CN1931840A - DNA target molecules and their application in inducing apoptosis and antagonizing tumor - Google Patents
DNA target molecules and their application in inducing apoptosis and antagonizing tumor Download PDFInfo
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- CN1931840A CN1931840A CNA2006101171313A CN200610117131A CN1931840A CN 1931840 A CN1931840 A CN 1931840A CN A2006101171313 A CNA2006101171313 A CN A2006101171313A CN 200610117131 A CN200610117131 A CN 200610117131A CN 1931840 A CN1931840 A CN 1931840A
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Abstract
The present invention relates to 8-oxo-8H-acenaphthene [1, 2-b] pyrrole derivative in the structure as shown and its use. The 8-oxo-8H-acenaphthene [1, 2-b] pyrrole derivative of the present invention in the concentration range of 0.01-10 microM can induce apoptosis of the cultured tumor cell and antagonize tumor growth in animal body, and is apoptosis inducer and antitumor compound with very high activity.
Description
Technical field
The present invention relates to class DNA target molecule and uses thereof, specifically, relate to also [1,2-b] pyrrole derivative and uses thereof of a kind of 8-oxygen-8H-acenaphthene.
Background technology
Thymus nucleic acid (DNA) is biological basic genetic material, is the carrier of genetic information.Many molecular energies combine with DNA, destroy the template action of DNA, influence gene regulating and expressive function, thereby bring out a lot of biological effects.The interaction of DNA and other molecule is the key areas in biological chemistry and the molecular biology.The interaction of research small-molecule substance and DNA helps the understanding of people to DNA and protein interaction mode, and significant to the research synthetic and the DNA higher structure of further instructing artificial nucleic acid.
In medical research, the research of DNA and targeted drug interaction of molecules not only can be illustrated the mechanism of causing a disease of vital process mechanism, disease from molecular level, and design that can guide drugs and synthetic, medicine in-vitro screening and the mechanism of curing the disease of inquiring into medicine.In addition, whether or identification particular sequence DNA indicator double-stranded DNA (or single stranded DNA) is had selective binding or have sequence-specific bonded targeted drug molecule to can be used as dna molecule hybridize, and this is an important content of DNA biosensor research.The design of novel DNA target compound all has very important theory significance and using value for chemistry, biology and medicine and pharmacology.In medical research and new drug design, in order to seek satisfactory DNA targeted drug molecule, must detailed understanding DNA and the intermolecular interaction of targeted drug.In living things system, the targeted drug molecule is complicated with combining of DNA, and binding pattern mainly comprises exterior static combination, the combination of ditch district and intercalation combination etc.It is a lot of to influence the interactional factor of targeted drug molecule and DNA, as the interaction of other molecule and targeted drug molecule or DNA in the character of the structure (shape, size, the flexibility that comprise molecule) of targeted drug molecule, the intermolecular interaction of targeted drug, solution and the solution etc.
Inserted type DNA targeted molecular has extremely important purposes in the interaction of studying small molecules and DNA and oncotherapy.As effective DNA intercalator, they generally all contain conjugated plane rigid structure and the alkaline side chain that πDian Zi lacks, its midplane fragrance or aromatic heterocycle system are easy to intercalation in the DNA base pair, alkaline side chain then can combine with DNA by electrostatic attraction, and alkaline side chain also plays extremely important effect to the cytotoxicity of compound simultaneously.The A.Ramos been reported is crossed the anti-tumor capacity of a series of naphthalimide compounds, the result shows forfeiture (the Curr.Med.Chem.-Anti Cancer Agents that will cause compound activity when the nitrogen-atoms in the side chain is replaced by carbon atom, sulphur atom and Sauerstoffatom, 2001,1:237).Stephanie Blanchard has also estimated the cytotoxic effect (J.Med.Chem of different compounds to the L1210 L-1210,2004,47:978), as expectation, relatively those do not have substituent compound, and the derivative that has N-dialkyl group side chain has lower IC
50Value.In addition, also have some other intercalator also to obtain same result, as anthra pyrazoles, pyrazoles acridine, anthracene-4-methane amide, mitoxantrone and its aza analogues etc. (J.Med.Chem, 2004,40:3749).
Summary of the invention
One of the object of the invention is, also [1,2-b] pyrrole derivative of a kind of novel 8-oxygen-8H-acenaphthene is provided;
Two of the object of the invention is, the also purposes of [1,2-b] pyrrole derivative of above-mentioned novel 8-oxygen-8H-acenaphthene is provided.
The present invention is at ZL 02148400.7 (inventing the red and Xiao Yi of the artificial money rising sun etc.) disclosed 8-oxygen-8H-acenaphthene also [1,2-b] on the basis of pyrroles-9-nitrile and derivative thereof, introduce new active group at parent, formulated DNA target molecule involved in the present invention (novel 8-oxygen-8H-acenaphthene also [1,2-b] pyrrole derivative), and through experiment confirm: 8-oxygen-8H-acenaphthene that the present invention formulated also [1,2-b] pyrrole derivative can be in the concentration range of 0.01 μ M～10 μ M, with the apoptosis of tumor cells and the remarkable animal tumor growth in vivo that suppresses of dose-dependent mode inducing culture, be very high cell death inducer and antineoplastic compound of a class activity.
