CN1872828A - Monocyclic polysubstitution cyclohexenol, and compound of ketones, and preparation method, and usage - Google Patents

Monocyclic polysubstitution cyclohexenol, and compound of ketones, and preparation method, and usage Download PDF

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CN1872828A
CN1872828A CN 200510074808 CN200510074808A CN1872828A CN 1872828 A CN1872828 A CN 1872828A CN 200510074808 CN200510074808 CN 200510074808 CN 200510074808 A CN200510074808 A CN 200510074808A CN 1872828 A CN1872828 A CN 1872828A
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hydroxyl
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CN1872828B (en
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崔承彬
李长伟
蔡兵
韩冰
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Institute of Pharmacology and Toxicology of AMMS
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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

The polysubstituted cyclohexenol of monocycle and ketone compounds and preparation method thereof and purposes
Technical field:
The present invention relates to the polysubstituted cyclohexenol of monocycle and ketone compounds and its production and use.Be specifically related to contain a polysubstituted cyclohexenol of the substituent monocycle of long chain hydrocarbon and ketone compounds, the extract that contains this compounds, extraction separation and purifying and obtain the purposes that the method for this compounds and described extract or compound are used to prepare cell cycle inhibitor, cell death inducer, inhibition of cell proliferation, cancer cells kill agent and antineoplastic agent.
Background technology:
Monocycle substituted cyclohexene alcohol or ketone compounds mean a series of compounds that are connected with hydroxyl or carbonyl and other different substituents on the carbon skeleton of tetrahydrobenzene.Such skeletal structure of compound is single, mainly forms different compounds because of substituting group kind and the position of substitution different.Wherein, contain a polysubstituted cyclohexenone compounds of the substituent monocycle of long chain hydrocarbon bibliographical information is arranged a bit.As document S.R.Johns, et al., Campnosperma Exudates.The opticallyactive long-chain 5-hydroxyclohex-2-enones and long-chainbicycle[3.3.1] nonane-3,7-diones:Aust.J.Chem., 40,79-96,1987, document E.F.Queiroz, et al., New dihydroalkylhexenones from Lannea edulis, J.Nat.Prod., 66,578-580,2003, document S.J.Correia, et al., Alkyl phenolsand derivatives from Tapirira obtuse:Phytochem., 56,781-784,2001 and document A.Groweiss, et al., Novel cytotoxic, alkylated hydroquinones fromLannea welwitschii, J.Nat.Prod., 60,116-121,1997 grades had all once been put down in writing several polysubstituted cyclohexenone compounds of monocycle that contain a long chain hydrocarbon groups (17 carbon or nineteen carbon).Document S.J.Correia, et al., Alkyl phenols and derivatives from Tapiriraobtuse:Phytochem., 56,781-784,2001 had also once reported 4 cyclohexenol analog derivatives that contain one 16 carbochain alkyl, but the other end of 16 carbochain alkyl also is connected with a phenyl ring in these 4 compounds.So far, polysubstituted cyclohexenol of monocycle or the ketone compounds with long chain hydrocarbon substituting group type identical with The compounds of this invention do not seen bibliographical information as yet.
Fructus Choerospondiatis Choerospondias axillaries (Roxb.) Burtt et Hill is an Anacardiaceae Fructus Choerospondiatis platymiscium, its bark and fruit medicine, bark treatment of ulcer, burn, eczema scrotum, fresh fruit is eliminating indigested food then, control the food stomachache that stagnates, the fruit stone detoxifcation of sobering up is controlled the wind poison and is played a pimple and become sore or ulcer carbuncle (the new medical college in Jiangsu volume, the Chinese medicine voluminous dictionary, the first volume, Shanghai, the Shanghai People's Press, 1977,397-398 page or leaf and the 1564th page).Dry fruit is as Chinese medicine " fructus choerospondiatis " income China version pharmacopeia (Chinese Pharmacopoeia Commission, Pharmacopoeia of People's Republic of China, an one, Beijing, Chemical Industry Press,, the 32nd page in 2000) in 2000.The existing report of polytype chemical ingredientss such as the flavones in the Fructus Choerospondiatis, phenolic acid, organic acid, amino acid, steroidal, the also existing research report of various active (Xiong Dongsheng etc. such as antibiotic, the anticoagulant of Fructus Choerospondiatis crude extract, total flavones or monomeric compound; Research overview and the application prospect thereof of Fructus Choerospondiatis plant aspect medicine: Guangdong pharmacy, 2000 the 10th the 5th phases of volume, 8-10 page or leaf).But polysubstituted cyclohexenol of the monocycle that the anti-tumor activity of Fructus Choerospondiatis and activeconstituents thereof especially the present invention relates to or ketone activeconstituents are not seen the research report so far.
Summary of the invention:
The present invention aims to provide a kind ofly to have cell cycle inhibition, apoptosis-inducing and the anti-tumor activities such as direct killing of cancer cells is contained a polysubstituted cyclohexenol of the substituent monocycle of long chain hydrocarbon or ketone compounds.
The inventor finds that first the crude extract of Fructus Choerospondiatis has good lethal cell toxicant, apoptosis-inducing, cell cycle inhibition to reach the anti-tumor activities such as inhibition to cancer cell multiplication to cancer cells, then its activeconstituents is studied, and is found that polysubstituted cyclohexenol of monocycle shown in the following formula I in the Fructus Choerospondiatis crude extract or ketone compounds have above-mentioned anti-tumor activity:
Figure A20051007480800051
Formula I
Wherein, R is hydrogen and hydroxyl (formation cyclohexenol), or is oxygen (formation cyclonene); R 1, R 2, R 3, R 4, R 5, R 6Be hydrogen, hydroxyl, amino, methoxyl group or the optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-20Alkyl, and, R 1-R 6In at least one is the described optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-C 20Alkyl.
