CN101104615A - Anticancer cytoskeleton inhibiting and cell elongation inducing compound and synthetic method thereof - Google Patents
Anticancer cytoskeleton inhibiting and cell elongation inducing compound and synthetic method thereof Download PDFInfo
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Abstract
The present invention discloses an anti-cancer cytoskeleton restraint and cell elongation attraction compound and the synthesis method, wherein, the compound is 7-chloro-6-piperidin-1-yl-quinoline-5, 8-dione (chemical name: 7-chloro-6-piperidin-1-yl-quinoline-5, 8-dione), and the chemical formula of the compound is C14H13ClN2O2, which is known as PT-262 in shortened form. The compound can stabilize actin filament to enable abnormal polymerization of actin filament, resulting in cancer cell elongation. In addition, PT-262 is provided with antitumor activity, which can block the survival path of Ras-ERK protein in cancer cells to result in cancer cell apoptosis, destroy mitochondrial membrane and accelerate the activation of caspase-3 protein. Moreover, PT-262 can inhibit the content of cyclic protein B1 and phosphorylation cdc2 protein (phospho-cdc2) to bring on the cessation of cancer cell cycle stop and inhibit the growth of cancer cells.
Description
Technical field
The present invention relates to a kind of carcinostatic agent and synthetic method, especially a kind of new synthetic compound 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione and its synthetic method, the anticancer cytoskeleton that abbreviates PT-262 as suppresses and cell elongation lures and draws compound and its synthetic method.
Background technology
Because diet, daily life system is improper and lack factor such as motion, the probability that the modern suffers from cancer is more and more high in recent years, and the patient who suffers from cancer more has the trend of rejuvenation gradually in recent years; Have only early discovery, early treatment to be only the sole mode of cure diseases, minimizing cancer lethality rate for this illness of cancer, therefore, how controlling the state of an illness fast, stopping to worsen possibility has been one of most important problem of modern medicine circle.
New discovery such as taxol and colchicine in the cancer inhibitor has a significant effect at present, relatively also has some problems to occur; As,
The taxol related description:
Research group's evidence taxol of the MonroeWall leader of national cancer institute (NCI) is effective in cure to multiple cancer, but the effect of treatment mammary cancer and ovarian cancer is best, in 40 advanced ovarian cancer patients that receive treatment in the first batch, have 12 patients' tumour to reduce to surpass 50%, inhibiting rate is 30%.
The skeleton of taxol is made up of the Taxan of 15 yuan of rings, carbon 4 and 5 in carbon at ring link with undersaturated 4 yuan of oxygen rings again, also be with a lateral chain of ester group in 13 in carbon, have 9 unsymmetrical carbons on taxane-ring and 4 yuan of oxygen rings, the structure-activity relation that taxol is carried out studies have shown that, though lateral chain of ester group has reduced the solubleness of taxol molecule in water, it is the key position that the taxol molecule has pharmacologically active.Change side chain and can both increase the water-soluble of molecule by removing any one group on 13 of the carbon eliminations, but it is actively sharply descended.The function of this kind taxol can't be elongated cancer cells in addition, lacks to stablize the cancer cells actin filament and suppress the effect that actin filament goes polymerization.
In addition, taxol still has many problems on using, and taxol mainly is present in the Pacific yew bast, and the content of taxol only is 0.01%～0.03% in the endothelium of doing.Extract the 1kg taxol and need strip the endothelium of 9000Kg, and treat 5～6 of the yew trees in 100 years of a needs of patients growth from 2000～3000 trees.Pacific yew is the rare seeds of a kind of poor growth, utilizes its bark paclitaxel produced meeting in next life that ecotope is done great damage.Therefore, must try every possible means to obtain taxol from other approach.
The colchicine related description:
Colchicum autumnale (ColchicumautumataL) is a Liliaceae Colchicum plant, the perennial herb flower bulbs.Bloom 8～October.Bud is spindle-like, when open like funnel, rose pink (or red-purple).Just begin to use colchicine treatment sciatica and sacroiliitis etc. during 19th-century, found again afterwards that it all had certain curative effect to the leukemia of mammary cancer, cervical cancer, acute lymphoblastic.Colchicine (antitumor drug) is evident in efficacy to mammary cancer, to cervical cancer, esophagus cancer, lung cancer certain curative effect may be arranged also.
