Pd(II) and Rh(III) Complexes with Isoquinoline Derivatives Induced Mitochondria-Mediated Apoptotic and Autophagic Cell Death in HepG2 Cells (2020)

Open AccessCCS ChemistryRESEARCH ARTICLE1 Jun 2021Pd(II) and Rh(III) Complexes with Isoquinoline Derivatives Induced Mitochondria-Mediated Apoptotic and Autophagic Cell Death in HepG2 Cells Noor Shad Gul†, Taj-Malook Khan†, Yan-Cheng Liu, Muhammad Iqbal Choudhary, Zhen-Feng Chen and Hong Liang Noor Shad Gul† State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004 , Taj-Malook Khan† State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004 , Yan-Cheng Liu State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004 , Muhammad Iqbal Choudhary International Center for Chemical and Biological Sciences, University of Karachi, Karachi 74270 , Zhen-Feng Chen *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004 and Hong Liang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004 https://doi.org/10.31635/ccschem.020.202000363 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Nonplatinum metal complexes of [Pd(L1)Cl2] ( C1), [Rh(L1)Cl3(DMSO)] ( C2), [Pd(L2)Cl2] ( C3), and [Rh(L3)Cl3(DMSO)] ( C4) with isoquinoline derivatives have been prepared and characterized. C1– C4 exhibited higher in vitro anticancer activity and lower toxicity than the corresponding ligands and cisplatin against HepG2 cells. The mechanistic studies revealed that C1 arrested the cell cycle at S-phase by regulation of cyclin and cyclin-dependent kinases. C1 was accumulated in mitochondria, which increased the generation of reactive oxygen species (ROS) and endoplasmic reticulum (ER)-stress response through mitochondrial dysfunction. Moreover, C1 stimulated Ca2+ release, activated the caspase cascade, and triggered mitochondria-mediated apoptosis. The in vivo studies of C1 demonstrated higher safety than cisplatin and effective tumor growth inhibition. C1 is a potential anticancer drug candidate. Download figure Download PowerPoint Introduction Platinum drugs, including cisplatin and its analogs carboplatin (Paraplatin) and oxaliplatin (Eloxatin), have been in clinical use to treat various solid tumors.1–3 Despite clinical success, drug resistance and severe side effects limit their wider use and effectiveness.4 Therefore, numerous studies have been carried out to develop new drugs with fewer side effects and better chemotherapeutic efficacy than platinum-based drugs.5 During the past decade, nonplatinum metal complexes such as KP1019, auranofin, NAMI-A, and padeliporfin (TOOKAD) possessing promising anticancer activity against various kinds of cancers with different anticancer mechanisms are reported. Some of these complexes are now in different phases of clinical trials.6,7 In addition, palladium(II), rhodium(III), and copper(II) complexes are shown to exhibit higher anticancer activity toward various human cancer cells than platinum-based drugs.8–10 In this regard, palladium(II) complexes presented different anticancer properties against breast, lung, and prostate cancers than cisplatin.11,12 The palladium complexes with ethyl and benzyl amines induced apoptosis and DNA damage by activating the p53 protein and causing cell cycle arrest.13–17 In addition, several palladium complexes with triazole, pyrazole, and pyrrole as ligands induced reactive oxygen species (ROS)-mediated apoptosis.18 In ROS-mediated apoptosis, the intrinsic pathway is responsible for the release of cytochrome C and the activation of caspases, which regulate the Bcl-2 family proteins and ultimately lead to cell death.19,20 Many palladium complexes with imidazole and pyridine ligands produce high levels of ROS and reactive nitrogen species (RNS) that not only increase the risk of mutation and inhibit cell division but also induce apoptosis if the stress persists.21 