cytotoxic activity began to be detected at concentrations above 12.5 micrograms per ml. Compound II predominantly inhibited the cell cycle of tsFT210 cells at G0/G1 at concentrations ranging from 3.12 micrograms per ml to 12.5 micrograms per ml, with predominantly apoptosis-inducing and some S-phase inhibitory activity beginning at concentrations above 25 micrograms per ml, and necrotic cytotoxic activity occurring at concentrations at or above 100 micrograms per ml. The flow cytometry analysis and detection results are basically consistent with the morphological detection results observed under the microscope.

And (3) morphological detection results: morphological features that when tsFT210 cells are treated with 0.78 microgram per ml of compound I and 25 microgram per ml of compound II, some cells begin to undergo apoptosis, and when treated with 1.56 microgram per ml of compound I or more and 50 microgram per ml of compound II, most cells in the field exhibit typical apoptotic morphological features, i.e. snowflake or membrane-bound cell debris; when treated with compound I at or above 12.5 micrograms per ml and compound II at or above 50 micrograms per ml, a proportion of the cancer cells began to exhibit morphological characteristics typical of necrotic cells, and as the concentrations of compounds I and II increased, necrotic cytotoxic activity also increased, indicating that compound I and compound II at high concentrations had direct cytotoxic activity against mammalian cancer cells.

Conclusion

The experimental results show that the compound I and the compound II can play the anti-tumor role of inhibiting cancer cell proliferation by inhibiting cell cycle, inducing cancer cell apoptosis or directly killing cytotoxic activity to the cancer cells. Therefore, the compound of the present invention can be used as an antitumor agent for treating tumors, and can also be used as a low molecular probe for inducing apoptosis or inhibiting cell cycle for life science research for exploring the essence of life phenomenon.

Claims

1. An extract characterized by containing at least one compound of formula I:

formula I

Wherein,

r is oxygen, R₁、R₂、R₆Is hydrogen, R₃Is 2-hydroxy- (Z) -14-ene nonadecyl,R₄、R₅is a hydroxyl group; or

R is beta-hydroxy and alpha-hydrogen, R₁And R₅Is hydroxy, R₂Is (Z) -14-enenonalkyl, R₃、R₄、R₆Is hydrogen.

2. A compound of formula I:

formula I

Wherein,

r is oxygen, R₁、R₂、R₆Is hydrogen, R₃Is 2-hydroxy- (Z) -14-enenonalkyl, R₄、R₅Is a hydroxyl group; or

3. A pharmaceutical composition comprising as active ingredient the extract of claim 1 or the compound of claim 2 together with one or more pharmaceutically acceptable carriers or excipients.

4. A method for preparing the extract of claim 1, which comprises extracting Choerospondias axillaris with an alcohol or aqueous alcohol to obtain the extract.

5. A process for the preparation of a compound of formula I according to claim 2, which comprises leaching choerospondias axillaris with an alcohol or aqueous alcohol to obtain a crude extract, and then separating and purifying the crude extract to obtain the compound of formula I.

6. Use of an extract according to claim 1 and/or a compound of formula I according to claim 2 for the preparation of a cell cycle inhibitor, an apoptosis-inducing agent, an inhibitor of cell proliferation or a tumor cell killing agent.

7. Use of an extract according to claim 1 and/or a compound of formula I according to claim 2 for the preparation of an antitumor medicament.