CN1872838A - Compound of monocyclic polysubstitution saturated cyclohexanones, prepartion method and usage - Google Patents
Compound of monocyclic polysubstitution saturated cyclohexanones, prepartion method and usage Download PDFInfo
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- CN1872838A CN1872838A CN 200510074805 CN200510074805A CN1872838A CN 1872838 A CN1872838 A CN 1872838A CN 200510074805 CN200510074805 CN 200510074805 CN 200510074805 A CN200510074805 A CN 200510074805A CN 1872838 A CN1872838 A CN 1872838A
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Abstract
This invention relates to a polysubstituted saturated cyclohexanone compound, the 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 agent.
Description
Technical field:
The present invention relates to monocycle polysubstitution saturated cyclohexanones compound and its production and use.Be specifically related to contain the substituent monocycle polysubstitution saturated cyclohexanones of long chain hydrocarbon compound, the extract that contains this compounds, extraction separation and a purifying and obtain the purposes that the method for this compounds and described extract and 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 saturated cyclohexanones compound means a series of compounds that are connected with different substituents on the carbon skeleton of hexanaphthene ketone.Such skeletal structure of compound is single, mainly forms different compounds because of substituting group kind and the position of substitution different.Wherein, contain some existing bibliographical information of the substituent replacement monocycle of long chain hydrocarbon saturated cyclohexanones compound.As document S.R.Johns, etal., Campnosperma Exudates.The optically active long-chain5-hydroxyclohex-2-enones and long-chain bicycle[3.3.1] nonane-3,7-diones:Aust.J.Chem., 40,79-96,1987 had once put down in writing several replacement monocycle saturated cyclohexanones compounds that contain a long chain hydrocarbon groups (17 carbon or nineteen carbon).But the compound of above-mentioned document record is artificial converted product.Not only number is few, to replace type limited for hitherto known this compounds, and does not appear in the newspapers so far about the anti-tumor activity of this compounds and the substituent monocycle polysubstitution saturated cyclohexanones of the long chain hydrocarbon compound that contains with identical replacement type with The compounds of this invention.
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 the monocycle polysubstitution saturated cyclohexanones activeconstituents 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 a kind ofly has cell cycle inhibition, apoptosis-inducing and to the substituent monocycle polysubstitution saturated cyclohexanones of the long chain hydrocarbon compound that contains of anti-tumor activities such as cancer cells direct killing.
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 monocycle polysubstitution saturated cyclohexanones compound has above-mentioned anti-tumor activity shown in the following formula I in the discovery Fructus Choerospondiatis crude extract:
Wherein,
The six-ring of pimelinketone is got
4C
1The chair form configuration;
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, all the other at least three for being selected from the oxygen-containing substituents of hydroxyl or methoxyl group.
Therefore, first aspect of the present invention relates to a kind of extract, it is characterized in that wherein comprising monocycle polysubstitution saturated cyclohexanones compound and the pharmacy acceptable salt thereof shown at least a formula I:
Wherein,
The six-ring of pimelinketone is got
4C
1The chair form configuration;
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, all the other at least three for being selected from the oxygen-containing substituents of hydroxyl or methoxyl group.
Second aspect of the present invention relates to monocycle polysubstitution saturated cyclohexanones compound and pharmacy acceptable salt thereof shown in the formula I:
Wherein,
The six-ring of pimelinketone is got
4C
1The chair form configuration;
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, all the other at least three for being selected from the oxygen-containing substituents of hydroxyl or methoxyl group.
The 3rd aspect of the present invention relates to the method for preparing the said extracted thing, and described method comprises with alcohol or aqueous alcohol to be extracted relevant plant material, for example Fructus Choerospondiatis, and getting crude extract is extract.
The 4th aspect of the present invention relates to the method for preparing above-mentioned formula I compound, described method comprise with alcohol or aqueous alcohol to relevant plant material, for example Fructus Choerospondiatis extract after the crude extract, obtain formula I compound through separation and purification again.
