Ruthenium-artesunate complex and preparation method and application thereof
阅读说明：本技术 一种钌-青蒿琥酯配合物及其制备方法与应用 (Ruthenium-artesunate complex and preparation method and application thereof ) 是由 李蓉涛 陈毕纯 叶瑞绒 刘丹 陈宣钦 李洪梅 卢俊健 马秀蓉 于 2020-12-21 设计创作，主要内容包括：本发明公开一种钌-青蒿琥酯配合物及其制备方法与应用,钌-青蒿琥酯配合物可利用钌配合物改善细胞对青蒿琥酯的摄取,进入细胞后,钌配合物与青蒿琥酯协同发挥抗肿瘤作用,本发明研究钌-青蒿琥酯配合物的抗肿瘤活性,发现其具有良好的抗肿瘤活性,相对于传统的有机小分子来说,钌配合物具有多配位构型,可以进行不同配体的修饰,实现其与相应底物的特异性结合。(The ruthenium-artesunate complex can improve the uptake of cells to artesunate by utilizing the ruthenium complex, and after the ruthenium complex and the artesunate enter the cells, the ruthenium complex and the artesunate cooperatively play an anti-tumor role.)
1. The ruthenium-artesunate complex is characterized in that the chemical formula is [ Ru (DIP) ]2bpy(4-CH3-4'-CH2OART)](PF6)2The structure is shown as the following formula I:
2. the preparation method of the ruthenium-artesunate complex as claimed in claim 1, which comprises the following steps:
s1, 7.6729mmol of bathophenanthroline and 3.836mmol of RuCl3·nH2Placing O and 38.36mmol of lithium chloride in a three-neck flask, adding 11mL of N, N-dimethylformamide, reacting for 8h at 153 ℃ under the protection of nitrogen, dropwise adding the reaction solution into 150mL of glacial acetone after the reaction is finished, then placing in a refrigerator at 4 ℃ overnight, performing suction filtration, and alternately washing with water and acetone which are used for precooling at 4 ℃ to obtain cis- [ Ru (DIP)2Cl2]·2H2O；
S2, 0.3144mmol of cis- [ Ru (DIP) obtained from S12Cl2]·2H2Placing O and 0.3144mmol of 4-hydroxymethyl-4 '-methyl-2-2' -bipyridine into a three-neck flask, adding 100mL of ethanol water solution with volume fraction of 75%, reacting at 85 ℃ for 8h under the protection of nitrogen, fully concentrating the reaction solution to 10mL after the reaction is finished, adding 1.2576mmol of ammonium hexafluorophosphate solid, ultrasonically precipitating a large amount of precipitate, performing suction filtration and vacuum drying, and separating and purifying the precipitate by silica gel column chromatography with acetonitrile/water/saturated sodium nitrate solution as eluent to obtain a reaction intermediate [ Ru (DIP)2bpy(4-CH3-4'-CH2OH)](PF6)2；
S3, placing 0.1592mmol of reaction intermediate, 15.92mmol of artesunate, 0.2388mmol of dehydrating agent and 0.1592mmol of catalyst in a 250mL round-bottom flask, adding 100mL of super-dry dichloromethane, reacting at room temperature for 3 days, concentrating the reaction solution to 5mL after the reaction is finished, dropwise adding the reaction solution into 20mL of diethyl ether, precipitating a large amount of precipitate, performing suction filtration and vacuum drying, and separating and purifying the obtained crude product by silica gel column chromatography with dichloromethane/methanol solution as eluent to obtain a ruthenium-artesunate complex [ Ru (DIP)2bpy(4-CH3-4'-CH2OART)](PF6)2。
3. The method for producing a ruthenium-artesunate complex according to claim 2, wherein the dehydrating agent is dicyclohexylcarbodiimide.
4. The method for producing a ruthenium-artesunate complex according to claim 2, wherein the catalyst is 4-dimethylaminopyridine.
5. The method for preparing a ruthenium-artesunate complex as claimed in claim 2, wherein the acetonitrile/water/saturated sodium nitrate solution is obtained by mixing acetonitrile, water, and a saturated sodium nitrate solution at a volume ratio of 100:9: 1.
6. The method for producing a ruthenium-artesunate complex according to claim 2, wherein the dichloromethane/methanol solution is prepared by mixing dichloromethane and methanol at a volume ratio of 60: 1.
