CN1616428A - Acenaphthene heterocyclic compound and its cell fading inducing and anti-tumor use - Google Patents
Acenaphthene heterocyclic compound and its cell fading inducing and anti-tumor use Download PDFInfo
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- CN1616428A CN1616428A CN 200410050449 CN200410050449A CN1616428A CN 1616428 A CN1616428 A CN 1616428A CN 200410050449 CN200410050449 CN 200410050449 CN 200410050449 A CN200410050449 A CN 200410050449A CN 1616428 A CN1616428 A CN 1616428A
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- acenaphthene
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- 230000001939 inductive effect Effects 0.000 title claims abstract description 14
- 230000000259 anti-tumor effect Effects 0.000 title abstract description 9
- CWRYPZZKDGJXCA-UHFFFAOYSA-N acenaphthalene Natural products C1=CC(CC2)=C3C2=CC=CC3=C1 CWRYPZZKDGJXCA-UHFFFAOYSA-N 0.000 title description 15
- HXGDTGSAIMULJN-UHFFFAOYSA-N acetnaphthylene Natural products C1=CC(C=C2)=C3C2=CC=CC3=C1 HXGDTGSAIMULJN-UHFFFAOYSA-N 0.000 title description 8
- -1 Acenaphthene heterocyclic compound Chemical class 0.000 title description 3
- 238000005562 fading Methods 0.000 title 1
- 150000001875 compounds Chemical class 0.000 claims abstract description 34
- 230000006907 apoptotic process Effects 0.000 claims abstract description 30
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 26
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 12
- 241001465754 Metazoa Species 0.000 claims abstract description 11
- 229910052736 halogen Chemical group 0.000 claims abstract description 5
- 230000004614 tumor growth Effects 0.000 claims abstract description 5
- 230000001093 anti-cancer Effects 0.000 claims abstract description 4
- 125000005843 halogen group Chemical group 0.000 claims abstract 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 49
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 12
- 230000030833 cell death Effects 0.000 claims description 10
- 125000000217 alkyl group Chemical group 0.000 claims description 9
- 230000022131 cell cycle Effects 0.000 claims description 8
- 238000001727 in vivo Methods 0.000 claims description 7
- 239000000411 inducer Substances 0.000 claims description 7
- 230000018199 S phase Effects 0.000 claims description 6
- BRNULMACUQOKMR-UHFFFAOYSA-N thiomorpholine Chemical compound C1CSCCN1 BRNULMACUQOKMR-UHFFFAOYSA-N 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- PVOAHINGSUIXLS-UHFFFAOYSA-N 1-Methylpiperazine Chemical compound CN1CCNCC1 PVOAHINGSUIXLS-UHFFFAOYSA-N 0.000 claims description 3
- 231100000673 dose–response relationship Toxicity 0.000 claims description 3
- 238000004043 dyeing Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 claims description 3
- 230000003442 weekly effect Effects 0.000 claims description 3
- 125000002757 morpholinyl group Chemical group 0.000 claims description 2
- 125000004193 piperazinyl group Chemical group 0.000 claims description 2
- 125000005936 piperidyl group Chemical group 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 claims 2
- 238000010253 intravenous injection Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 29
- 230000000694 effects Effects 0.000 abstract description 8
- 125000003277 amino group Chemical group 0.000 abstract 1
- 230000001419 dependent effect Effects 0.000 abstract 1
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- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 12
- LWLSVNFEVKJDBZ-UHFFFAOYSA-N N-[4-(trifluoromethoxy)phenyl]-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide Chemical compound FC(OC1=CC=C(C=C1)NC(=O)N1CCC(CC1)CC1=CC(=CC=C1)OC1=NC=C(C=C1)C(F)(F)F)(F)F LWLSVNFEVKJDBZ-UHFFFAOYSA-N 0.000 description 10
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- 239000002904 solvent Substances 0.000 description 9
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 8
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- 235000015097 nutrients Nutrition 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 230000025084 cell cycle arrest Effects 0.000 description 3
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- 238000001816 cooling Methods 0.000 description 3
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- 150000003141 primary amines Chemical class 0.000 description 3
- 150000003335 secondary amines Chemical class 0.000 description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 101710183280 Topoisomerase Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 150000002367 halogens Chemical group 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012434 nucleophilic reagent Substances 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 235000015320 potassium carbonate Nutrition 0.000 description 2
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- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
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- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
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- MAFQLJCYFMKEJJ-UHFFFAOYSA-N ethyl 4-aminobutanoate Chemical compound CCOC(=O)CCCN MAFQLJCYFMKEJJ-UHFFFAOYSA-N 0.000 description 1
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Abstract
The present invention relates to a kind of amino group or halogen substituted 8-oxy-8H-acenaphthenee[1, 2-b] pyrrolyl-9-nitrile derivatives and their biological use. They are applied mainly in intracorporeal and extracorporeal inducing cell apoptosis and as anticancer compound. These compounds are 3, 6-disubstituted, 3-disubstituted or 6-substituted 8-oxy-8H-acenaphthenee[1, 2-b] pyrrolyl-9-nitrile derivatives. They can induce tumor cell apoptosis and block cell period in the dosage dependent mode within the wide concentration range of 0.01-10 micro mole. The highest activity compound B1 has IC50=0.17 micro mole (0.56 micro gram). When they are used in animal tumor model body, they can inhibit tumor growth obviously in inducing cell apoptosis mode. Therefore, the present invention is one kind of apoptosis inducing agent and antitumor compound with high activity.
