CN105017335B - A kind of 2,3- bisbenzimidazoles pyridine cobalt (II) complex and its application in pharmacy - Google Patents
A kind of 2,3- bisbenzimidazoles pyridine cobalt (II) complex and its application in pharmacy Download PDFInfo
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- CN105017335B CN105017335B CN201510485810.5A CN201510485810A CN105017335B CN 105017335 B CN105017335 B CN 105017335B CN 201510485810 A CN201510485810 A CN 201510485810A CN 105017335 B CN105017335 B CN 105017335B
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- AWCRMEQAQYXASF-UHFFFAOYSA-N cobalt(2+);pyridine Chemical compound [Co+2].C1=CC=NC=C1 AWCRMEQAQYXASF-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 201000007270 liver cancer Diseases 0.000 claims abstract description 9
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 9
- INAAIJLSXJJHOZ-UHFFFAOYSA-N pibenzimol Chemical compound C1CN(C)CCN1C1=CC=C(N=C(N2)C=3C=C4NC(=NC4=CC=3)C=3C=CC(O)=CC=3)C2=C1 INAAIJLSXJJHOZ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000013078 crystal Substances 0.000 claims abstract description 8
- 230000012010 growth Effects 0.000 claims abstract description 6
- 239000003814 drug Substances 0.000 claims description 14
- 229940079593 drug Drugs 0.000 claims description 10
- 230000002401 inhibitory effect Effects 0.000 claims description 7
- 125000004429 atom Chemical group 0.000 claims description 5
- 230000005764 inhibitory process Effects 0.000 claims description 4
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 4
- 230000006907 apoptotic process Effects 0.000 abstract description 20
- 230000010337 G2 phase Effects 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 59
- -1 Benzimidazoles compound Chemical class 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 229910052697 platinum Inorganic materials 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 238000009739 binding Methods 0.000 description 5
- PLTOVEJYEUZSKT-UHFFFAOYSA-N cobalt;pyridine Chemical compound [Co].C1=CC=NC=C1 PLTOVEJYEUZSKT-UHFFFAOYSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 230000001093 anti-cancer Effects 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 229940041181 antineoplastic drug Drugs 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000018199 S phase Effects 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229910017052 cobalt Inorganic materials 0.000 description 2
- 239000010941 cobalt Substances 0.000 description 2
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 2
- 150000004696 coordination complex Chemical class 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
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- 238000012549 training Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- HYZJCKYKOHLVJF-UHFFFAOYSA-N 1H-benzimidazole Chemical compound C1=CC=C2NC=NC2=C1 HYZJCKYKOHLVJF-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 229930003779 Vitamin B12 Natural products 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 150000003851 azoles Chemical class 0.000 description 1
- 125000003785 benzimidazolyl group Chemical group N1=C(NC2=C1C=CC=C2)* 0.000 description 1
- 125000005605 benzo group Chemical group 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000011095 buffer preparation Methods 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000025084 cell cycle arrest Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 150000004700 cobalt complex Chemical class 0.000 description 1
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005564 crystal structure determination Methods 0.000 description 1
- 238000002447 crystallographic data Methods 0.000 description 1
- 239000000824 cytostatic agent Substances 0.000 description 1
- 230000001085 cytostatic effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/06—Cobalt compounds
- C07F15/065—Cobalt compounds without a metal-carbon linkage
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/13—Crystalline forms, e.g. polymorphs
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of 2,3 bisbenzimidazole pyridine cobalt (II) complexs, crystal structure category anorthic system, space group P1, molecular formula C38H24CoN10.Co (II) atom is pentacoordinate.The invention also discloses application of 2,3 described bisbenzimidazole pyridine cobalt (II) complexs in pharmacy.Of the present invention 2,3 bisbenzimidazole pyridine cobalt (II) complexs can inhibit human liver cancer cell Hep G2 growths, induce the apoptosis of human liver cancer cell Hep G2, retardance human liver cancer cell Hep G2 also played an important role of to act on DNA with non-classical inserted mode in the G2 phases.
Description
Technical field
The present invention relates to a kind of 2,3- bisbenzimidazoles pyridine cobalt (II) complex and its applications in pharmacy.