7. The use of the ruthenium-artesunate complex according to claim 1 for the preparation of an antitumor agent.
Technical Field
The invention relates to a ruthenium-artesunate complex, a preparation method thereof and an anti-tumor effect thereof, belonging to the field of chemical biology.
Background
The ruthenium complex is in a 6-coordination octahedral configuration, and the unique three-dimensional structure of the ruthenium complex can provide various coordination modes, so that the ruthenium complex can be better matched with a three-dimensional structure cavity of protein, and the selectivity of the metal complex to the protein is improved; the ruthenium polypyridine complex also has abundant photochemical and photophysical characteristics, and can be used for conveniently carrying out cell positioning and mechanism research. Clarke et al discovered the complex Cl (NH) in 19803)5Ru (III) has anticancer activity on tumor-bearing rats. Zasinovich et al found cis- [ RuCl ] containing DMSO ligands2(DMSO)4]Has activity on mouse lung cancer cells, the activity of the medicine is slightly weaker than that of cisplatin, but the side effect of the medicine is smaller. Currently, two ruthenium complexes NAMI-A, KP1019 have successfully entered clinical trials. Coordination-saturated ruthenium polypyridine complexes are another class of compounds that have been extensively studied with anticancer promise. Polypyridine refers to pyridine derivatives having a multidentate coordination structure, which, when coordinated to ru (ii), can impart photoluminescent properties to the complex by metal-ligand charge transfer. And related documents report that ruthenium polypyridine complexes play an antitumor role through DNA damage and mitochondrial pathways. Gasser et al reported that a ruthenium polypyridine complex with carboxyl can specifically target tumor cell mitochondria, has an antitumor activity equivalent to that of cisplatin, and can overcome the drug resistance of cisplatin. The ruthenium complex belongs to an ionic compound, the solubility of the ruthenium complex is improved compared with that of an organic compound, the hydrophilic-lipophilic coefficient of the compound, the mode of entering cells and the kinetics of the compound are greatly influenced, and the uptake amount and the uptake mode of the metal complex by the cells can be improved.
Artesunate is one of artemisinin derivatives, and has higher antimalarial activity than artemisinin. Artesunate is reported in the literature to have anti-tumor activity. Chulwon et al reported that artesunate can inhibit the growth of tumor cells and induce apoptosis in a mouse model of chronic myeloid leukemia. Meanwhile, artesunate can effectively reduce the level of RAD51 in ovarian cancer cells and glioma cells (RAD51 is a key protein for mediating the sensitivity of cancer cells to DNA damaging agents), and further enhance the killing power of the DNA damaging agents on the cancer cells. However, the artesunate tablet has poor water solubility, obvious first-pass effect and liver drug enzyme induction effect when being taken orally, and low bioavailability; and the artesunate powder injection has the advantages of fast blood concentration reduction after intravenous injection, half-life period of about 30 minutes, fast in vivo clearance rate, and the above defects limit the application of the artesunate powder injection in various diseases.
The metal-organic drug complex can improve the uptake of small molecule drugs by cells by using the metal complex, and even solve the drug resistance problem of drugs. Guo Zijia et al reported that tetravalent platinum-artesunate complex targeting RAD51 has strong antitumor activity against BRCA mutated ovarian cancer and breast cancer. Aspirin is bonded to tetravalent platinum by Liu Yang Zhong and Liu Yang teams to synthesize the Asplatin, and the compound can effectively overcome the drug resistance of cisplatin. The ruthenium polypyridine complex has high lipid solubility, and the combination of the ruthenium polypyridine complex and the artesunate can improve the amount of the artesunate taken by cells. After entering cells, the ruthenium complex and the artesunate cooperatively play an anti-tumor role. Meanwhile, the ruthenium polypyridine complex has photochemical and photophysical characteristics, and can be used for conveniently carrying out cell positioning and mechanism research.
Disclosure of Invention
The invention aims to provide a ruthenium-artesunate complex, a preparation method thereof and application of the ruthenium-artesunate complex as an anti-tumor drug.