Similarly, rhodium(III) complexes with polyaromatic ligands have potent anticancer activities against various kinds of cancers, such as colon and breast cancers.22 Mechanistic studies revealed that these complexes target DNA as well as induce mitochondrial damage that leads to cell apoptosis.23 Rhodium complexes with trichlorido-5,6-dimethyl phenanthroline ligands display high cytotoxicity toward Jurkat leukemia cells and induce apoptosis by overproduction of ROS.24 Furthermore, cellular metabolic studies of the rhodium complexes demonstrated that the apoptotic cell death is time dependent with moderate induction of ROS, which indicated the involvement of an activated intrinsic mitochondria-mediated pathway.25 Moreover, rhodium complexes with phenylquinoline ligands are known as potent kinase C (PKCδ) inhibitors, and kinase C is regarded as a critical regulator for various cellular functions.26 The racemic cyclometalated rhodium(III) complexes are promising inhibitors of JAK2 kinase activity, which acts as the critical cell signaling mechanism for cell proliferation and apoptosis. The abnormal activities of these kinases could be correlated with irregular cell growth and survival.27 The formation of transition metal complexes with biologically active ligands is an effective approach in the development of anticancer drugs. However, one major drawback of metal complexes is their instability in the cellular environment.28 Therefore, the selection of an appropriate bidentate chelating ligand of oxygen and nitrogen, such as quinoline, bipyridine, phenanthroline, and their derivatives, has been proposed to improve the stability and cytotoxic activity of metal complexes.29–31 Isoquinoline is a prerogative heterocyclic compound exhibiting anticancer, anti-Alzheimer, anti-inflammatory, anticonvulsant, and antimicrobial properties.32 Recently, the tetrahydroisoquinoline derivative trabectedin was approved by the United States Food and Drug Administration (USFDA (2007)) and the European Commission (2015) for the treatment of soft tissue sarcoma.33 Also, we previously reported that gold complexes with bioactive isoquinoline derivatives display high anticancer activity, selectivity, and synergistic effects against different cancer cell lines.34 Inspired by the anticancer activity of metal complexes with isoquinolines, here, we synthesized palladium(II) and rhodium(III) complexes with isoquinoline derivatives and investigated their in vitro anticancer activity through the ROS/endoplasmic reticulum (ER)-stress-mediated and the autophagic cell death pathways. The in vivo anticancer activity of [Pd(L1)Cl2] ( C1) was also conducted on tumor xenograft model of mice bearing HepG2. Experimental Methods Materials The chemicals were purchased from Alfa Aesar (Ward Hill, MA) and Sigma-Aldrich (St. Louis, MO) and used without further purification. Tris-buffered saline (TBS) solution (50 mM NaCl, 5 mM Tris) of pH 7.4 was prepared in double-distilled water. For the in vitro cytotoxicity assays, 2 mM stock solution of metal complexes was prepared in dimethyl sulfoxide (DMSO). Similarly, 2 mM solution of cisplatin was prepared in 0.9% NaCl saline. The working solutions were prepared from stock solution by serial dilution with TBS. Elemental analysis was used to determine the purity of each complex, which were ≥95%. Instrumentation A PerkinElmer 2400 Series II elemental analyzer was used for elemental analysis. Electrospray ionization mass spectrometry (ESI-MS) spectra were recorded on a Bruker HCT Spectrometer. A Bruker AV–600 nuclear magnetic resonance (NMR) spectrometer was used for the recording of 1H and 13C NMR spectra. Synthesis and characterization Ligands synthesis 1-(2′-Aminophenyl)-6,7-dimethoxy-isoquinoline (L1), 1-(2′-aminophenyl)-6-methoxy-3,4-dihydroisoquinoline (L2), and 1-(2′-aminophenyl)-6-methoxy-isoquinoline (L3) were synthesized by the reported Bischler–Napieralski reaction.35 Spectroscopic data of L1 Infrared (IR) (KBr, cm−1): 3328 (N–H), 2927 (C–H), 2852 (Ar–H), 1623 (C=N), 1577, 1500 (C=C), 1247, 1115 (C–C), 872, 756, 653. 