CN1267670A - Two aporphine-benzyl isoquinoline compounds with antineoplastic activity - Google Patents
Two aporphine-benzyl isoquinoline compounds with antineoplastic activity Download PDFInfo
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Abstract
By using the surface part of thalictrum atriplex as material and through extraction with specific solvent, are obtained two parts, B and C, which are separated with silica gel or silica gel column and eluted with specific solvent to obtain pure compounds. After TLC test and spectrascopic analysis, the new compounds are named Thaliatraplextine and Thaliatraplexine. Pharmacodynamic test shows that the two compounds have obvious suppression to cancer of human body and cancer cell strain of mouse.
Description
The invention belongs to a class and have Ah the pouncing on's phenanthrene-bisindole alkaloid compound of anti-tumor activity.
Ah pouncing on's phenanthrene-benzyl isoquinoline compounds has stronger restraining effect to kinds of tumor cells.The U.S. began in 1975 thalictrine (thalicarpine) has been carried out the I clinical trial phase, at maximum tolerance (1400mg/m
2) and intravenously administrable (1100mg/m
2) time, occur all that brachialgia, central nervous system suppress, feel sick, vomiting, elevation of blood pressure or to reduce sinus rate uneven, and with side effects such as ECG change.Began in 1980 it is carried out the II clinical trial phase, more side effect occurs, and finally cause the termination of antitumor clinical trial.The nineties, searching and antitumor screening to A Piaofei such as thalictrine-benzylisoquinoline new alkaloids are still continuing, effect is better than parental cell to discovery thalictrines such as Chen.G to the drug-fast rat ovary tumour cell of cis-platinum tool to those, the prompting thalictrine can replace plus cisplatin in treatment those to the anti-medicine of cis-platinum and with Hyperthermic malignant tumour, also the synergy and the mechanism of action of thalictrine and multiple antitumour drug compared systematic research simultaneously.Discovery thalictrines such as Todorov DK have proliferation function to two kinds of human glioma cells.The composition thaifarajine that Wu YW etc. tells from root and Rhizome of Javan Meadowrue (Tjavanicum) is inhibited to five kinds of tumour cells.Lin L-Z etc. tells multiple alkaloid from Da Ye grass of meadow rue (T.faberi Ulbr.) all have strong cytotoxicity to tumour cells such as P-388.Up to the present, it is individual to separate the A Piaofei-benzylisoquinoline dimer alkaloid nearly more than 80 that obtains from vegitabilia, they mainly are distributed in Thalictrum, and in Katsuratree genus and the part berberis, many these constituents have obvious restraining effect to tumour cell.But the new compound of two tool anti-tumor activities involved in the present invention up to now, still finds no patent or bibliographical information.
The present invention's purpose is to make full use of the plant resources that contains A Piaofei-bisindole alkaloid class of China's abundant, deeply research and develop, searching has new chemical structure, and anti-tumor activity is strong, and this compounds that toxic side effect is little is in the hope of the clinical new type antineoplastic medicine that provides is provided.
Technical scheme of the present invention comprises following steps according to this:
With narrow preface grass of meadow rue (Tatriptex Finet et Gagnep) over-ground part (11.5Kg) is the raw material (see figure 1), through with 95% alcohol reflux twice, each two hours; The dregs of a decoction are used 70% alcohol reflux twice again, and each two hours, behind the merging extracted twice liquid, reclaim solvent, get medicinal extract, the acetate dissolution with 5%, the sour water suspension is used NH
4The OH alkalization, PH9-10 filters, and must leach extracted with diethyl ether 4 times of thing A-1 (27g) buck suspension, behind the ether extraction liquid decompression and solvent recovery, gets extractum A-2 (64.5g).Merge A-1 and A-2, with the chloroform dissolving, insolubles discards, and chloroform solution 5%NaOH aqueous solution extraction 4 times after chloroform layer reclaims solvent, get medicinal extract B (82.5g); Buck layer NH
4Cl is saturated, uses extracted with diethyl ether, obtains ether extraction liquid, behind the recovery solvent, obtains medicinal extract C (9g).