Description
Technical field
The present invention relates to a class 8-oxygen-8H-acenaphthene also the amino of [1,2-b] pyrroles-9-nitrile or halogen substituted derivative and in vivo, external cell death inducing effect and as the application of anticancer compound.
Background technology
Along with the research that pair cell death process and mechanism deepen continuously, apoptosis becomes the new target spot of antitumor drug research.Because the characteristic of tumour cell is to resist apoptosis by escaping the organism signals-modulating, reach " immortality ".So effectively inducer of apoptosis will fundamentally suppress the existence of tumour cell and the continuation abnormal shapeization of precancerous lesion.No matter as the instrument of oncology fundamental research or the medicine of clinical treatment tumour, inducer of apoptosis all has great value.At present, comprise Calbiochem (German), Merck (German), Promage how tame biotech firms such as (USA) has all developed and has continued exploitation apoptosis induction product.There are some inducer of apoptosis to become the clinical chemotherapeutics of using, for example camptothecine (Y Pommier.Diversity ofDNA topoisomerases I and inhibitors.Biochimie, 1998,80; 255-270.).These products all belong to vitamin A, non-steroidal anti-inflammatory drugs, polyphenol, vanilla element and topoisomerase enzyme inhibitor, as seen they are still based on the chemosynthesis product of natural drug or its analogue, all exist the source not enough, cost an arm and a leg, problem such as selectivity is low, side reaction is big, effect is undesirable.In order to find new chemotherapeutics, The National Cancer Institute random screening anti-tumor activity (the John N.Weinstein of 300000 different sorts compounds, Timothy GMyers, Patrick M.O ' Connor, et al.Aninformation-intensive approach to the molecular pharmacology of cancer.Science, 1997,275; 3430-349.).In recent years, Qian Xuhong etc. also synthesizes, detects, has reported multiple anticancer compound.The target of this research field be find new texture, lower median lethal concentration, be to act on the compound that all has anti-tumor activity by way of, vivo and vitro with apoptosis-induced.
Summary of the invention
The present invention is successfully preparing new fluorescent chromophore 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile and derivative (ZL021484007 thereof, Qian Xuhong, on basis Xiao Yi), at parent 8-oxygen-8H-acenaphthene also [1,2-b] introduce new active group on pyrroles-9-nitrile, formulated novel cell inducer of apoptosis and antineoplastic compound involved in the present invention.