The said 8-oxygen of the present invention-8H-acenaphthene also [1,2-b] pyrrole derivative has structure shown in the formula (1):
In the formula (1), R
1, R
2Be selected from H respectively, contain 1～3 N, S or/and five yuan of O or hexa-member heterocycle base or by the group shown in the formula (2), and R
1And R
2Be not H simultaneously; R
3Be CN or COOR
4, R wherein
4Be H, C
1～C
6Alkyl or C
1～C
6Haloalkyl;
In the formula (2), R
5Be H or C
1～C
6Alkyl; R
6For containing five yuan or the hexa-atomic aromatic heterocyclic of S,
Or
Wherein: R
7Be H or C
1～C
6Alkyl, R
8, R
9Be selected from C respectively
1～C
6Alkyl in a kind of, n=1～6.Preferred version of the present invention is: work as R
3During for CN, R
1By H, contain 1～3 N, S or/and five yuan of O or hexa-member heterocycle base or by the group shown in the formula (2) [it is described identical with preamble to be comprised substituent implication in the formula (2)], R
2For H or contain 1～3 N, S or/and five yuan or the hexa-member heterocycle base of O, and R
1And R
2Be not H simultaneously;
Preferred scheme is: work as R
3During for CN, R
1For H, contain 1～3 N, S or/and the hexa-member heterocycle base of O (as morpholinyl, parathiazan base or piperazinyl etc.) or by the group shown in the formula (2), in the formula (2), R
5Be H or C
1～C
3Alkyl; R
6For containing five yuan of aromatic heterocyclics of S,
Or
Wherein: R
7Be H or C
1～C
3Alkyl, R
8, R
9Be selected from C respectively
1～C
3Alkyl in a kind of, n=1～4; R
2For H or contain 1～3 N, S or/and the hexa-member heterocycle base of O (as morpholinyl, parathiazan base or piperazinyl etc.), and R
1And R
2Be not H simultaneously.
Another preferred version of the present invention is: work as R
3Be COOR
4(R wherein
4Be H, C
1～C
6Alkyl or C
1～C
6Haloalkyl) time, R
1, R
2Be selected from H respectively, contain 1～3 N, S or/and five yuan of O or hexa-member heterocycle base or by the group shown in the formula (2), and R
1And R
2Be not H simultaneously; In the formula (2), R
5Be H or C
1～C
6Alkyl; R
6For containing five yuan or the hexa-atomic aromatic heterocyclic of S,
Or
Wherein: R
7Be H or C
1～C
6Alkyl, R
8, R
9Be selected from C respectively
1～C
6Alkyl in a kind of, n=1～6;
Preferred scheme is: work as R
3Be COOR
4(R wherein
4Be H, C
1～C
3Alkyl or C
1～C
3Single haloalkyl) time, R
1, R
2Be selected from H respectively, contain 1～3 N, S or/and the hexa-member heterocycle base of O (as morpholinyl, parathiazan base or piperazinyl etc.) or by the group shown in the formula (2), and R
1And R
2Be not H simultaneously; In the formula (2), R
5Be H or C
1～C
3Alkyl; R
6For containing five yuan of aromatic heterocyclics of S,
Or
Wherein: R
7Be H or C
1～C
3Alkyl, R
8, R
9Be selected from C respectively
1～C
3Alkyl in a kind of, n=1～4.
The said 8-oxygen of the preparation the present invention-8H-acenaphthene also method of [1,2-b] pyrrole derivative comprises the steps:
With 8-oxygen-8H-acenaphthene also [1,2b] pyrroles-9-nitrile (it prepares referring to ZL 02148400.7) is starting raw material, be placed on organic solvent (as acetonitrile, tetrahydrofuran (THF), pyridine, N, dinethylformamide or dimethyl sulfoxide (DMSO)) in, with corresponding uncle or secondary amine (nucleophilic reagent) in 20～100 ℃, reaction (fragrant hydrogen nucleophilic substitution reaction) 0.5～24 hour promptly gets one of target compound after steaming solvent.
With
Earlier with 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile places by the vitriol oil and oleum and forms acid (temperature is controlled at 0～5 ℃), 15～25 ℃ of reactions 10～20 hours 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-formic acid, carry out esterification with corresponding alcohol or haloalkane then and obtain corresponding ester (8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-manthanoate), at last with 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-manthanoate promptly get two of target compound after being substituted reaction as stated above with corresponding uncle or secondary amine.
Description of drawings
Fig. 1～Fig. 5 be 3-parathiazan base-8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile (compound 7) hatch altogether after flow cytometer detected result figure with training oxygen cell.
Wherein: X-coordinate is a DNA concentration, and ordinate zou is a cell count, and compound 7 is hatched altogether with training oxygen cell, and its concentration is respectively 0,0.1,1,5,10 μ M.