1H NMR [500 MHz, (CD3)2SO, δ]: 8.43 (d, J = 4.6 Hz, 1H), 7.68 (d, J = 4.6 Hz, 1H), 7.44 (s, 1H), 7.22 (m, 2H), 7.18 (s, 1H), 6.91 (d, J = 6.3 Hz, 1H), 6.75 (td, J = 0.9, 6.0 Hz, 1H), 5.11 (s, 2H) 3.98 (s, 3H), 3.75 (s, 3H). 13C NMR [100 MHz, (CD3)2SO, δ]: 157.1, 157.0, 152.9, 150.1, 146.9, 141.1, 131.2, 129.5, 123.3, 122.7, 119.0, 116.4, 116.2, 105.9, 105.5, 56.2, 55.6 ( Supporting Information Figures S1–S3). Spectroscopic data of L2 IR (KBr, cm−1): 3409 (O–H), 3293 (N–H), 3184 (C–H), 2966, 2833 (Ar–H), 1606 (C=N), 1556, 1486, 1450 (C=C), 1252, 1107 (C–C), 833, 757, 682, 728. 1H NMR [500 MHz, (CD3)2·SO, δ]: 2.72 (t, J = 7.2 Hz, 2H), 3.73 (t, J = 7.2 Hz, 2H), 3.83 (s, 3H), 5.83 (s, 2H), 6.58 (t, J = 7.1 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.85 (dd, J = 2.5, 8.6 Hz, 1H), 6.93 (d, J = 2.3 Hz, 1H), 7.04–7.14 (m, 3H). 13C NMR [125 MHz, (CD3)2SO, δ]: 26.7, 47.2, 55.9, 112.3, 113.4, 115.4, 116.5, 121.1, 122.7, 129.8, 130.0, 131.0, 141.4, 148.4, 161.3, 166.4 ( Supporting Information Figures S4–S6). Spectroscopic data of L3 IR (KBr, cm−1): 3461 (O–H), 3329 (N–H), 3202, 3048 (C–H), 2922 (Ar–H), 1617 (C=N), 1553, 1456, 1410 (C=C), 1256, 1116 (C–C), 833, 682, 628. 1H NMR [500 MHz, (CD3)2SO, δ]: 8.50 (d, J = 4.7 Hz, 1H), 7.77 (m, 3H), 7.44 (d, J = 2.1 Hz, 1H), 7.26 (dd, J = 2.1, 8.0 Hz, 1H), 7.22 (m. 1H) 7.12 (dd, J = 1.1, 6.2 Hz, 1H), 6.74 (m, 1H), 5.09 (s, 2H) 3.96 (s, 3H). 13C NMR [100 MHz, (CD3)2SO, δ]: 160.6, 159.1, 146.9, 142.9, 139.2, 131.3, 129.6, 123.1, 122.6, 120.3, 119.5, 116.2, 116.1, 108.1, 105.4, 56.0 ( Supporting Information Figures S7–S9). General procedure for the formation of complexes Metal salts (1 mmol) and ligands (0.5 mM) were dissolved in an equal volume of dichloromethane and methanol, and the total volume of 5 mL solvent was placed into a 25 cm-long glass Pyrex glass tube. The mixture was frozen by liquid nitrogen for 5 min, and the air was removed using a vacuum pump. The tube was sealed by fire torch. After 72 h of constant heating at 80 °C, block crystals were harvested. A suitable crystal was selected for single-crystal X-ray diffraction analysis. All the refinement description and crystal data are listed in Supporting Information Table S2. [Pd(L1)Cl2] After formation of the complex via the general procedure, block orange crystals of C1 were harvested. A suitable crystal was selected for single-crystal X-ray diffraction analysis. Yield: 70%. Elemental analysis calcd for C17H16Cl2N2O2Pd (%): C, 44.62; H, 3.52; N, 6.12; found (%): C, 44.71; H, 3.35; N, 6.23. 1H NMR (600 MHz, DMSO-d6, δ): 8.66 (d, J = 12.0 Hz, 1H), 7.88–7.86 (m, 1H), 7.63–7.60 (m, 1H), 7.58 (s, 1H), 7.56 (d, J = 12.0 Hz, 1H), 7.47 (dd, J = 18.0, 12.0 Hz, 2H), 7.38 (s, 3H), 4.00 (s, 3H), 3.82 (s, 3H). 13C NMR (150 MHz, DMSO-d6, δ): 154.7, 153.1, 151.4, 144.0, 139.1, 135.4, 133.2, 132.5, 132.1, 125.1, 122.4, 121.5, 106.5, 106.1, 56.8, 56.1, 49.1. IR (KBr, cm–1): 2971 (–OCH3), 3128 (C–H), 1089 (C–N), 2381 (C=N), 3429 (N–H), 1614 (C=C), <400 (Pd–Cl), 412 (Pd–N). ESI-MS: m/z = 480.95 [M + Na]+ ( Supporting Information Figures S10–S13). [Rh(L1)Cl3(DMSO)] After formation of the complex via the general procedure, block yellow crystals of Rh(L1)Cl3(DMSO) ( C2) were harvested. A suitable crystal was selected for single-crystal X-ray diffraction analysis. Yield: 65%. Elemental analysis calcd for C19H22Cl3N2O3RhS (%): C, 40.20; H, 3.91; N, 4.93; S, 5.65; found (%): C, 40.33; H, 4.01; N, 5.04; S, 5.76. 