CN105601676A - Ruthenium complex and application thereof - Google Patents
Ruthenium complex and application thereof Download PDFInfo
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- CN105601676A CN105601676A CN201610067339.2A CN201610067339A CN105601676A CN 105601676 A CN105601676 A CN 105601676A CN 201610067339 A CN201610067339 A CN 201610067339A CN 105601676 A CN105601676 A CN 105601676A
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- 239000012327 Ruthenium complex Substances 0.000 title claims abstract description 36
- YNPNZTXNASCQKK-UHFFFAOYSA-N Phenanthrene Natural products C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 claims abstract description 15
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 claims abstract description 15
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 10
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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
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0046—Ruthenium compounds
- C07F15/0053—Ruthenium compounds without a metal-carbon linkage
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a ruthenium complex and application thereof. The cation part of the ruthenium complex is [Ru(phen)2(HIPMP)]<2+>, and the anion part is (ClO4)[-] or Cl[-]. The mononuclear ruthenium complex has favorable anticancer activities, and has obvious treatment effects on cervical cancer cells, liver cancer cells, lung cancer cells nasopharyngeal carcinoma cells and other cancers. The tumor cell apoptosis induction of the mononuclear ruthenium complex mainly relates to endogenous endoplasmic reticulum channels, and has important meanings for researching high-efficiency ruthenium antineoplastic drugs.
Description
Technical field
The present invention is specifically related to a kind of ruthenium complex and application thereof.
Background technology
Cancer is one of topmost disease threatening at present human health and life security. The whole world approximately has 1,270 ten thousand people to be diagnosed as cancer patient every year, and the number that cancer is died from the whole world every year accounts for 13% of total death toll. China approximately has 1,500,000 people to die from cancer every year, and presents ascendant trend year by year. Since cis-platinum is found to have active anticancer, the application and research of Platinum Anti-tumor Drugs is developed rapidly. Wherein platinum medicine Anticancer Effect and Mechanism is mainly taking DNA as main target, destroys copying and suppress cell division etc. and being used for inhibition tumor cell and growing of DNA. But platinum medicine is long-term uses that to have toxic and side effect large, the shortcoming such as the strong and anticancer spectrum of drug resistance is narrow. Therefore, finding efficient, high selection and the little cancer therapy drug of side effect is the main direction of cancer therapy drug exploitation.
Ruthenium complex, compared with cis-platinum, has relatively low toxicity, and high selectivity easily absorbs and drained very soon and overcome the feature such as cells resistance of platinum medicine in vivo, be considered to one of the most promising antineoplastic (Coord.Chem.Rev.,2002,232,69). Up to the present, two kinds of ruthenium complex NAMI and KP1019 have entered the phase ii clinical trial stage, the former has obvious inhibitory action to the metastatic tumor of muroid, and the latter has obvious result for the treatment of to colon cancer, can suppress the inoperative tumor growth of some cis-platinums. But the antitumor mechanism of ruthenium complex is mainly the antitumor mechanism of the inducing apoptosis of tumour cell taking DNA as target or taking mitochondria as target spot at present. There is the large and low shortcoming such as selective of toxic and side effect in such ruthenium complex.
Summary of the invention
The object of the present invention is to provide a kind of preparation and application thereof of ruthenium complex.
The technical solution used in the present invention is:
A kind of ruthenium complex, is made up of cation and anion, and described cation is [Ru (phen)2(HIPMP)]2+, structural formula is as follows:
。
Preferably, described anion is inorganic ion.
Preferably, described inorganic salts anion is (ClO4)－Or Cl－。
The preparation method of described ruthenium complex, comprises the following steps:
1), by 1,10-Phendione, ammonium acetate and 5-cresotinic acid aldehyde, under inert gas shielding, fully reaction obtains intermediate productHIPMP；
2) intermediate product step 1) being obtainedHIPMPWithcis-[Ru(phen)2Cl2]·2H2O, fully reaction under inert gas shielding, is cooled to the saturated solution that slowly drips anionic inorganic salt after room temperature and produces precipitation, and precipitation is further purified dry ruthenium complex.
Preferably, react added 1 in step 1), 10-Phendione, ammonium acetate and 5-cresotinic acid aldehyde amount of substance are than being 1:20:1.
Preferably, the solvent that reacts used in step 1) is glacial acetic acid.
Preferably, the reaction of step 1) is carried out with the method that adds hot reflux.