The present invention is at 8-oxygen-8H-acenaphthene also [1,2-b] 3 or 6 of pyrroles-9-nitrile introduce various primary amine and secondary amine and halogen, generate 3,6-two replaces, 8-oxygen-8H-acenaphthene that 3-replaces or 6-replaces also [1,2-b] pyrroles-9-nitrile, this is the compound that a class has cell death inducing effect and anti-tumor activity, its general formula of molecular structure:
(1)R
1＝NHR
4，R
2＝H，
A, R
4=X SR
5, X=C
2-C
6The straight or branched alkyl, R
5=CH
3, C
2H
5
B, R
4=YCOO (CH
2)
mCH
3, Y=C
2-C
6The straight or branched alkyl, m=0-3;
c、R
4＝(CH
2)
nAr，n＝1-3，Ar＝2’-CF
3Ph-，3’-CF
3Ph-，4’-CF
3Ph-，2’-FPh-，
3’--FPh-，4’-FPh-；
d、R
4＝(CH
2)
xCF
3，x＝2，3；
E, R
4=X S (O) R
6, X=C
2-C
6The straight or branched alkyl, R
6=CH
3, C
2H
5
F, R
4=X S (O2) R
6, X=C
2-C
6The straight or branched alkyl, 6
5=CH
3, C
2H
5
(2) R
2=pyrrolidyl, piperidyl, piperazinyl, N '-methylpiperazine base, morpholinyl, parathiazan base;
(3)R
1＝F，Cl，Br，R
2＝H；
(4)R
1＝H，R
2＝F，Cl，Br；
(5)R
1＝R
2＝F，Cl，Br。
The present invention is with 8-oxygen-8H-acenaphthene with good rigidity coplanarity and strong electron deficiency also [1,2-b] pyrroles-9-nitrile (ZL021484007) is raw material, with the nucleophilic reagent primary amine or stretch amine fragrant hydrogen nucleophilic substitution reaction takes place, obtain 3,6-two replaces, 8-oxygen-8H-acenaphthene that 3-replaces or 6-replaces is [1,2-b] pyrroles-9-nitrile (general formula of molecular structure A) also:
Also [1,2-b] pyrroles-9-nitrile is in solvent (tetrahydrofuran (THF), acetonitrile, pyridine, dimethyl formamide or dimethyl sulfoxide (DMSO)) for 8-oxygen-8H-acenaphthene, and with the primary amine of equimolar amount, secondary amine class nucleophilic reagent reacts, and temperature is 20-100 ℃, 0.5～24 hour reaction times.After removing partial solvent under reduced pressure after the cooling, filter or directly column chromatography can make also [1,2-b] pyrroles-9-nitrile of product 3 or the single substituted-amino of 6--8-oxo-8H-acenaphthene.
And 3, the two substituted-aminos of 6--8-oxygen-8H-acenaphthene also [1,2-b] synthetic method of pyrroles-9-nitrile then is: 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile is at solvent (tetrahydrofuran (THF), acetonitrile, pyridine, dimethyl formamide or dimethyl sulfoxide (DMSO)) in the secondary amine of excessive (4～5 times of molar weights) for a long time through reaction in 6～48 hours, temperature is 20-100 ℃, replaces product with obtaining two after the above-mentioned same processing.
3 or 8-oxygen-8 hydrogen-acenaphthene of replacing of 6-halogen [1,2-b] pyrroles-9-nitrile (5,6 among general molecular formula A also, 7) synthetic method is the base catalysis ring-closure reaction: raw material 2-(2-oxo-2 hydrogen-halogenated acenaphthylene)-propane dinitrile, at solvent (tetrahydrofuran (THF), acetone, pyridine, dimethyl formamide, acetonitrile, ethanol, methyl alcohol or dimethyl sulfoxide (DMSO)) in, basic catalyst (salt of wormwood, triethylamine or pyridine) exists down, temperature is 40-120 ℃, stirs 10-100 minute.After removing partial solvent under reduced pressure after the cooling, filtration can make product.
The B series compound of structure is disclosed as fluorescent chromophore among A series compound and the patent ZL021484007:
(1) R
1=parathiazan base, pyrrolidyl, R
2=H;
(2) R
1=NHR
4, R
2=H, R
4=C
1-C
12Branched-chain alkyl;
(3) R
1=R
2=pyrrolidyl, the parathiazan base;
(4)R
1＝NH(CH
2)
yNR
5，y＝2，3，R
5＝CH
3，C
2H
5，R
2＝H；
(5) B1 (works as R
1=parathiazan base, R
2During=H)
As in the body, the application of cell in vitro inducer of apoptosis and antineoplastic compound.Method is: at first measure IC by mtt assay
50Selecting the HELA cell is target cell, becomes density to be about 5 * 10 cell dilution with the RPMI1640 nutrient solution
5The cell suspension of individual/ml adds in the 96 porocyte culture plates, and every hole adds 200 μ l tumour cell suspensions.The 100%DMSO solution that adds compd A, B respectively, final concentration 100,50,10,5,0.5,0.1,0.05,0.01 μ M establishes cell control group and positive drug cyclophosphamide-a control group simultaneously.CO
2Every hole adds MTT solution 20 μ l after hatching 24hrs in the incubator, measures optical density(OD) (OD) value under the 570nm with microplate reader, according to optical density value, calculates the survival rate of each hole tumour cell.