Background technology
In current treatment of cancer, the use of platinum-containing anticancer drug accounts for the 70% of anticancer drug, representativeness medicine therein
Object cis-platinum can effectively treat kinds cancer, be to treat one of testis and the maximally efficient anticancer drug of oophoroma, but it
There is certain limitation, drug resistance is easily generated in tumour cell, there is serious side effect to body, such as nausea, diarrhea, kidney
With liver failure etc..Therefore, there is an urgent need to develop more effective, toxicity smaller or the resisting to combination with DNA target by people
Cancer drug.Cobalt is micro elements needed by human, participates in internal vitamin B12 synthesis, stimulates marrow hemopoiesis and adjusting red blood cell new
The important physiological functions such as old metabolism, and the growth of the malignant cell in body can be inhibited.Benzimidazoles compound has
There is unique active anticancer feature：(1) mitosis of tumour cell, (2) are inhibited using tubulin as main target molecule
Mediate by Caspase is protease activated and cromoci release characterized by Apoptosis, (3) inhibit hypoxia-inducible factor and
The release of vascular endothelial growth factor, (4) benzimidazole are the important feature units of many DNA minor groove bindings, can be with ditch
The mode that face combines acts on DNA.By Co (II) complex for synthesizing containing double benzene and imidazoles, it is desirable to can search out low toxicity,
The metal complex that active anticancer is high, anticancer spectrum is wide, to replace the anticancer therapy of platinum medicine.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of 2,3- bisbenzimidazoles pyridine cobalt (II) complex and its answer
With the result of the external anti-human liver cancer cell Hep-G2 of acquisition 2,3- bisbenzimidazole pyridine cobalt (II) complex.
The present invention solves above-mentioned technical problem and is adopted the technical scheme that：
One kind 2,3- bisbenzimidazole pyridine cobalt (II) complex, crystal structure be,
Application of described 2,3- bisbenzimidazole pyridine cobalt (II) complex in pharmacy.
Application in the pharmacy, including preparing the application in the drug for inhibiting human tumor cells Hep-G2 growths.
The drug for inhibiting human tumor cells Hep-G2 growths, inhibition effective concentration are 20~500 μm of ol/L.
2,3- bisbenzimidazoles pyridine cobalt (II) complex of the present invention can inhibit human liver cancer cell Hep-G2 lifes
Long, the apoptosis of induction human liver cancer cell Hep-G2, retardance human liver cancer cell Hep-G2 also had with non-classical insertion side in the G2 phases
Formula acts on the effect of DNA.
Description of the drawings
Fig. 1 is that 2,3- bisbenzimidazoles pyridine cobalt (II) complex of the present invention makees the inhibition of Hep-G2 cells
With cis-platinum is used as with reference to (n=4).
After Fig. 2 acts on Hep-G2 cells 48h for 2,3- bisbenzimidazoles pyridine cobalt (II) complex of the present invention
Streaming apoptosis figure.
After Fig. 3 acts on Hep-G2 cells 48h for 2,3- bisbenzimidazoles pyridine cobalt (II) complex of the present invention
Streaming apoptosis rate block diagram.
Fig. 4 is the cycle that 2,3- bisbenzimidazoles pyridine cobalt (II) complex of the present invention acts on Hep-G2 cells
Distribution.
Fig. 5 is the cycle that 2,3- bisbenzimidazoles pyridine cobalt (II) complex of the present invention acts on Hep-G2 cells
Distributive law block diagram.
Fig. 6 be 2,3- bisbenzimidazoles pyridine cobalt (II) complex of the present invention and DNA interaction it is ultraviolet-
It can be seen that spectrogram.
Fig. 7 is the infrared spectrogram of 2,3- bisbenzimidazoles pyridine cobalt (II) complex of the present invention.
Specific embodiment
Specific embodiment is described further technical scheme below in conjunction with the accompanying drawings.
1. 2,3 bisbenzimidazole pyridine cobalt (II) complex (C38H24CoN10) preparation
CoCl2·6H2The aqueous solution (5mL) of O 0.1189g (0.5mmol) is added to 2,3- bisbenzimidazole pyridines
After 0.0770g (0.25mmol) is stirred to react 3min in DMF solution (3mL), after adding concentrated ammonia liquor 5mL stirrings 30s, transfer
It into autoclave, is reacted 2 days at 180 DEG C, at the end of reaction, room temperature is down to the velocity gradient of 5 DEG C/h, filtered, go
Ion water washing obtains darkviolet bulk crystals.
2. infrared spectrum detects
Complex is with KBr tablettings in 4000-400cm-1Scope measures infared spectrum, as shown in Figure 7.
