CN100347163C - Cyclohexenone analog bicyclo (condensed ring) compound and its preparation method and uses - Google Patents
Cyclohexenone analog bicyclo (condensed ring) compound and its preparation method and uses Download PDFInfo
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- CN100347163C CN100347163C CNB2005100991096A CN200510099109A CN100347163C CN 100347163 C CN100347163 C CN 100347163C CN B2005100991096 A CNB2005100991096 A CN B2005100991096A CN 200510099109 A CN200510099109 A CN 200510099109A CN 100347163 C CN100347163 C CN 100347163C
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Abstract
The present invention relates to a cyclo-ketene double-ring (condensed ring) compound containing a long chain hydrocarbon substituting group, an extract containing the compound, a method for preparing the compound and the extract, and the purpose of the compound and the extract, wherein the cyclohexanone saturated three-ring (bridged ring) compound is disclosed in the formula I, and R1 and R2 are defined in the specification. The cyclo-ketene double-ring (condensed ring) compound of the present invention can be used as a cell cycle inhibiting agent, an apoptosis inductive agent, a cell proliferation inhibiting agent and an anti-tumor agent, and can be used for anti-tumor treatment.
Description
Technical field
The present invention relates to cyclohexenone analog bicyclo (condensed ring) compound and preparation method thereof and purposes.The purposes that is specifically related to contain a substituent cyclohexenone analog bicyclo of long chain hydrocarbon (condensed ring) new compound, the extract that contains this compounds and described compound and preparation method of extract and is used to prepare cell cycle inhibitor, cell death inducer, inhibition of cell proliferation, cancer cells kill agent and antineoplastic agent.
Background technology
Cyclohexenone analog bicyclo (condensed ring) compound mainly forms condensed ring by the six-membered carbon ring of cyclonene and another carbocyclic ring or heterocycle, and connects various substituting groups form on this condensed ring skeleton.In this compounds, form condensed ring and on this condensed ring skeleton, be connected with some existing bibliographical information of compound of different substituents by the six-membered carbon ring of cyclonene and another saturated five yuan of oxa-rings.As document C.Nishino, et al., Halleridone, a cytotoxic constituent from Cornuscontroversa:J.Nat.Prod., 51 (6), 1281-1282,1988 had once put down in writing 9 these compounds.But wherein only 1 be natural compounds, all the other 8 the artificial derivatives that are this natural product, and the dicyclo skeleton structure of all these compounds of being put down in writing and to replace type all different fully with compound of the present invention with substituting group.So far, identical with skeletal structure of compound of the present invention and be connected with the substituent cyclohexenone analog bicyclo of long chain hydrocarbon (condensed ring) compound and do not see 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 cyclohexanone analog dicyclo (condensed ring) active compound that the anti-tumor activity of Fructus Choerospondiatis and activeconstituents thereof especially the present invention relates to is not seen the research report so far.
Summary of the invention
The present invention aims to provide new the having cell cycle inhibition, apoptosis-inducing and the anti-tumor activities such as direct killing of cancer cells are contained a substituent cyclohexenone analog bicyclo of long chain hydrocarbon (condensed ring) compound of a kind of chemical structure.
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 has been found cyclohexenone analog bicyclo shown in the following formula I (condensed ring) new compound:
Wherein, R
1, R
2, R
3, R
4Be hydrogen, hydroxyl, amino, sulfonic group, carboxyl,-oxyl, amido, acyl group, acyloxy or amide group, perhaps for having or do not have substituent C such as hydroxyl, amino, sulfonic group, carboxyl
15～C
20The straight or branched alkyl, wherein, at least one is for having or do not have substituent (Z)-12-alkene-heptadecyls such as hydroxyl.