The invention provides a ruthenium-artesunate complex with a chemical formula of [ Ru (DIP) ]2bpy(4-CH3-4'-CH2OART)](PF6)2The structure is shown as the following formula I:
the invention also provides a preparation method of the ruthenium-artesunate complex, which comprises the following specific steps:
s1, 7.6729mmol DIP and 3.836mmol RuCl3·nH2O and 38.36mmol lithium chloride are placed in a three-neck flask, 11mL of N, N-dimethylformamide is added, reaction is carried out for 8h at 153 ℃ under the protection of nitrogen, after the reaction is finished, the reaction solution is dropwise added into 150mL of glacial acetone,then, the mixture is placed in a refrigerator at 4 ℃ overnight, filtered, washed alternately with water precooled at 4 ℃ and acetone to obtain cis- [ Ru (DIP)2Cl2]·2H2O；
S2, 0.3144mmol of cis- [ Ru (DIP) obtained from S12Cl2]·2H2Placing O and 0.3144mmol of 4-hydroxymethyl-4 '-methyl-2-2' -bipyridine into a three-neck flask, adding 100mL of ethanol water solution with volume fraction of 75%, reacting at 85 ℃ for 8h under the protection of nitrogen, fully concentrating the reaction solution to 10mL after the reaction is finished, adding 1.2576mmol of ammonium hexafluorophosphate solid, ultrasonically precipitating a large amount of precipitate, performing suction filtration and vacuum drying, and separating and purifying the precipitate by silica gel column chromatography with acetonitrile/water/saturated sodium nitrate solution as eluent to obtain a reaction intermediate [ Ru (DIP)2bpy(4-CH3-4'-CH2OH)](PF6)2，Ru-1；
S3, placing 0.1592mmol of reaction intermediate obtained in S2, 15.92mmol of ART, 0.2388mmol of dehydrating agent and 0.1592mmol of catalyst in a 250mL round bottom flask, adding 100mL of ultra-dry dichloromethane, reacting at room temperature for 3 days, concentrating reaction liquid to 5mL, dropwise adding the reaction liquid into 20mL of diethyl ether, precipitating a large amount of precipitate, performing suction filtration and vacuum drying, and separating and purifying the obtained crude product by silica gel column chromatography with dichloromethane/methanol solution as eluent to obtain ruthenium-artesunate complex [ Ru (DIP) ]2bpy(4-CH3-4'-CH2OART)](PF6)2，Ru-2。
The dehydrating agent is dicyclohexylcarbodiimide.
The catalyst is 4-dimethylamino pyridine.
The acetonitrile/water/saturated sodium nitrate solution is obtained by mixing acetonitrile, water and a saturated sodium nitrate solution according to the volume ratio of 100:9: 1.
The dichloromethane/methanol solution is obtained by mixing dichloromethane and methanol according to the volume ratio of 60: 1.
The DIP is phenanthroline; 4-hydroxymethyl-4 '-methyl-2-2' -bipyridine as bpy (4-CH)3-4'-CH2OH); ru-1 is a reaction intermediate; ru-2 is a ruthenium-artesunate complex; ART is artesunate.
The invention also provides application of the ruthenium-artesunate complex in preparing antitumor drugs, and researches show that the ruthenium-artesunate complex has good antitumor activity.
The anti-tumor effect of the ruthenium-artesunate complex is verified by an MTT colorimetric method, and the tumor cells are HeLa (human cervical cancer cell strain), HepG2 (human hepatoma cell strain) and A549R (drug-resistant human lung cancer cell strain). The uptake level and the uptake mechanism of the ruthenium-artesunate complex in tumor cells and Western Blot research are carried out in HeLa cells.
Compared with the prior art, the invention has the following excellent effects:
(1) the compound with the anti-tumor effect is a ruthenium complex which has a multi-coordination configuration and can be modified by different ligands to realize the specific combination of the compound and a corresponding substrate.
(2) The ruthenium polypyridine complex has high lipid solubility, can improve the uptake of cells to artesunate by combining the ruthenium polypyridine complex with the artesunate, and synergistically plays an anti-tumor role after entering the cells.
Drawings
FIG. 1 is a drawing of compound Ru-11H-NMR spectrum;
FIG. 2 is an ESI-MS spectrum of compound Ru-1;
FIG. 3 is a drawing of compound Ru-21H-NMR spectrum;
FIG. 4 is an ESI-MS spectrum of compound Ru-2;
FIG. 5 is a confocal laser microscopy study of Ru-2 uptake in HeLa cells;
FIG. 6 shows confocal laser microscopy on the uptake mechanism of Ru-2 in HeLa cells;
FIG. 7 shows the study of anti-tumor mechanism of Ru-2 in HeLa cells by Western Blot.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings and specific examples, but the scope of the present invention is not limited thereto, and the materials used in the examples can be obtained by market purchase or prepared by conventional methods without specific description.