3. crystal structure determination
By scale is suitable, surface is flawless and smooth crystal measures on Agilent G8910A CCD diffractometers.
Under temperature 293 (2) K, diffraction light sources are the Mo-K alpha rays by graphite monochromator monochromatizationUsingScan pattern is scanned, and diffraction data is corrected with the Lp factors and its empirical absorption.Crystal structure is by SHELXL -97
Program parses.
The crystal parameter of table 1 2,3- bisbenzimidazole pyridine cobalt (II) complex and structure refinement (T=293 (2))
4. 2,3 bisbenzimidazole pyridine cobalt complex [C38H24CoN10] crystal structure and analysis
2,3 bisbenzimidazole pyridine cobalt complex category anorthic systems, space group P1, molecular formula C38H24CoN10。Co
(II) atom is pentacoordinate, as Co1 atoms coordinate coordination with the N atoms of the benzimidazolyl of three different ligands respectivelyWith two benzimidazole pyridyl groups
Pyridine ring on N atoms coordinate coordinationTheir bond angle N7-
Co1-N10A=117.15 (11) °, N1-Co1-N10A=94.61 (11) °, N1-Co1-N2=79.04 (11) °, N7-Co1-N6
=77.14 (11) °).The N atoms of Co (II) atoms and coordination not totally one plane.Its bond distance and bond angle are as shown in table 2.
The bond distance of table 2 2,3- bisbenzimidazole pyridine cobalt (II) complexWith bond angle (°)
5. cell survival rate is detected using mtt assay
Drug is grouped：Five concentration gradients are set, their final concentration is followed successively by 500 μm of ol/L, 100 μm of ol/L, 20 μ
mol/L、4μmol/L、0.8μmol/L；Positive controls：Cis-platinum, if five concentration gradients, final concentration be followed successively by 500 μm of ol/L,
100μmol/L、20μmol/L、4μmol/L、0.8μmol/L；Blank control group：Drug is not added with, only celliferous 1640 training completely
Support base；Acellular control group：Containing only 1640 complete mediums (being not added with drug and cell).
Operating method：The cell of exponential phase is selected, is first rinsed twice with PBS phosphate buffers, then with containing in right amount
0.25% 1~1.5min of Trypsin Induced of EDTA, makes attached cell become individual cells suspension, and cell counting count board carries out
It counts.The concentration for adjusting cell is 5 × 104A/mL is inoculated into 96 orifice plates, and the volume added in per hole is 100 μ L.It is put into and contains
5%CO237 DEG C of constant incubators in cultivate for 24 hours after, in each hole add in 100 μ L various concentration gradients testing drug.Often
The concentration gradient of a test sample sets 4 multiple holes parallel tests, each testing drug 3 repeated experiments.Training is put into after adding medicine
It supports and 48h is cultivated in case, take out and add in MTT test fluids (PBS is formulated as 5mg/mL), per 20 μ L of hole, continue after being incubated 4h, it will thereon
Clear carefully to remove and DMSO150 μ L are added in into each hole, 96 orifice plates of vibration, which allow, crystallizes fully dissolving, in enzyme-linked immunosorbent assay instrument
The upper OD values chosen 570nm wavelength and measure every hole.By the calculating to cell inhibitory rate, half cytostatic concentration is obtained in mapping
(IC50).Cell inhibitory rate calculation formula is as follows：
3 2,3- bisbenzimidazole pyridine cobalt complexes of table are to the inhibitory action (n=4) of Hep-G2 cells
Fig. 1 is block diagram of 2,3- bisbenzimidazole pyridine cobalt (II) complexs to the inhibitory action of Hep-G2 cells, suitable
Platinum is used as with reference to (n=4).
6. influence of the 2,3 bisbenzimidazole pyridine cobalt complexes to Hep-G2 Apoptosis and cycle
6.1 flow cytometer detection Apoptosis
The cell of exponential phase is selected, it is 2 × 10 to count cell density5A/mL is inoculated into 6 orifice plates, is added in per hole
2mL.It is put into containing 5%CO237 DEG C of constant incubators in cultivate for 24 hours after, replace the complete medium of isometric drug containing.Administration
After 48h, the cell of six orifice plates is collected with 0.25% trypsase without EDTA, 800r/min centrifugation 5min discard culture solution,
The PBS of precooling is washed twice, and the Binding Buffer of 500 μ L are added in the cell that PBS is washed floats cell again, adds 5 μ L
5 μ LPI are added after Annenxin V-FITC and cell mixing reaction 5min, mixing is protected from light 15min at room temperature.Transfer
Into in streaming pipe, detected on flow cytometer.