Therefore, first aspect of the present invention relates to (condensed ring) compound of cyclohexenone analog bicyclo shown in the formula I or its pharmacy acceptable salt:
Wherein, R
1, R
2, R
3, R
4Be hydrogen, hydroxyl, amino, sulfonic group, carboxyl,-oxyl, amido, acyl group, acyloxy or amide group, perhaps for having or do not have substituent C such as hydroxyl, amino, sulfonic group, carboxyl
15～C
20The straight or branched alkyl, wherein, at least one is for having or do not have substituent (Z)-12-alkene heptadecyls such as hydroxyl.
Second aspect of the present invention relates to a kind of extract, it is characterized in that wherein comprising cyclohexenone analog bicyclo (condensed ring) compound or its pharmacy acceptable salt shown at least a above-mentioned described formula I in first aspect.
The 3rd aspect of the present invention relates to the method for preparation above-mentioned first aspect described 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 4th aspect of the present invention relates to preparation above-mentioned second aspect described method that contains the extract of formula I compound, described method comprise with alcohol or aqueous alcohol to relevant plant material for example Fructus Choerospondiatis extract, must crude extract be extract.
The 5th aspect of the present invention relates to the pharmaceutical composition that contains as described formula I cyclohexenone analog bicyclo (condensed ring) compound in first aspect of activeconstituents and one or more pharmaceutical carriers or vehicle.
The 6th aspect of the present invention relates to the purposes that the described formula I cyclohexenone analog bicyclo in first aspect (condensed ring) compound is used to prepare cell cycle inhibitor, cell death inducer, inhibition of cell proliferation cancer, cell killing agent and antineoplastic agent.
In an embodiment of the invention, the present invention relates to (condensed ring) compound of the cyclohexenone analog bicyclo shown in the formula I or its pharmacy acceptable salt:
Wherein, R
1, R
2, R
3, R
4Be hydrogen, hydroxyl, amino, sulfonic group, carboxyl,-oxyl, amido, acyl group, acyloxy or amide group, perhaps for having or do not have substituent C such as hydroxyl, amino, carboxyl or sulfonic group
15～C
20The straight or branched alkyl, wherein, at least one is for having or do not have substituent (Z)-12-alkene-heptadecyls such as hydroxyl.
In the above-mentioned definition, term "-oxyl " is meant that alkyl combines the group that forms with Sauerstoffatom.
Term " amido " is meant amino (NH
2) replaced formed group by alkyl list or two.
Term " acyl group ", " acyloxy " and " amide group " are meant that respectively (CO-), (OCO-), carbonylamino (NHCO-) is replaced formed group by alkyl to carbonyl oxygen base to carbonyl.
" alkyl " in the above-mentioned definition is if not specialize carbon number, then be meant any group of forming by carbon atom and hydrogen atom, for example can comprise as alicyclic radicals such as fatty group, cycloalkyl, aromatic bases etc. such as alkyl with no more than 20 carbon atoms, thiazolinyl, alkynyl, perhaps their combination.Alkyl in the fatty group, thiazolinyl, alkynyl can be straight chain or side chain.Alicyclic radical can right and wrong fragrance the saturated or unsaturated cyclic alkyl of monocycle, condensed ring, bridged ring or volution.Aromatic base is phenyl, naphthyl, phenanthryl, xenyl etc. for example.The combination of above-mentioned group is cycloalkylalkyl, cycloalkyl thiazolinyl, cycloalkyl alkynyl, cycloalkenyl alkyl, cycloalkenyl group thiazolinyl, cycloalkenyl group alkynyl, alkyl-cycloalkyl, thiazolinyl cycloalkyl, alkynyl cycloalkyl, alkyl cycloalkenyl group, thiazolinyl cycloalkenyl group, alkynyl cycloalkenyl group, aralkyl, arylalkenyl, sweet-smelling alkynyl, alkylaryl, alkenyl aryl, alkynyl aryl etc. for example.Especially, described " C
15～C
20The straight or branched alkyl " refer to straight or branched alkyl, alkenyl or alkynyl with 15～20 carbon atoms.