Embodiment
Confirm the said 8-oxygen of the present invention-8H-acenaphthene also [1,2-b] pyrrole derivative experimental technique of can be used as cell death inducer and antineoplastic compound (comprise in the body and external) be at first to measure IC by methods such as MTT
50
Suppress experiment as the HeLa cells in vitro and use mtt assay.The MTT colorimetric test is a kind of method that detects cell survival and growth, has been widely used in screening anti-tumor medicine, cell toxicity test etc. at present.Concrete steps are as follows:
1) inoculating cell:, be made into the individual cells suspension with the RPMI1640 nutrient solution that contains 10% foetal calf serum, with every hole 10 with HeLa cell 0.25% tryptic digestion
3～10
4Individual cell inoculation in 96 well culture plates, every pore volume 200 μ l.
2) culturing cell: culture plate is moved into CO
2In the incubator, 37 ℃, 5%CO
2And cultivate under the saturated humidity after 24 hours, the adding compound concentration, continues to cultivate 72 hours to 100 μ M from 1 μ M.
3) colour generation: every hole adds MTT solution (5mg/ml) 20 μ l, hatches 4 hours, stops cultivating, and careful the suction abandoned culture supernatant in the hole.Then, every hole adds 150 μ l DMSO, vibrates 10 minutes, and crystallisate is fully dissolved.
4) colorimetric: select the 570nm wavelength, on microplate reader, measure each hole absorbance value, the record result.Calculate cell survival rate: test group absorbance value/control group absorbance value * 100%, with cell survival rate to compound dosage logarithm mapping obtain IC
50Value (cell proliferation rate is reduced to 50% o'clock required compound concentration).
The P388 cells in vitro suppresses experiment and also uses mtt assay, concrete grammar is as follows: by different tumor growth rates, the tumour cell 90 μ l holes that some amount are in logarithmic phase are inoculated in the 96 hole microtest plates, add soup 10 μ l/ holes after cultivating 24h, to each cell strain, each concentration is three multiple holes.If establishing acellular zeroing hole medicine, other have color will do the acellular zeroing of relative medicine concentration hole.Tumour cell is at 37 ℃, 5%CO
2Cultivate after 48 hours under the condition, add MTT (Sigma) liquid 5mg/ml and prepare 20 μ l/ holes with physiological saline; Continue to cultivate after 4 hours, (the 50 μ l/ holes of 10%SDS-5% isopropylcarbinol-0.01mol/lHCl) are in CO to add three liquid
2Spend the night in the incubator.Survey the OD570 value with microplate reader then.Calculate the inhibiting rate of analyte by following formula to growth of cancer cells:
Tumor control rate=(control group OD value-treatment group OD value)/control group OD value * 100%.
The A549 cells in vitro suppresses experiment and uses sulphonyl rhodamine B (Sulforhodamine B, SRB) protein staining method method, concrete grammar is as follows: according to cell growth rate, the tumour cell that will be in logarithmic phase is inoculated in 96 well culture plates with 90 μ l/ holes, and adherent growth 24 hours is dosing 10 μ l/ holes again.Each concentration is established three multiple holes.And the physiological saline solvent of establishing respective concentration contrasts and acellular zeroing hole.Tumour cell is at 37 ℃, 5%CO
2Cultivated 72 hours under the condition, the nutrient solution that inclines then with 10% cold TCA fixed cell, is placed for 4 ℃ and is used distilled water wash 5 times, seasoning in the air after 1 hour.Add SRB (Sigma) the 4mg/ml solution 100 μ l/ holes by the preparation of 1% Glacial acetic acid then, dyeing is 15 minutes in the room temperature, removes supernatant liquor, with 1% acetic acid washing 5 times, dry air.The Tris solution that adds 150 μ l/ holes at last, microplate reader 520nm wavelength are measured the A value down.Calculate the inhibiting rate of analyte by following formula to growth of cancer cells:
Tumor control rate=(A
540 control wells-A
540 dosing holes)/A
540 control wells* 100%.
The active experimental technique of anti-tumor in vivo is: at first train oxygen 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.Inoculate after 5 days, the knurl piece forms, and beginning is according to the concentration gradient 30%DMSO solution of vein or local injection compound 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, and the observation animals survived time.The result shows that the compound among the present invention all has the effect that suppresses tumor growth in various degree, prolongs the tumor model survival time of animals.
The present invention also by flow cytometer detected the said 8-oxygen of the present invention-8H-acenaphthene also [1,2b] pyrrole derivative in vivo, external evoked apoptotic effect.In in vitro tests, 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 appointed compound 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 simultaneously and is contrast, and 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 5.1-33.8% between 0.01-10 μ M for the compound of best results.
The invention will be further described below by embodiment, and its purpose only is better to understand content of the present invention and unrestricted protection scope of the present invention:
Embodiment 1
8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-nitrile (compound 1) also
In 500mL single port flask, add the 0.1mol acenaphthenequinone successively, the 0.11mol propane dinitrile, the 150ml acetonitrile is heated to backflow, reaction 4h, reaction mixture is become orange red transparent by faint yellow muddiness.After being chilled to room temperature, filter, collect orange red filter cake, get 1-dicyano methylene radical-2-oxygen-acenaphthene, thick productive rate 96%.