B partly uses silica gel column chromatography, with chloroform-methanol system wash-out, every part of 100ml collects 210 parts altogether, 72-119 part (9.8g) wherein, use silica gel column chromatography once more, with chloroform-methanol system wash-out, every part of 50ml, collect 60 parts altogether, wherein 19-20 part obtains a white amorphous powder, and TLC checks to single spot, is Compound I (60mg).
C partly uses the silica gel H column chromatography, with chloroform-methanol system wash-out, obtain 105 parts, wherein 40-43 part is used silica gel column chromatography once more, with vinyl acetic monomer-methyl alcohol system wash-out, obtains 30 parts, 15-29 part wherein, obtain a white amorphous powder, TLC checks to single spot, is Compound I I (110mg).
Compound I, the unformed powder of white, mp 83-85 ℃, the Dragendorff reagent react positive, UV spectrum (MeOH) nm:202,222 (sh), 280,304, (see figure 2) is identical with known thalifaberine compounds, and the structural framework of determining this compound is thalifaberibe type A Piao phenanthrene-bisindole alkaloid.IR spectrum (see figure 3) shows 3440cm
-1Hydroxyl absorbs.The high resolution mass spectrometry determining molecular weight is m/z 682.3314, determines that its molecular formula is C
40H
46O
8N
2(calculated value is 682.3327) removed in the FAB-MS (see figure 4) and provided m/z 683 (M
++ 1) outside the fragment, base peak is m/z 206, according to the cleavage of mass spectrum rule of this compounds, illustrates that the isoquinoline 99.9 ring of benzylisoquinoline one side has two OCH
3 1H-NMR (CDCl
3) (see figure 5) δ 2.29,2.52ppm two unimodal methyl signals, be n-formyl sarcolysine base signal, δ 3.56,3.78,3.79,3.82,3.91,3.98ppm six methoxyl group signals are arranged, δ 6.02-7.81ppm has seven fragrant hydrogen signals, and wherein (2H is d.J=8.6Hz) with δ 6.96 (2H for δ 6.76, d.J=8.6Hz) be AA ' BB ' system, can be summed up as four symmetric fragrant hydrogen on benzylisoquinoline part and the phenyl ring that oxo bridge links to each other, δ 7.81 (1H, s) characteristic signal of 11 of compounds hydrogen for this reason.
13C-NMR spectrum (seeing Table 1) shows in low place (135-157ppm) has nine oxygen to replace carbon signal on the phenyl ring, except that methoxyl group and oxo bridge, supposition should have a hydroxyl, infer that from mass spectral m/z 369 fragments hydroxyl is in the luxuriant and rich with fragrance side of A Piao, NOE experiment (see figure 6) shows when shining δ 3.91 and 3.78ppm signal respectively, all there is gain at δ 7.81ppm peak, during irradiation δ 7.81ppm signal, δ 3.91 and 3.78ppm signal all have gain, determine that δ 3.78 and 3.91ppm peak lay respectively at 1 and 10 carbon signal of the luxuriant and rich with fragrance side of A Piao, during irradiation δ 3.82ppm signal, δ 6.53ppm signal has obvious gain, determine that δ 3.82ppm signal is 6 ' a methoxyl group, δ 6.53ppm signal is 5 ' hydrogen signal, the fragrant hydrogen of the methoxyl group of definite δ 3.56 and δ 6.01ppm should be respectively 7 ' and 8 ' signal simultaneously, when irradiation δ 3.95ppm signal, δ 3.78 and 3.79ppm signal have gain, determine that δ 3.95ppm signal is 2 a methoxyl group, δ 3.79ppm signal is 3 a methoxyl group, and hydroxyl should be connected in 9.HMBC spectrum (see figure 7) δ 2.29 and two n-formyl sarcolysine bases of 2.52ppm signal have long-range relevantly with δ 52.8 and 46.2ppm respectively, determine that two n-formyl sarcolysine bases are respectively 6 and 2 '.This just verifies further that above-mentioned supposition is rational.Final determine that Compound I is the new thalifaberine type A Piao phenanthrene-bisindole alkaloid of 1.2.3.10.6 ' .7 '-hexa methoxy-9-hydroxyl, its structure is suc as formula shown in the I, called after thaliatriplexine.(S, S) thalifaberine is identical, thereby determines that this compound 6 and 1` position chiral carbon are S, the S configuration with known compound of the same type for its CD spectrum.
