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

Abstract

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 †N. S. Gul and T.-M. Khan contributed equally to this work.Google Scholar More articles by this author , Taj-Malook Khan† State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004 †N. S. Gul and T.-M. Khan contributed equally to this work.Google Scholar More articles by this author , Yan-Cheng Liu State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004 Google Scholar More articles by this author , Muhammad Iqbal Choudhary International Center for Chemical and Biological Sciences, University of Karachi, Karachi 74270 Google Scholar More articles by this author , 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 Google Scholar More articles by this author 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 Google Scholar More articles by this author 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 microplate reader.39 The cytotoxicity was assessed using the absorbance ratio of the treated cells and the control cells. Bliss method (n = 5) was used to measure the IC50 values, which show the sensitivity of cells toward complexes and ligands. Cell cycle determination Commercially available 70 mm plates were used for cell culturing. The cells were treated with C1 for 24 h. The cells were collected after 24 h, then washed with PBS, and fixed in 70% ethanol for one night at –20 °C. After resuspension in RNAs and propidium iodide (PI), fluorescence-activated cell sorting (FACS) was used for analysis of the cells. Mod Fit LT (version 3.3; Variety Software House, Topsham, Maine, USA) software was used for cell cycle calculation. Apoptosis assessment The cells were cultivated for 24 h in a six-well plate. After treated with C1, the cells were collected, washed, and incubated with PI (5 µL) and annexin V (5 µL) for 25 min at 30 °C. The samples were protected from light during incubation with dyes. FACS was used for the analysis of cell apoptosis. Mitochondrial membrane potential analysis A lipophilic fluorescent (JC-1) dye was used for measurement of membrane potential of mitochondria. The cells were exposed to C1 at different concentrations for 24 h. After the exposure with C1, the cells were washed, collected, and incubated for 25 min with JC-1 dye. Flow cytometry was used for the detection of membrane potential of mitochondria. The damage to the mitochondrial membrane was shown by the orange fluorescence accumulated in the mitochondria, whereas the dye monomeric form produced green fluorescence, which accumulated in the cytosol at the event of mitochondrial membrane damage. Confocal microscopic studies of ROS generation in ER HepG2 cells were seeded on the commercially available coverslip of poly-1-lysine with the density of 2 × 106 in six-well plates with complete medium. Six hours after treatment with C1, the cells were washed and incubated for 20 min with 25 mM of 2′,7-dichlorodihydrouorescein diacetate (H2DCFDA) (488 nm/515 nm) at 37 °C. H2DCFDA was replaced with prewarmed ER-Tracker Red (587 nm/615 nm) and the cells were incubated for 20 min. The cells were washed with ultrapure water, and the coverslips were placed on the slides and immediately analyzed with confocal microscopy by using suitable objective lenses and Zeiss FLUOWIEW viewer (Carl Zeiss (Oberkochen, Germany)). Caspases determination The cells were treated with C1 for 24 h. Activated caspases were measured by using CaspGLOW kit after the treatment. The cells were then washed and incubated for 20 min with 0.5 mL of FITC-LEHD-FMK in the dark and immediately analyzed with FACS. The data were compared with the control. Determination of intracellular Ca2+ levels The cells were treated with C1 and cisplatin for 24 h. The intracellular level of Ca2+ was determined by using Fluo-3Am (fluorescent dye). Afterwards, the cells were washed and incubated for 25 min with (5 µM) Fluo-3Am in the dark and immediately analyzed with FACS at 525 nm wavelength of excitation. The dye was broken down into Am (Acetoxymethyl) and Fluo-3 by intracellular esterase when crossing the cell membrane. The Fluo-3 bound with cellular Ca2+ resulting in a strong fluorescence on excitation wavelength of 488 nm. Western blot analysis The total protein was collected by incubating the cells in lysis buffer, and the BCA (the bicinchoninic acid assay) Protein Assay kit was used for the determination of protein concentration. Equal amount (25 μg) of protein was loaded in the lanes of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (10%). The proteins were then transferred to poly(vinylidene difluoride) (PVDF) membrane for 1 h at 120 V after the electrophoresis were done. The membrane was placed in a solution containing the primary antibody at 4 °C for 8 h and incubated in nonfat milk dissolved in TBST (Tris-Buffered Saline, 0.1% Tween) for 2.5 h at pH 7.4. The membrane was then incubated for 2 h with a secondary antibody conjugated with horseradish peroxidase at room temperature. TBST was used for washing the membrane, and signals were obtained by using the enhanced chemiluminescent kit (Western Blotting Kit of Peirce ECL). In vivo anticancer activity Nude mice from Peking Union Medical College (Beijing, China) were used in this study. All the mice were housed at Guangxi Medical University facilitation center for experimental animals in a controlled environment of