The 5th aspect of the present invention relates to and containing as the formula I monocycle polysubstitution saturated cyclohexanones compound of activeconstituents and the pharmaceutical composition of one or more pharmaceutical carriers or vehicle.
The 6th aspect of the present invention relates to the purposes that formula I monocycle polysubstitution saturated cyclohexanones compound is 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 following formula I monocycle polysubstitution saturated cyclohexanones compound and pharmacy acceptable salt thereof:
Wherein,
The six-ring of pimelinketone is got
4C
1The chair form configuration;
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, all the other at least three for being selected from the oxygen-containing substituents of hydroxyl or methoxyl group.
In a preferred embodiment of the invention, the present invention relates to following formula I monocycle polysubstitution saturated cyclohexanones compound and pharmacy acceptable salt thereof:
Wherein,
R
1And R
2For hydroxyl or (Z)-and 14-alkene nonadecyl, R
3, R
4, R
5, R
6Be hydroxyl or hydrogen.
In the further preferred embodiment of the present invention, the R among the formula I
1Be (Z)-14-alkene nonadecyl, R
2, R
4, R
6Be hydroxyl, R
3, R
5Be hydrogen.
In another further preferred embodiment of the present invention, the R among the formula I
2Be (Z)-14-alkene nonadecyl, R
1, R
3, R
6Be hydroxyl, R
4, R
5Be hydrogen.
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 relevant vegetable material, for example Fructus Choerospondiatis are extracted, 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 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, 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 is by suppressing the cell cycle turnover, bring out cancer cell-apoptosis or mode such as directly kill and wound demonstrates the biologic activity that suppresses tumor cell proliferation, thereby has 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 (1.0mg/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 deuterated methanol
1The H nuclear magnetic resonance spectrum;
Fig. 9 is that Compound I I is in deuterated methanol
13The C nuclear magnetic resonance spectrum;
Figure 10 is circular double dispersion (CD) spectrum of Compound I I in methanol solution (1.0mg/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, six-ring is got
4C
1Configuration (absolute configuration is 3S4R6R), R
1Be (Z)-14-alkene nonadecyl, R
2, R
4, R
6Be hydroxyl, R
3, R
5Be hydrogen; 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, six-ring is got
4C
1Configuration (absolute configuration is 3S4S6S), wherein R
2Be (Z)-14-alkene nonadecyl, R
1, R
3, R
6Be hydroxyl, R
4, R
5Be hydrogen:
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-810Spectropolarimeter.
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 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 fraction 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).(5.0cm * 22cm), with chloroform (C)-ethyl acetate (Ac), acetone, methyl alcohol system wash-out, merge fraction 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 72 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 104mg of Compound I.
Compound I white crystals type powder, fusing point 75.1-76.2 ℃, [α]
D 31+ 37.7 ° of (c1.0, CHCl
3), molecular formula C
25H
46O
4Positive ion TOF-MSm/z:428[M+NH
4]
+, 411[M+H]
+, 393[M+H-H
2O]
+, 375[M+H-2H
2O]
+Negative ion TOF-MSm/z:455[M-H+HCOOH]
-, 445[M+Cl]
-, 391[M-H-H
2O]
-Positive ion HR-TOF-MSm/s: measured value 411.3475[M+H]