Term " pharmacy acceptable salt " can be medicinal inorganic or organic salt.Have basic group in the formula I compound of the present invention and can form pharmaceutical salts with mineral acid, for example vitriol, hydrochloride, hydrobromate, phosphoric acid salt etc. as amino, amido etc.; 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.Have acidic-group such as carboxyl or sulfonic compound among the formula I of the present invention and also can form pharmaceutical salts with basic metal or alkaline-earth metal, preferred but be not limited to sodium salt, sylvite, magnesium salts or calcium salt etc.
Preferably, each group has as giving a definition in formula I cyclohexenone analog bicyclo (condensed ring) compound of the present invention or its pharmacy acceptable salt:
R
1And R
2Be hydrogen, hydroxyl, amino, sulfonic group, carboxyl,-oxyl, amido, acyl group, acyloxy or amide group, R
3And R
4For hydrogen, have or do not have substituent C such as hydroxyl
15～C
20Straight or branched alkyl or (Z)-12-alkene-heptadecyl.
In the further preferred embodiment of the present invention, R
1And R
2Be hydroxyl, R
3Be hydrogen, R
4Be (Z)-12-alkene-heptadecyl, promptly following Compound I I:
Another embodiment of the present invention is the extract that contains above-mentioned formula I compound.Extract of the present invention is meant the extract of the natural drug that contains formula I compound.Extract described in the present invention, as long as wherein contain above-mentioned formula I compound, and desired result that can production I compound (as suppressing cell cycle, cell death inducing, killing and wounding cancer cells etc.), not only comprise plant milk extract, also comprise the extract of animal under the possible situation or fungi, bacterium etc., or vegetable cell, fungi, bacterium are cultivated the extract of the culture that obtains.
Plant milk extract can be the extract of plant integral body or the extract of indivedual tissue, organ or part, as root, skin, leaf, flower, fruit, seed, fruit stone etc.Can use the raw material or the dried raw material of fresh acquisition during extraction.
The extract of gained can be that plant material is for example directly extracted resulting initial extract, medicinal extract or carried out preliminary purification such as solvent-extracted bulk processing thing etc., as long as wherein contain formula I compound, also contain other does not have materially affect to formula I compound activity material simultaneously.
Containing the extract of above-mentioned formula I compound and pure formula I compound can prepare by the following method: for example Fructus Choerospondiatis or culture etc. extract to relevant vegetable material with alcohol or aqueous alcohol, 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 can be lower alcohols such as methyl alcohol, ethanol, preferred alcohol; Pure content can be about 60～95% in the described aqueous alcohol, the aqueous ethanol of preferred alcohols content 60～95%.
Described extraction can be adopted modes such as lixiviate, diafiltration or backflow, obviously, extraction time, number of times, the amount of employed alcohol can be according to the mode that is adopted with temperature and different, so does not intend this is made special qualification among the present invention.For example, the room temperature lixiviate can soak at every turn 4～8 days, extract 3～6 times, the room temperature diafiltration can be extracted operation 5～10 days continuously, the each sustainable backflow of refluxing extraction 1～3 hour, extracts 3～6 times.
Described separation and purification comprises the ordinary method and the means of 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, thin-layer chromatography, high performance liquid phase and recrystallizing and refining can carry out repeatedly repeatedly, column chromatography and thin-layer chromatography can use sorbent materials such as silica gel well known in the art, aluminum oxide, polymeric amide, macroporous resin, in addition, also can use reverse phase silica gels such as RP-18, RP-8 as sorbent material.
The inventor etc. adopt 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 demonstrates the biologic activity that suppresses cell proliferation to tumour cell, thereby has antitumor action by the turnover of inhibition cell cycle, the modes such as cancer cell-apoptosis or direct killing of bringing out.
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.
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 suction, rectal application, nasal cavity applied medicine, cheek medication, local application, non-enterally administer, as in subcutaneous, vein, intramuscular, intraperitoneal, the sheath, in the ventricle, in the breastbone or 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.Extract of the present invention and compound also can be made slowly-releasing and controlled release preparation according to conventional methods and use.