The result shows the IC of the compound B-11 that cytotoxicity is the strongest
50=0.17 μ M (0.56 μ g).Other Compound I C
50Value is between 0.4-6.8 μ M.
The active experimental technique of anti-tumor in vivo is: at first cultivate hepatoma cell line H22, it is subcutaneous only to be inoculated in the right armpit of mouse by 200 μ l/, is prepared into animal model for tumour.In back 5 days of inoculation, the knurl piece formed.Beginning is according to the concentration gradient 30%DMSO solution of vein or local injection compd A, B once a day.Experimental session is measured tumour major diameter (a) and perpendicular minor axis (b), by formula 1/2ab twice weekly
2It is long-pending to calculate the knurl block.Observe the animals survived time.The result shows that A series compound and B series compound all have and suppresses tumor growth in various degree, prolongs the effect of tumor model survival time of animals.
The present invention also by flow cytometer detected A, B series compound in vivo, external evoked apoptosis and the effect of retardance cell cycle.Method is in the in vitro tests, and selecting the HeLa cell is target cell, becomes density to be about 5 * 10 cell dilution with the RPMI1640 nutrient solution
5The cell suspension of individual/ml adds in the 96 porocyte culture plates, and every hole adds 200 μ l tumour cell suspensions.Add compd A, B respectively by concentration gradient, establish cell control group and positive drug cyclophosphamide-a control group simultaneously.CO
2After hatching 24 hours in the incubator, 70% cold ethanol is fixed; In the in vivo test, the tumor tissues of the tumor model animal of the compound treatment of learning from else's experience, the preparation single cell suspension is set undressed tumor model animal groups simultaneously and is contrast.70% cold ethanol is fixed.Flush away stationary liquid before the last machine testing, propidium iodide dyeing, flow cytometer detects.Computer automatically to the position at each peak, peak heights, cell cycle each the time phase per-cent analyze.The result shows that with dose-dependent mode cell death inducing, apoptosis rate is at 9%-31% between 0.01-10 μ M for the compound B-11 of best results.Cell-cycle arrest is in the S phase.Other compound all relies on mode inducing culture apoptosis with dosage.
A, B series compound be all as antineoplastic compound or cell death inducer, with the mode of cell death inducing in vivo, the vitro inhibition growth of tumor.
Description of drawings
Fig. 1: flow cytometer detected result.X-coordinate is a DNA concentration, and ordinate zou is a cell count.Compound B-11 and culturing cell are hatched altogether, final concentration 0.01 μ M.After 24 hours, flow cytometer detects, computer for analysis DNA concentration and cell cycle.Diagram: visible apoptotic peak, apoptosis ratio 9.4%, cell cycle analysis result show that S phase cell accounts for 6.7%.
Fig. 2: flow cytometer detected result.Compound B-11 and culturing cell are hatched altogether, final concentration 0.1 μ M.After 24 hours, flow cytometer detects.The result: visible apoptotic peak, apoptosis ratio 15.6%, cell cycle analysis result show that S phase cell accounts for 26.5%.
Fig. 3: flow cytometer detected result.Compound B-11 and culturing cell are hatched altogether, final concentration 10 μ M.After 24 hours, flow cytometer detects.The result: visible apoptotic peak, apoptosis ratio 31.3%, cell cycle analysis result show that S phase cell accounts for 32.4%.
Fig. 4 a: flow cytometer detected result.Subcutaneous tumor-bearing mice after the compound B-11 processing, is got the knurl piece and is prepared cell suspension, and flow cytometer detects.Result: visible apoptotic peak, apoptosis ratio 13.1%.
Fig. 4 b: flow cytometer detected result.The subcutaneous tumor-bearing mice of control group (not passing through compound treatment) is got the knurl piece and is prepared cell suspension, and flow cytometer detects.Result: do not have apoptotic peak.