+, calculated value 411.3474 ([M+H]
+).UV λ
MaxNm (log ε) in MeOH:201 (3.64, end absorption).IR(KBr)v
maxcm
-1：3394，3282(OH)，2920，2850(CH
3&CH
2)，1718(CO)，1465，1332，1166(C-O)，1070，1004，651。CDλ
maxnm(mdeg)in MeOH at 1.0mg/ml：317(-21.3715)，283.7(+58.3594)，206.6(-30.5037)，202.6(-1.16184)。
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 | δ H(J in Hz) | COSY b) | HMBC c) | δc |
| 1 2 3 4 5 6 7 8 9-17 18 19 20 21 22 23 24 25 3-0 H 4-0 H 6-0 H | - Ha 2.98dd(12.8，11.5) He 2.69ddd(12.8，4.5，0.9) 3.98m 4.20m Ha 1.70dd(14.9，3.5) He 2.43dd(14.9，3.9) - Ha 2.07ddd(14.2，12.3，4.5) Hb 1.77ddd(14.22，12.4，4.6) Ha 1.39m Hb 1.02m 1.22-1.30 d)m 1.29-1.35 f)m 2.02(2H)m 5.35AB type 5.35AB type 2.02(2H)m 1.29-1.35 f)m 1.29-1.35 f)m 0.90(3H)t(7.1) 2.42brs 2.46brs 3.58s | He-2，H-3 Ha-2，H-3，H-4 Ha-2，He-2，3-OH，H-4 He-2，H-3，Ha-5，He-5 H-4，He-5 H-4，Ha-5 Hb-7，Ha-8，Hb-8 Ha-7，Ha-8，Hb-8 Ha-7，Hb-7，Hb-8 Ha-7，Hb-7，Ha-8 Ha-8，Hb-8，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-3 H-4 | C-1，C-3，C-4，C-6 C-1，C-3，C-4 C-6 C-6，C-7 C-1，C-3，C-4，C-6，C-7 C-5，C-6，C-8，C-9 C-1，C-5，C-6，C-8，C-9 C-9，C-10 C-9，C-10 C-8，9～C-17，18，19 C-16，C-17，C-19，C-20 C-17，C-18，C-20，C-21 C-19，C-22 C-19，C-22 C-20，C-22，C-23，C-24 C-21，C-22，C-24，C-25 C-22，C-23 C-23，C-24 C-3，C-4 C-3，C-4，C-5 C-1，C-5，C-6，C-7 | 210.98s 41.81t 71.96d 68.50d 40.45t 77.40s 39.64t 23.09t 29.5-29.8 e)t 29.5-29.8 e)t 26.89 h)t 129.91 f)d 129.82 i)d 27.18 h)t 22.33t 31.96t 14.00q - - - |
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.H), i): 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 Ha-2, on He-2, Ha-7, Hb-7, observe the NOE signal respectively; When irradiation H-3, on He-2, H-4, Ha-5, He-5,3-OH, observe the NOE signal respectively; When irradiation Ha-5, on H-3, H-4, He-5, observe the NOE signal respectively; When irradiation Ha-7, on Ha-2, He-5, Hb-7, Hb-8, H-9, observe the NOE signal respectively; When irradiation He-7, on Ha-7, 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: contain the preparation of the thick component of chromatography of Compound I I
Get aminoform 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 cut according to thin layer detection and active testing result, obtain 6 component Fr-1 (3.3g, V
P: V
A100: 1 wash-out parts), Fr-2 (2.1g, V
P: V
A=50: 1 wash-out part), Fr-3 (2.2g, 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 cut 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-5-5 is the thick component of chromatography that contains Compound I I.
The purifying of third step: Compound I I and refining
Fr-5-5 (1.9g) is dissolved with an amount of chloroform-methanol (1: 1), last SephadexLH-20 post (post bed 3.8cm * 36cm), with chloroform-methanol (1: 1) wash-out, obtain component Fr-5-5-1 (0.8g), Fr-5-5-2 (0.4g) and Fr-5-5-3 (0.7g) successively by elution order.Compound I I mainly concentrates among the Fr-5-5-2, therefore, Fr-5-5-2 is separated by preparation silica gel thin-layer chromatography (chloroform-methanol launches at 7: 1), obtains the crude product (230mg) of Compound I I.This Compound I I crude product is in the anti-phase analysis high performance liquid chromatography of RP-18 (203nm detection), when with 83% methanol-eluted fractions, located to provide the single elution peak of Compound I I in 59 minutes in retention time, then carry out the same terms amplification test separation and purification, separate through the anti-phase preparation high performance liquid chromatography of RP-18 (203nm detection), collect corresponding elution fraction, concentrate and in methyl alcohol the recrystallization operation obtain the pure product 135mg of Compound I I.