In 500mL single port flask, add 1-dicyano methylene radical-2-oxygen-acenaphthene crude product 0.05mol successively, salt of wormwood 1g, the 200ml acetonitrile is heated to backflow, reaction 4h, a large amount of khaki color solids are separated out.After being chilled to room temperature, filter, washing is collected filter cake and is got compound 1, yield 95%.
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)，8.04-8.08(t，J＝8.0Hz，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
+(253，m/z)。
Embodiment 2
8-oxygen-8H-acenaphthene is synthetic (compound 2) of [1,2-b] pyrroles-9-formic acid also
In 50mL single port flask, add the 60mL vitriol oil or 25mL oleum, under 0～5 ℃, add also [1,2-b] pyrroles-9-nitrile of 0.05mol 8-oxygen-8H-acenaphthene in batches, add the back and continue reaction 16 hours in room temperature.Carefully slowly it is splashed in the trash ice then, simultaneously violent stirring after dripping off, is left standstill, careful filtration, and filter cake is washed with massive laundering, is neutral until filtrate, gets compound 2, yield 95%, M.p.245 ℃ behind the filtration cakes torrefaction.
1H NMR(400MHz，DMSO-d
6)δ＝11.41(s，1H，OH
*)，8.95(d，1H，J＝7.2Hz)，8.55(d，1H，J＝8.0Hz)，8.53(d，1H，J＝7.2Hz)，8.48(d，1H，J＝8.0Hz)，7.96(dd，1H，J
1＝8.0Hz，J
2＝7.2Hz)，7.88(dd，1H，J
1＝8.0Hz，J
2＝7.2Hz).
IR(KBr)cm
-1：3208，1774，1700，1636，1583，1570.
MS(API-ES)m/z(M-H)
-248.1。
Embodiment 3
8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-methyl-formiate (compound 3) also
In the 100mL round-bottomed flask, add also [1,2-b] pyrroles-9-formic acid of 0.01mol8-oxygen-8H-acenaphthene successively, the 50mL acetonitrile is a solvent, excessive greatly methyl iodide under the nitrogen protection, is heated to 35 ℃ of reactions.After reaction finished, pressure reducing and steaming acetonitrile, thick product separated (methylene chloride-methanol 80: 1) through silica gel column chromatography and get compound 3, yield 98%.M.p.202 ℃.
1H NMR(400MHz，CDCl
3)：δ＝9.12(d，1H，J＝7.2Hz)，8.75(d，1H，J＝7.6Hz)，8.31(d，1H，J＝8.0Hz)，8.25(d，1H，J＝8.0Hz)，7.87(dd，1H，J
1＝8.0Hz，J
2＝7.2Hz)，7.77(dd，1H，J
1＝8.0Hz，J
2＝7.6Hz)，3.20ppm(s，3H，CH
* 3).
IR(KBr)cm
-1：3068，2933，1765，1712，1645，1586，1571.
HRMS (ESI) m/z (M+H)
+Calculate C
16H
10NO
3264.0661, experimental value 264.0672.
Embodiment 4
3-(N, N '-diethylin-ethylamino-)-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-nitrile (compound 4) also
In 50mL single port flask, add also [1,2-b] pyrroles-9-nitrile and N of 0.5mmol 8-oxygen-8H-acenaphthene successively, N '-diethyl ethylenediamine 2mmol, the 20mL acetonitrile is made solvent, and at room temperature stirring reaction is 1 hour.After reaction finished, pressure reducing and steaming acetonitrile, thick product separated (methylene chloride-methanol 10: 1) through silica gel column chromatography and get compound 4.Yield 42%.m.p.＞300℃。
1H NMR(400MHz，DMSO-d
6)δ8.97(d，1H，J＝7.6Hz)，8.61(d，1H，J＝7.6Hz)，7.98(d，1H，J＝9.2Hz)，7.91(dd，1H，J＝8.0，7.6Hz)，7.06(1H，d，J＝9.2Hz)，3.79(br s，2H，-NHCH
2)，2.93(brs，2H，-NHCH
2CH
2)，2.38-2.41(m，4H，N(CH
2CH
3)
2)，1.12-1.15ppm(m，6H，N(CH
2CH
3)
2).
IR(KBr)cm
-1：3313，2968，2213，1324，1574，1522.
HRMS (ESI) m/z (M-H)
-Calculate C
21H
21N
4O 343.1559, experimental value 343.1557.