1H NMR (600 MHz, DMSO-d6, δ): 9.33 (d, J = 6.0 Hz, 1H), 8.39 (d, J = 12.0 Hz, 1H), 7.92 (d, J = 12.0 Hz, 1H), 7.73 (d, J = 6.0 Hz, 1H), 7.58 (s, 1H), 7.48 (s, 1H), 7.43 (dd, J = 12.0, 6.0 Hz, 1H), 7.34 (dd, J = 12.0, 6.0 Hz, 2H), 5.76–5.71 (m, 1H), 4.03 (s, 3H), 3.81 (s, 3H), 3.53 (s, 3H), 3.43 (s, 3H). 13C NMR (150 MHz, DMSO-d6, δ): 156.2, 154.6, 150.7, 145.8, 142.0, 135.6, 134.3, 131.8, 130.4, 125.5, 124.1, 123.1, 120.2, 107.4, 105.8, 56.7, 56.0. IR (KBr cm−1): 2919 (–OCH3), 3017 (C–H), 1099 (C–N), 2323 (C=N), 3461 (N–H), 1614 (C=C), 1425 (S=O), 626 (Rh–S), <400 (Rh–Cl), 433 (Rh–N). ESI-MS: m/z = 588.93 [M + Na]+ ( Supporting Information Figures S14–S17). [Pd(L2)Cl2] After formation of the complex via the general procedure, block orange crystals of [Pd(L2)Cl2] ( C3) were harvested. A suitable crystal was selected for single-crystal X-ray diffraction analysis. Yield: 70%. Elemental analysis calcd for C16H16Cl2N2OPd (%): C, 44.42; H, 3.95; N, 6.09; found (%): C, 44.53; H, 3.79; N, 6.18. 1H NMR (600 MHz, DMSO-d6, δ): 7.68 (d, J = 12.0 Hz, 1H), 7.61–7.58 (m, 1H), 7.50 (d, J = 12.0 Hz, 1H), 7.35–7.34 (m, 2H), 7.16 (d, J = 6.0 Hz, 1H), 7.02 (s, 2H), 6.93–6.91 (m, 1H), 4.79 (d, J = 18.0 Hz, 1H), 3.87 (s, 3H), 3.46–3.42 (m, 1H), 2.82–2.79 (m, 2H). 13C NMR (150 MHz, DMSO-d6, δ): 166.9, 163.3, 141.9, 139.7, 133.5, 133.1, 132.8, 131.9, 124.9, 121.2, 121.0, 113.4, 113.1, 56.2, 52.3. IR (KBr, cm−1): 2913 (–OCH3), 3004 (C–H), 1024 (C–N), 2230 (C=N), 3425 (N–H), 1634 (C=C), <400 (Pd–Cl), 419 (Pd–N). ESI-MS: m/z = 426.9 [M–H]– ( Supporting Information Figures S18–S21). [Rh(L3)Cl3(DMSO)] After formation of the complex via the general procedure, block yellow crystals of Rh(L3)Cl3(DMSO) ( C4) were harvested. A suitable crystal was selected for single-crystal X-ray diffraction analysis. Yield: 60%. Elemental analysis calcd for C18H20Cl3N2O2RhS (%): C, 40.21; H, 3.75; N, 5.21; S, 5.96; found (%): C, 40.35; H, 3.71; N, 5.36; S, 5.35 1H NMR (600 MHz, DMSO-d6, δ): 9.39 (d, J = 6.0 Hz, 1H), 8.44 (s, 1H), 8.09 (d, J = 6.0 Hz, 1H), 7.97 (d, J = 12.0 Hz, 1H), 7.57 (d, J = 6.0 Hz, 1H), 7.52 (d, J = 6.0 Hz, 1H), 7.44–7.41 (m, 2H), 7.34–7.31 (m, 2H), 5.73 (s, 1H), 4.01 (s, 3H), 3.53 (s, 3H), 3.43 (s, 3H), 3.35 (s, 3H). 13C NMR (150 MHz, DMSO-d6, δ): 162.4, 158.7, 146.9, 142.1, 140.6, 134.9, 131.6, 131.4, 130.5, 125.4, 124.0, 122.6, 121.5, 120.6, 105.3, 56.5, 42.1, 41.8. IR (KBr, cm−1): 3013 (–OCH3), 3108 (C–H), 1019 (C–N), 2350 (C=N), 3440 (N–H), 1617 (C=C), 1411 (S=O), 679 (Rh–S), <400 (Rh–Cl), 413 (Rh–N). ESI-MS: m/z = 558.92 [M + Na]+ ( Supporting Information Figures S22–S25). Single-crystal X-ray crystallographic analysis A Bruker Smart APAX II was used at room temperature to collect X-ray data of complexes C1– C4. The monochromatic Mo-Kα radiation graphite source with wavelength of (λ = 0.71073 Å) was used for analysis. The direct method for the solvation of crystal structure was applied by using SHELXS-97.36 And for refining all nonhydrogen atoms, a method of full-matrix least-squares on F2 was applied with the thermal parameters of anisotropy by using SHELXL-97.37 The atoms of hydrogen were isotropically located at calculated positions. Cellular uptake of complexes The cells were placed in a cell culture plate (100 mm) at 37 °C in a 5% CO2 atmosphere for 24 h. Approximately 10 µM each of C1– C4 complexes was added and incubation continued for 24 h. After 24 h the cells were washed, collected, and digested with HNO3. Dilution was carried out by adding double-distilled water to 5% HNO3. Inductively coupled plasma mass spectrometry (ICP-MS) was used for the measurement of metal content in the diluted solution.38 The experiment was repeated three times and represented as mean ± SD. For the determination of metal content in the mitochondria, nucleus, and cytoplasm, the cells were placed in a 70 mm plate at 37 °C for 24 h in a 5% CO2 and 95% humidified atmosphere. The cells were treated and