Compound I I white crystals type powder, fusing point 92-94 ℃, [α]
D 31+ 31.4 ° of (c1.0, CHCl
3), molecular formula C
25H
46O
4Positive ion TOF-MSm/z:428[M+NH
4]
+, 411[M+H]
+, 393[M+H-H
2O]
+, 375[M+H-H
2O]
+Negative ion TOF-MSm/z:469[M-H+CH
3COOH]
-, 455[M-H+HCOOH]
-, 445[M+Cl]
-, 391[M-H-H
2O]
-Positive ion HR-TOF-MSm/z: measured value 411.3487[M+H]
+, calculated value 411.3474 ([M+H]
+).UV λ
MaxNm (log ε) in MeOH:200 (3.69, end absorption).IR(KBr)v
maxcm
-1：3383(OH)，2922，2850(CH
3&CH
2)，1726(CO)，1465，1457，1366，1080(C-O)，699，644，621。CDλ
maxnm(mdeg)in MeOHat 1.0mg/ml：319.5(-16.6417)，284.4(+107.044)，210.6(+9.10091)，204.8(+30.7266)。
1H reaches
13C NMR data see Table 2.
The 600MHz of table 2 Compound I I in deuterated methanol
1H and 400MHz
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 11-16 17 18 19 20 21 22 23 24 25 | - Ha 2.64dd(13.5，7.1) He 2.77dd(13.5，3.9) 3.84m 3.95m Ha 1.79dd(14.2，6.4) He 2.11dd(14.2，4.1) - Ha 1.89ddd(13.5，12.7，4.2) Hb 1.62ddd(13.5，12.7，4.1) Ha 1.37m Hb 1.17m 1.25-1.30 d)m 1.25-1.30 d)m 1.25-1.30 d)m 1.25-1.30 d)m 1.30-1.36 h)m 2.02(2H)m 5.33AB type 5.33AB type 2.02(2H)m 1.30-1.36 g)m 1.30-1.36 g)m 0.90(3H)t(6.9) | He-2，H-3 Ha-2，H-3 Ha-2，He-2，H-4 H-3，Ha-5，He-5 H-4，He-5 H-4，Ha-5 Hb-7，Ha-8，Hb-8 Ha-7，Ha-8，Hb-8 Ha-7，Hb-7，Hb-8 Ha-7，Hb-7，Ha-8 Ha-8，Hb-8 H-9，H-11 H-10，H-12 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 | C-1，C-3，C-4，C-6 C-1，C-3，C-4 C-1，C-4，C-5 C-2，C-3，C-6 C-1，C-3，C-4，C-6，C-7 C-11，C-3，C-4，C-6，C-7 C-5，C-6，C-8 C-1，C-5，-6，C-8 C-7，C-9，C-10 C-7，C-9，C-10 C-7，C-10，C-11 C-9，C-11，C-12 C-8，C9～C-17，18 C-15，16，C-18，C-19 C-6，C-17，C-19，C-20 C-17，C-18，C-20，C-21 C-19，C-22 C-19，C-22 C-20，C-21，C-23，C-24 C-21，C-22，C-24 C-22，C-23 C-23，C-24 | 212.24s 43.70t 75.08d 70.96d 42.02t 78.76s 39.22t 24.17t 30.62 a)t 30.32 a)t 30.6-30.8 f)t 31.20t 30.6-30.8 f)t 27.89 h)t 130.80d 130.80d 28.11 h)t 23.37t 33.14t 14.31q |
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), g f)): this signal is because of definitely belonging to overlapped the failing of other signal.E), i): have the identical signal ownership exchange between two signals of target of going up.
The NOE difference spectrum Measurement results of Compound I I: when irradiation He-2, on Ha-2 and H-3, observe the NOE signal respectively; When irradiation H-3, on He-2 and Ha-5, observe the NOE signal respectively; When irradiation H-4, on Ha-2 and He-5, observe NOE respectively; When irradiation Ha-5, on H-3, He-5, Ha-7, Hb-7, observe the NOE signal respectively; When irradiation He-5, on H-4, Ha-5, Ha-7, observe the NOE signal respectively; When irradiation Ha-7, on He-5, Hb-7, Ha-8, H-9, observe the NOE signal respectively; When irradiation He-7, on Ha-5, Ha-7, Ha-8, H-9, observe the NOE signal.