Compound I I, yellow granular crystal (acetone), mp 205-207 ℃, the Dragendorff reagent react positive, the UV (see figure 8) is identical with known thalifaberine compounds, determine that its structural framework is a thalifaberine type alkaloid, high resolution FAB-MS (see figure 9) determining molecular weight is m/z 638.3059 (experimental value), determines that its molecular formula is C
38H
42O
7N
2(calculated value is 638.3065) removes m/z 639 (M among the FAB-MS
++ 1) outside, base peak is m/z 192, according to the cleavage of mass spectrum rule of this type compound, illustrates that the isoquinoline 99.9 ring substituents of benzylisoquinoline one side has a hydroxyl and a methoxyl group.
1H-NMR (CDC
3) (see figure 10) δ 2.30 and 2.48ppm be two n-formyl sarcolysine base signals, δ 3.68,3.78, and 3.87 and 3.95ppm
I R
1＝R
2＝OCH
3
II R
1=H, R
2=OH shows four methoxyl groups, and δ 6.04-8.01ppm shows eight aromatic ring hydrogen signals, wherein δ 6.76 (2H, d.J=8.6Hz) and 6.96ppm (2H, d.J=8.6Hz), two groups of peaks are AA ' BB ' system, be benzylisoquinoline part with phenyl ring that oxo bridge links to each other on four fragrant hydrogen of symmetry.δ 8.01ppm (1H, s) characteristic signal of 11 of compounds hydrogen for this reason.
13C-NMR spectrum (seeing Table 1) shows have eight oxygen to replace carbon signal on the phenyl ring at low place (δ 135-157ppm), except that the hydroxyl of methoxyl group, oxo bridge and benzylisoquinoline one side, infers that the luxuriant and rich with fragrance side of A Piao also has monohydroxy.NOE experiment (seeing Figure 11) is when shining δ 3.95 and 3.68ppm peak respectively, δ 8.01ppm has gain, during irradiation δ 8.01ppm, there is gain at δ 3.95 and 3.68ppm peak, illustrate that δ 3.95 and 3.68ppm are respectively 10 and 1 's methoxyl group, during irradiation δ 3.87ppm peak, the signal of δ 6.58ppm has obvious gain, during irradiation δ 3.58ppm signal, there is gain at δ 3.87ppm peak, illustrates that δ 3.87ppm signal is 2 a methoxyl group, and the signal of δ 3.58 is 3 a hydrogen, during irradiation δ 3.78ppm peak, the signal of δ 6.50ppm has gain, and during irradiation δ 6.50ppm signal, there is gain at δ 3.78ppm peak, thereby determine that δ 3.78ppm is 6 ' a methoxyl group, the signal of δ 6.50 is 5 ' hydrogen, and analysis-by-synthesis and in conjunction with FAB-MS determines that two hydroxyls should be respectively at 9 and 7 ', the structure of inferring Compound I I at last is the new thalifaberine type A Piao phenanthrene-bisindole alkaloid of 1.2.10.6 '-tetramethoxy-9.7 '-dihydroxyl, and its structure is suc as formula shown in the II.Its CD spectrum and similar known compound (S, S)-thalifaberine is identical, determines that 1 ' and 6 chiral carbon is S, S configuration, called after thaliatriplextine
Table 1 Compound I and II's
1346.7 2 146.2 144.5 4 ' 22.7 27.3 3 150.0 110.8 4,64.9 1a 124.0 124.0 1 ' a 127.0 128.8 1b, 128.0 129.5 3 ' 46.2, C-NMR data C I II C I II 1 150.0 145.1 1 ' 64.9 ' a 124.3 126.8 3a 150.0 122.0 5 ' 111.1,114.0 4 21.0 25.2 6 ' 147.0,144.0 5 52.8 53.1 7 ' 147.0,145.7 6a, 62.2 61.9 8 ' 111.0,111.0 7 24.0 28.8 9 ' 133.6,133.4 7a, 122.5 122.9 10 ' 132.0,115.0 11 108.2 108.6 14 ' 132.0,156.5 10 146.3 143.5 13 ' 115.0,115.0 9 137.8 138.3 12 ' 157.0,130.8 8 138.8 138.8 11 ' 115.0,130.8 11a, 124.0 124.0 α, 40.5 40.5 N-CH343.5 43.4 N-CH
342.6 42.4 1-OCH
361.6 55.8 6 '-OCH
356.4 60.2 2-OCH
361.9 55.9 7 '-OCH
3--56.4--3-OCH
3--61.6--10-OCH
355.8 56.3
Through pharmaceutical research, Compound I and II all have stronger anti tumor activity in vitro.