Therefore, first aspect of the present invention relates to a kind of extract, it is characterized in that wherein comprising the polysubstituted cyclohexenol of the monocycle shown at least a formula I and ketone compounds and pharmacy acceptable salt thereof:
Figure A20051007480800061
Formula I
Wherein, R is hydrogen and hydroxyl, or is oxygen; R 1, R 2, R 3, R 4, R 5, R 6Be hydrogen, hydroxyl, amino, methoxyl group or the optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-20Alkyl, and, R 1-R 6In at least one is the described optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-C 20Alkyl.
Second aspect of the present invention relates to the polysubstituted cyclohexenol of the monocycle shown in the formula I and ketone compounds and pharmacy acceptable salt thereof:
Figure A20051007480800062
Formula I
Wherein, R is hydrogen and hydroxyl, or is oxygen; R 1, R 2, R 3, R 4, R 5, R 6Be hydrogen, hydroxyl, amino, methoxyl group or the optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-20Alkyl, and, R 1-R 6In at least one is the described optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-C 20Alkyl.
The 3rd aspect of the present invention relates to the method for preparing the said extracted thing, described method comprise with alcohol or aqueous alcohol to relevant plant material for example Fructus Choerospondiatis extract, crude extract be an extract
The 4th aspect of the present invention relates to by described extract and separating and the method for purifying formula I compound, and described method is passed through separation and purification again and obtained formula I compound after comprising that with alcohol or aqueous alcohol for example Fructus Choerospondiatis extracts to relevant plant material.
The 5th aspect of the present invention relates to and containing as the polysubstituted cyclohexenol of formula I monocycle of activeconstituents and the pharmaceutical composition of ketone compounds and one or more pharmaceutical carriers or vehicle.
The 6th aspect of the present invention relates to the purposes that the polysubstituted cyclohexenol of formula I monocycle and ketone compounds are used to prepare cell cycle inhibitor, cell death inducer, inhibition of cell proliferation cancer cells kill agent and antineoplastic agent.
In an embodiment of the invention, the present invention relates to the polysubstituted cyclohexenol of the monocycle shown in the formula I and ketone compounds and pharmacy acceptable salt thereof:
Figure A20051007480800071
Formula I
Wherein, R is hydrogen and hydroxyl, or is oxygen; R 1, R 2, R 3, R 4, R 5, R 6Be hydrogen, hydroxyl, amino, methoxyl group or the optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-20Alkyl, and, R 1-R 6In at least one is the described optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-C 20Alkyl.
In a preferred embodiment of the invention, the present invention relates to the polysubstituted cyclohexenol of formula I monocycle and ketone compounds and pharmacy acceptable salt thereof:
Figure A20051007480800081
Formula I
Wherein, R is hydrogen and hydroxyl, or is oxygen; R 1, R 2, R 3And R 4Be hydroxyl or optional (the Z)-14-alkene nonadecyl that is replaced by substituting groups such as hydroxyl, amino, and, R 2Or R 3At least one is optional (the Z)-14-alkene nonadecyl that is replaced by substituting groups such as hydroxyl, amino; R 5, R 6Be hydroxyl or hydrogen.
In the further preferred embodiment of the present invention, the R among the formula I is an oxygen, R 1, R 2, R 6Be hydrogen, R 3Be 2-hydroxyl-(Z)-14-alkene nonadecyl, R 4, R 5Be hydroxyl (absolute configuration is 4R5S8R).
In another further preferred embodiment of the present invention, the R among the formula I is beta-hydroxy and α-hydrogen, R 1And R 5Be hydroxyl, R 2Be (Z)-14-alkene nonadecyl, R 3, R 4, R 6Be hydrogen (absolute configuration is 1R4S6S).
Containing the extract of above-mentioned formula I compound and pure formula I compound can prepare by the following method: with alcohol or aqueous alcohol to relevant vegetable material for example Fructus Choerospondiatis extract, crude extract be an extract, and then separation and purification and obtain formula I compound from crude extract.
According to the present invention, the alcohol that is adopted in the aforesaid method is ethanol; Described aqueous alcohol is 60～95% aqueous ethanol; Described separation and purification comprises ordinary method and the means of utilizing Separation of Natural Products purifying well known to those skilled in the art, as liquid-liquid extraction, column chromatography, thin-layer chromatography, high performance liquid chromatography and recrystallization etc.Wherein column chromatography, high performance liquid phase and recrystallizing and refining can carry out repeatedly repeatedly.
The present invention adopts lissamine rhodamine B (sulforhodamine B, SRB) method and flow cytometry detect the method for cell morphological characteristic in conjunction with microscopically, have tested formula I compound to cell inhibitory effect, cell cycle inhibition and the apoptosis-inducing of mouse breast cancer tsFT210 cell, human large intestine cancer HCT-15 cell, HeLa Cells, human breast carcinoma MCF-7 cell, human ovarian cancer A2780 cell and to the effects such as direct killing of cancer cells.Through experiment confirm, formula I compound can be by suppressing the cell cycle turnover, bring out cancer cell-apoptosis or mode such as directly kill and wound shows the biologic activity that suppresses tumor cell proliferation to tumour cell, thereby have antitumor action.