Embodiment 5
3-(4 butyric acid ethyl ester-butylamine base)-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-nitrile (compound 5) also
In 50mL single port flask, add 0.5mmol 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile and 4-aminobutyric acid ethyl ester 2mmol successively, the 20mL acetonitrile is made solvent, and at room temperature stirring reaction is 4 hours.After reaction finished, pressure reducing and steaming acetonitrile, thick product separated (methylene chloride-methanol 15: 1) through silica gel column chromatography and get compound 5.Yield 45%.m.p.138.5-139.5℃
1H NMR(400MHz，DMSO-d
6)δ9.58(br s，1H，-NH)，8.90(d，1H，J＝8.0Hz)，8.55(d，1H，J＝8.0Hz)，7.93(d，1H，J＝9.2Hz)，7.87(dd，1H，J＝8.0，7.6Hz)，7.02(d，1H，J＝8.8Hz)，4.05(q，2H，OCH
2CH
3，J＝6.8Hz)，3.61(q，2H，NHCH
2CH
2，J＝7.2Hz)，2.52-2.50(m，2H，CH
2COO)，1.99-1.96(m，2H，-NHCH
2CH
2)，1.68ppm(t，3H，OCH
2CH
3，J＝6.8Hz).
13C NMR(100MHz，DMSO-d6)δ177.1，172.5，156.0，138.8，132.4，131.1，1129.6，128.0，127.0，125.8，122.1，116.1，114.3，111.4，108.1，63.0，45.2，30.7，30.6，17.0ppm；
IR(KBr)cm
-1：3326，2979，2218，1723，1627，1576，1531.
HRMS (ESI) m/z (M+H)
+Calculate C
21H
18N
3O
3360.1348, experimental value 360.1349.
Embodiment 6
3-(thienyl-2-base-methylamino-)-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-nitrile (compound 6) also
In 50mL single port flask, add 0.5mmol 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile and 2-methylamino thiophene 2mmol successively, the 20mL acetonitrile is made solvent, and at room temperature stirring reaction is 12 hours.After reaction finished, pressure reducing and steaming acetonitrile, thick product separated (methylene chloride-methanol 12: 1) through silica gel column chromatography and get compound 6.Yield 38%.m.p.250 ℃.
1H NMR(400MHz，d
6-DMSO)：δ＝9.79(br s，1H，NH
*)，8.94(d，1H，J＝8.0Hz)，8.67(d，1H，J＝8.8Hz)，8.10(d，1H，J＝7.6Hz)，7.86(dd，1H，J
1＝7.6Hz，J
2＝8.0Hz)，7.50(d，1H，J＝3.6Hz)，7.23(d，1H，J＝3.6Hz)，7.08(d，1H，J＝8.8Hz)，7.02(dd，1H，J
1＝3.6Hz，J
2＝3.6Hz)，4.98(s，2H，CH
* 2).
IR(KBr)cm
-1：3320，2920，2217，1628，1572，1492.
HRMS (ESI) m/z (M-H)
-Calculate C
20H
10N
3OS 340.0545, experimental value 340.0546.
Embodiment 7
3-parathiazan base-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-nitrile (compound 7) also
In 50mL single port flask, add 0.5mmol 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile and 2mmol parathiazan successively, the 20mL acetonitrile is made solvent, and at room temperature stirring reaction is 8 hours.After reaction finished, pressure reducing and steaming acetonitrile, thick product separated (methylene dichloride-acetone 60: 1) through silica gel column chromatography and get compound 7.Yield 35%; M.p.232 ℃.
1H NMR(400MHz，DMSO-d
6)δ8.61(d，2H，J＝8.0Hz)，8.12(d，1H，J＝8.8Hz)，7.94(dd，1H，J＝8.0，8.0Hz)，7.40(d，1H，J＝8.4Hz)，3.94(t，4H，J＝4.8Hz，N(CH
2CH
2)
2S)，2.97ppm(t，4H，J＝4.8Hz，N(CH
2CH
2)
2S).
IR(KBr)cm
-1：2219，1624，1572，1499.
HRMS (ESI) m/z (M+H)
+Calculate C
19H
14N
3OS 332.0858, experimental value 332.0861.
Embodiment 8
3-(thienyl-2-base-methylamino-)-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-methyl-formiate (compound 8) also
In the 50mL round-bottomed flask, add 0.5mmol 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-manthanoate and 2-methylamino thiophene 2.5mmol successively, the 20mL acetonitrile is a solvent, is heated to 60 ℃ of reactions.TLC tracking, reaction finish back pressure reducing and steaming acetonitrile, separate (methylene chloride-methanol 12: 1) through twice silica gel column chromatography and get compound 8.Yield 41%.M.p.240 ℃.
1H NMR(400MHz，d
6-DMSO)：8＝9.71(br s，1H，NH
*)，8.87(d，1H，J＝8.0Hz)，8.77(d，1H，J＝8.8Hz)，8.60(d，1H，J＝7.6Hz)，7.86(dd，1H，J
1＝7.6Hz，J
2＝8.0Hz)，7.46(d，1H，J＝3.6Hz)，7.23(d，1H，J＝3.6Hz)，7.09(d，1H，J＝8.8Hz)，7.02(dd，1H，J
1＝3.6Hz，J
2＝3.6Hz)，4.98(s，2H，CH
* 2)，2.97ppm(s，3H，COOCH
* 3).
IR(KBr)cm
-1：3298，2923，1748，1710，1693，1624，1563，1525.
HRMS (ESI) m/z (M+Na)
+Calculate C
21H
14N
2NaO
3S 397.0623, experimental value 397.0614.