External pharmacodynamics test method:
The oncocyte cultivation is put into the incubator that contains 5%CO2 in RPMI 1640 substratum of the Streptomycin (Streptomycin sulphate) that contains 10% calf serum and 100U/ml Pemcillin (penicillin) and 100 μ g/ml cultivate.Measure allied compound to human oral JEG-3 (KB cell) with MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide) method, human oophoroma cell line (A2780), human large intestine cancer cell strain (HCT-8), and the lethal effect of mouse lymphocyte leukemia cell line (P-388).Half-inhibition concentration (IC50, the i.e. 50%Inhibition Concentration) expression that its fragmentation effect suppresses with tumor cell proliferation, concrete grammar is as follows:
Collect well-grown tumour cell, be mixed with 1 * 10 with the RPMI1640 substratum that contains 10% calf serum
4/ ml cell suspension, in 96 well culture plates, 100 μ l (containing 1000 tumour cells) are inoculated in every hole, put 37 ℃, cultivated 24 hours in the 5%CO2 incubator, the administration group adds the RPMI1640 substratum that contains the different concns medicine, every hole 100 μ l, if 4-5 concentration, 3 parallel holes in every hole, control group adds 1640 substratum that contain the equal-volume solvent, every hole 100 μ l, put 37 ℃, 5%CO
2Cultivated 4 days in the incubator.Discard nutrient solution, every hole adds MTT solution 100 μ l (0.4mg/ml is with RPMI 1640 preparations), hatches 4 hours for 37 ℃.Abandoning supernatant, every hole adding DMSO 150 μ l, dissolving Fomazan particle after the slight concussion, with BIO-RAD 550 type microplate reader, is being broadcast mensuration OD value under the long 540nm condition.
The result calculates: cough with the inhibiting rate mapping of the different concns of medicine and pair cell and obtain dose response curve, therefrom obtain half-inhibition concentration (IC
50).
Experimental result:
Compound I is at the external IC that above-mentioned tumor cell growth is suppressed
50Be respectively 2.65,2.34,2.81 and 0.7 μ g/ml;
Compound I I is at the external IC that above-mentioned tumor cell growth is suppressed
50Be respectively 2.78,2.46 and 2.77 μ g/ml (the P388 cell is not surveyed). see table 2 for details.
The external half-inhibition concentration (IC of table 2 to growth of cancer cells
50)
IC
50(μg/ml)
Sample KB A2780 HCT-8 P-388
I 2.65 2.34 2.81 0.7
II 2.78 2.46 2.77 does not survey
The result shows: Compound I and II have obvious effect to above-mentioned knurl strain.
Advantage of the present invention and positively effect are, resulting two new compound I and II are because to relevant human cancer cell strain tool obvious in-vitro suppression effect, thereby can make full use of the plant resources that contains A Piaofei-bisindole alkaloid class of China's abundant, the a large amount of extraction prepares, so that deeply carry out pharmacology, clinical study, create conditions for developing good effect and the little new antitumoral medicine of toxic side effect.