Formula I compound of the present invention and various medicine acceptable carrier, vehicle or supplementary product compatibility can be made into antitumor drug, are used for tumor treatment.
Term among the present invention " pharmacy acceptable salt " can be medicinal inorganic or organic salt.The compound that has basic group among the formula I of the present invention can form pharmaceutical salts with mineral acid, for example vitriol, hydrochloride, hydrobromate, phosphoric acid salt; Also can form pharmaceutical salts, for example acetate, oxalate, Citrate trianion, gluconate, succinate, tartrate, tosilate, mesylate, benzoate, lactic acid salt, maleate etc. with organic acid.The compound that has acidic-group among the formula I of the present invention can form pharmaceutical salts with basic metal or alkaline-earth metal, and is preferred but be not limited to sodium salt, sylvite, magnesium salts or calcium salt.
The compounds of this invention can be separately or with the form administration of pharmaceutical composition.Route of administration can be oral, non-enteron aisle or topical.Pharmaceutical composition can be made into various suitable formulations according to route of administration.
The pharmaceutical composition of The compounds of this invention can be used with following any-mode: oral, spraying sucks, rectal application, nasal cavity applied medicine, cheek medication, local application, non-enterally administer, as subcutaneous, vein, intramuscular, intraperitoneal is in the sheath, in the ventricle, breastbone interior and intracranial injection or input, or by the medication of a kind of outer planting reservoir.Wherein preferred oral, intraperitoneal or intravenous administration mode.
When medicine for oral use, The compounds of this invention can be made into oral acceptable dosage form arbitrarily, includes but not limited to tablet, capsule, the aqueous solution or aqeous suspension.Wherein, the carrier that tablet uses generally comprises lactose and W-Gum, also can add lubricant such as Magnesium Stearate in addition.The thinner that capsule preparations uses generally comprises lactose and dried corn starch.Aqueous suspension preparation then normally mixes use with activeconstituents with examples of suitable emulsifiers and suspension agent.Randomly, also can add some sweeting agents, perfume compound or tinting material in the above oral preparations form.
When topical application, The compounds of this invention can be made into suitable ointment, lotion or creme dosage form, wherein activeconstituents is suspended or is dissolved in one or more carriers.The spendable carrier of ointment formulation includes but not limited to: mineral oil, Albolene, white vaseline, propylene glycol, polyoxyethylene, polyoxytrimethylene, emulsifying wax and water; The spendable carrier of lotion or creme includes but not limited to: mineral oil, sorbitan monostearate, polysorbate60, n-Hexadecane ester type waxes, cetene are fragrant and mellow, 2-Standamul G, benzyl alcohol and water.
The all right aseptic injection preparation form medication of The compounds of this invention comprises aseptic injection water or oil suspension or aseptic injectable solution.Wherein, spendable carrier and solvent comprise water, Ringer's solution and isotonic sodium chlorrde solution.In addition, the fixed oil of sterilization also can be used as solvent or suspension medium, as direactive glyceride or two glyceryl ester.
It may be noted that in addition, The compounds of this invention using dosage and using method depend on all multifactor, comprise activity intensity, Time of Administration, metabolic rate, the severity of illness and diagnosis and treatment doctor's the subjective judgement of patient's age, body weight, sex, natural health situation, nutritional status, compound.Preferred using dosage is between the 0.01-100mg/kg body weight/day.
Formula I compound of the present invention also can be used as and suppresses the cell cycle or bring out apoptotic low molecular biosciences probe to be used for life science.When wushu I compound is used for life science as cell cycle inhibitor or cell death inducer, dissolve in methyl alcohol or the aqueous methanol, also dissolve in the aqueous solution of dimethyl sulfoxide (DMSO) and be applied.
Description of drawings:
Fig. 1 is the ultra-violet absorption spectrum of Compound I in methanol solution (40 μ g/ml);
Fig. 2 is the infrared absorption spectrum (KBr) of Compound I;
Fig. 3 is that Compound I is in deuterochloroform 1The H nuclear magnetic resonance spectrum;
Fig. 4 is that Compound I is in deuterochloroform 13The C nuclear magnetic resonance spectrum;
Fig. 5 is circular double dispersion (CD) spectrum of Compound I in methanol solution (0.1mg/ml);
Fig. 6 is the ultra-violet absorption spectrum of Compound I I in methanol solution (40 μ g/ml);
Fig. 7 is the infrared absorption spectrum (KBr) of Compound I I;
Fig. 8 is that Compound I I is in deuterochloroform 1The H nuclear magnetic resonance spectrum;
Fig. 9 is that Compound I I is in deuterochloroform 13The C nuclear magnetic resonance spectrum;
Figure 10 is circular double dispersion (CD) spectrum of Compound I I in methanol solution (0.1mg/ml);
Figure 11 is the fluidic cell histogram that mouse breast cancer tsFT210 cell records after 17 hours with the Compound I processing;
Figure 12 is the fluidic cell histogram that mouse breast cancer tsFT210 cell records after 17 hours with Compound I I processing.
Embodiment:
The following example will further specify the present invention, but the present invention will not be construed as limiting.