Preferably, step 1) is reacted the intermediate product obtainingHIPMPBe further purified, comprise the following steps: to dissolve with a small amount of ethanol, fill post with silica gel, ethanol, as eluent, is collected yellow color component, and rotary evaporation obtainsHIPMPYellow powder.
Preferably, step 2) in reaction addHIPMP withcis-[Ru(phen)2Cl2]·2H2The amount of substance of O is than being 1:1.
Preferably, step 2) in reaction solvent used be ethanol or ethylene glycol.
Preferably, step 2) in reaction solvent used be ethanol.
Preferably, step 2) reaction carry out with the method that adds hot reflux.
Preferably, step 2) in inorganic salts anion be (ClO4)－Or Cl－。
Preferably, step 2)cis-[Ru(phen)2Cl2]·2H2O andHIPMPThan joining in ethanol for 1:1, under inert gas shielding, add hot reflux 8 hours according to amount of substance, obtain red clear liquid, slowly drip NaClO after being cooled to room temperature4Saturated solution or directly revolve steaming, produces reddish brown precipitation.
The application of ruthenium complex in antineoplastic described in above-mentioned any one.
Preferably, described cancer comprises cervical carcinoma, liver cancer, lung cancer or nasopharyngeal carcinoma.
The invention has the beneficial effects as follows:
The disclosed ruthenium complex of this patent, is a kind of ruthenium complex strong inhibition tumor cell toxicity, taking endoplasmic reticulum as target spot inducing apoptosis of tumour cell that has, and has important meaning for the efficient ruthenium antineoplastic of research.
Ruthenium complex of the present invention be a kind of novel structure have antitumaous effect, containing p-cresol structure monokaryon ruthenium (II) complex.
Monokaryon ruthenium of the present invention (II) complex has good active anticancer, especially remarkable to treatment of cancer effects such as human cervical carcinoma cell, HCC, lung carcinoma cell or nasopharyngeal carcinoma cells.
Monokaryon ruthenium of the present invention (II) complex inducing apoptosis of tumour cell relates generally to endogenic endoplasmic reticulum path.
Monokaryon ruthenium of the present invention (II) complex has lower normal cell toxicity and high selectivity.
Brief description of the drawings
Fig. 1 is complex [Ru (phen)2(HIPMP)](ClO4)2Synthetic route chart;
Fig. 2 is that ruthenium complex is to Cytostatic to tumor cell lab diagram;
Fig. 3 is ruthenium complex induction HeLa Apoptosis flow cytometer detection figure;
Fig. 4 dyes rear cell imaging figure altogether with ER-Tracker, Mito-Tracker and LYSO-Tracker after HeLa cell and ruthenium complex are cultivated 2h;
Fig. 5 is the affect figure of ruthenium complex on endoplasm Netcom road protein expression.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited to this.
Embodiment 1 monokaryon ruthenium complex [Ru (phen)
2
(HIPMP)](ClO
4
)
2
Synthetic
(1) by 1; 10-Phen-5; 6-diketone, ammonium acetate and 5-cresotinic acid aldehyde are 1:20:1 according to amount of substance ratio; be dissolved in appropriate glacial acetic acid, under inert gas shielding, Hybrid Heating refluxes 4 hours, and mixture adds water after being cooled to room temperature; use 25% ammonia neutralization; suction filtration, obtains yellow mercury oxide water and ether washing, obtains corresponding crude product. Crude product dissolves with a small amount of ethanol, and with silica gel (60-100 order) dress post, ethanol, as eluent, is collected yellow color component, and rotary evaporation obtains yellow powder 2-(1H-Imidazo-[4,5-f] [1,10] phenanthrolin-2-yl)-4-methylphenol (is intermediate productHIPMP). Productive rate: 83%. Anal.CalcdforC20H14N4O:C,73.61;H,4.32;N,17.17.Found:C,73.14;H,4.61;N,17.32%.FAB-MS:m/z=327(M+1)；
（2）cis-[Ru(phen)2Cl2]·2H2O andHIPMPThan joining in appropriate ethanol for 1:1, under inert gas shielding, add hot reflux 8 hours according to amount of substance, obtain red clear liquid, slowly drip NaClO after being cooled to room temperature4Saturated solution, produces reddish brown precipitation. Suction filtration, product is crossed the mixed liquor that post acetonitrile and ethanol volume ratio are 10:1 and is rinsed, and vacuum drying obtains end-product [Ru (phen)2(HIPMP)](ClO4)2. Synthetic route chart is shown in Fig. 1.