And colchicine also is to have many problems on using, for example: the toxic side effect of colchicine is bigger, gastrointestinal reactions such as common nausea,vomiting,diarrhea, stomachache are serious symptoms in early stage of poisoning, it has direct repression to marrow in addition, causes agranulocytosis, aplastic anemia easily; Same, the function of this kind colchicine can't be elongated cancer cells, lacks equally to stablize the cancer cells actin filament and suppress the effect that actin filament goes polymerization.
In order to develop the anticancer drugs that make new advances, and solve aforementioned issues associated, increase anticancer effect, a kind of anticancer cytoskeleton inhibition of the special proposition of the present invention and cell elongation lure draws compound, can not only replace taxol and colchicine, and it has more tangible anticancer effect.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, provide a kind of anticancer cytoskeleton inhibition and cell elongation to lure and draw compound 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione, being called for short aforesaid compound at this is PT-262.
For solving the problems of the technologies described above, the present invention adopts the basic design of technical scheme to be: a kind of anticancer cytoskeleton inhibition and cell elongation lure draws compound, it is characterized in that: this compound is 7-chloro-6-piperi din-1-yl-quinoline-5,8-dione, its chemistry 7-chloro-6-six hydrogen by name are than pyridine-1-base-quinoline-5,8-diketone, chemical formula are C
14H
13ClN
2O
2Abbreviate PT-262 as, it can stablize the actin filament effect, causes the unusual polymerization of actin filament, causes the cell of cancer cells to elongate; In addition, it has antitumour activity, can block the cancer cells survival route, causes cancer cell-apoptosis; PT-262 can cause the cancer cells cycle to stop to grow with anticancer in addition.
Described compound 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione, its chemistry 7-chloro-6-six hydrogen by name are than pyridine-1-base-quinoline-5, and the 8-diketone is the related derivatives on basis.
Described as cytoskeleton inhibitor and cell elongation attractant, via stablizing the cancer cells actin filament and suppressing actin filament and go polymerization, cause the cancer cells elongation; And suppress little GTPase protein simultaneously, comprise Ras, Rac, Rho and cdc42, and suppress them and regulate and control downstream albumen.
Described inhibitor as cdc2 protein kinase and cdc25, and suppress the relevant downstream albumen that cdc2 protein kinase and cdc25 are controlled.
Described as the proteic blocker of Ras-ERK, the survival route of anticancer comprises shutting mutually and downstream albumen of being controlled, and avoids survival, hyperplasia and the transfer of cancer cells.
Described as apoptotic attractant, can destroy a line body, caspase-3 proteolytic enzyme and shutting mutually and downstream albumen in the activation cancer cells.
Described conduct is stablized the cancer cells actin filament and is suppressed actin filament and go polymerization, influences extracellular matrix extracellular matrix albumen, suppresses hematoblastic agglutination, and anticoagulant effect is arranged.
Described conduct is stablized the actin filament of other cell and is promoted the polymerization of actin filament, can be used as the attractant of dividing a word with a hyphen at the end of a line of neurocyte and vascular cell, help neurocyte contact conduction neurotransmission, and the newborn angiogenesis of blood vessel.
A kind of anticancer cytoskeleton suppresses and cell elongation lures the synthetic method of drawing compound, and its synthetic method is as follows:
Dropwise add triethylamine triethylamine to containing 6,7-dichloroquinoline-5 stirs in the benzole soln of 8-dione and piperidine, uses rotary distiller with solvent removal afterwards, to form brown solid; Brown solid is dissolved in 50% ethyl acetate ethyl acetate/ hexane hexanes, by purifying output PT-262.
Dropwise add triethylamine triethylamine 0.56ml, 5.1mmol on demand to including 6,7-dichloroquinoline-5,8-dione 1.00g, 4.4mmol and piperidine 0.50ml, 5.1mmol the 150ml benzole soln in, stirring at room 5 minutes, use rotary distiller with solvent removal afterwards, to form brown solid.
By chromatograph tubing string chromatography purifying, produce and contain 0.72g, 59%PT-262 and 6-chloro-7-piperidin-1-yl-quinoline-5,8-dione contains 0.48g, 40% work in-process, further isolates PT-262 again.
After adopting technique scheme, the present invention compared with prior art has following beneficial effect.
PT-262 has anticancer activity, comprises having the death (comprising lung cancer, breast cancer and cervical cancer etc.) of bringing out various human cancer cells, and causes the cell cycle to stop and the cell growth-inhibiting.PT-262 stablizes the cancer cells skeleton and causes the irreversible prolongation of cell, and suppresses the activity of existence signal protein Ras-ERK and mitotic circulating protein B1 and cdc2, and destroys grain line body and start the apoptosis path of caspase-3.