incubated for 24 h with C1– C4 at a concentration of 10 µM. After the incubation, cells were washed three times with phosphate-buffered saline (PBS), and the Solarbio kit was used to extract mitochondria and nuclei, per the procedure given by the manufacturer. All the samples were treated with concentrated HNO3 and dilution was done by adding double-distilled water up to 5% HNO3 concentration. By using ICP-MS, the content of metal in the nucleus and mitochondria was measured. All these experiments were repeated three times and represented as mean ± SD. Determination of lipophilicity The flask-shaking method was used for the determination of lipophilicity of C1– C4. C1 is used as an example to demonstrate the procedure here. A mixture of an equal volume of saturated stock solution of C1 in octanol (saturated with NaCl of 0.9% W/V) and NaCl (0.9% W/V) aqueous (saturated with octanol) was shaken for 24 h on the oscillator. Both phases of water and oil were collected and were gently separated and dried under vacuum after centrifugation at 5000g for 5 min. The substance obtained was dissolved in 300 μM HNO3 (65%) and diluted by Milli-Q water containing 10 parts per billion indium, to 5 milliliters. The concentration of C1 (concentration of C1 in octanol (Co) and concentration of C1 in water (Cw)) was determined by ICP-MS using In as internal standard. The log Po/w values were calculated from Co/Cw. Cell culturing and treatment Dulbecco's modified eagle medium (DMEM) with fetal bovine serum (10%) was used for culturing cells. The cells were held in a 5% CO2 humidified environment at 37 °C. The stock solution of C1 (2 mM) was prepared in DMSO, and serial dilutions were performed to obtain the required working solutions in PBS. Assay of in vitro cytotoxicity The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used for the evaluation of the in vitro cytotoxicity. The cells with the density of 4 × 103 were cultivated in a flat-bottomed 96-well plate. The cells were treated with different concentrations of the complexes, ligands, and cisplatin for 48 h. Cisplatin was dissolved in PBS and media containing <1% DMSO and used as the positive control. Negative control cells were supplemented with DMEM. MTT solution was added after the treatment with drugs and incubated for another 4 h. After adding DMSO to dissolve the formazan crystals and discarding the supernatant, the absorbance at 490/650 nm was measured on a The cytotoxicity was using the absorbance of the treated cells and the control cells. method = was used to the which the of cells toward complexes and Cell cycle determination 70 mm were used for cell The cells were treated with C1 for 24 h. The cells were collected after 24 washed with and in for one at °C. After in and cell was used for analysis of the cells. was used for cell cycle The cells were cultivated for 24 h in a plate. After treated with the cells were collected, washed, and incubated with and for 25 at °C. The samples were from incubation with was used for the analysis of cell apoptosis. potential analysis A was used for measurement of potential of The cells were to C1 at different concentrations for 24 h. After the with the cells were washed, collected, and incubated for 25 with was used for the of potential of The damage to the mitochondrial was shown by the orange accumulated in the mitochondria, the which accumulated in the at the of mitochondrial studies of ROS generation in HepG2 cells were on the of with the density of 2 × in with after treatment with the cells were washed and incubated for with 25 mM of at 37 °C. was with and the cells were incubated for min. The cells were washed with and the were placed on the and with by using suitable and determination The cells were treated with C1 for 24 h. were measured by using kit after the The cells