CN101548974B - Compound having fluorescent characteristic and anti-cancer activity, preparation thereof and application thereof - Google Patents
Compound having fluorescent characteristic and anti-cancer activity, preparation thereof and application thereof Download PDFInfo
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- CN101548974B CN101548974B CN2009100520683A CN200910052068A CN101548974B CN 101548974 B CN101548974 B CN 101548974B CN 2009100520683 A CN2009100520683 A CN 2009100520683A CN 200910052068 A CN200910052068 A CN 200910052068A CN 101548974 B CN101548974 B CN 101548974B
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Abstract
The invention discloses a compound having fluorescent characteristic and anti-cancer activity. Chemical name thereof is 1,3-diphenyl-4-pyrazolyl formaldehyde isoniazide, and chemical structural formula thereof is as follows. The compound is prepared from reaction of 1,3-diphenyl-4-pyrazolyl formalde and isoniazide in absolute ethyl alcohol. The compound is activated by wide frequency band, has strong fluorescent characteristic, has median inhibiting dose IC50 less than 10ng/mL to K562 leukaemia cancer cells, can be used as a lead compound of anti-cancer medicament, especially as a lead compound of anti-leukaemia medicament, has cell tag identifying function, will be beneficial to further studying action mechanism between the compound and DNA and observing distribution of the compound in cells so as to trace kinetic process of intake and discharge thereof, and has obvious social effect and economic benefit.
Description
Technical field
The present invention relates to a kind of chemical compound and preparation and application with active anticancer, specifically, relates to a kind of chemical compound and preparation and application that had not only had fluorescence but also had active anticancer.
Background technology
The data show that International Union Against Cancer announces, the annual cancer mortality number in the whole world is higher than the dead patient's of acquired immune deficiency syndrome (AIDS), malaria and tuberculosis summation at present, the annual newly-increased cases of cancer 1,100 ten thousand in the whole world, the patient nearly 8,000,000 who dies from cancer.As not adopting an effective measure, to the year two thousand thirty, the annual newly-increased cases of cancer in the whole world will increase to 1,600 ten thousand, and the patient who dies from cancer will be increased to 1,150 ten thousand.Obviously rise in the past 20 years of the incidence rate of China's malignant tumor and mortality rate, in some main big cities of China, malignant tumor has occupied the first place in the dead cause of disease, has become the principal disease of harm people ' s health and life.The cancer morbidity of Sheng Gaoing makes researcher constantly seek new and effective anticancer lead compound day by day.
In order to break away from the synthetic blindness of cancer therapy drug, all be devoted to study the mechanism of action of various anticancer complexes and DNA both at home and abroad.The report of research anticancer complex and the DNA mechanism of action is a lot, and past attempts is used biochemical means more, is colony's cell after the homogenate but biochemical means study, can not keep the living cells state, and can not study at individual cells according to the heterogeneity of cell; Use the fluorescence means in recent years mostly, select for use some fluorescence molecule as probe, the mechanism of action of coming researching DNA and micromolecule and medicine by the change in fluorescence of probe mark molecule, thus inquire into pathogenesis and screening and the new high-efficiency low-toxicity medicine of design.Research report is arranged at present: the anticarcinogen amycin has red autofluorescence under the suitableeest green exciting light, can observe this medicine whereby in intracellular distribution and follow the trail of the dynamic process that it is taken in, discharges.As seen, a kind of chemical compound with fluorescence, self can have the cell marking recognition function.
In sum, study a kind of chemical compound that had not only had fluorescence but also had active anticancer, can and observe this chemical compound in intracellular distribution and follow the trail of its dynamic process of taking in, discharging convenient way is provided to the mechanism of action of this chemical compound of further research and DNA, will have far reaching significance the research and development paces of thoroughly breaking away from synthetic blindness of cancer therapy drug and quickening PTS.
Summary of the invention
The purpose of this invention is to provide a kind of chemical compound and preparation and application with fluorescence and active anticancer.