Example 1
7.6729mmol DIP, 3.836mmol RuCl3·nH2Placing O and 38.36mmol of lithium chloride in a three-neck flask, adding 11mL of N, N-dimethylformamide, reacting for 8h at 153 ℃ under the protection of nitrogen, dropwise adding the reaction solution into 150mL of glacial acetone after the reaction is finished, then placing in a refrigerator at 4 ℃ overnight, performing suction filtration, and alternately washing with water and acetone which are used for precooling at 4 ℃ to obtain cis- [ Ru (DIP)2Cl2]·2H2O；
0.3144mmol of cis- [ Ru (DIP)2Cl2]·2H2Placing O and 0.3144mmol of 4-hydroxymethyl-4 '-methyl-2-2' -bipyridine into a three-neck flask, adding 100mL of ethanol water solution with volume fraction of 75%, reacting at 85 ℃ for 8h under the protection of nitrogen, fully concentrating the reaction solution to 10mL after the reaction is finished, adding 1.2576mmol of ammonium hexafluorophosphate solid, ultrasonically precipitating a large amount of precipitate, performing suction filtration and vacuum drying, and separating and purifying the precipitate by silica gel column chromatography with acetonitrile/water/saturated sodium nitrate solution (100:9:1, v/v/v) as eluent to obtain a reaction intermediate [ Ru (DIP) ]2bpy(4-CH3-4'-CH2OH)](PF6)2Ru-1, 83.64% yield.
FIGS. 1 and 2 show the compound Ru-11H-NMR and ESI-MS spectra; as shown in the drawings,1H NMR(600MHz,d6-DMSO)δ8.82(d,J＝18.8Hz,2H),8.35(s,2H),8.25(d,J＝21.3Hz,6H),7.95(d,J＝5.1Hz,2H),7.89–7.49(m,24H),7.45(s,1H),7.36(s,1H),5.78(s,1H),4.77(s,2H),2.56(s,3H)。MS(ESI):m/z:965.3[M-2PF6-H]+。
placing 0.1592mmol reaction intermediate, 15.92mmol ART, 0.2388mmol dehydrating agent dicyclohexylcarbodiimide and 0.1592mmol catalyst 4-dimethylamino pyridine in a 250mL round bottom flask, adding 100mL ultra-dry dichloromethane, reacting at room temperature for 3 days, concentrating the reaction solution to 5mL after the reaction is finished, dropwise adding the reaction solution into 20mL diethyl ether, precipitating a large amount of precipitate, filtering, and vacuum dryingSeparating and purifying the obtained crude product by silica gel column chromatography with dichloromethane/methanol solution (60:1, v/v) as eluent to obtain ruthenium-artesunate complex [ Ru (DIP)2bpy(4-CH3-4'-CH2OART)](PF6)2Ru-2, yield 48.5%.
FIGS. 3 and 4 show the compounds Ru-21H-NMR and ESI-MS spectra; as shown in the drawings,1H NMR(600MHz,DMSO)δ8.87–8.80(m,2H),8.34(s,2H),8.28–8.20(m,6H),7.95(d,J＝5.3Hz,2H),7.82(d,J＝5.6Hz,1H),7.77(s,2H),7.72–7.62(m,21H),7.47(d,J＝5.9Hz,1H),7.38(d,J＝5.8Hz,1H),5.63(d,J＝4.2Hz,1H),5.50(d,J＝8.3Hz,1H),5.37(s,2H),2.76(dd,J＝18.2,4.4Hz,4H),2.57(s,3H),2.25(s,1H),2.14(s,1H),1.96(d,J＝14.3Hz,1H),1.79(s,1H),1.48(s,3H),1.34(s,3H),1.21(d,J＝9.4Hz,3H),1.14(s,1H),0.85(s,4H),0.67(s,3H)。MS(ESI):m/z:666.2[M-2PF6]2+。
example 2
The ruthenium-artesunate complex is tested by an anti-tumor activity experiment:
HepG2, A549R and HeLa cells were cultured in DMEM medium containing 10% fetal calf serum and 1% penicillin-streptomycin solution (10% fetal calf serum and 1% penicillin-streptomycin by volume) at 37 deg.C with 5% CO2Culturing in a cell culture box, digesting the cells into single cell suspension with 0.25% trypsin by mass percent, counting the living cells by adopting a blood counting chamber, and adjusting the concentration of the living cells to be 5 multiplied by 104PermL, inoculating in 96-well culture plate, culturing for 24 hr at 160 μ L per well, adding different amounts of drugs to final concentrations of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.125 μ M, and 1.5625 μ M, standing at 37 deg.C and containing 5% CO2The incubation was carried out for 48 hours, MTT (20. mu.L/well) was added 4 hours before the end, the supernatant was discarded 4 hours after the end, DMSO (150. mu.L/well) was added, and the mixture was shaken for 15 minutes, after which the absorbance at a wavelength of 570nm was measured.