When topical application, The compounds of this invention can be made into suitable plaster, 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 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 embodiment 1 gained compound (Compound I I) in methanol solution (40 μ g/ml);
Fig. 2 is the infrared absorption spectrum (KBr) of Compound I I;
Fig. 3 is that Compound I I is in deuterochloroform
1The H nuclear magnetic resonance spectrum;
Fig. 4 is that Compound I I is in deuterochloroform
13The C nuclear magnetic resonance spectrum;
Fig. 5 is circular double dispersion (CD) spectrum of Compound I I in methanol solution (0.1mg/ml);
Fig. 6 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.
Need to prove that 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, thermometer is not calibrated.Specific rotatory power is measured with JSASCO P-1020 type polarimeter.Negative ions ESI-MS and HR-ESI-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 JEOL Eclips-600 type NMR spectrometer with superconducting magnet (600MHz
1H-NMR, 150MHz
13C-NMR) measure.Circular double dispersion (CD) spectrum is measured with JASCOJ-810 Spectropolarimeter.
Embodiment 1
Get the dry bark (3.2kg) of Fructus Choerospondiatis (Choerospondias axillaries (Roxb.) Burtt et Hill) 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, uses chloroform extraction, use 3 liters of chloroforms at every turn, coextraction 4 times.The combined chloroform extraction liquid is concentrated into driedly, obtains extract 60g.
Silicagel column (post bed 7.5cm * 18.5cm), carry out gradient elution chromatography with sherwood oil-acetone (100: 1 → 2: 1) solvent systems as eluent reduces pressure on extract obtained (60g) dry method.In elution process, come gradient to increase progressively the polarity (V of eluting solvent by the consumption that increases acetone (A) in the sherwood oil (P)
P: V
A=100: 1,50: 1,10: 1,5: 1 etc.).Merge flow point according to thin layer detection and active testing result, in V
P: V
A=5: 1 wash-out partly obtains the 10.8g extract, is extract Fr-4.
Silicagel column (post bed 5.0cm * 20cm), use chloroform (C)-acetone (A) gradient elution (V of system reduces pressure on extract Fr-4 (10.8g) dry method
C: V
A=30: 1,15: 1,10: 1,5: 1 etc.), merge flow point according to thin layer detection and active testing result, in V
C: V
A=10: 1 wash-out partly obtains the 3.8g extract, is extract Fr-4-3.
Extract Fr-4-3 (3.8g) in methyl alcohol on Sephadex LH-20 post (post bed 3.8cm * 36cm), and use methanol-eluted fractions, obtain containing the chromatography component of Compound I I, this component is separated through preparation silica gel thin-layer chromatography (sherwood oil-ether-propyl carbinol-acetate 2: 1: 0.2: 0.1 launches), obtains Compound I I crude product.This Compound I I crude product was located to provide the single elution peak of Compound I I in 67 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 peak collected, through concentrated the pure product 167mg of Compound I I.
Through structure elucidation, Compound I I has following structure (4S5R8S absolute configuration):
Wherein,
R
1And R
2Be hydroxyl;
R
3Be hydrogen;
R
4Be (Z)-12-alkene-heptadecyl.
Arabic numerals on carbocyclic ring and the long-chain are represented the mark of corresponding carbon atom.
Its various physico-chemical properties and spectral data are as follows:
Compound I I, molecular formula C
25H
42O
4, oily compound
Positive ion ESI-MS m/z:429[M+Na]
+, 424[M+NH
4]
+, 407[M+H]
+, 389
[M+H-H
2O]
+，371[M+H-2H
2O]
+；
Negative ion ESI-MS m/z:405[M-H]
-, 387[M-H-H
2O]
-
Positive ion HR-ESI-MS m/z: measured value 407.3171[M+H]
+, calculated value 407.3161 (C
25H
43O
4[M+H]
+)
[α]
D 31-63.4°(c 1.0，CHCl
3)
UV λ
MaxNm (log ε) in MeOH:213.6 (3.96), 202 (3.97) (referring to Fig. 1)
IR (KBr) ν
MaxCm
-1: 3384 (OH), 3005 (=CH-), 2925,2854 (CH
3﹠amp; CH
2), 1683 (conjugation CO), 1460,1340,1298,1269,1203,1138,1070,1018 (C-O), 922,719,646 (referring to Fig. 2)
CD λ
MaxNm (mdeg) in MeOH at 0.1mg/ml:330.8 (6.25309), 245.7 (21.3081), 205.4 (+28.3949) (referring to Fig. 5).