Use 5 among the PT-262 compound, 8-quinolinediones and 6,7-dihaloquinoline-5,8-diones can be used as the precursor of making biologically active.The derivative of Quinolinediones has anticancer and anti-microbial effect.6-Anilino-5,8-quinolinedione (LY83583) is the inhibitor of cyclase (guanylyl cyclase), has the ability that cell proliferation reached and lured the cancer cells caducity that suppresses.And 6-Chloro-7-(2-morpholin-4-ylethylamino) quinoline-5,8-dione (NSC 663284) is the inhibitor of cdc25 phosphoprotein phosphatase (phosphatases), can suppress the propagation of human breast cancer cell.In addition, lavendamycin is the quinolinedione derivative, has antitumour activity.
Importantly, PT-262 of the present invention is 5, and the novel derivative of 8-quinolinedione can change the structure of cytoskeleton, obviously causes cell elongation.
Microtubule of cytoskeleton (microtubulin) and actin filament (F-actin) have been considered to effective target of cancer chemotherapy, for example, taxol (paclitaxel) can the stabilize microtubules structure, cause the expendable polymerization of microtubule, the carrying out of blocking-up cell mitogen (mitosis).On the contrary, vinca alkaloids (vinca alkaloids) and colchicine (colchicine) are to suppress the polymerization (polymerization) of microtubule, can't form Spindle fibre (spindle) and inhibition mitotic division.The effect of chalone (cytochalasin B) is to be connected with the anode of actin filament, reduces the quantity of actin filament, and prevents the polymerization of actin filament.Yet what phalloidin (phalloidin) can be stablized actin filament and suppress actin filament goes polymerization (depolymerization).
PT-262 and phalloidin effect have the actin filament of stable cancer cells equally, and the polymerization of going that suppresses actin filament, cause the elongation of cancer cells; The polymerization of actin filament can be controlled by little GTPase protein, and for example Ras, Rac, Rho and cdc42 regulate and control these paths, can influence the stable of cytoskeleton; PT-262 has the Ras of inhibition protein expression ability, shows that PT-262 can suppress Ras and other little GTPase protein path, the dimerization effect of modulate actin silk and the prolongation of cell.
Blocking-up cancer cells survival route is the Critical policies of treatment cancer; The message path of the signal adjusting protein kinases (ERK) that Ras albumen institute trigger cell is outer can make cancer cells survival, hyperplasia and transition; PT-262 has the Ras-ERK of inhibition albumen bang path, blocks this cancer cells survival route, and PT-262 is provided anticancer effect; The cdc2 protein kinase combines with circulating protein (cyclin) B1, can control that cancer cells is mitotic to carry out; The activation of cdc2 and circulating protein B1 is that mitotic division institute is necessary, the activation of cdc2 mainly via the activated protein kinase (CAK) of cdc2 in the proteic Thr161 phosphorylation of cdc2 (phosphorylation), and by the dephosphorylation (dephosphorylation) of cdc25 phosphoprotein phosphatase; PT-262 can suppress phosphorylated protein cdc2 (Thr-161) and reduce circulating protein B1, the carrying out in blocking-up cancer cells cycle; In addition, cancer therapy drug can see through apoptosis (apoptosis) path that starts cancer cells, and reaches antitumous effect; PT-262 can destroy a line body, causes a line body membrane potential to descend, and makes the caspase-3 in the cancer cells protease activated, brings out cancer cell-apoptosis.
Chemical name manifestation mode of the present invention is 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione (7-chloro-6-six hydrogen are than pyridine-1-base-quinoline-5,8-diketone), and chemical formula of the present invention: C
14H
13ClN
2O
2
Synthetic method of the present invention is as follows: dropwise add triethylamine (triethylamine) (0.56ml, 5.1mmol) to containing 6,7-dichloroquinoline-5,8-dione (1.00g, 4.4mmol) and piperidine (0.50ml, 5.1mmol) the 150ml benzole soln in, stirring at room 5 minutes, use rotary distiller with solvent removal afterwards, to form brown solid, brown solid is dissolved in ethyl acetate (ethyl acetate)/hexane (hexanes) of 50%,, produces the PT-262 of 0.72g (59%) and the 6-chloro-7-piperidin-1-yl-quinoline-5 of 0.48g (40%) by chromatograph tubing string (chromatography) purifying, 8-dione (PT-261), further isolate PT-262 again, PT-262 is a brown solid, chemical structure such as Fig. 1, the chemical structure of PT-26-2, further with the evaluation structure of spectrum 2D nucleus magnetic resonance (NMR) and X-ray crystallization diffraction, the molecular weight 276.0666 of PT-262, fusing point are 145-146 ℃.