Description of drawings:
Fig. 1: the preparation flow of new A Piao phenanthrene-bisindole alkaloid Compound I and II;
Fig. 2: the UV spectrum of Compound I;
Fig. 3: the IR spectrum of Compound I;
Fig. 4: the FAB-MS spectrum of Compound I;
Fig. 5: Compound I
1The H-NMR spectrum;
Fig. 6: the NOE difference spectrum of Compound I;
Fig. 7: the HMBC spectrum of Compound I;
The UV spectrum of Fig. 8: Compound I I;
The FAB-MS spectrum of Fig. 9: Compound I I;
Figure 10. Compound I I's
1The H-NMR spectrum;
The NOE difference spectrum of Figure 11: Compound I I.
Claims (2)
1. have two new A Piao phenanthrene-benzyl isoquinoline compounds of anti-tumor activity, it is characterized in that
(1). the structure of compound is shown below:
In the formula: R
1Be H or OCH
3
R
2Be OH or OCH
3
(2). the preparation of compound comprises following steps successively:
Get aerial part of Thalictrum atriplex top and be divided into raw material, alcohol reflux gets medicinal extract, through acetate dissolution, the NH4OH alkalization is filtered, must leach thing A-1, get extractum A-2, merge A-1 and A-2 through extracted with diethyl ether, through the chloroform dissolving, discard insolubles, NaOH aqueous solution extraction chloroform solution, get medicinal extract B, with the saturated buck layer of NH4Cl, extracted with diethyl ether, medicinal extract C, through silicagel column or silica gel H post chromatography repeatedly, finally obtain desired compound again.
(3). the half-inhibition concentration (IC50) that suppresses with cultured tumor cells in vitro propagation adopts mtt assay to measure the lethal effect of compound to human oral cavity JEG-3 (KB cell), human oophoroma cell line (A2780), human large intestine cancer cell strain (HCT-8) and mouse lymph leukemia cell line (P-388) as index.
2. according to the described new A Piao phenanthrene-benzyl isoquinoline compounds of claim 1, it is characterized in that R in structure
1=R
2=OCH
3The time, being the new A Piaofei of 1.2.3.10.6 ' .7 '-hexa methoxy-9-hydroxyl-bisindole alkaloid Compound I, its molecular formula is C
40H
46O
8N
2, molecular weight is 682.3327 (calculated values), is that white is unformed Powdered; Work as R
1=H, R
2During=OH, be the new A Piaofei of 1.2.10.6 '-tetramethoxy-9.7 '-dihydroxyl-bisindole alkaloid Compound I I, molecular formula is C
38H
42O
7N
2, molecular weight is 638.3065 (calculated values), is yellow granular crystal.
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| CN1113869C CN1113869C (en) | 2003-07-09 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101480394B (en) * | 2008-01-09 | 2012-07-04 | 湘北威尔曼制药股份有限公司 | Anti-tumor pharmaceutical composition |
| CN101278989B (en) * | 2008-05-16 | 2013-12-11 | 重庆邮电大学 | Method for extracting Thalictrum aquilegifolium total alkaloids and total saponins from Thalictrum aquilegifolium |
| CN105315208A (en) * | 2014-07-17 | 2016-02-10 | 中国科学院兰州化学物理研究所 | Aporphine type alkaloid and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3976652A (en) * | 1971-12-07 | 1976-08-24 | Research Corporation | Isoquinoline compounds |
| US4013664A (en) * | 1973-06-20 | 1977-03-22 | Research Corporation | Synthesis of hernandaline |
-
2000
- 2000-03-23 CN CN00103296A patent/CN1113869C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101480394B (en) * | 2008-01-09 | 2012-07-04 | 湘北威尔曼制药股份有限公司 | Anti-tumor pharmaceutical composition |
| CN101278989B (en) * | 2008-05-16 | 2013-12-11 | 重庆邮电大学 | Method for extracting Thalictrum aquilegifolium total alkaloids and total saponins from Thalictrum aquilegifolium |
| CN105315208A (en) * | 2014-07-17 | 2016-02-10 | 中国科学院兰州化学物理研究所 | Aporphine type alkaloid and preparation method thereof |
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