were washed and incubated for with mL of in the and with The data were with the control. Determination of Ca2+ levels The cells were treated with C1 and cisplatin for 24 h. The of Ca2+ was determined by using the cells were washed and incubated for 25 with in the and with at nm wavelength of The was into and by the cell The with cellular Ca2+ in a on wavelength of analysis The total protein was collected by the cells in and the Assay kit was used for the determination of protein concentration. of protein was in the of The proteins were to for h at after the were The was placed in a solution containing the at 4 °C for h and incubated in dissolved in for h at pH The was incubated for 2 h with a with at room was used for the and were obtained by using the kit of In vivo anticancer activity mice from were used in this All the mice were at Guangxi University for in a environment of and h and was to the The studies were performed per Guangxi for the and of Laboratory mice were into containing mice in each The cisplatin was dissolved in 0.9% saline. Similarly, a saline solution of 0.9% was used for control. the of the tumor the cisplatin C1 was The cisplatin (2 and C1 and 10 were 2 The volume of tumor was calculated in by using the given volume of tumor = × × The growth of were as the of after the treatment against the tumor The mice were after of The tumor was collected and and was applied for in The given was used to the tumor growth = tumor of the control mean tumor of the tumor of the control was used for data including the was and Synthesis and characterization of ligands and complexes ligands of isoquinoline derivatives and L3 were synthesized using the reported L1 and L3 only in the and of the L1 has at and L3 has only one at L2 is a which has only one at The ligands were by NMR and IR All the complexes, including and C4 were prepared by the ligand and corresponding metal in a Pyrex glass tube containing equal volume of dichloromethane and as the The tube was sealed by fire torch. After 72 h of constant heating at 80 °C, block crystals were harvested. The complexes were by elemental and In addition, the stability of complexes C1– C4 was determined under of pH 7.4 in and by and liquid The indicated that complexes C1– C4 are under for 48 h ( Supporting structure of complexes The crystal of the complexes were determined by single-crystal X-ray diffraction analysis and are reported in Supporting Information Table C1 and In complexes, atoms of and the heterocyclic nitrogen of bidentate isoquinoline ligand L1 and L2 are with In the palladium and nitrogen are = and = the for palladium and are = and = In the palladium and nitrogen are = and = the for palladium and are = and = The and C4 complexes have in which one bidentate L1 three atoms, and one from DMSO are with The and of the bidentate ligands and and are located to each and the three atoms and and are to the of the The corresponding are as = = = and = for complexes Download figure Download PowerPoint In vitro cytotoxicity The cytotoxicity of C1– C4 was investigated by MTT assay against cancer cell including and human cell with cisplatin as a positive control. The values obtained from the cell assay are listed in Table treated with was on cell in cell was found in the of complexes C1– C4 with values from to µM. Complexes C1– C4 higher cytotoxicity in the cancer cells than the corresponding ligands and metal the complexes, C1 exhibited the in vitro anticancer activity against the cell which is higher than that of C1 cytotoxicity toward human cell with and complexes C1 and exhibited lower which that the and of the on the isoquinoline the cytotoxicity of the is with the that C1 and have higher lipophilicity Po/w than and C4 ( Supporting Table of and Complexes Cell and Normal Cell HepG2 L1 L2 L3 C1 ± ± ± ± ± ± ± ± ± ±