In view of the above, PT-262 of the present invention has that the cancer cells of making prolongation, irreversible stabilized cell skeleton, cell cycle stop, growth-inhibiting and apoptosis, except having anticancer function, also can be used as the inhibitor of cytoskeleton and the attractant of cell elongation.In addition, stablize the cancer cells actin filament and suppress actin filament and go polymerization, influence the structure of cytoskeleton, can influence extracellular matrix protein (extracellular matrix) and act on, suppress hematoblastic aggegation, produce anticoagulant effect.The polymerization of stabilized cell actin filament and promotion actin filament, can be used as the attractant of dividing a word with a hyphen at the end of a line of neurocyte and vascular cell, help the contact conduction (neurotransmission) between neurocyte, and new life's effect (angiogenesis) of blood vessel.
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Description of drawings
Fig. 1 is a chemical structural drawing of the present invention;
Fig. 2 A, 2B and 2C figure are the dead data plots that PT-262 of the present invention brings out three-type-person's quasi-cancer cell;
Fig. 3 is the growth figure of PT 262 anticancer of the present invention;
Fig. 4 is the stagnation figure that PT-262 of the present invention causes the cancer cells cycle;
Fig. 5 is that PT-262 of the present invention suppresses phosphorylation cdc2 protein kinase, circulating protein B1, Ras albumen and phosphorylation ERK protein expression spirogram;
Fig. 6 A, 6B are the apoptotic effect figure that PT-262 of the present invention brings out cancer cells, and wherein, Fig. 6 A is for suppressing the grain line body membrane potential figure of A549 pneumonocyte, and Fig. 6 B is for bringing out the proteic spirogram that contains of activated state caspase-3;
Fig. 7 A, 7B are polymerisation of actin filaments effect and the cell elongation figure that PT 262 of the present invention brings out cancer cells, and wherein, Fig. 7 A is microscopic examination figure, and Fig. 7 B is with Leica confocal software analysis image cell length figure;
Fig. 8 is the comparison diagram of PT-262 of the present invention and other cytoskeleton inhibitor;
Fig. 9 is the comparison diagram of PT-262 and various cytoskeleton inhibitor.
Embodiment
The experimental cell that below is described in detail PT-262 of the present invention is cultivated, kinds of experiments content and experiment gained result, and arranges in pairs or groups and graphicly be described as follows one by one:
Three kinds of experimental cells are cultivated (Cell culture)
The A549 cell strain was from 58 years old male lung cancer (lung carcinoma) patient, and the H1299 cell strain is the human non-small cell lung cancer of p53 afunction.
The MCF-7 cell strain is 69 years old white people women's a mammary cancer (breast adenocarcinoma).
The adenocarcinoma of cervix (cervical carcinoma) of (HeLa) cell strain from 31 years old women drawn in the sea, these cell cultures are at the RPMI-1640 substratum, and add 10% serum, 100 μ g/ml penicillin (Penicillin), 100 μ g/ml Streptomycin sulphates (Streptomycin), with 0.03%w/v bran propylhomoserin (L-glutamine), and cell is cultivated in 37 ℃ and 5% carbonic acid gas.
Cytotoxicity analysis (MTT assay)
With 1 * 10
4Individual cell is implanted the cell cultures dish in 96 holes, in CO2gas incubator, cultivated 16-20 hour, the PT-262 that adds different concns handled in nutrient solution 24 hours, treatment time finishes, the nutrient solution that will contain medicine is removed, clean with phosphate buffer soln (PBS), add nutrient solution again, continue to cultivate after 48 hours, remove nutrient solution in the dish, adding contains the nutrient solution of 500 μ g/ml MTT, cultivates 4 hours, removes to contain the MTT nutrient solution, add DMSO at last with cytolysis, measure the absorbancy of wavelength 565nm with ferment immunity analysis instrument (ELISA reader).
Cell growth analysis (Cell growth assay)
With 5 * 10
5Cell cultures in the p100 culture dish, after incubator is cultivated 16-20 hour, the PT-262 that adds different concns handled 24 hours, after the nutrient solution that will contain medicine removes, and clean with phosphate buffer soln, handle with Trypsin ferment (trypsin), make cell become suspended state, utilize cell counter in microscopically counting cells number.