Embodiment 9
8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-formic acid-2-bromo-ethyl ester (compound 9) also
In the 100mL round-bottomed flask, add also [1,2-b] pyrroles-9-formic acid of 0.01mol 8-oxygen-8H-acenaphthene successively, the 50mL acetonitrile is a solvent, excessive greatly glycol dibromide under the nitrogen protection, is heated to 35 ℃ of reactions.After reaction finished, pressure reducing and steaming acetonitrile, thick product separated (methylene chloride-methanol 70: 1) through silica gel column chromatography and get compound 9.Yield 18%.m.p.205-206 ℃.
1H NMR(400MHz，CDCl
3)：δ＝9.10(d，1H，J＝7.6Hz)，8.74(d，1H，J＝7.6Hz)，8.31(d，1H，J＝7.6Hz)，8.26(d，1H，J＝8.0Hz)，7.86(dd，1H，J
1＝7.6Hz，J
2＝7.6Hz)，7.77(dd，1H，J
1＝8.0Hz，J
2＝7.6Hz)，4.13(t，2H，J＝6.4Hz，CH
* 2CH
2Br)，3.66(t，2H，J＝6.4Hz，CH
2CH
* 2Br).
13C NMR(100MHz，CDCl
3)：δ＝178.7，168.7，166.3，142.9，137.2，136.5，134.2，132.7，132.3，131.6，129.3，128.1，127.5，125.3，121.3，39.5，28.3.
IR(KBr)cm
-1：3063，2921，1769，1712，1647，1585，1572.
HRMS (ESI) m/z (M+Na)
+Calculate C
17H
10BrNNaO
3377.9742, experimental value 377.9730.
Embodiment 10
3-parathiazan base-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-formic acid-2-bromo-ethyl ester (compound 10) also
In the 50mL round-bottomed flask, add also [1,2-b] pyrroles-9-formic acid-2-bromo-ethyl ester of 0.01mol 8-oxygen-8H-acenaphthene, the 50mL acetonitrile is a solvent, excessive greatly parathiazan under the nitrogen protection, is heated to 60 ℃ of reactions.After reaction finished, pressure reducing and steaming acetonitrile, thick product separated (methylene chloride-methanol 70: 1) through silica gel column chromatography and get compound 10.Yield 35%.m.p173 ℃.
1H NMR(400MHz，CDCl
3)：δ＝9.03(d，1H，J＝8.4Hz)，8.78(d，1H，J＝7.2Hz)，8.44(d，1H，J＝8.0Hz)，7.82(dd，1H，J
1＝7.2Hz，J
2＝8.4Hz)，7.20(d，1H，J＝8.0Hz)，4.11((t，2H，J＝6.4Hz，OCH
* 2)，3.64(t，2H，J＝6.4Hz，CH
* 2Br)，3.72(br s，4H，-N(CH
* 2CH
2)
2S)，3.00ppm(br s，4H，-N(CH
2CH
* 2)
2S).
IR(KBr)cm
-1：2916，1753，1703，1628，1601，1570.
HRMS (ESI) m/z (M+H)
+Calculate C
21H
18BrN
2O
3S 457.0222, experimental value 457.0211.
Embodiment 11
3,6-dimorpholine base-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-nitrile (compound 11) also
In 50mL single port flask, add 0.5mmol 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile and 5mmol morpholine successively, the 20mL acetonitrile is made solvent, and at room temperature stirring reaction is 10 hours.After reaction finished, pressure reducing and steaming acetonitrile, thick product separated (methylene dichloride-acetone 50: 1) through silica gel column chromatography and get compound 11.Yield 45%; M.p.180 ℃.
1H NMR(400MHz，DMSO-d
6)δ8.21(d，1H，J＝10Hz)，8.05(d，1H，J＝8.8Hz)，7.59(d，1H，J＝10Hz)，7.31(d，1H，J＝8.8Hz)，3.88(br s，4H，N(CH
2CH
2)
2O)，3.82(br s，4H，-N(CH
2CH
2)
2O)，3.54(br s，4H，N(CH
2CH
2)
2O)，3.46ppm(br s，4H，N(CH
2CH
2)
2O).
IR(KBr)cm
-1：2219，1610，1546，1502.
HRMS (ESI) m/z (M+H)
+Calculate C
23H
21N
4O
3401.1614, experimental value 401.1606.
Embodiment 12
6-parathiazan base-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-nitrile (compound 12) also
In 50mL single port flask, add 0.5mmol 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile and 5mmol morpholine successively, the 20mL acetonitrile is made solvent, and at room temperature stirring reaction is 8 hours.After reaction finished, pressure reducing and steaming acetonitrile, thick product separated (methylene dichloride-acetone 60: 1) through silica gel column chromatography and get compound 12.Yield 12%; M.p.247 ℃.
1H NMR(400MHz，DMSO-d6)δ8.36(d，1H，J＝7.6Hz)，8.31(d，1H，J＝9.6Hz)，8.24(d，1H，J＝7.6Hz)，7.81(d，1H，J＝9.6Hz)，7.73(dd，1H，J＝7.6，7.6Hz)，3.81(br s，4H，-N(CH
2CH
2)
2S)，2.88ppm(br s，4H，-N(CH
2CH
2)
2S).