Chemical compound with fluorescence and active anticancer provided by the invention, its chemical name is: 1,3-diphenyl-4-pyrazoles formaldehyde isoniazid that contracts, its chemical structural formula is:
The preparation method of described chemical compound with fluorescence and active anticancer is as follows: with 1,3-diphenyl-4-pyrazoles formaldehyde is dissolved in a certain amount of dehydrated alcohol, and the concentration that makes solution is 0.1～0.2mol/L; Add the glacial acetic acid of isoniazid and catalytic amount, make isoniazid and 1, the mol ratio of 3-diphenyl-4-pyrazoles formaldehyde is (1.1～1.2): 1; Be heated to backflow while stirring; Back flow reaction 2～8 hours is to TLC (V
Ethyl acetate: V
Petroleum ether=1: 1) detection reaction is finished; Naturally cool to room temperature, filter,, promptly get object gained crude product ethyl alcohol recrystallization.
Described 1,3-diphenyl-4-pyrazoles formaldehyde is method preparation described in the 791st page of reference literature " organic chemistry " 2007 the 27th the 6th phase of volume and getting.
Because, belonging to wideband at 250～430nm, the excitation spectrum wave-length coverage of described chemical compound with fluorescence and active anticancer excites; Its emission spectra peak value has intensive fluorescent characteristic at the 464nm place; And described chemical compound is to the half amount of suppression IC of K562 leukaemia cancer cell
50Less than 10ng/mL, therefore chemical compound of the present invention is expected the lead compound as cancer therapy drug, especially is expected the lead compound as the leukemia cancer drug.
Compared with prior art, beneficial effect of the present invention is as follows:
1, because of chemical compound of the present invention not only has good active anticancer, to the half amount of suppression IC of K562 leukaemia cancer cell
50Less than 10ng/mL, and self has strong fluorescent properties, belonging to wideband excites, has the cell marking recognition function, therefore chemical compound of the present invention is as the lead compound of cancer therapy drug, will help further studying the mechanism of action of this compounds and DNA and observe this compounds in intracellular distribution and follow the trail of that it is taken in, the dynamic process of discharge.
2, preparation method of the present invention is simple, mild condition, and no toxic and harmful produces, the recyclable utilization of solvent for use.
Description of drawings
Fig. 1 is the infrared spectrum of chemical compound of the present invention;
Fig. 2 is the hydrogen spectrogram of chemical compound of the present invention;
Fig. 3 is the carbon spectrogram of chemical compound of the present invention;
Fig. 4 is the mass spectrum of chemical compound of the present invention;
Fig. 5 is the exciting light spectrogram of chemical compound of the present invention;
Fig. 6 is the emission spectra figure of chemical compound of the present invention;
Fig. 7 is the concentration inhibition curve chart of chemical compound of the present invention to the K562 leukaemia cancer cell;
Fig. 8 is the inverted microscope photo of the K562 leukaemia cancer cell before compound effects of the present invention;
Fig. 9 is the inverted microscope photo of the K562 leukaemia cancer cell after compound effects of the present invention.
The specific embodiment
Below in conjunction with embodiment the present invention is done further detailed, complete explanation, used 1 among the embodiment, 3-diphenyl-4-pyrazoles formaldehyde is method preparation described in the 791st page of reference literature " organic chemistry " 2007 the 27th the 6th phase of volume and getting.
Embodiment 1: the preparation of chemical compound of the present invention
1) preparation 1-Phenylethanone. phenylhydrazone
0.33mol 1-Phenylethanone. and 0.33mol phenylhydrazine are dissolved in the 150mL dehydrated alcohol, splash into several glacial acetic acids, reflux 2h, cooling is filtered, and uses ethyl alcohol recrystallization, and vacuum drying gets object-1-Phenylethanone. phenylhydrazone, productive rate: 92%, fusing point: 101～103 ℃.
2) preparation 1,3-diphenyl-4-pyrazoles formaldehyde
Stir down in 24ml is cooled to 0 ℃ DMF and drip the new POCl that steams of 15g
3, temperature is controlled at below 10 ℃, after dropwising, fully stirs 0.2h under this temperature; 10.2g 1-Phenylethanone. phenylhydrazone is dissolved among the 30ml DMF, and temperature control is being added drop-wise in the said mixture below 10 ℃, continues to stir 0.4h under this temperature; At room temperature stir 0.4h then, be warming up to 60 ℃, stir 0.8h; Pour in the 30g trash ice after naturally cooling to room temperature, be neutralized to pH=8 with solid sodium carbonate; Sucking filtration washes with water, and drying is used ethyl alcohol recrystallization, gets object-1,3-diphenyl-4-pyrazoles formaldehyde, and yellow crystals, productive rate: 85.4%, fusing point: 147～149 ℃.