1H reaches
13C NMR data 1 (referring to Fig. 3, the Fig. 4) that see the following form.
The 600MHz of table 1 Compound I I in deuterochloroform
1H and 150MHz
13The C nuclear magnetic resonance data
A)
| Mark | δ H(J in Hz) | COSY b) | HMBC c) | δc |
| 1 2 3 4 5 6 7 8 9 10 11 12-16 17 18 19 20 21 22 23 24 25 4-OH 5-OH | — 5.95d(10.2) 6.73d(10.2) — — Ha 2.72d(16.0) He 2.86d(16.0) Ha 2.23dd(13.2，6.1) Hb 1.71dd(13.2，9.3) 4.43m Ha 1.57m Hb 1.33m Ha 1.41m Hb 1.28m 1.23-1.28 d)m 1.23-1.28 d)m 1.23-1.28 d)m 1.29-1.35 f)m 2.02(2H)m 5.35ABtype 5.35ABtype 2.02(2H)m 1.29-1.35 f)m 1.29-1.35 f)m 0.90(3H)t(7.1) 3.95br s 2.96br s | H-3 H-2 He-6 Ha-6 Hb-7，H-8 Ha-7，H-8 Ha-7，Hb-7 Ha-10 Hb-10 Ha-9 Hb-9，H-11 Hb-10 Hb-10， H-18 H-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-1，3，4，5，6 C-1，2，4，5 C-1，2，4，5，7 C-1，2，4，5，7 C-4，5，6，8，9 C-4，5，6，8， C-10 C-7，8，10，11 C-7，8，10，11 C-8，9，11 C-8，9，11 C-10，12，13 C-11～18 C-15，16，18，19 C-16，17，19，20 C-17，20，21 C-19，22 C-19，22 C-20，21，23，24 C-21，22，24，25 C-22，23，25 C-23，24 C-3，4，5 C-4，5，6，7 | 196.83s 126.78d 126.75d 146.04d 145.98d 99.49s 78.68s 46.98t 43.58t 43.55t 77.33d 35.99t 25.58t 29.73t 29.5-29.6 e)t 29.28t 29.5-29.6 e)t 26.88 g)t 129.87d 129.84d 27.17 h)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 (
IrJ
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): the signal ownership is interchangeable between these two signals.
The NOE difference spectrum Measurement results of Compound I I: when irradiation Ha-6, on He-6 and Hb-7, observe NOE; When irradiation He-6, on Ha-6, Ha-7 and Hb-7, observe NOE; When irradiation Ha-7, on He-6, Hb-7 and H-8, observe NOE; When irradiation Hb-7, on He-6, Ha-6 and Ha-7, observe NOE; When irradiation H-8, on Ha-7, Ha-9, Hb-9, Ha-10 and Hb-10, observe NOE.
Embodiment 2. biological activity tests
Laboratory sample and experimental technique
The preparation of sample solution: get the pure product of Compound I I of separation and purification in the foregoing description 1, 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 I is to human cancer cell inhibition of proliferation activity
In the srb assay test, Compound I I demonstrates in various degree inhibition activity to the propagation of human large intestine cancer HCT-15 cell, HeLa Cells, human ovarian cancer A2780 cell, human breast carcinoma MCF-7 cell, and the Compound I I of different concns the results are shown in Table 2 to these human cancer cell inhibition of proliferation active testings.