Synthetic yield is 70%. Anal.CalcdforC44H30N8Cl2O9Ru:C,53.56;H,3.06;N,11.36%;Found:C,53.87;H,2.92;N,11.58%.ES-MS[CH3CN,m/z]:788.2([M–2ClO4–H]+),394.1([M–2ClO4]2+).1HNMR(400MHz,DMSO-d6)ppm:9.18(d,J=8.0Hz,1H),9.03(d,J=6.0Hz,1H),8.78(d,J=3.8Hz,2H),8.77(d,J=6.0Hz,2H),8.40(s,4H),8.16(d,J=6.0Hz,2H),8.12(d,J=6.0Hz,2H),8.08(d,J=12.0Hz,2H),8.04(t,J=6.0Hz,2H),7.88(d,J=8.0Hz,2H),7.78(m,2H),7.78(dd,J=8.0Hz,2H),7.39(d,J=8.0Hz,2H),7.14(d,J=7.0Hz,1H),2.36(s,3H).。
The inhibitory action of embodiment 2 monokaryon ruthenium (II) complexs to tumour cell HeLa, A549, HepG2 and CNE-1 propagation
Cytotoxicity experiment: adopt mtt assay to study the in vitro toxicity experiment of complex. First experimental cell is placed in to 37 DEG C, 5.0%CO2In incubator, grow to logarithmic phase, 0.25% trypsinization collecting cell, adjusts concentration of cell suspension, makes cell density greatly about 1 × 104Individual/mL, every hole 100mL is inoculated in 96 orifice plates, and cell density is about 3-5 × 103Individual/hole, is placed in 37 DEG C, 5%CO2Incubator in cultivate 24h. Change liquid, add the medicine of variable concentrations gradient, each concentration is done 3 Duplicate Samples, blank zeroing group (culture medium, MTT, DMSO) is set, blank group (the medicine dissolving medium of culture medium, cell, same concentrations, MTT, DMSO), positive controls (cis-platinum of culture medium, cell, variable concentrations, MTT, DMSO). Be placed in 37 DEG C, 5%CO2Incubator in continue cultivate 48h. Suck supernatant, every hole adds 90 μ l fresh mediums, then adds 10 μ lMTT solution (5mg/mL, i.e. 0.5%MTT), continues to cultivate 4h. Stop cultivating, discard nutrient solution in hole, every hole adds 150 μ lDMSO, is placed in low-speed oscillation 30min on shaking table, and crystal is fully dissolved. Enzyme-linked immunosorbent assay instrument detects the absorbance OD in each hole of 490nm wavelength.
The inhibiting rate of relevant cell proliferation and half-inhibition concentration (IC50) use formula below to calculate: growth inhibition ratio=(ODContrast-ODExperiment）/（ODContrast-ODBlank), all OD values all deduct blank zeroing group OD value. By inhibiting rate and drug concentration mapping, draw dose-effect curve, therefrom calculate IC50Value. Monokaryon ruthenium complex [Ru (phen)2(HIPMP)](ClO4)2(embodiment 1 compound) is to tumour cell HeLa(human cervical carcinoma cell), A549(lung carcinoma cell), HepG2 (HCC) or CNE-1(nasopharyngeal carcinoma cell) IC of inhibited proliferation50Value is in table 1, and inhibiting rate and drug concentration graph of relation are shown in Fig. 2.
Table 1[Ru (phen)2(HIPMP)](ClO4)2To the IC of tumour cell50Value
Result shows, monokaryon ruthenium complex [Ru (phen)2(HIPMP)](ClO4)2To the IC of tumour cell HeLa, A549, HepG2 and CNE-150Value and cis-platinum quite, and toxicity maximum to HeLa tumour cell. All than it, the cytotoxicity to cancer cell is little to the cytotoxicity of normal cell LO2 for mononuclear complex, illustrates that complex has lower normal cell toxicity.