Embodiment
Embodiment 1
1 gram 8-oxygen-8H acenaphthene also adds 0.46 gram 3-thiopurine methyltransferase-propylamine, stirring at normal temperature 2 hours in 50 milliliters of acetonitriles of [1,2-b] pyrroles-9-nitrile, the evaporation section solvent, separate out also [1,2-b] pyrroles-9-nitrile A1 of product 3-(3 '-thiopurine methyltransferase-propyl group) amino-8-oxygen-8H-acenaphthene, yield 60%.M.p.266-268℃；
1H?NMR(400M，DMSO)：δ9.604(br?s，-NH-，1H)，8.5-8.54(d，J＝7.6Hz，1H)，8.2-8.21(d，J＝7.2Hz，1H)，7.75-79(d，J＝8.8Hz，1H)，7.57-7.6(t，J＝7.8Hz，1H)，6.9-6.92(d，J＝9.2Hz，1H)，3.24(br?s，-NHCH
2CH
2-，2H)，2.82-2.84(m，-NHCH
2CH
2CH
2-，2H)，2.42(br?s，-SCH
3-，3H)，1.89-1.91(m，-NHCH
2CH
2CH
2-，2H)；ESI-MS：[M+H]
-(334m/z).
Embodiment 2
1 gram 8-oxygen-8H acenaphthene also adds 0.54 gram 4-aminobutyric acid ethyl ester, stirring at normal temperature 30 minutes in 50 milliliters of acetonitriles of [1,2-b] pyrroles-9-nitrile, the evaporation section solvent, separate out also [1,2-b] pyrroles-9-nitrile A2 of product 3-(3 '-carboxylic acid, ethyl ester propyl group) amino-8-oxygen-8H-acenaphthene, yield 65%.M.p.＞300℃；
1H?NMR(400M，DMSO)：δ9.20(br?s，-NH-，1H)，8.84-8.86(d，J＝7.6Hz，1H)，8.64-8.66(d，J＝7.2Hz，1H)，7.95＝7.97(d，J＝8.8Hz，1H)，7.74-7.78(t，J＝7.8Hz，1H)，6.80-6.82(d，J＝9.2Hz，1H)，4.14-4.19(q，-COOCH
2CH
3，2H)，3.62-3.67(m，-NHCH
2CH
2CH
2-，2H)，2.52-2.55(m，-NHCH
2CH
2CH
2COO-，2H)，2.10-2.13(m，-NHCH
2CH
2CH
2COO-，2H)，1.35-1.38(t，-COOCH
2CH
3，3H)；ESI-MS：[M+H]
-(360m/z).
Embodiment 3
1 gram 8-oxygen-8H acenaphthene also [1,2-b] add 0.45 gram parathiazan in 50 milliliters of acetonitriles of pyrroles-9-nitrile, stirring at normal temperature 2 hours, column chromatography for separation goes out product 3-parathiazan base-8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile and 6-parathiazan base-8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile A3, yield is respectively 40% and 30%.A3:M.p.225 ℃ of decomposition;
1H NMR (400M, DMSO): 8.69-8.71 (d, J=8.0Hz, 1H), 8.42-8.45 (d, J=8.2Hz, 1H), and 7.98-7.96 (d, J=8.0Hz, 1H), 7.88-7.92 (t, J=8.0Hz, 1H), and 7.04-7.02 (d, J=8.2Hz, 1H), 3.68 (br s ,-N (CH
2CH
2)
2S, 4H), 3.05 (br s ,-N (CH
2CH
2)
2S, 4H); ESI-MS:[M-H]
-(330m/z).
Embodiment 4
1 gram 8-oxygen-8H acenaphthene also adds 0.43 gram N methyl piperazine in 50 milliliters of acetonitriles of [1,2-b] pyrroles-9-nitrile, and stirring at normal temperature 2 hours, column chromatography for separation go out also [1,2-b] pyrroles-9-nitrile A4 of product 6-(N '-methylpiperazine base)-8-oxygen-8H-acenaphthene, yield 30%.A4:M.p.240 ℃ of decomposition;
1H NMR (400M, DMSO): 8.69-8.71 (d, J=8.0Hz, 1H), 8.42-8.45 (d, J=8.2Hz, 1H), and 7.98-7.96 (d, J=8.0Hz, 1H), 7.88-7.92 (t, J=8.0Hz, 1H), and 7.04-7.02 (d, J=8.2Hz, 1H), 3.45 (br s ,-N (CH
2CH
2)
2NCH
3, 4H), 2.95 (br s ,-N (CH
2CH
2)
2NCH
3, 4H), 2.43 ((br s ,-N (CH
2CH
2)
2NCH
3, 3H); ESI-MS:[M+H]
-(329m/z).