3) preparation 1,3-diphenyl-4-pyrazoles formaldehyde isoniazid that contracts
With 5mmol 1,3-diphenyl-4-pyrazoles formaldehyde is dissolved in the dehydrated alcohol of 30ml, adds the glacial acetic acid of 5.5mmol isoniazid and several (catalytic amounts); Be heated to backflow while stirring; Back flow reaction 2～8 hours is to TLC (V
Ethyl acetate: V
Petroleum ether=1: 1) detection reaction is finished; Naturally cool to room temperature, filter, with gained crude product ethyl alcohol recrystallization, must object-1,3-diphenyl-4-pyrazoles formaldehyde isoniazid that contracts, shallow green powder shape solid, productive rate: 79%, fusing point: 132～134 ℃.
IR (KBr tabletting, cm
-1): 3174 (N-H), 1657 (C=O), 1600 (C=N), detailed spectrum is as shown in Figure 1.
1H NMR (DMSO-d
6, 400MHz): (ArH), 9.04 (CH=N), 11.94 (NH), detailed spectrum as shown in Figure 2 for s, 1H for s, 1H for m, 15H for δ 7.36～8.79.
13C NMR (DMSO-d
6, 400MHz): δ 161.8 (C=O), 152 (C=N), 151.2,142.9,141.2,139.7,132.6,130.3,129.5,129.4,129.2,127.9,127.7,122.2,119.6,117.3 (Ar), detailed spectrum is as shown in Figure 3.
MS
+=368.0, detailed spectrum as shown in Figure 4.
As seen from Figure 5: the excitation spectrum wave-length coverage of chemical compound of the present invention belongs to wideband and excites at 250～430nm.
As seen from Figure 6: the emission spectra peak value of chemical compound of the present invention has intensive fluorescent characteristic at the 464nm place.
Embodiment 2: the active anticancer experiment (mtt assay) of chemical compound of the present invention
(1) principle: MTT is the yellow tetrazolium salts of a kind of water solublity, and chemical name is bromination [3-(4,5-dimethylthiazole-2-)-2, a 5-diphenyl] tetrazole, is commonly called as tetrazolium bromide.The cardinal principle of MTT colorimetry is that the dehydrogenase (as succinate dehydrogenase and diaphorase etc.) on the mitochondrial respiratory chain can be reduced into water miscible yellow MTT water-fast bluish violet crystal substance-first Za (Formazan) in the living cells, and dead cell does not then have this function.The growing amount of Jia Za is directly proportional with viable count under normal conditions, and the depth of the color of solution is directly proportional with the amount of contained Formazan.Absorbance (OD value) at the 570nm place after the Formazan dissolving has the good absorption value, therefore can understand the ability of medicine inhibition or killing tumor cell according to the OD value reflection cell growth and the survival condition at 570nm place.
(2) reagent agent: 1,3-diphenyl-4-pyrazoles formaldehyde isoniazid that contracts.
(3) for the examination cell: the K562 leukaemia cancer cell
(4) preparation of reagent concentration: with 1,3-diphenyl-4-pyrazoles formaldehyde isoniazid that contracts is made into 10mg/mL, 1mg/mL respectively with DMSO, 0.5mg/mL, 0.1mg/mL, 0.01mg/mL, draw each Concentraton gradient medicine 10 μ L in aseptic EP pipe, be diluted to 0.1mL with RPMI-1640; Matched group does not add test sample [1,3-diphenyl-4-pyrazoles formaldehyde contract isoniazid].
(5) preparation of cell suspension: in disposable centrifuge tube, add the 10mL1640 culture fluid with pipette, extract one bottle of cell culture fluid, the centrifugal 5min of 2000rpm pours out supernatant, transfers to 1mL with 1640, is transferred in the aseptic EP pipe, carries out cell counting.