In following examples, have following structure hereinafter referred to as the The compounds of this invention of Compound I through evaluations such as nucleus magnetic resonance: formula I compound, wherein R is an oxygen, R 1, R 2, R 6Be hydrogen, R 3Be 2-hydroxyl-(Z)-14-alkene nonadecyl, R 4, R 5Be hydroxyl (absolute configuration is 4R5S8R); The compounds of this invention hereinafter referred to as Compound I I has following structure through evaluations such as nucleus magnetic resonance: formula I compound, wherein R is beta-hydroxy and α-hydrogen, R 1And R 5Be hydroxyl, R 2Be (Z)-14-alkene nonadecyl, R 3, R 4, R 6Be hydrogen (absolute configuration is 1R4S6S):
Figure A20051007480800111
Formula I
Arabic numerals on carbocyclic ring and the long-chain are represented the mark of corresponding carbon atom
In the structural research of compound, fusing point is measured with the accurate micro melting point apparatus of the world, Beijing space science and technology limited Company X-4 type, and temperature is not proofreaied and correct.Specific rotatory power is measured with JSASCO P-1020 type polarimeter.Negative ions TOF-MS and HR-TOF-MS measure with the American AB I API of company 3000 type liquid chromatograph-mass spectrometers and the Britain LCT of Micromass company type mass spectrograph respectively, UV spectrum is measured infrared spectra Nicolet Magna-IR with Shimadzu UV2501PC type uv-spectrophotometric instrument TM550 type determination of infrared spectroscopy, nuclear magnetic resonance spectrum JEOLEclips-600 type NMR spectrometer with superconducting magnet (600MHz 1H-NMR, 150MHz 13C-NMR) measure.Circular double dispersion (CD) spectrum is measured with JASCO J-810 Spectropolarimeter.
The extraction and the separation and purification of embodiment 1. Compound I
First step: contain the preparation of the extract of Compound I
Get the dry bark (3.2kg) of Fructus Choerospondiatis (picking up from area, Menla, Yunnan in August, 1999) and pulverize the back, soaked 7 days at every turn, carry altogether 4 times with 25 liters of room temperature lixiviates of ethanol.United extraction liquid, concentrate drying gets ethanol extraction 750g.Ethanol extraction 750g is suspended in 3 premium on currency, use chloroform (3 liters), ethyl acetate (3 liters), propyl carbinol (3 liters) respectively to extract successively 4 times, obtain chloroform extract 60g, ethyl acetate extract 310g, n-butanol extract 300g and water layer residue 80g respectively.Wherein, chloroform extract is the extract that contains Compound I.
Second step: contain the preparation of the thick component of chromatography of Compound I
Silicagel column (post bed 7.5cm * 18.5cm), carry out wash-out with sherwood oil (P)-acetone (A), methanol solvate system reduces pressure on chloroform extract (60g) dry method.Come gradient to increase progressively the polarity of eluting solvent or be replaced with the polarity that methyl alcohol strengthens eluting solvent by the consumption that increases acetone (A) in the sherwood oil (P).Merge stream part according to thin layer detection and active testing result, obtain 6 component Fr-1 (3.2g, V P: V A=100: 1 wash-out part), Fr-2 (2.3g, V P: V A=50: 1 wash-out part), Fr-3 (2.0g, V P: V A=10: 1 wash-out part), Fr-4 (10.8g, V P: V A=5: 1 wash-out part), Fr-5 (13.6g, V P: V A=2: 1 wash-out partly) and Fr-6 (25g, methanol-eluted fractions part).(strain bed 5.0cm * 22cm), with chloroform (C)-ethyl acetate (Ac), acetone, methyl alcohol system wash-out, merge stream part according to thin layer detection and active testing result obtains 6 component Fr-5-1 (4.1g, V with the silicagel column that reduces pressure on Fr-5 (13.6g) dry method C: V Ac=8: 1 wash-out part), Fr-5-2 (980mg, V C: V Ac=4: 1 wash-out part), Fr-5-3 (3.7g, V C: V Ac=2: 1 wash-out part), Fr-5-4 (1.1g, eluent ethyl acetate part), Fr-5-5 (1.8g, acetone wash-out part), Fr-5-6 (0.8g, methanol-eluted fractions part).Wherein, Fr-5-3 is the thick component of chromatography that contains Compound I.
Third step: the purifying of Compound I and refining
Fr-5-3 (3.7g) is gone up Sephadex LH-20 post (post bed 3.8cm * 36cm), with chloroform-methanol (1: 1) wash-out, mainly contained the chromatography component of Compound I.This component when using methanol-eluted fractions, located to provide the elution fraction that contains Compound I in 8 minutes in retention time in the anti-phase analysis high performance liquid chromatography of RP-18 (203nm detection).Separate through the anti-phase preparation high performance liquid chromatography of the same terms RP-18 (203nm detection), with methanol-eluted fractions and collect corresponding elution fraction, obtain the Compound I crude product.This Compound I crude product was located to provide the single elution peak of Compound I in 58 minutes in retention time in the anti-phase analysis high performance liquid phase (203nm detection) with the RP-18 of 80% methanol-eluted fractions.Separate through the anti-phase preparation high performance liquid chromatography of the same terms RP-18 (203nm detection), with 80% methanol-eluted fractions and the corresponding elution fraction collected, concentrated and in chloroform-methanol recrystallization get the pure product 98mg of Compound I.