Cell cycle analysis (cell cycle analysis)
With 1 * 10
6The cell branch plant to the p60 culture dish, cultivated 16-20 hour, the PT-262 that adds different concns handled 24 hours, after the nutrient solution that will contain medicine removes, clean, handle with the Trypsin ferment again with phosphate buffer soln, make cell become suspended state, collect in the centrifuge tube of inserting 15 milliliters; Through 1500 rotating speeds centrifugal 5 minutes, remove supernatant liquor, the alcohol fixation cell with 70% is inserted-20 ℃ of refrigerations at least two hours.With 1500 rotating speeds centrifugal 5 minutes again, collecting cell added and contains 1%Triton X-100, and 0.1mg/ml rnase (RNaseA) and 4 μ g/ml propidiumiodine stains mix, after 37 ℃ of dry type incubators are cultivated 30 minutes.Sample uses flow cytometer (flowcytometer) analysis, and with ModFit LT software (Ver.2.0), the per-cent of each cell cycle of quantitative analysis.
Grain line body membrane potential (Mitochondrial membrane potential)
With 5 * 10
5The cell branch plant to the p60 culture dish, cultivated 16-20 hour, the PT-262 that adds different concns handled after 24 hours, will clean with phosphate buffer soln, handle with the Trypsin ferment again, make cell become suspended state, cell is collected the alcohol fixation cell with 70% via centrifugal back, insert-20 ℃ of refrigerations two hours, centrifugal collecting cell with the DiOC6 fluorescein stain of cell and 0.5 μ M, was cultivated 30 minutes again.With cell centrifugation, remove supernatant liquor then, add the PBS of 0.5ml ice again, last, the DiOC6 fluorescence intensity is with at flow cytometry analysis.
West ink dot method (Western blot)
After drug treating finishes, cell is incorporated in the extraction damping fluid that contains proteinase inhibitor, Protein content in the analytic sample, utilize the electrophoretic analysis of 10-12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) again, with the gel behind the electrophoresis, change and steep pvdf membrane, film is dipped in 5% skimmed milk that contains one-level antibody, reacted 24 hours down at 4 ℃, under room temperature, cleaned 5-15 minute with the TTBS damping fluid, repeated washing 3 times, pvdf membrane is dipped in 5% skimmed milk that contains secondary antibody again, at room temperature reacted 1-2 hour, then with TTBS buffer solution for cleaning 5-15 minute, repeated washing 3 times utilizes chemical cold light method with the automatic radiography of X-ray sheet.
Cytoskeleton dyeing and confocal microscopical analysis (Cytoskeleton staining and confocalmicroscope)
Cover glass is positioned in the culture dish of p60, with cell seeding on cover glass, cultivated in the incubator of carbonic acid gas 16 ~ 20 hours, after cell do not handled or handling PT-262,, clean 3 times with phosphate buffered saline buffer again 37 ℃ of following fixed cells 60 minutes with 4% (v/v) paraformaldehyde solution; Actin filament and tubulin dyeed 30 minutes down at 37 ℃ with the tubulin antibody of 20 U/mlBODIPYFLphallacidin and Cy3 mark respectively; At last, nucleus is by 2.5 μ g/mlHoechst 33258 dyeing 30 minutes, add 80% glycerine at last after, use the nail varnish mounting, again with the microscopic examination of Leica confocal.
Statistical study
All results are at least from the experiment more than 3 times, and data is by using student t test (Student ' st test) to come analytic statistics difference, p value＜0.05 just to have statistics in statistical discrepancy.
The test result of PT-262 of the present invention
Cytotoxic test result, by the MTT analysis to measure, the result obtains from 3-14 experiment, wherein, * represent p value＜0.05, * represents p value＜0.01, for more not handling and handle PT-262, add the PT-262 of 1-10 μ M, handle cancer cells after 24 hours, improve with drug level, can cause A549 lung carcinoma cell (consulting Fig. 2 A), MCF-7 breast cancer cell (consulting Fig. 2 B), and HeLa cervical cancer cell (consulting Fig. 2 C), cell survival rate then increases with concentration and descends, and the concentration value (IC50) that suppresses 50% cell survival (cell viability) is 2-4 μ M (consulting Fig. 2 A-Fig. 2 C).
Clearly demonstrate embodiments of the present invention for asking, as shown in Figure 3, analyze the growth-inhibiting situation of PT-262 to cancer cells with the A549 lung carcinoma cell, the result obtains from 3 experiments, and * represents p＜0.05, and * * represents p＜0.01, for more not handling and handle PT-262.PT-262 through 5 or 10 μ M handled cancer cells after 24 hours, improved with drug level, obviously suppressed the growth of A549 pneumonocyte, and the PT-262 of 10 μ M handles, and almost completely causes growth of cancer cells to stop.