Embodiment 3 monokaryon ruthenium (II) complexs adopt AnnexinV and the two method research HeLa cell apoptosis assays that dye of PI
Alexafluor?The two methods of dying of 488annexinV/PI are a kind of sxemiquantitative Apoptosis analytical method (Vermes that detect with flow cytometer, C.Haanen, H.Steffens-NakkenandC.Reutellingsperger, J.Immunol.Methods, 1995,184,39-51.), its specific experiment step is as follows: collect logarithmic phase cell, adjust concentration of cell suspension, every hole 1 × 10 in 6 orifice plates5Individual cell. Be placed in 37 DEG C, 5%CO2Incubator in cultivate about 24h. Change liquid, add 1mL variable concentrations (10,20,40 μ M) medicine, three parallel holes of each concentration arrange the blank hole that adds same concentrations medicine dissolving medium simultaneously, continue to cultivate 24h. Carefully suck supernatant, 0.25% trypsinization collecting cell, adjusts concentration of cell suspension, makes cell density greatly about 1 × 104Individual/mL, 1000rpm, 5min is centrifugal, removes supernatant, adds 100 μ lPBS resuspended, uses strainer filtering. 1000rpm, 5min is centrifugal, removes supernatant, adds 100 μ l1 × BindingBuffer re-suspended cells, adds respectively 5 μ lAlexafluor?488annexinV and 1 μ lPI, incubated at room 15min, adds 400 μ l1 × BindingBuffer, and with the detection of FACSCantoII flow cytometer, experimental result is as shown in Figure 3. Experimental result presents good concentration dependent. When the concentration of complex is during at 10 μ M, 18.3% HeLa cell is in early apoptosis state, and 14.3% HeLa cell is in apoptotic state in late period. When the concentration of effect is while reaching 40 μ M, always have 88.2% cell in apoptotic state (early stage and late period apoptosis). Along with the raising of concentration, the quantity of the apoptotic cell of complex induction rises thereupon, and complex can cause HeLa Apoptosis.
Embodiment 4 monokaryon ruthenium (II) complexs are distributional analysis experiment in cell
HeLa cell is containing cultivating in the DMEM culture medium of 10% hyclone, cell (5 × l08/ L) be seeded in 12 orifice plates, approximately 2 × l0 of every hole4Individual cell. Be placed in 5%CO2Under 95% air conditions, 37 DEG C of cultivations, adherent growth 24 hours. Change liquid, add 1mL monokaryon ruthenium (II) complex (20 μ M), three parallel holes of same concentration arrange the blank hole that adds same concentrations medicine dissolving medium simultaneously, continue to cultivate 24h. Sucking-off nutrient solution, then with PBS buffer solution washing 3-4 time, every hole adds respectively the 50nMMito-Tracker that contains of 1mL37 DEG C of incubation, the PBS of ER-Tracker and LYSO-Tracker dyeing liquor, hatch 30min, observe and pictures taken by OlympusIX71 inverted fluorescence microscope, exciting light adopts ruddiness and green glow binary channels to excite.
Experimental result is shown in Fig. 4, and as we can see from the figure, monokaryon ruthenium complex is under laser excitation, and the cell after complex is painted is all to present bright redness, and is distributed in endoplasmic reticulum, well coincide with commercial endoplasmic reticulum dyestuff. Complex and commercial mitochondria dyestuff and lysosome dyestuff well do not coincide. Illustrate that complex is positioned in endoplasmic reticulum.
The signal path research of embodiment 5 monokaryon ruthenium (II) complex inducing apoptosis of tumour cell effects
(1) protein sample preparation, collects in logarithmic phase HeLa cell, adjusts concentration of cell suspension, every hole 1 × 10 in 6 orifice plates5Individual cell, is placed in incubator overnight incubation.
(2) change liquid, add ruthenium complex, concentration is 10,20,40 μ M, with co-culture of cells 24h.
(3) hatch while end, collecting cell, extracts albumen. Get appropriate protein, add sample-loading buffer in the ratio of 4:1,100 DEG C of metal bath heating 5min make albuminous degeneration, and 20,000rpm is centrifugal, gets supernatant stand-by.
(4) SDS-PAGE electrophoresis, installs vertical electrophoresis apparatus, prepares the separation gel of desired concn, encapsulating.
(5) after treating that PAGE gelling is admittedly complete, take off glass plate, be contained in electrophoresis tank by flag sequence, pull out comb. Dilute the 5 × electrophoretic buffer having prepared with ultra-pure water, mix, add electrophoresis tank.
(6) loading, plugs sample injector, and with special loading rifle head application of sample, general marker3 μ l, provides the comb of sample applied sample amount 25 μ l(1mm thickness for oneself), can adjust as required the applied sample amount of sample.