temperature, humidity, and 12 h light and dark cycle. Commercially available food was provided to the animals. The studies were performed per Guangxi Medical University’s Guide for the Caring and Use of Laboratory Animals. Tumor-bearing mice were divided into four groups containing six mice in each group. The cisplatin was dissolved in 0.9% saline. Similarly, a saline solution of 0.9% was used for vehicle control. When the diameter of the tumor reached 1.4 cm (day 0), the cisplatin or C1 was injected intraperitoneally. The cisplatin (2 mg/kg) and C1 (5 and 10 mg/kg) were administrated every 2 days. The volume of tumor was calculated in mm3 by using the given formula: volume of tumor = 0.5 × (longest diameter) × (shortest diameter)2. The growth curves of tumors were drawn as the number of days after the first treatment against the average tumor volume. The mice were sacrificed after 14 days of treatment. The tumor was collected and weighed, and formalin was applied for fixing in paraffin embedding. The given formula was used to calculate the tumor growth inhibition rate (IRT): IRT = 100% − (mean tumor weight of the control group − mean tumor weight of the experimental group)/mean tumor weight of the control group.40 Statistics SPSS (version 13.0, Armonk, New York, USA) was used for data processing, including the Student’s t test. p ≤ 0.05 was considered statistically significant. Results and discussion Synthesis and characterization of ligands and complexes Three ligands of isoquinoline derivatives L1, L2, and L3 were synthesized using the reported procedure. Ligand L1 and L3 differ only in the number and position of the methoxy group. L1 has two methoxy groups at position 6 and 7, whereas L3 has only one methoxy group at position 6. L2 is a dihydroisoquinoline, which has only one methoxy group at position 6.41 The ligands were characterized by NMR spectroscopy and IR spectroscopy. All the complexes, including C1, C2, C3, and C4 were prepared by mixing the ligand and corresponding metal salt in a thick Pyrex glass tube containing equal volume of dichloromethane and methanol as the solvent. The tube was sealed by fire torch. After 72 h of constant heating at 80 °C, block crystals were harvested. The complexes were characterized by elemental analysis, NMR, IR, and ESI-MS (Scheme 1). In addition, the stability of complexes C1– C4 was determined under physiological conditions of pH 7.4 in TBS and Tris–KCl–HCl buffer by UV–visible spectroscopy and high-performance liquid chromatography (HPLC). The results indicated that complexes C1– C4 are stable under physiological conditions for 48 h ( Supporting Information). Crystal structure of complexes The crystal structures of the four complexes were determined by single-crystal X-ray diffraction analysis (Figure 1). Selected bond angles and bond lengths are reported in Supporting Information Table S1. C1 and C3 showed distorted square planar geometry. In both complexes, two atoms of chlorine and the heterocyclic nitrogen of bidentate isoquinoline ligand L1 and L2 are coordinated with Pd(II). In C1, the bond lengths between palladium and nitrogen are Pd–N1 = 2.031 Å and Pd–N2 = 2.036 Å, whereas the bond lengths for palladium and chlorine are Pd–Cl1 = 2.282 Å and Pd–Cl2 = 2.305 Å. In C3, the bond lengths between palladium and nitrogen are Pd–N1 = 2.035 Å and Pd–N2 = 2.033 Å, whereas the bond lengths for palladium and chlorine are Pd–Cl1 = 2.993 Å and Pd–Cl2 = 2.984 Å. The C2 and C4 complexes have six-coordinated distorted octahedral geometry in which one bidentate L1 or L3, three chlorine atoms, and one S from DMSO are coordinated with Rh(III). The Rh–N2 (Rh1–N2 and Rh2–N2) of the bidentate ligands (L1 or L3) and Rh–S1 (Rh1–S1 and Rh2–S1) are located perpendicular to each other, and the three chlorine atoms and Rh–N1 (Rh1–N1 and Rh2–N1) are positioned to the basal part of the square. The corresponding bond lengths are as follows: Rh1–N2 = 2.13 Å, Rh2–N2 = 2.09 Å, Rh1–S1 = 2.27 Å, and Rh2–S1 = 2.25 Å. Scheme 1 | Synthetic routes for complexes C1–C4. Download figure Download PowerPoint In vitro cytotoxicity The cytotoxicity of C1– C4 was investigated by MTT assay against seven cancer cell lines, including T-24, SKOV-3, HepG2, MDA-MB216, MGC-803, A549, HeLa, and normal human cell line WI-38, with cisplatin as a positive control. The IC50 values obtained from the cell viability assay are listed in Table 1. When treated with L1–L3, no significant effect was observed on cell viability in any cell line, whereas significant impact was found in the case of complexes C1– C4 with IC50 values ranging from 1.05 to 22.04 µM. Complexes C1– C4 had higher cytotoxicity in the cancer cells than the corresponding ligands and metal salts. Among the four complexes, C1 exhibited the highest in vitro anticancer activity against the tested cell lines, which is higher than that of cisplatin. Notably, C1 showed low cytotoxicity toward human normal cell line WI-38. Compared with C3 and C4, complexes C1 and C2 exhibited lower IC50 values, which implies that the number and position of the methoxy group on the isoquinoline scaffold affect the cytotoxicity of the complexes. This result is consistent with the fact that C1 and C2 have higher lipophilicity (log Po/w values) than C3 and C4 ( Supporting Information). Table 1 | IC50 (µM) Values of L1–L3 and Complexes C1–C4 Against Seven Cancer Cell Lines and One Normal Cell Compounds WI-38 T-