As shown in Figure 4, cause the stagnation figure in cancer cells cycle for PT-262; The present invention analyzes the influence that PT-262 carried out the cancer cells cycle with two kinds of lung cell A549s (having normal p53 gene) and H1299 (lacking normal p53 gene), the result is obtained by 3 to 4 experiments, * represent p＜0.05, * * represents p＜0.01, for more not handling and handle PT-262.After the PT-262 processing,, the cancer cells of G0/G1 phase is reduced, and make cell stagnate the phase in G2/M at two kinds of lung carcinoma cells.
As shown in Figure 5, described PT-262 suppresses shown in phosphorylation cdc2 protein kinase, circulating protein B1, Ras albumen and the phosphorylation ERK protein expression spirogram, the present invention analyzes PT-262 to the proteic influence in the cancer cells with western ink dot method, and Fig. 5 is after handling PT262, the expression amount of analyzing proteins.The result shows after the PT-262 of 5 to 20 μ M handles 24 hours, obviously reduces the content of phosphorylation cdc2 protein kinase and circulating protein B1.And Ras and the proteic expression amount of phosphorylation ERK also obviously reduce after PT-262 handles.The proteic activation of ERK is the phosphorylation via it, and ERK-2 albumen is albumen in contrast, and expression ERK total protein content can not change.
Shown in Fig. 6 A, Fig. 6 B, then be that the present invention analyzes the variation of PT-262 to the grain line body membrane potential of lung carcinoma cell, after handling PT-262, cell utilizes DiOC6 dyeing, via flow cytometry analysis (consulting Fig. 6 A), the result is 3-4 experiment, and * * represents p＜0.01, for more not handling and handle PT-262.Improve with drug level, obviously suppress the grain line body membrane potential (consulting Fig. 6 A) of A549 pneumonocyte.Analyze PT-262 to the proteic influence of caspase-3 in the cancer cells with western ink dot method, the result shows with the drug level raising, obviously brings out the proteic content of activated state caspase-3 (consulting Fig. 6 B), and expression PT-262 can cause apoptosis.
See also polymerisation of actin filaments effect and cell elongation figure that the described PT-262 of the present invention of Fig. 7 A, Fig. 7 B brings out cancer cells again; Among the figure with 'beta '-tubulin, actin filament and nucleus, the 'beta '-tubulin antibody of being demarcated with Cy3 fluorescence respectively, BODIPY FL phallacidin and Hoechst 33258 carry out fluorescent dye, with the microscopic examination of Leica confocal, shown in Fig. 7 A, the blue nucleus of representing the A549 lung carcinoma cell, red fluorescence is represented 'beta '-tubulin, the green actin filament of representing, the result shows that PT-262 has changed cytoskeletal structure and obviously lured and draws cell elongation that arrow indication place the polymerization of actin filament occurs and grows most advanced and sophisticated thing (spike), with Leica confocal software analysis image, calculate cell length, after handling 2 μ M PT-262, the cell mean length becomes 65.30 microns (μ m) (consulting Fig. 7 B) by 39.15; Some cell length rises to 160 microns, and PT-262 can cause the prolongation of other various cancer cells equally.
The diversity ratio that then compares PT-262 and known cytoskeleton inhibitor, consult the comparison diagram of the described PT-262 of Fig. 8 and other cytoskeleton inhibitor, the A549 cell is handled PT-262 respectively, taxol (paclitaxel), colchicine (colchichine), phalloidin (phalloidin) and chalone (cytochalasin B).As shown in Figure 8, handle the 50nM taxol after 24 hours, the tubulin of meeting stabilized cell skeleton promotes the microtubule polymerization effect, makes and represents the red fluorescence intensity of 'beta '-tubulin obviously to strengthen; On the contrary, the 50nM colchicine is handled, and can suppress tubulin polymerization, the red fluorescence of 'beta '-tubulin reduces, and after handling phalloidin (0.5U/ml), promotes the polymerization of actin filament, represent the green fluorescence intensity of actin filament to increase, and cause cell to elongate.The PT-262 that handles 2 μ M similarly makes the polymerization of actin filament, and the A549 lung carcinoma cell is elongated.