6.2 flow cytometer detection cell cycles
The cell of exponential phase is selected, it is 5 × 10 to count cell density5A/mL is inoculated into 6 orifice plates, is added in per hole
2mL.It is put into containing 5%CO237 DEG C of constant incubators in cultivate for 24 hours after, replace the complete medium of isometric drug containing.Administration
After 48h, the cell of six orifice plates is collected with 0.25% trypsase containing EDTA, 800r/min centrifugation 5min abandon supernatant, room temperature
PBS wash twice, be slowly added into 70% ethyl alcohol of precooling -20 DEG C it is fixed for 24 hours after, centrifugation cell is abandoned ethyl alcohol and is fixed
Liquid adds in room temperature PBS, and cell is made to stand hydration 15min, and supernatant is removed in centrifugation.RNaseA is added in into cell suspension in water-bath 37
After DEG C being incubated 30min, add in PI and dyed, be protected from light 30min at room temperature, cell cycle is detected on flow cytometer
Variation.Fig. 2 acts on the streaming apoptosis figure after Hep-G2 cells 48h for 2,3 bisbenzimidazole pyridine cobalt complexes.
4 2,3 bisbenzimidazole pyridine cobalt complex of table acts on the streaming apoptosis rate after Hep-G2 cells 48h
Fig. 3 acts on the streaming apoptosis rate cylindricality after Hep-G2 cells 48h for 2,3- bisbenzimidazole pyridine cobalt complexes
Figure, as shown in figure 3,2,3 bisbenzimidazole pyridine cobalt complexes, which choose final concentration of 25,50,100 μm of ol/L, acts on Hep-G2
Cell sets blank control group.It can be drawn by Fig. 3, with the increase of 2,3 bisbenzimidazole pyridine cobalt complex concentration, Hep-
Total apoptosis rate of G2 cells also gradually increases.Total apoptosis rate of blank control group is 2.67% ± 0.52%, 2,3 pairs of benzo miaows
When azoles pyridine cobalt complex concentration is 25 μm of ol/L, total apoptosis rate of Hep-G2 cells is 10.45% ± 0.36%, when concentration is passed
Increasing to 50 μm of ol/L, total apoptosis rate of Hep-G2 cells slightly rises to 15.75% ± 0.83%, when continuing to increase with concentration,
Total apoptosis rate of Hep-G2 cells increases the 44.66% ± 1.22% of D groups by the 15.75% ± 0.83% of C groups, with blank
Control group is many compared to be higher by.The necrosis rate of Hep-G2 cells increases also with the increase of concentration, but Hep-G2 on the whole
The total apoptosis rate of cell is higher than necrosis rate, so 2,3 bisbenzimidazole pyridine cobalt complexes mainly inhibit Hep- in a manner of apoptosis
The multiplication of G2 cells.Total apoptosis rate of administration group cell and total apoptosis rate of blank control group cell there were significant differences (P ＜
0.05)。
Influence of the 6.3 2,3 bisbenzimidazole pyridine cobalt complexes to the tumour cell Hep-G2 cycles
Fig. 4 is the period profile that 2,3- bisbenzimidazole pyridine cobalt complexes act on Hep-G2 cells.
5 2,3- bisbenzimidazole pyridine cobalt complexes of table act on the period profile rate of Hep-G2 cells
Fig. 5 is the period profile rate column diagram that 2,3- bisbenzimidazole pyridine cobalt complexes act on Hep-G2 cells, such as
Shown in Fig. 5,2,3 bisbenzimidazole pyridine cobalt complexes choose final concentration of 25,50,100 μm of ol/L and act on Hep-G2 cells,
Blank control group is set.As shown in Figure 5, after 2,3 bisbenzimidazole pyridine cobalt complexes act on Hep-G2 cells 48h, with
Administration concentration is incremented by, and the cell quantity of G1 phases shows downward trend, by 68.39% ± 0.77% drop of blank control group
To 100 μm of ol/L groups 39.73% ± 0.78%, the S phases cell quantity without significant change, and G2 phases cell quantity is presented and risen
Trend rises to the 41.62% ± 0.45% of 40 μm of ol/L groups by the 15.98% ± 0.35% of blank control group, shows 2,3 pairs
Benzimidazole pyridine cobalt complex induction Hep-G2 Apoptosis be by by Hep-G2 cell-cycle arrests in the G2 phases.Administration group
Cycle Arrest effect with blank control group has significant difference (except the S phases of 25 μm of ol/L groups) (P ＜ 0.05).