Embodiment 5
1 gram 8-oxygen-8H acenaphthene also [1,2-b] add 0.82 gram 2-(4 '-trifluoromethyl) ethamine in 50 milliliters of acetonitriles of pyrroles-9-nitrile, stirring at normal temperature 30 minutes, the evaporation section solvent, separate out product 3-2-(4 '-trifluoromethyl) ethylamino-8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile A5, yield 55%.A5：M.p.254-256℃；
1H?NMR(400M，DMSO)：δ9.13-9.11(d，J＝7.6Hz，-NH-，1H)，9.03-9.01(d，J＝7.6Hz，1H)，8.54-8.52(d，J＝7.2Hz，1H)，7.90-7.87(d，J＝9.2Hz，1H)，7.87-7.83(d，J＝8.0Hz，2H)，7.27-7.25(d，J＝8.0Hz，2H)，7.22-7.20(t，J＝8.0Hz，1H)，7.09-7.07(d，J＝9.2Hz，1H)，3.60-3.59(br?s，-NHCH
2CH
2-，2H)，2.80-2.78(d，-NHCH
2CH
2，2H)；ESI-MS：[M+H]
-(418m/z).
Embodiment 6
1 gram 8-oxygen-8H acenaphthene also adds 0.50 gram 2-trifluoromethyl ethamine, stirring at normal temperature 30 minutes in 50 milliliters of acetonitriles of [1,2-b] pyrroles 9-nitrile, the evaporation section solvent, separate out also [1,2-b] pyrroles-9-nitrile A6 of product 3-(2 '-trifluoromethyl ethyl) amino-8-oxygen-8H-acenaphthene, yield 55%.M.p.266-268℃；
1H?NMR(400M，DMSO)：δ9.450(br?s，-NH-，1H)，8.6-8.62(d，J＝7.6Hz，1H)，8.42-8.45(d，J＝7.2Hz，1H)，7.80-7.82(d，J＝8.8Hz，1H)，7.57-7.6(t，J＝7.8Hz，1H)，7.12-7.14(d，J＝9.2Hz，1H)，3.16(br?s，-NHCH
2CH
2-，2H)，2.32-2.34(m，-NHCH
2CH
2CF
3-，2H)；ESI-MS：[M+H]
-(342m/z).
Embodiment 7
1 gram 2-(2-oxo-2 hydrogen-acenaphthene)-propane dinitrile, 0.2 gram salt of wormwood, 10 milliliters of dimethyl sulfoxide (DMSO) are heated to 100 ℃, stir 15 minutes, and crystal is separated out in cooling.Filter, washing and drying gets also [1,2-b] pyrroles-9-nitrile A7 of product yellowish brown crystal 8-oxo-8 hydrogen-acenaphthene, yield, 80%.
1H?NMR(400M，DMSO)：δ8.71-8.69(d，J＝8.0Hz，1H)，8.67-8.65(d，J＝7.6Hz，1H)，8.64-8.62(d，J＝8.0Hz，1H)，8.42-8.40(d，J＝7.6Hz，1H)，7.99-7.95(t，J＝7.8Hz，1H)；
13C?NMR(100M，DMSO)：δ177.48，138.26，137.73，134.40，132.72，131.82，131.37，128.91，127.94，127.37，126.13，122.22，119.72，113.82，113.38；IR(KBr)cm
-1：2231，1643，1577；ESI-MS：M+Na
+(331，m/z).