Compound I white crystals type powder, fusing point 66-68 ℃, [α] D 31-64.9 (c1.0, CHCl 3), molecular formula C 25H 44O 4, positive ion TOF-MS m/z:431[M+Na] +, 409[M+H] +, 391[M+H-H 2O] +, 373 [M+H-2H 2O] +Negative ion TOF-MS m/z:453[M-H+HCOOH] -, 443[M+Cl] -, 407[M-H] -, 389[M-H-H 2O] -Positive ion HR-TOF-MS m/z: measured value 409.3329[M+H] +, calculated value 409.3318 (C 25H 45O 4[M+H] +).UVλ max nm(logε)in MeOH：215(3.82)。IR (KBr) ν MaxCm -1: 3429 (OH), 3004 (=CH-), 2925,2852 (CH 3﹠amp; CH 2), 1683 (conjugation CO), 1635,1460 (C=C), 1456,1340,1271,1083,1020,1047 (C-O), 920,848,721.CDλ max nm(mdeg)in MeOH at 0.1 mg/ml：311(+1.92777)，237.6(-52.6351)，208.7(+59.1803)。 1H reaches 13C NMR data see Table 1.
The 600MHz of table 1 Compound I in deuterochloroform 1H and 400MHz 13The C nuclear magnetic resonance data a)
Mark δH(J in Hz) COSY b) HMBC c) δ c
1 2 3 4 5 6 7 8 9 10 11 12-16 17 18 19 20 21 22 23 24 25 O HO HO H —— 6.02dm(10.1) 6.86dd(10.1，2.3) 4.56dd(2.8，2.3) —— Ha 2.56dd(16.0，0.9) He 2.90brd(16.0) Ha 1.89dd(14.9，11.2) Hb 1.61dd(14.9，1.6) 4.03m 1.46(2H)m Ha 1.33m Hb 1.26m 1.28(2H)m 1.23-1.30 d)m 1.30-1.37 f)m 1.30-1.37 f)m 2.02(2H)m 5.35ABtype 5.35ABtype 2.02(2H)m 1.30-1.37 f)m 1.30-1.37 f)m 0.90(3H)t(7.1) 2.44brs 3.43brs 4.68brs H-3，4，He-6 H-2，4 H-2，3 He-6，Ha-7 H-2，Ha-6 Ha-6，Hb-7，H-8 Ha-7，H-8 Ha-7，Hb-7，H 2-9 H-8，H 2-10 H 2-9，Hb-10，H-11 H 2-9，Ha-10，H-11 Ha-10，Hb-10，H-12 H-11，17 H-16，18 H-17，19 H-18，20 H-19，21 H-20，22 H-21，23 H-22，24 H-23，25 H-24 C-4，6 C-1，5 C-2，3，5～7 C-1，C-4，5，7 C-1，2，C-4，5，7 C-4，5，8，9 C-4～6，C-8，9 C-8，7，10～12 C-11，12 C-11，12 C-10，12，13 C-10～18 C-15，16，18，19 C-16，17，19，20 C-17，18，20，21 C-19，22 C-19，22 C-20，21，23，24 C-21，22，24 C-22，23 C-23，24 198.28s 129.23d 149.78d 74.19d 77.61s 49.05t 37.17t 69.12d 38.71t 29.29t 25.11t 29.4-29.7 e)t 31.93t 23.32t 26.88 g)t 129.85d 129.85d 27.17 g)t 22.32t 31.93t 13.98q —— —— ——
A): signal is according to DEPT, PFG in the table 1H- 1H COSY, PFG HMQC, PFG HMBC and NOE difference spectrum analysis result are belonged to.B): the code name in this hurdle is illustrated respectively in PFG 1H- 1In the H COSY spectrum with same rower position in proton provide the proton of coupling coherent signal.C): the code name in this hurdle be illustrated respectively in PFG HMBC spectrum ( LrJ CH=8.3 or 4Hz) in same rower position in proton provide the carbon atom of HMBC coupling coherent signal.D), f e)): this signal is because of definitely belonging to overlapped the failing of other signal.G): it is interchangeable to have the identical signal ownership that goes up between two signals of target.
The NOE difference spectrum Measurement results of Compound I: when irradiation H-2, on H-3, observe NOE; When irradiation H-3, on H-2 and H-4, observe NOE respectively; When irradiation H-4, on H-3, Ha-6, Ha-7, Hb-7, H-8, observe NOE respectively; When irradiation Ha-6, on H-4, He-6, observe NOE; When irradiation He-6, on Ha-6 and H-8, observe the NOE signal.
Extraction and the separation and purification of embodiment 2. Compound I I
First step: contain the preparation of the extract of Compound I I
With reference to the first step among the embodiment 1, with method operation, the main extract that has been obtained to contain Compound I I by 6 kilograms of Fructus Choerospondiatis raw materials is chloroform extract 110 grams.
Second step: the separation and purification of Compound I I in the chloroform extract
Get chloroform extract 60 grams that contain Compound I I, the silicagel column that reduces pressure on the dry method (post bed 7.5cm * 18.5cm), carry out wash-out with sherwood oil (P)-acetone (A), methanol solvate system.Come gradient to increase progressively the polarity of eluting solvent or be replaced with the polarity that methyl alcohol strengthens eluting solvent by the consumption that increases acetone (A) in the sherwood oil (P).Merge stream part according to thin layer detection and active testing result, obtain 6 component Fr-1 (3.3g, V P: V A=100: 1 wash-out part), Fr-2 (2.1g, V P: V A=50: 1 wash-out part), Fr-3 (2.2 g, V P: V A=10: 1 wash-out part), Fr-4 (11.0g, V P: V A=5: 1 wash-out part), Fr-5 (14.0g, V P: V A=2: 1 wash-out partly) and Fr-6 (24.5g, methanol-eluted fractions part).(post bed 5.0cm * 22cm), with chloroform (C)-ethyl acetate (Ac), acetone, methyl alcohol system wash-out, merge stream part according to thin layer detection and active testing result obtains 6 component Fr-5-1 (4.3g, V with the silicagel column that reduces pressure on Fr-5 (14.0g) dry method C: V Ac=8: 1 wash-out part), Fr-5-2 (1.0g, V C: V Ac=4: 1 wash-out part), Fr-5-3 (3.8g, V C: V Ac=2: 1 wash-out part), Fr-5-4 (1.0g, eluent ethyl acetate part), Fr-5-5 (1.9g, acetone wash-out part), Fr-5-6 (0.9g, methanol-eluted fractions part).Wherein, Fr-5-4 is the thick component of chromatography that contains Compound I I.