7. cobalt complex and the uv-visible absorption spectra and binding constant of DNA interactions
Experimental method
(1) Tris-HCl buffer preparations
Tris 0.3028g, NaCl 0.5265g are weighed, first with a certain amount of deionized water dissolving, then with appropriate concentrated hydrochloric acid
PH value about 7.2 is adjusted, is then settled to 500mL.
(2) preparation of CT-DNA solution and concentration determine
It weighs 1.00mg filiforms CT-DNA and is dissolved in 1.00ml Tris-HCl buffer solutions and be made into the molten of 1.00mg/ml
Liquid, jiggled under the conditions of 4 DEG C makes its dissolving overnight, and a small amount of mother liquor is taken to survey it at 260nm and 280nm after diluting 50 times
Absorbance, if A260/A280=1.8~1.9, then illustrate that meeting experiment will substantially free of protein, phenol and RNA in solution
It asks, it is not necessary that processing is further purified.The concentration of DNA is calculated with the molar concentration of base-pair, mole suction at 260nm
Backscatter extinction logarithmic ratio is 6600M-1cm-1.The concentration calculation formula of DNA is as follows：
[DNA] be DNA molar concentration, A260For the light absorption value of the concentration DNA solution at 260nm, K is extension rate.
(3) preparation of complex and ligand solution
Using DMSO as solvent, under conditions of ultrasonic dissolution assisting, respectively by each metal complex and ligand of corrresponding quality
Compound concentration is 2.285 × 10-3Mol/L solution, stand for standby use.
(4) uv-visible absorption spectra detection complex and DNA effect curves
In uv-visible absorption spectra instrument, 2970 μ l Tris-HCl are added in into the cuvette of reference cell and sample cell
Buffer solution, zeroing.30 μ l complex solutions are added in sample cell, 30 μ l DMSO are added in reference cell, then every time respectively to
The CT-DNA solution of 3 μ l is added in sample cell and sample cell, after the micro sample-adding rifle piping and druming mixing of 1000 μ l, stands 3min,
Change in the range detection absorption spectrum of 200-700nm, and record the absorbance at each absorption spectrum curve maximum absorption band.
(5) complex and DNA binding constants KbCalculating
Complex and the binding constant calculation formula that DNA interacts are as follows：
εAFor the molar absorption coefficient of solution under any DNA concentration, εBIt is mole suction when complex is bonded completely with DNA
Backscatter extinction logarithmic ratio.εFNot add in the molar absorption coefficient of pure complex before DNA.With-[DNA]/(εA-εF) ordinate is done to [DNA] work
Figure draws slope and cuts ratio, that is, binding constant K of evidence, slope and interceptb.Fig. 6 coordinates for 2,3- bisbenzimidazole pyridines cobalt
Object and the ultraviolet-visible spectrogram of DNA interactions, [Complex 7]:2.285μmol/L；[DNA]:2.77,5.54,8.31,
11.08,13.85,16.62,19.39,22.16,24.93μmol/L。
From fig. 6 it can be seen that work as when fixing the concentration of 2,3 bisbenzimidazole pyridine cobalt complexes, with DNA concentration
Increase, the intensity of absworption peak can be gradually reduced, and show hypochromic effect, and slight red shift occurs, according to what is fitted
Equation y=5.3939x+144.52, R2=0.9844, calculate complex and the binding constant K of DNAbFor 3.73 × 103M-1, say
Bright complex is interacted with non-classical inserted mode with DNA.
Claims (3)
1. one kind 2,3- bisbenzimidazole pyridine cobalt (II) complex, crystal structure are,
Described 2,3 bisbenzimidazole pyridine cobalt (II) complex category anorthic systems, space group P1, molecular formula are
C38H24CoN10；Co (II) atom is pentacoordinate, and the N atoms of Co (II) atoms and coordination not totally one plane.
2. 2,3- bisbenzimidazoles pyridine cobalt (II) complex described in claim 1 is preparing inhibition human liver cancer cell Hep-G2
Application in the drug of growth.
3. application according to claim 2, which is characterized in that the medicine for inhibiting human liver cancer cell Hep-G2 growths
Object, inhibition effective concentration are 20~500 μm of ol/L.
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