Embodiment 8
The anti tumor activity in vitro experiment.Adopt cell culture method that A, B series compound are carried out the anti-tumor activity experiment.At first measure IC by mtt assay
50Selecting the HeLa cell is target cell, becomes density to be about 5 * 10 cell dilution with the RPMI1640 nutrient solution
5The cell suspension of individual/ml adds in the 96 porocyte culture plates, and every hole adds 200 μ l tumour cell suspensions.The 100%DMSO solution that adds A, B series compound respectively, final concentration 100,50,10,5,1,0.5,0.1,0.05,0.01 μ M sets up culture of tumor cell control group and cancer therapy drug endoxan positive controls simultaneously.CO
2Every hole adds MTT solution 20 μ l after hatching 24hrs in the incubator, measures optical density(OD) (OD) value under the 570nm with microplate reader, according to optical density value, calculates the survival rate of each hole tumour cell.Experiment repeats 3 times, gets its mean value.According to formula logIC
50=Xm-I[P-1/4 (3-Pm-Pn)] calculating IC
50
Wherein, Xm:log peak concentration, I:log (peak concentration/relative concentration), P: positive rate sum, Pm: maximum positive rate, Pn: minimum positive rate.The result shows the IC of the compound B-11 that cytotoxicity is the strongest
50=0.17 μ M (0.56 μ g).(table 1).Other Compound I C
50Value is between 0.4-6.8 μ M.
Detect apoptosis ratio and cell-cycle arrest by flow cytometer again.Selecting the HELA cell is target cell, becomes density to be about 5 * 10 cell dilution with the RPMI1640 nutrient solution
5The cell suspension of individual/ml adds in the 96 porocyte culture plates, and every hole adds 200 μ l tumour cell suspensions.Add compound respectively, establish cell control group and positive drug cyclophosphamide-a control group simultaneously.CO
2After hatching 24 hours in the incubator, cold ethanol is fixed.Flush away stationary liquid before the last machine is got single cell suspension, propidium iodide dyeing, and flow cytometer detects.Computer automatically to the position at each peak, peak heights, cell cycle each the time phase per-cent analyze.The result shows that with dose-dependent mode cell death inducing, apoptosis rate is at 9%-31% between 0.01-10 μ M for the compound B-11 of best results.Cell-cycle arrest is at S phase (accompanying drawing 1-3).
Other compound all relies on mode inducing culture apoptosis with dosage, and the apoptosis ratio is about 15%.
Embodiment 9
Anti-tumor in vivo is active to be detected.Select the Chinese kunming mice of raising, random packet, 10 every group.The liver cancer cell H22 that cultivates, it is subcutaneous only to be inoculated in the right armpit of mouse by 200 μ l/.After 5 days, the subcutaneous tumors piece forms in the lotus knurl.The 30%DMSO solution of beginning vein or local injection compd A, B.With the compound B-11 is example, is divided into 0.03mg/kg body weight group and 0.3mg/kg body weight group.Experimental session is measured tumour major diameter (a) and perpendicular minor axis (b), by formula 1/2ab twice weekly
2Calculate gross tumor volume. observe the animals survived time.Tested the 14th day, and pressed gross tumor volume and calculate tumour inhibiting rate.0.03mg/kg body weight group tumour inhibiting rate 50%, 0.3mg/kg body weight group tumour inhibiting rate 70%.Other compound all has the effect that suppresses tumor growth in various degree, and inhibiting rate is about 20-50%.
The control animals mean survival time is 18 days, the mean lifetime of compound group 26 days.Statistical procedures result shows P＜0.05, thinks that these compounds have the effect that prolongs the tumour survival time of animals.
Test after 7 days, strip the mouse Subcutaneous tumor, with physiological saline tissue homogenate is prepared cell suspension, the same method cells were tested by flow cytometry apoptosis rate by 1: 3 volume.The result show these compounds all have induced tumor histocyte effect of apoptosis (accompanying drawing 4a, 4b).The apoptosis ratio is between 10%-50%.