(7) electrophoresis, plugs in, and constant voltage 80V is set, and sample arrives concentrated glue separation gel interface 120V afterwards, can adjust as required time length.
(8) transferring film, pvdf membrane steeps 30 minutes with absolute methanol, storing order (Hei-Bai): sponge-filter paper-glue-pvdf membrane-filter paper-sponge. According to black, to black (negative pole), white is placed on transferring film clip in electrophoresis tank the order of red (positive pole), puts into ice chest simultaneously, fills it up with fresh transferring film liquid (being stored in 4 DEG C), and constant current 250mA/h is set.
(9) immuning hybridization, transferring film finishes rear taking-up film, with TBST flushing one time, is immersed in confining liquid, at room temperature slowly shake on shaking table, sealing 1h. Film is taken out from confining liquid, with TBST flushing one time, be immersed in the primary antibodie solution preparing, be placed in 4 DEG C of overnight incubation. Primary antibodie is hatched after end, washes film 3 times with TBST, each 6min. Select corresponding two to resist according to the source of primary antibodie, two anti-dilutions are with using confining liquid, and general 1:2000 dilutes, and room temperature shaking table is hatched 1h.
(10) exposure imaging, two anti-hatching after end, wash film 3 times, each 5min with TBST. Blot after TBST with paper handkerchief, ECL is evenly added drop-wise to pvdf membrane, preservative film in covering, is placed in post-exposure in exposure box. Select the time for exposure according to the power of destination protein signal. Adjust the time for exposure to obtain optimum efficiency according to destination protein Band signal is strong and weak, scan film, preserves analysis result.
Westernbolt laboratory test results is shown in Fig. 5, p-PERK, and p-eIF2 α and CHOP protein expression increase, and Caspase-7 and PARP albumen rupture. Result shows, can there is phosphorylation by inducible protein kinase beta sample endoplasmic reticulum kinases (PERK) in monokaryon ruthenium complex, then activate downstream signaling molecule Eukaryotic Initiation Factor 4 2(enkaryoticinitiation2 α, eIF2 α), raise er stress specific transcription factor CHOP simultaneously, thus active cell apoptotic signal path. Under the induction of CHOP, Caspase-7 is sheared activation, and then its substrate molecule of cutter activation PARP, the apoptotic marker molecule of PARP, and its Activation markers cell irreversible apoptosis is occurred. In sum, this monokaryon ruthenium complex can pass through er stress apoptosis pathway induced tumor cell generation apoptosis.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (10)
1. a ruthenium complex, is made up of cation and anion, it is characterized in that: described cation is [Ru (phen)2(HIPMP)]2+, structural formula is as follows:
。
2. ruthenium complex according to claim 1, is characterized in that: described anion is inorganic ion.
3. ruthenium complex according to claim 1, is characterized in that: described inorganic salts anion is (ClO4)－Or Cl－。
4. the preparation method of ruthenium complex described in claim 1, is characterized in that, comprises the following steps:
By 1,10-Phendione, ammonium acetate and 5-cresotinic acid aldehyde, under inert gas shielding, fully reaction obtains intermediate productHIPMP；
The intermediate product that step 1) is obtainedHIPMPWithcis-[Ru(phen)2Cl2]·2H2O, fully reaction under inert gas shielding, is cooled to the saturated solution that slowly drips anionic inorganic salt after room temperature and produces precipitation, and precipitation is further purified dry ruthenium complex.
5. preparation method according to claim 4, is characterized in that: in step 1), react added 1,10-Phendione, ammonium acetate and 5-cresotinic acid aldehyde amount of substance are than being 1:20:1.
6. preparation method according to claim 4, is characterized in that: the solvent that reacts used in step 1) is glacial acetic acid.
7. preparation method according to claim 4, is characterized in that: step 2) in reaction addHIPMPWithcis-[Ru(phen)2Cl2]·2H2The amount of substance of O is than being 1:1.
8. preparation method according to claim 4, is characterized in that: step 2) in reaction solvent used be ethanol or ethylene glycol.
9. the application of ruthenium complex in antineoplastic described in claim 1-3 any one.
10. application according to claim 9, is characterized in that: described cancer comprises cervical carcinoma, liver cancer, lung cancer or nasopharyngeal carcinoma.
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| CN110857310A (en) * | 2018-08-12 | 2020-03-03 | 南京大学 | Polyamphidine ruthenium complex with photoactivity and application thereof |
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