Fr-5-4 is separated through repeatedly preparation thin-layer chromatography (chloroform-methanol launches at 7: 1), and recrystallization gets the pure product 87mg of Compound I I in chloroform-methanol.
Compound I I white crystals type powder, fusing point 60-62 ℃, [α] D 31+ 46.4 (c1.0, CHCl 3), molecular formula C 25H 46O 3Positive ion TOF-MS m/z:433[M+K] +, 417[M+Na] +, 412[M+NH 4] +, 395[M+H] +, 394[M] +, 377[M+H-H 2O] -, 359[M+H-2H 2O] +Negative ion TOF-MS m/z:439[M-H+HCOOH] -, 429[M+Cl] -Positive ion HR-TOF-MS m/z: measured value 395.3523[M+H] +, calculated value 395.3525 (C 25H 47O 3[M+H] +).UV λ MaxNm (log ε) in MeOH:200 (3.77, end absorption).IR(KBr)ν max cm -1：3367(OH)，2923，2852(CH 3 & CH 2)，1460，1457(C＝C)，1379(CH 3)，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 reaches 13The CNMR data see Table 2.
Table 2 Compound I I at deuterium for the 600MHz in the fluoroform 1H and 400MHz 13The C nuclear magnetic resonance data a)
Mark δH(J in Hz) COSY b) HMBC c) δ C
1 2 3 4 5 6 7 8 9 10 11-16 17 18 19 20 21 22 23 24 25 1-O H 4-O H 6-O H 4.078dd(7.3，1.9) 5.61ddd(10.1，2.2，1.9) 5.85ddd(10.1，1.4，1.1) 4.50brs Ha 1.45dd(13.3，9.3) He 2.29ddd(13.3，5.5，1.1) —— 1.60(2H)m Ha 1.36m Hb 1.29-1.35 d)m 1.30m 1.22-1.29 e)m 1.22-1.29 e)m 1.30m 1.29-1.35 d)m 2.02(2H)m 5.35ABtype 5.35ABtype 2.02(2H)m 1.29-1.35 d)m 1.29-1.35 d)m 0.90(3H)t(7.0) 2.12d(7.3) 1.59brs 1.99brs 1-O H H-3，H-4 H-2 H-2，Ha-5，He-5 H-4，He-5 H-4，Ha-5 Ha-8，Hb-8 H-7，Hb-8 H-7，Ha-8 Ha-8，Hb-8，H-10 H-9 H-10，H1-17 H-16，H-18 H-17，H-19 H-18，H-20 H-19，H-21 H-20，H-22 H-21，H-23 H-22，H-24 H-23，H-25 H-24 H-1 C-2，3 C-4，6 C-1 C-1，4 C-1，3，4，6，7 C-1，5，6，8，9 C-7，9，10 C-9，10 C-8，10，11 C-8，11，12 C-9～C-18 C-15，16，18 C-16，17，19，20 C-17，20，21 C-19 C-22 C-20，21，23，24 C-21，22，24 C-22，23 C-23，24 C-1，6 C-5 C-1，5，6 70.00d 69.96d 129.61d 132.75d 65.57d 65.54d 40.90t 74.21s 39.41t 23.52t 30.11t 29.31t 29.5-29.7 f)t 30.11t 29.5-29.7 f)t 26.89 g)t 129.90 h)d 129.87 h)d 129.84 h)d 129.83 h)d 27.18 g)t 22.33t 31.95t 14.00q ——— ——— ———
A): signal is according to DEFT, PFG in the table 1H- 1H COSY, PFG HMQC, PFG HMBC and NOE difference spectrum analysis result are belonged to.B): the code name in this hurdle is illustrated respectively in PFG 1H- 1In the H COSY spectrum with same rower position in proton provide the proton of coupling coherent signal.C): the code name in this hurdle be illustrated respectively in PFG HMBC spectrum ( LrJ CH=8.3 or 4Hz) in same rower position in proton provide the carbon atom of HMBC coupling coherent signal.D), f e)): this signal is because of definitely belonging to overlapped the failing of other signal.G), h): it is interchangeable to have the identical signal ownership that goes up between two signals of target.
The NOE difference spectrum Measurement results of Compound I I: when irradiation H-1, at 1-OH, H-2 observes NOE on Ha-5, the Ha-7; When irradiation H-4, on H-3,4-OH, He-5,6-OH, observe NOE respectively; When irradiation Ha-5, at H-1, He-5, H 2Observe NOE on-7 respectively; When irradiation He-5, at H-4, Ha-5, H 2-7, observe the NOE signal on the Ha-8.
Embodiment 3. biological activity tests
Laboratory sample and experimental technique
The preparation of sample solution: get the Compound I I of separation and purification among the Compound I of separation and purification in the foregoing description 1 and the embodiment 2, precision takes by weighing in right amount, is mixed with the solution of desired concn with methyl alcohol, and is active for surveying.