Table 2 Compound I I suppresses the srb assay test result of human cancer cell propagation
| Cell strain | The Compound I I of different concns is to human cancer cell inhibition of proliferation rate (%) | IC 50(μm) | ||||
| 200μM | 100μM | 10μM | 1μM | 0.1μM | ||
| HCT-15 HeLa A2780 MCF-7 | 83.3％ 71.5％ 62.1％ 77.1％ | 84.6％ 86.6％ 59.6％ 78.9％ | 68.4％ 57.0％ 36.0％ 62.5％ | 27.0％ 31.1％ 10.5％ 4.9％ | 3.4％ 30.9％ — 7.4％ | 3.2 9.2 43.9 8.6 |
Table 2 is the result show, in srb assay test, Compound I I is to the antitumour activity of the anticancer propagation of the equal showed different of cancer cells of being tested.
The flow cytometry result
As seen from Figure 6, the tsFT210 cell check and analysis have been carried out with the Compound I I processing back of different concns with flow cytometry.The result shows: Compound I I mainly shows the cell cycle to the concentration range of every milliliter 1.56 microgram every milliliter 0.39 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 3.125 microgram; When concentration when every milliliter 25 microgram is above, begin to detect the gangrenosum acne cytotoxic activity.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 tsFT210 cell begins to detect the morphological specificity that more cell is apoptotic cell when the Compound I I with every milliliter 1.56 microgram handles, when handling with the Compound I I more than every milliliter 3.125 microgram, most cells are typical apoptosis morphological specificity in the visual field, and promptly cell snowflake flap or film are being wrapped up in cell debris; When handling with every milliliter 25 microgram or this Compound I I more than concentration, cell count significantly reduces, and the part cell is the morphological specificity of gangrenosum acne cell, shows that Compound I I has direct killing sexual cell cytotoxic activity to mammalian cancer cells when high density.
Conclusion
Above-mentioned experimental result shows that formula I compound of the present invention 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 (9)
1. formula I compound or its pharmacy acceptable salt:
Wherein, R
1And R
2Be hydrogen, hydroxyl, amino,-oxyl, amido, acyl group, acyloxy or amide group, R
3And R
4For hydrogen or have or do not have the C of hydroxyl substituent
15～C
20Straight or branched alkyl or (Z)-12-alkene-heptadecyl.
2. the described compound of claim 1, wherein, R
1And R
2Be hydroxyl, R
3Be hydrogen, R
4Be (Z)-12-alkene-heptadecyl.
3. extract is characterized in that wherein containing at least a claim 1 or 2 described compounds or its pharmacy acceptable salt.
4. pharmaceutical composition contains claim 1 or 2 described compounds or the described extract of claim 3 and one or more pharmaceutical carriers or vehicle as activeconstituents.
5. the preparation method of claim 1 or 2 described compounds, this method comprise with alcohol or aqueous alcohol lixiviate plant material, obtain extract, separation and purification and obtaining from extract then.
6. the described preparation method of extract of claim 3, this method comprise with alcohol or aqueous alcohol lixiviate plant material, obtain extract.
7. claim 5 or 6 method, described alcohol is ethanol, and described aqueous alcohol is 60～95% aqueous ethanol, and described separation and purification comprises liquid-liquid extraction, column chromatography, thin-layer chromatography, high performance liquid chromatography and recrystallization.
8. claim 1 or 2 described compounds or the described extract of claim 3 are used to prepare the antiblastic of cell cycle inhibitor, cell death inducer, cell or the purposes of tumor cytotoxicity agent.
9. claim 1 or 2 described compounds or the described extract of claim 3 are used to prepare the purposes of antitumor drug.
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| Title |
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| HALLERIDONE,A CYTOTOXIC CONSTITUENT FROMCORNUS CONTROVERSA. CHIKAO NISHINO et al..Journal of Natural Products,Vol.51 No.6. 1988 * |
| 南酸枣植物在药物方面的研究概况及其应用前景 熊冬生等.广东药学，第10卷第5期 2000 * |
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