IR(KBr)cm
-1：3431，2924，2852，2224，1618，1600.
HRMS (ESI) m/z (M+H)
+Calculate C
19H
14N
3OS 332.0858, experimental value 332.0851.
Embodiment 13
The anti tumor activity in vitro experiment.Adopt cell culture method that the series compound among the present invention is carried out the anti-tumor activity experiment, 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.Add the 100%DMSO solution of appointed compound respectively by concentration gradient, final concentration 100,50,10,5,0.5,0.1,0.05,0.01 μ M establishes culturing cell tumour control group and antitumor drug endoxan positive controls simultaneously.After hatching 24 hours in the CO2 incubator, every hole adds MTT solution 20 μ l, with optical density(OD) (OD) value under the enzymatic determination 570nm, calculates the survival rate of each hole tumour cell according to optical density value.The experiment triplicate is got its mean value.With cell survival rate IC is obtained in compound dosage logarithm mapping
50Value (cell proliferation rate is reduced to 50% o'clock required compound concentration) the results are shown in Table 1.
The different antitumor activity of compound of table 1
| Compound | Cytotoxicity (IC 50，μM) | ||
| Hela | P388 | A549 | |
| Compound 1 | 21.2 | --- | --- |
| Compound 2 | 24.2 | --- | --- |
| Compound 3 | 40.9 | 0.80 | 0.45 |
| Compound 4 | 6.8 | --- | --- |
| Compound 5 | 4.8 | --- | --- |
| Compound 6 | 1.1 | --- | --- |
| Compound 7 | 0.17 | --- | --- |
| Compound 8 | 1.3 | 11.3 | 6.69 |
| Compound 9 | 1.3 | 1.34 | 5.51 |
| Compound 10 | 1.2 | 1.41 | 8.03 |
| Compound 11 | ＞100 | --- | --- |
| Compound 12 | 2.8 | --- | --- |
Embodiment 14
The compound that has detected indication of the present invention by flow cytometer in vivo, external evoked apoptotic effect.In in vitro tests, 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 the 100%DMSO solution of appointed compound respectively by concentration gradient, final concentration 100,50,10,5,0.5,0.1,0.05,0.01 μ M establishes culturing cell tumour control group and antitumor drug endoxan positive controls simultaneously.After hatching 24 hours in the CO2 incubator, 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, with dose-dependent mode cell death inducing, apoptosis rate (is seen Fig. 1-Fig. 5) at 5.1-33.8% to the compound of best results between 0.01-10 μ M.As seen apoptotic peak, apoptosis rate is respectively 3.2%, 5.1%, 7.9%, 15.5%, 33.8%.
Embodiment 15
The anti-tumor in vivo active testing.Select compound 7 to carry out the living animal experiment.Select the Chinese kunming mice of raising, grouping immediately, 10 every group.Adopt the method for the subcutaneous lotus knurl of right lower extremity, get the mouse cancer ascites that contains the H22 cell that went down to posterity 7 days,, get 0.2ml in the right lower extremity subcutaneous vaccination by dilution in 1: 2, after the lotus knurl 4 days, see that lotus knurl position has unguiculus size tumour to begin administration, successive administration was drawn materials after 7 days, got 4 for every group, get blood, liver, kidney, other animal is observed lifetime.
Recording knurl after drawing materials heavily reaches the knurl volume and sees Table 2.
Table 2 anti-tumor in vivo active testing
| Grouping | The average knurl volume in treatment back | Average knurl is heavy | Body weight before the treatment-treatment back body weight |
| Control group 0.3mg/kg 0.1mg/kg 0.03mg/kg | 0.36 0.43 0.28 0.98 | 0.61 0.62 0.39 1.1 | 0.43 -8.9 -1.06 0.27 |
Claims (9)
1, a kind of 8-oxygen-8H-acenaphthene [1,2-b] pyrrole derivative also, it has structure shown in the formula (1):
In the formula (1), R
1, R
2Be selected from H respectively, contain 1～3 N, S or/and five yuan of O or hexa-member heterocycle base or by the group shown in the formula (2), and R
1And R
2Be not H simultaneously; R
3Be CN or COOR
4, R wherein
4Be H, C
1～C
6Alkyl or C
1～C
6Haloalkyl;
In the formula (2), R
5Be H or C
1～C
6Alkyl; R
6For containing five yuan or the hexa-atomic aromatic heterocyclic of S,
Or
Wherein: R
7Be H or C
1～C
6Alkyl, R
8, R
9Be selected from C respectively
1～C
6Alkyl in a kind of, n=1～6.
2, as the said 8-oxygen of claim 1-8H-acenaphthene [1,2-b] pyrrole derivative also, it is characterized in that, wherein work as R
3During for CN, R
1For H, contain 1～3 N, S or/and five yuan of O or hexa-member heterocycle base or by the group shown in the formula (2), R
2For H or contain 1～3 N, S or/and five yuan or the hexa-member heterocycle base of O, and R
1And R
2Be not H simultaneously.