Clone and cell cultures: active testing adopts mouse breast cancer tsFT210 clone, human large intestine cancer HCT-15 clone, HeLa Cells system, human breast carcinoma MCF-7 clone, human ovarian cancer A2780 clone.
Cell is with the RPMI-1640 substratum that contains 10%FBS, at 32 ℃ (tsFT210 cells) or 37 ℃ (HCT-15, HeLa, MCF-7 and A2780 cell) succeeding transfer culture in the incubator that feeds 5% carbonic acid gas.
Cell inhibitory effect activity test method (lissamine rhodamine B method is a srb assay): the human large intestine cancer HCT-15 cell in the vegetative period of taking the logarithm, HeLa Cells, human breast carcinoma MCF-7 cell, human ovarian cancer A2780 cell, being mixed with density with fresh RPMI-1640 substratum is every milliliter 2 * 10 5The cell suspension of individual cell is inoculated in 96 orifice plates by every hole 200 microlitres, and every hole adds the sample or the blank solution of 2 microlitre different concns, cultivates 24 hours down for 37 ℃.Get it filled under the thing effect cell after cultivating is at first observed the morphological change that drug treating causes under opticmicroscope, judge to have or not the cell cycle to suppress the morphological feature of apoptosis or necrocytosis.Then inhale and remove supernatant liquor and in every porocyte, add 20% Tricholroacetic Acid, 50 microlitres, place 4 ℃ to fix 1 hour, water flushing 5 times and dry air.Every hole adds acetum 50 microlitres of 0.4%SRB and left standstill 30 minutes in room temperature.Clean 4 times with 1% acetic acid water, remove unconjugated free SRB dyestuff.Every hole adds 150 microlitre Tris damping fluids (10mmol/L, pH 10.5) soluble protein combination dye and utilizes MD company to produce SPECTRA MAX Plus type microplate reader and measure optical density(OD) (OD) value of every hole at the 520nm place.Each concentration of sample all is provided with three holes in same 96 orifice plate, and other establishes three hole blanks, gets the average OD value in three holes by IR%=(OD Blank-OD Sample)/OD Blank* 100% formula is calculated the cell proliferation inhibition rate (IR%) under each concentration.
The flow cytometry testing method: the tsFT210 cell in the vegetative period of taking the logarithm, being mixed with density with fresh RPMI-1640 substratum is every milliliter 2 * 10 5The cell suspension of individual cell is inoculated in 24 orifice plates by 0.5 milliliter in every hole, and every hole adds the sample solution of 5 microlitre different concns, cultivates 17 hours down for 32 ℃.Get it filled under the thing effect cell after cultivating is at first observed the morphological change that drug treating causes under opticmicroscope, judge to have or not the cell cycle to suppress that the morphological feature of apoptosis or necrocytosis is taken pictures in case of necessity.Then cell is transferred to 1.5 milliliters of Eppendorf centrifuge tubes from 24 orifice plates respectively, 4 ℃ following 3000 rev/mins centrifugal 3 minutes, supernatant liquor is removed in suction, add 0.5 milliliter of phosphate buffer solution (PBS) concussion washing once, centrifugal collecting cell under the same terms, add 150 microlitre propidium iodide (PI) aqueous solution (in 100 ml waters, contain 5 milligrams of PI, 100 milligrams of citric acids are received and 200 milligrams of NP-40), dyeing is after 30 minutes under 4 ℃, add 150 microlitre PBS dilution, measure the content distribution of DNA in the cell with flow cytometry analysis.
Experimental result
Compound I and Compound I I are to human cancer cell inhibition of proliferation activity
In the srb assay test, Compound I and Compound I I have all shown certain inhibition activity to the cell proliferation of human large intestine cancer HCT-15 cell, and the Compound I of different concns and Compound I I the results are shown in Table 3 to the inhibition active testing of human large intestine cancer HCT-15 cell proliferation.
Table 3 Compound I and Compound I I suppress the srb assay test result of human large intestine cancer HCT-15 cell proliferation
Compound Compound I and II the inhibiting rate (%) that inclines of different concns to HCT-15 cell proliferation IC 50(μM)
200μM 100μM 10μM 1μM 0.1μM 0.01μM
Compound I Compound I I 79.0％ 83.2％ 77.7％ 83.1％ 63.0％ 48.0％ 26.0％ 39.9％ 4.7％ 38.6％ 1.8％ 36.8％ 4.1 11.4
In the srb assay test, Compound I also demonstrates in various degree inhibition activity to the propagation of HeLa Cells, human ovarian cancer A2780 cell, human breast carcinoma MCF-7 cell, and the Compound I of different concns the results are shown in Table 4 to these human cancer cell inhibition of proliferation active testings.
Table 4 Compound I suppresses the srb assay test result of human cancer cell propagation
Cell strain The Compound I of different concns is to human cancer cell inhibition of proliferation rate (%) IC 50(μM)
200μM 100μM 10μM 1μM 0.1μM
HeLa A2780 MCF-7 72.0％ 65.1％ 75.7％ 84.6％ 75.4％ 74.4％ 64.8％ 46.9％ 70.3％ 30.6％ 20.2％ 16.8％ 23.8％ 11.1％ 8.6％ 4.2 11.4 3.5
Table 3 and table 4 result show that in the srb assay test, Compound I and Compound I I have all shown the antitumour activity of anticancer propagation in various degree to the cancer cells of being tested.