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 respectively, 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, pH10.5) soluble protein combination dye and utilize 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 | The Compound I of different concns and II are to the inhibiting rate (%) of HCT-15 cell proliferation | IC 50(μM) | |||||
| 200μM | 100μM | 10μM | 1μM | 0.1μM | 0.01μM | ||
| Compound I Compound I I | 78.1％ 85.3％ | 76.5％ 84.0％ | 33.3％ 46.7％ | 9.6％ 43.9％ | 4.9％ 45.3％ | 4.3％ 47.7％ | 18.3 16.4 |
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 | 85.7％ 71.2％ 87.7％ | 95.5％ 64.4％ 86.6％ | 55.6％ 8.8％ 27.5％ | 40.3％ 2.3％ 28.1％ | 26.7％ 2.0％ 21.6％ | 9.4 51.1 19.1 |
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 12.5 microgram every milliliter 3.1 microgram and suppresses active, and cell cycle of tsFT210 cell is suppressed at the G0/G1 phase; And when concentration is above above every milliliter 12.5 microgram, mainly present the apoptosis-inducing activity, and all detecting the obvious apoptosis peak in the sub-G0/G1 district, and increase with concentration, apoptotic peak is remarkable further; When concentration when every milliliter 50 microgram is above, begin to detect certain 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 6.2 microgram to the concentration range of every milliliter 25 microgram, also the more weak G2/M phase of demonstration suppresses active simultaneously when every milliliter 25 microgram is above, suppress active when concentration begins to present certain S phase when every milliliter 50 microgram is above, 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 cell of lesser amt began the morphological specificity of apoptosis cells when the tsFT210 cell was handled as the Compound I I with the Compound I of every milliliter 12.5 microgram and 25 micrograms, when handling with the Compound I more than every milliliter 25 microgram, most forms that present typical apoptotic cell are that snowflake flap cell or film are being wrapped up in cell debris in the visual field; And when handling with the above Compound I I of 50 micrograms, the number of apoptotic cell also increases to some extent in the visual field, but the specific activity Compound I of Compound I I cancer cell specific induction of apoptosis is weak significantly a lot.When handling with Compound I more than every milliliter 50 microgram and Compound I I, some cancer cells begins to be the morphological feature of typical gangrenosum acne cell in various degree, and along with the concentration of Compound I and II increases, the gangrenosum acne cytotoxic activity also strengthens, and shows that Compound I has certain 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,
The six-ring of pimelinketone is got
4C
1The chair form configuration;
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, all the other at least three for being selected from the oxygen-containing substituents of hydroxyl or methoxyl group.
2。Formula I compound or its pharmacy acceptable salt class:
Formula I
Wherein,
The six-ring of pimelinketone is got
4C
1The chair form configuration;
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, all the other at least three for being selected from the oxygen-containing substituents of hydroxyl or methoxyl group.
3. the described formula I compound of claim 2, wherein, R
1And R
2For hydroxyl or (Z)-14-alkene nonadecyl; R
3, R
4, R
5, R
6Be hydroxyl or hydrogen.
4. claim 2 or 3 described formula I compounds, wherein, R
1Be (Z)-14-alkene nonadecyl, R
2, R
4, R
6Be hydroxyl, R
3, R
5Be hydrogen.
5. claim 2 or 3 described formula I compounds, wherein, R
2Be (Z)-14-alkene nonadecyl, R
1, R
3, R
6Be hydroxyl, R
4, R
5Be 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 the relevant plant material of aqueous alcohol lixiviate Fructus Choerospondiatis for example, obtain extract.
8. after the preparation method of the described formula I compound of claim 2, this method comprised that with alcohol or the relevant plant material of aqueous alcohol lixiviate for example Fructus Choerospondiatis obtains crude extract, separation and purification obtained formula I compound.
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, tumour 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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