(6) preparation of cultivating system: add the cell culture fluid of amount of calculation in the aseptic EP pipe, make that cell concentration is 10
5Individual/mL, 20% Ox blood serum, 200 μ L, medicine 10 μ L use the RPMI-1640 polishing to 1mL.
(7) be inoculated in 96 orifice plates, every hole 100 μ L, and establish matched group, 8 every group multiple holes, 37 ℃, saturated humidity, 5%CO
2Cultivated 44 hours, and added 10 μ L, 0.5% MTT solution (the yellow tetrazolium salts of a kind of water solublity, chemical name are bromination [3-(4,5-dimethylthiazole-2-)-2,5-diphenyl] tetrazole, are commonly called as tetrazolium bromide) continued to hatch 4 hours.
(8) add 10%SDS100 μ L in 96 orifice plates, stopped reaction placed room temperature 8 hours, measured the absorbance (A of each hole at the 570nm place with enzyme-linked immunosorbent assay instrument
570), be calculated as follows inhibitory rate of cell growth: suppression ratio (%)=(1-A experiment/A contrast) * 100%.
50% individual effectively dosage is called median effective dose, uses ED
50Expression, IC commonly used in experiment in vitro
50The half amount of suppression is represented.General IC when synthetic drug
50＜10 μ gml
-1, judgement sample has active anticancer.
Table 1MTT colorimetry detects the inhibition percentage rate (%) of coordination compound to the K562 growth of cancer cells cycle
By table 1 and Fig. 7 as seen: chemical compound of the present invention has good biological activity to the K562 leukaemia cancer cell, along with the increase of consumption, the inhibitory action of the growth cycle of K562 leukaemia cancer cell is strengthened; When dosage is 0.01 μ gml
-1The time, still be 52% to the suppression ratio of the growth cycle of K562 leukaemia cancer cell; And all Concentraton gradient empty experimental cells grow fine; Show that chemical compound of the present invention can effectively suppress the growth of K562 leukaemia cancer cell.
Fig. 8 and Fig. 9 are the inverted microscope photos at the forward and backward K562 leukaemia cancer cell of compound effects of the present invention, as seen: before compound effects of the present invention, K562 leukaemia cancer cell, big or small homogeneous; Through after the compound effects of the present invention, the K562 leukaemia cancer cell almost all is killed; Show that chemical compound of the present invention has very strong toxicity to the K562 leukaemia cancer cell.
In sum: chemical compound of the present invention has active anticancer, is expected the lead compound as leukemia cancerous cell medicine.
Claims (3)
1. the chemical compound with fluorescence and active anticancer is characterized in that, its chemical name is:
1,3-diphenyl-4-pyrazoles formaldehyde isoniazid that contracts, its chemical structural formula is:
2. described preparation method of claim 1 with chemical compound of fluorescence and active anticancer, it is characterized in that described method is: with 1,3-diphenyl-4-pyrazoles formaldehyde is dissolved in a certain amount of dehydrated alcohol, and the concentration that makes solution is 0.1～0.2mol/L; Add the glacial acetic acid of isoniazid and catalytic amount, make isoniazid and 1, the mol ratio of 3-diphenyl-4-pyrazoles formaldehyde is (1.1～1.2): 1; Be heated to backflow while stirring; Back flow reaction 2～8 hours is to V
Ethyl acetate: V
Petroleum ether=1: the 1TLC detection reaction is finished; Naturally cool to room temperature, filter,, promptly get object gained crude product ethyl alcohol recrystallization.
3. the described application of chemical compound in the preparation anti-leukemia medicine of claim 1 with fluorescence and active anticancer.
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| EP2495561B1 (en) * | 2009-10-30 | 2016-12-14 | Keio University | Anticancer agent sensitivity-determining marker |
| CN110128402B (en) * | 2019-05-30 | 2022-03-22 | 河南师范大学 | Branched fluorescent dye compound containing pyrazole ring |
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Non-Patent Citations (2)
| Title |
|---|
| 田晓红等.1_苯基_3_芳基_4_甲酰基吡唑衍生物的合成及其荧光性能.《有机化学》.2007,第27卷(第6期),第790-795页. |
| 田晓红等.1-苯基-3-芳基-4-甲酰基吡唑衍生物的合成及其荧光性能.《有机化学》.2007,第27卷(第6期),第790-795页. * |
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