3, as the said 8-oxygen of claim 2-8H-acenaphthene [1,2-b] pyrrole derivative also, it is characterized in that, wherein R
1For H, contain 1～3 N, S or/and the hexa-member heterocycle base of O or by the group shown in the formula (2), R
2For H or contain 1～3 N, S or/and the hexa-member heterocycle base of O, and R
1And R
2Be not H simultaneously; And in the formula (2), R
5Be H or C
1～C
3Alkyl, R
6For five yuan of aromatic heterocyclics containing S,
Or
Wherein: R
7Be H or C
1～C
3Alkyl, R
8, R
9Be selected from C respectively
1～C
3Alkyl in a kind of, n=1～4.
4, as the said 8-oxygen of claim 3-8H-acenaphthene also [1,2-b] pyrrole derivative, it is characterized in that, said derivative is: 3-(N, N '-diethylin-ethylamino-)-8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile, 3-(4 butyric acid ethyl ester-butylamine base)-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-nitrile also, 3-(thienyl-2-base-methylamino-)-8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-nitrile, 3-parathiazan base-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-nitrile also, 3,6-dimorpholine base-8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-nitrile or 6-parathiazan base-8-oxygen-8H-acenaphthene [1,2-b] pyrroles-9-nitrile also also.
5, as the said 8-oxygen of claim 1-8H-acenaphthene [1,2-b] pyrrole derivative also, it is characterized in that, wherein work as R
3Be COOR
4The time, R wherein
4Be H, C
1～C
6Alkyl or C
1～C
6Haloalkyl, R
1, R
2Be selected from H respectively, contain 1～3 N, S or/and five yuan of O or hexa-member heterocycle base or by the group shown in the formula (2), and R
1And R
2Be not H simultaneously; In the formula (2), R
5Be H or C
1～C
6Alkyl; R
6For containing five yuan or the hexa-atomic aromatic heterocyclic of S,
Or
Wherein: R
7Be H or C
1～C
6Alkyl, R
8, R
9Be selected from C respectively
1～C
6Alkyl in a kind of, n=1～6.
6, as the said 8-oxygen of claim 5-8H-acenaphthene [1,2-b] pyrrole derivative also, it is characterized in that, wherein R
4Be H, C
1～C
3Alkyl or C
1～C
3Single haloalkyl, R
1, R
2Be selected from H respectively, contain 1～3 N, S or/and the hexa-member heterocycle base of O or by the group shown in the formula (2), and R
1And R
2Be not H simultaneously; In the formula (2), R
5Be H or C
1～C
3Alkyl; R
6For containing five yuan of aromatic heterocyclics of S,
Or
Wherein: R
7Be H or C
1～C
3Alkyl, R
8, R
9Be selected from C respectively
1～C
3Alkyl in a kind of, n=1～4.
7, as the said 8-oxygen of claim 6-8H-acenaphthene also [1,2-b] pyrrole derivative, it is characterized in that said derivative is: 8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-formic acid, 8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-methyl-formiate also, 3-(thienyl-2-base-methylamino-)-8-oxygen-8H-acenaphthene also [1,2-b] pyrroles-9-methyl-formiate, 8-oxygen-8H-acenaphthene is [1,2-b] pyrroles-9-formic acid-2-bromo-ethyl ester or 3-parathiazan base-8-oxygen-8H-acenaphthene [1,2-b] pyrroles-9-formic acid-2-bromo-ethyl ester also also.
8, as any described 8-oxygen-8H-acenaphthene in the claim 1～7 also [1,2-b] pyrrole derivative as cell death inducer.
9, as any described 8-oxygen-8H-acenaphthene in the claim 1～7 also [1,2-b] pyrrole derivative as antineoplastic compound.
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|---|---|---|---|
| CNA2006101171313A CN1931840A (en) | 2006-10-13 | 2006-10-13 | DNA target molecules and their application in inducing apoptosis and antagonizing tumor |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006101171313A CN1931840A (en) | 2006-10-13 | 2006-10-13 | DNA target molecules and their application in inducing apoptosis and antagonizing tumor |
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| Publication Number | Publication Date |
|---|---|
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Family
ID=37877885
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|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN101633637B (en) * | 2009-08-18 | 2011-09-28 | 华东理工大学 | 8-oxo-8H-acenaphtho[1,2-b]pyrrole derivative |
| 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 |
| WO2012100368A1 (en) * | 2011-01-25 | 2012-08-02 | 大连理工大学 | Method for screening tumor cells and use thereof. |
-
2006
- 2006-10-13 CN CNA2006101171313A patent/CN1931840A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN101633637B (en) * | 2009-08-18 | 2011-09-28 | 华东理工大学 | 8-oxo-8H-acenaphtho[1,2-b]pyrrole derivative |
| 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 |
| WO2012100368A1 (en) * | 2011-01-25 | 2012-08-02 | 大连理工大学 | Method for screening tumor cells and use thereof. |
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