The cells are divided into a blank control group, a positive control group, a negative control group and an experimental group, wherein the blank control group is a culture medium containing 1% DMSO, the positive control group adopts cisplatin (cissplatin), the negative control group adopts Ru-1, Ru-1+ ART and ART, and the experimental group adopts Ru-2.
IC50The concentration is calculated by the sps and is the half inhibitory effective concentration.
The test results are shown in table 1 below, and the experimental data are the average values obtained after three parallel experiments.
As can be seen from Table 1, the IC of Ru-2 was determined at the same concentration50The lowest, it indicates that Ru-2 has the best anti-tumor effect.
TABLE 1
Example 3
The uptake of the ruthenium-artesunate complex in HeLa cells was investigated, and this example shows the uptake of the ruthenium-artesunate complex in HeLa cells by confocal laser microscopy:
HeLa cells were cultured in 35mm Corning confocal laser culture dishes, and when the density was 70%, 5. mu.M Ru-2 was added thereto and incubated for 1 hour, 2 hours, 3 hours, and 4 hours, respectively, and then washed twice with PBS and immediately observed with a confocal laser microscope.
As a result of the study, as shown in FIG. 5, the amount of Ru-2 uptake by the cells increased in a time-dependent manner.
Example 4
The uptake mechanism of the ruthenium-artesunate complex in HeLa cells was studied, and this example investigated the uptake mechanism of the ruthenium-artesunate complex in HeLa cells by confocal laser microscopy:
HeLa cells were cultured in 35mm Corning confocal laser culture dishes, and when the density was 70%, 30. mu.M of CCCP (CCCP, carbonyl cyanide metachlorobenzhydrazone, metabolic inhibitor) and 50. mu.M of Chloroquine (Chloroquine, endocytosis inhibitor) were added for preincubation for 1 hour, after which the culture solution was replaced with a culture solution containing 5. mu.M of Ru-2 (DMEM as the medium), and after further incubation for 4 hours, the cells were washed twice with PBS and immediately observed with a laser confocal microscope.
The research result is shown in fig. 6, after being treated by 4 ℃ or a metabolic inhibitor CCCP, the uptake efficiency of the HeLa cells to the complex is reduced, and after being pretreated by an endocytosis inhibitor chloroquine, the uptake efficiency of the HeLa cells to the complex is not obviously changed; the research result shows that the ruthenium-artesunate complex enters HeLa cells through an energy-dependent pathway.
Example 5
The research on the anti-tumor mechanism of the ruthenium-artesunate complex comprises the following steps:
HeLa cells were cultured in a 60mm Corning dish, and when the density thereof reached 70%, Ru-2 (the concentration of Ru-2 was set to 0. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, respectively) was added, and the mixture was incubated in an incubator at 37 ℃ for 24 hours, after which RIPA lysate was added, the cells were lysed on ice, centrifuged, and the supernatant was collected and the total protein concentration was measured with a BCA protein assay kit.
Separating target protein by SDS-polyacrylamide gel electrophoresis, transferring the target protein to a PVDF membrane, and soaking the PVDF membrane in a confining liquid and shaking for 2h at room temperature. Followed by shaking with the corresponding primary antibody (. beta. -actin,. beta. -caspase-3, PARP, Bax, Bcl-2) overnight at 4 ℃ and finally incubation with horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. Signal detection was performed using ECL chemiluminescent substrate kit.
caspase-3 is a critical executive molecule for apoptosis; PARP is a marker of apoptosis; bcl-2 can enhance the resistance of cells to most DNA damage factors and inhibit apoptosis caused by chemotherapeutic drugs; the Bax gene is the main apoptosis gene of human body, and the encoded Bax protein can form heterodimer with Bcl-2 to generate repression on the Bcl-2.
As shown in FIG. 7, after Ru-2 treatment, caspase-3 cleavage can be induced, and PARP is sheared, so that Bcl-2 expression in cells can be reduced, and Bax expression can be improved.
The experimental results show that the ruthenium-artesunate complex can play the role of anti-tumor by inducing HeLa cells to undergo apoptosis.