The flow cytometry result
Flow cytometry analytical results: the tsFT210 cell has been carried out check and analysis with the Compound I and the Compound I I processing back of different concns with flow cytometry.The result shows: Compound I mainly shows the cell cycle to the concentration range of every milliliter 0.78 microgram every milliliter 0.19 microgram and suppresses active, and cell cycle of tsFT210 cell is suppressed at the G0/G1 phase; And from the concentration of every milliliter 0.78 microgram, present the apoptosis-inducing activity simultaneously, all detect the obvious apoptosis peak, and increase with concentration in the sub-G0/G1 district, apoptotic peak is remarkable further, begins mainly to be the apoptosis-inducing activity from every milliliter 0.78 microgram; When concentration when every milliliter 12.5 microgram is above, begin to detect the gangrenosum acne cytotoxic activity.Compound I I then mainly is suppressed at the G0/G1 phase with the cell cycle of tsFT210 cell every milliliter 3.12 microgram to the concentration range of every milliliter 12.5 microgram, mainly be apoptosis-inducing and certain S phase is suppressed activity from beginning more than every milliliter 25 microgram, when concentration every milliliter 100 microgram or the gangrenosum acne cytotoxic activity when this concentration is above, occurs.The morphologic detection result of above-mentioned flow cytometry detected result and the observation of following microscopically is identical substantially.
Morphologic detection result: under the optics inverted microscope, observe, the part cell began to occur the morphological specificity of apoptosis when the tsFT210 cell was handled as the Compound I I with the Compound I of every milliliter 0.78 microgram and every milliliter 25 microgram, when handling with the Compound I I more than the Compound I more than every milliliter 1.56 microgram and every milliliter 50 microgram, most cells is typical apoptosis morphological specificity in the visual field, and promptly cell snowflake flap or film are being wrapped up in cell debris; When with every milliliter 12.5 microgram or this Compound I and every milliliter 50 microgram or this Compound I I more than concentration when handling more than concentration, some cancer cells begins to be the morphological specificity of typical gangrenosum acne cell, and along with the concentration of Compound I and II increases, the gangrenosum acne cytotoxic activity also strengthens, and shows that Compound I and Compound I I have direct killing sexual cell cytotoxic activity to mammalian cancer cells when high density.
Conclusion
Above-mentioned experimental result shows that Compound I and Compound I I can be by suppressing the cell cycle, bring out cancer cells generation apoptosis or the direct killing sexual cell cytotoxic activity of cancer cells being brought into play the antitumor action that its anticancer is bred.Therefore, compound of the present invention can be used as antineoplastic agent and is used for tumor treatment, also can be used as the life science that the low molecular probe that brings out apoptosis or suppress the cell cycle is used for exploring biological phenomena essence.

Claims (10)

1. extract is characterized in that wherein containing at least a formula I compound or its pharmacy acceptable salt class:
Formula I
Wherein, R is hydrogen and hydroxyl, or is oxygen; R 1, R 2, R 3, R 4, R 5, R 6Be hydrogen, hydroxyl, amino, methoxyl group or the optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-20Alkyl, and, R 1-R 6In at least one is the described optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-C 20Alkyl.
2. formula I compound or its pharmacy acceptable salt class:
Figure A2005100748080002C2
Formula I
Wherein, R is hydrogen and hydroxyl, or is oxygen; R 1, R 2, R 3, R 4, R 5, R 6Be hydrogen, hydroxyl, amino, methoxyl group or the optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-20Alkyl, and, R 1-R 6In at least one is the described optional saturated or unsaturated straight or branched C that is replaced by substituting groups such as hydroxyl, amino 15-C 20Alkyl.
3. the described formula I compound of claim 2, wherein, R is hydrogen and hydroxyl, or is oxygen; R 1, R 2, R 3And R 4Be hydroxyl or optional (the Z)-14-alkene nonadecyl that is replaced by substituting groups such as hydroxyls, and, R 2Or R 3At least one is optional (the Z)-14-alkene nonadecyl that is replaced by substituting groups such as hydroxyls; R 5, R 6Be hydroxyl or hydrogen.
4. claim 2 or 3 described formula I compounds, wherein, R is an oxygen, R 1, R 2, R 6Be hydrogen, R 3Be 2-hydroxyl-(Z)-14-alkene nonadecyl, R 4, R 5Be hydroxyl.
5. claim 2 or 3 described formula I compounds, wherein, R is beta-hydroxy and α-hydrogen, R 1And R 5Be hydroxyl, R 2Be (Z)-14-alkene nonadecyl, R 3, R 4, R 6Be hydrogen.
6. contain as each the described compound of claim 2-5 of activeconstituents and the pharmaceutical composition of one or more pharmaceutical carriers or vehicle.
7. the described preparation method of extract of claim 1, this method comprise with alcohol or aqueous alcohol to relevant plant material for example Fructus Choerospondiatis extract, obtain extract.
8. the preparation method of the described formula I compound of claim 2, this method comprise that with alcohol or the relevant plant material of aqueous alcohol lixiviate Fructus Choerospondiatis for example obtain crude extract, separation and purification obtains formula I compound from crude extract then.
9. the described formula I compound of described extract of claim 1 and/or claim 2 is used to prepare the antiblastic of cell cycle inhibitor, cell death inducer, cell or the purposes of tumor cytotoxicity agent.
10. the described formula I compound of described extract of claim 1 and/or claim 2 is used to prepare the purposes of antitumor drug.
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