What deserves to be mentioned is that Fig. 9 is the comparison diagram of PT-262 and various cytoskeleton inhibitor, wherein, a, b suppress the function of Spindle fibre, cause mitotic division to stop, but can not cause the cancer cells elongation; C suppresses cytokinesis (cytokinesis), but can not cause cell elongation via the polymerization of going of Actin muscle; D, f cause the elongation of cancer cells via the abnormal polymerization effect of Actin muscle.Existing taxol can the stabilize microtubules structure, causes the polymerization that promotes microtubule, the carrying out of blocking-up cell mitogen.Another commonly uses colchicine is see through to suppress the polymerization of microtubule, can't form Spindle fibre and suppresses mitotic division.Another effect of commonly using chalone is to be connected with actin filament, and suppresses the polymerization of actin filament.
Above-mentioned taxol, colchicine and chalone can not elongate cancer cells, yet phalloidin is to see through the polymerization of going of stablizing actin filament and suppressing actin filament.PT-262 of the present invention and phalloidin effect are similar, by stablizing the cancer cells actin filament and suppressing actin filament and go polymerization, cause the cancer cells elongation.
Therefore, compound of the present invention and its synthetic method not only cause the cancer cells elongation, more can stablize the cancer cells actin filament and suppress actin filament and go polymerization, influence the structure of cytoskeleton, can influence extracellular matrix protein (extracellular matrix) effect, suppress hematoblastic aggegation, produce anticoagulant effect, other function that will illustrate especially is as follows:
1. the present invention causes the cancer cells elongation via stablizing the cancer cells actin filament and suppressing actin filament and go polymerization.Simultaneously, can suppress little GTPase protein, comprise Ras, Rac, Rho and cdc42, and suppress them and regulate and control downstream albumen.
2. as the inhibitor of cdc2 protein kinase and cdc25, and suppress the relevant downstream albumen that cdc2 protein kinase and cdc25 are controlled.
3. as the proteic blocker of Ras-ERK, the survival route of anticancer comprises shutting mutually and downstream albumen of being controlled, and avoids survival, hyperplasia and the transfer of cancer cells.
4. as apoptotic attractant, can destroy a line body, caspase-3 proteolytic enzyme and shutting mutually and downstream albumen in the activation cancer cells.
5. stablize the cancer cells actin filament and suppress actin filament and go polymerization, influence extracellular matrix (extracellular matrix) albumen, suppress hematoblastic agglutination, anticoagulant effect is arranged.
6. stablize the polymerization of the actin filament and the promotion actin filament of other cell, can be used as the attractant of dividing a word with a hyphen at the end of a line of neurocyte and vascular cell, help neurocyte contact conduction (neurotransmission), and the new life of blood vessel (angiogenesis).
In addition, also comprise with 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione (chemical name: 7-chloro-6-six hydrogen are than pyridine-1-base-quinoline-5,8-diketone) be the basis related derivatives with the above-mentioned every effect person of tool.
In sum, the present invention is that a kind of anticancer cytoskeleton suppresses and cell elongation lures and draws compound and its synthetic method, the brand-new synthetic compound of the present invention is 7-chloro-6-piperidin-1-yl-quinoline-5, (chemical name: 7-chloro-6-six hydrogen are than pyridine-1-base-quinoline-5 for 8-dione, the 8-diketone), its chemical formula is C
14H
13ClN
2O
2, abbreviating PT-262 as, it has stable actin filament effect, causes the unusual polymerization of actin filament, causes the cell of cancer cells to elongate; In addition, PT-262 has antitumour activity, can block the survival route of Ras-ERK albumen in the cancer cells, causes the effect of apoptosis of cancer cells, destroys grain line body film, promotes the proteic activation of caspase-3; PT-262 can cause the cancer cells cycle to stop to grow with anticancer in addition.
Claims (11)
1. an anticancer cytoskeleton suppresses and cell elongation lures and draws compound, it is characterized in that: this compound is 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione, and its chemistry 7-chloro-6-six hydrogen by name are than pyridine-1-base-quinoline-5,8-diketone, chemical formula are C
14H
13ClN
2O
2Abbreviate PT-262 as, it can stablize the actin filament effect, causes the unusual polymerization of actin filament, causes the cell of cancer cells to elongate; In addition, it has antitumour activity, can block the cancer cells survival route, causes cancer cell-apoptosis; PT-262 can cause the cancer cells cycle to stop to grow with anticancer in addition.
2. according to claim 1-kind of anticancer cytoskeleton inhibition and cell elongation lure draws compound, it is characterized in that: described compound 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione, its chemistry 7-chloro-6-six hydrogen by name are than pyridine-1-base-quinoline-5, and the 8-diketone is the related derivatives on basis.