Table 1 compound B-11 is to cultivating the HeLa cell inhibiting
| Concentration (μ M) | 0.01 | ?0.05 | ?0.1 | ?0.2 | ?0.4 | ?0.5 | ?1 | ?2 |
| Inhibiting rate (%) (3 times) | 2 | ?6.7 | ?31.5 | ?53.5 | ?68 | ?78 | ?83.8 | ?89.8 |
| 1.2 | ?6 | ?31 | ?52.2 | ?67.5 | ?76.6 | ?83.4 | ?87.8 | |
| 1.4 | ?7.1 | ?31.4 | ?51.2 | ?67.6 | ?76.4 | ?83.6 | ?88.2 | |
| Average inhibiting rate (%) | 1.53 | ?6.6 | ?31.3 | ?52.3 | ?67.7 | ?77.0 | ?83.6 | ?88.6 |
Claims (3)
1, the also amino or the halogen substituted derivative of [1,2-b] pyrroles-9-nitrile of a class 8-oxygen-8H-acenaphthene is characterized in that this derivative is 3, and 6-two replaces, and 8-oxygen-8H-acenaphthene that 3-replaces or 6-replaces is [1,2-b] pyrroles-9-nitrile also, general formula of molecular structure A:
Among the general formula A, (1) R
1=NHR
4, R
2=H,
A, R
4=XSR
5, X=C
2-C
6The straight or branched alkyl, R
5=CH
3, C
2H
5
B, R
4=YCOO (CH
2)
nCH
3, Y=C
2-C
6The straight or branched alkyl, n=0-3;
c、R
4＝(CH
2)
xAr，x＝1-3，Ar＝2’-CF
3Ph-，3’-CF
3Ph-，4’-CF
3Ph-，2’-FPh-，
3’--FPh-，4’-FPh-；
d、R
4＝(CH
2)
yCF
3，y＝2，3；
E, R
4=XS (O) R
6, X=C
2-C
6The straight or branched alkyl, R
6=CH
3, C
2H
5
F, R
4=XS (O
2) R
6, X=C
2-C
6The straight or branched alkyl, R
6=CH
3, C
2H
5
(2) R
2=pyrrolidyl, piperidyl, piperazinyl, N '-methylpiperazine base, morpholinyl, parathiazan base, R
1=H;
(3)R
1＝F，Cl，Br，R
2＝H；
(4)R
1＝H，R
2＝F，Cl，Br；
(5)R
1＝R
2＝F，Cl，Br。
2, according to the application of the described compd A of claim 1 and compd B as cell death inducer, it is characterized in that: the 100%DMSO solution of A, B two compounds, hatched altogether 24 hours with the mammalian tumor cell of cultivating, concentration gradient is under the condition of 0.01 μ M, 0.1 μ M, 1 μ M, 10 μ M, fix through 70% ethanol, after the propidium iodide dyeing, flow cytometer detects, the result proves that they can be with dose-dependent mode inducing apoptosis of tumour cell, the apoptosis ratio and can be blocked the cell cycle in the S phase between 9%-31%.
3, according to the application of the described compd A of claim 1 and compd B as anticancer compound, it is characterized in that: the 30%DMSO solution of A, B two compounds, local injection or intravenous injection are in the tumor model animal body, through about 30 days observation period, prove that they can prolong the lifetime of tumor model animal (P＜0.05), the mean lifetime of experimental group was at 26 days, the mean lifetime of 30%DMSO control group was at 18 days, measure tumour major diameter (a) and perpendicular minor axis (b), by formula 1/2ab twice weekly in the observation process
2Calculate gross tumor volume, the result proves that they can suppress the growth of tumor tissues (P＜0.01); Observe and finish back execution animal, strip tumor tissues, detect by flow cytometer after the homogenate, prove their cell death inducing in vivo, the apoptosis ratio is between 10%-50%.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1940563B (en) * | 2005-09-28 | 2010-04-28 | 华东理工大学 | Fluorescent marking reagent |
| WO2010054575A1 (en) * | 2008-11-11 | 2010-05-20 | 大连理工大学 | Acenaphtho heterocycle compounds, cyclodextrin inclusion compounds and complexes, and uses in the manufactures of bh3 protein analogue, bcl-2 family protein inhibitors thereof |
| CN101723907B (en) * | 2009-07-25 | 2011-09-14 | 大连理工大学 | O-dicyano-acenaphtho pyrazine compound and anti-tumor application thereof |
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| WO2012012941A1 (en) * | 2010-07-28 | 2012-02-02 | 大连理工大学 | Bcl-2 family protein inhibitors, their cyclodextrin inclusion compounds, complexes, and uses in manufacture of bcl-2 family protein inhibitors thereof |
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| CN101723907B (en) * | 2009-07-25 | 2011-09-14 | 大连理工大学 | O-dicyano-acenaphtho pyrazine compound and anti-tumor application thereof |
| CN102336700A (en) * | 2010-07-28 | 2012-02-01 | 大连理工大学 | Amino-substituted acenaphthylene heterocyclic compounds and application thereof |
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| CN111551416B (en) * | 2020-03-22 | 2021-08-06 | 华南理工大学 | Bacterial apoptosis evaluation method based on cell membrane phosphatidylserine fluorescent staining |
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