3. a kind of anticancer cytoskeleton inhibition according to claim 1 and 2 and cell elongation lure draws compound, it is characterized in that: described as cytoskeleton inhibitor and cell elongation attractant, via stablizing the cancer cells actin filament and suppressing actin filament and go polymerization, cause the cancer cells elongation; And suppress little GTPase protein simultaneously, comprise Ras, Rac, Rho and cdc42, and suppress them and regulate and control downstream albumen.
4. a kind of anticancer cytoskeleton inhibition according to claim 1 and 2 and cell elongation lure draws compound, it is characterized in that: described inhibitor as cdc2 protein kinase and cdc25, and suppress the relevant downstream albumen that cdc2 protein kinase and cdc25 are controlled.
5. a kind of anticancer cytoskeleton inhibition according to claim 1 and 2 and cell elongation lure draws compound, it is characterized in that: described as the proteic blocker of Ras-ERK, the survival route of anticancer, comprise shutting mutually and downstream albumen of being controlled, avoid survival, hyperplasia and the transfer of cancer cells.
6. a kind of anticancer cytoskeleton inhibition according to claim 1 and 2 and cell elongation lure draws compound, it is characterized in that: described as apoptotic attractant, can destroy a grain line body, caspase-3 proteolytic enzyme and shutting mutually and downstream albumen in the activation cancer cells.
7. a kind of anticancer cytoskeleton inhibition according to claim 1 and 2 and cell elongation lure draws compound, it is characterized in that: described conduct is stablized the cancer cells actin filament and is suppressed actin filament and go polymerization, influence extracellular matrix extracellular matrix albumen, suppress hematoblastic agglutination, anticoagulant effect is arranged.
8. a kind of anticancer cytoskeleton inhibition according to claim 1 and 2 and cell elongation lure draws compound, it is characterized in that: described conduct is stablized the actin filament of other cell and is promoted the polymerization of actin filament, can be used as the attractant of dividing a word with a hyphen at the end of a line of neurocyte and vascular cell, help neurocyte contact conduction neurotransmission, and the newborn angiogenesis of blood vessel.
9. an anticancer cytoskeleton inhibition and a cell elongation lures the synthetic method of drawing compound, and its synthetic method is as follows:
Dropwise add triethylamine triethylamine to containing 6,7-dichloroquinoline-5 stirs in the benzole soln of 8-dione and piperidine, uses rotary distiller with solvent removal afterwards, to form brown solid; Brown solid is dissolved in 50% ethyl acetate ethyl acetate/ hexane hexanes, by purifying output PT-262.
10. lure the synthetic method of drawing compound according to described anticancer cytoskeleton inhibition of claim 9 and cell elongation, it is characterized in that: dropwise add triethylamine triethylamine 0.56ml, 5.1mmol on demand to including 6,7-dichloroquinoline-5,8-dione 1.00g, 4.4mmol and piperidine 0.50ml, 5.1mmol the 150ml benzole soln in, stirring at room 5 minutes, use rotary distiller with solvent removal afterwards, to form brown solid.
11. lure the synthetic method of drawing compound according to described anticancer cytoskeleton inhibition of claim 10 and cell elongation, it is characterized in that: by chromatograph tubing string chromatography purifying, produce and contain 0.72g, 59%PT-262 and 6-chloro-7-piperidin-1-yl-quinoline-5,8-dione contains 0.48g, 40% work in-process, further isolates PT-262 again.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160570A (en) * | 2011-12-09 | 2013-06-19 | 彩虹天健康科技研究(北京)有限责任公司 | Discovery for interaction mechanism of UNC-51 kinase and PP1 phosphatase |
| CN103364570A (en) * | 2013-08-02 | 2013-10-23 | 青岛农业大学 | Method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses |
| CN108531453A (en) * | 2017-03-01 | 2018-09-14 | 中国科学院动物研究所 | A method of converting non-neuronal cell to neuronal cell |
-
2006
- 2006-07-13 CN CNA2006100993058A patent/CN101104615A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160570A (en) * | 2011-12-09 | 2013-06-19 | 彩虹天健康科技研究(北京)有限责任公司 | Discovery for interaction mechanism of UNC-51 kinase and PP1 phosphatase |
| CN103364570A (en) * | 2013-08-02 | 2013-10-23 | 青岛农业大学 | Method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses |
| CN108531453A (en) * | 2017-03-01 | 2018-09-14 | 中国科学院动物研究所 | A method of converting non-neuronal cell to neuronal cell |
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