In vitro and in vivo anticancer activity of novel Rh(III) and Pd(II) complexes with pyrazolopyrimidine derivatives.

{"full_text": " Bioorganic Chemistry 141 (2023) 106838\n\n\n Contents lists available at ScienceDirect\n\n\n Bioorganic Chemistry\n journal homepage: www.elsevier.com/locate/bioorg\n\n\n\n\nIn vitro and in vivo anticancer activity of novel Rh(III) and Pd(II) complexes\nwith pyrazolopyrimidine derivatives\nYun-Qiong Gu a, b, 1, Meng-Xue Ma a, 1, Qi-Yuan Yang a, b, Kun Yang a, Huan-Qing Li a, Mei-Qi Hu a,\nHong Liang a, *, Zhen-Feng Chen a, *\na\n State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources\n(Ministry of Education of China), Collaborative Innovation Center for Guangxi Ethnic Medicine, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal\nUniversity, Guilin 541004, China\nb\n School of Environment and Life Science, Nanning Normal University, Nanning 530001, China\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: Six pyrazolopyrimidine rhodium(III) or palladium(II) complexes, [Rh(L1)(H2O)Cl3] (1), [Rh(L2)(CH3OH)Cl3] (2),\nRh(III)/Pd(II) complexes [Rh(L3)(H2O)Cl3] (3), [Rh2(L4)Cl6]\u0387CH3OH (4), [Rh(L5)(CH3CN)Cl3]\u03870.5CH3CN (5), and [Pd(L5)Cl2] (6), were\nAnticancer activity synthesized and characterized. These complexes showed high cytotoxicity against six tested cancer cell lines.\nCell cycle arrest\n Most of the complexes showed higher cytotoxicity to T-24 cells in vitro than cisplatin. Mechanism studies indi\u00ad\nER-stress\nApoptosis\n cated that complexes 5 and 6 induced G2/M phase cell cycle arrest through DNA damage, and induced apoptosis\n via endoplasmic reticulum stress response. In addition, complex 5 also induced cell apoptosis via mitochondrial\n dysfunction. Complexes 5 and 6 showed low in vivo toxicity and high tumor growth inhibitory activity in mouse\n tumor models. The inhibitory effect of rhodium complex 5 on tumor growth in vivo was more pronounced than\n that of palladium complex 6.\n\n\n\n\n1. Introduction biological activities [17\u201319], such as anticancer [20], antibacterial [21]\n and antifungal activities [22]. The different modes of interactions be\u00ad\n Cancer has become one of the main causes of disease death, which is tween Rh(III) complexes and biomolecular targets may be related to\ncaused by the abnormal proliferation of cells that grow out of control in their unique molecular structures [23,24]. Various rhodium poly\u00ad\nthe body. It involves cross-talk among multiple genes and signaling pyridine complexes bind DNA via covalent and noncovalent in\u00ad\npathways, DNA alterations, gene transcription and translation [1,2] and teractions, to improve the uptake of the drug by cells and enhance\nit is an extremely complicated and highly lethal disease [3\u20135]. Metal- cytotoxicity by increasing the rigid plane and hydrophobicity of the\nbased drugs such as cisplatin and its analogues have been widely used polypyridine ligand [25]. Quercetin rhodium(III) complex exhibits\nto treat different types of cancer for more than 40 years [6], including potent anti-proliferative activity and a higher safety profile for normal\novarian, bladder, cervical, testicular, and lung cancers, as well as lym\u00ad cells, through triggering apoptosis and causing cell cycle arrest in the\nphoma, myeloma, and melanoma [7\u201311]. However, cisplatin has serious sub G0 phase [26]. Palladium(II) complexes efficiently bind to HSA\ntoxicity and often develops drug resistance [12]. Thus, overcoming these (human serum albumin) molecules, revealing a rational model for\ndisadvantages has become a top priority in the development of metal- anticancer drug design, transport, and toxicity reduction [27]. These\nbased antitumor drugs through the discovery of agents with stronger rhodium and palladium complexes have shown significant anticancer\nantitumor activity, less toxic side effects and lower tendency to induce activities with few side effects and they have the potential to be devel\u00ad\ndrug resistance [13]. oped as platinum drug substitutes.\n Among them, rhodium(III) and palladium(II) complexes have Our previous studies have found that Rh(III)/Pd(II) complexes with\nattracted much interest due to their unique chemical and pharmaco\u00ad isoquinoline derivatives induced HepG2 cell death by mitochondria-\nlogical properties [14\u201316]. The rhodium complexes exhibit a variety of mediated apoptotic and autophagic pathways [28]. Some rhodium\n\n\n\n * Corresponding authors.\n E-mail addresses: hliang@gxnu.edu.cn (H. Liang), chenzf@gxnu.edu.cn (Z.-F. Chen).\n 1\n Yun-Qiong Gu and Meng-Xue Ma contributed equally to this work.\n\nhttps://doi.org/10.1016/j.bioorg.2023.106838\nReceived 23 July 2023; Received in revised form 22 August 2023; Accepted 4 September 2023\nAvailable online 16 September 2023\n0045-2068/\u00a9 2023 Elsevier Inc. All rights reserved.\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\n(III)-picolinamide complexes inhibited cell proliferation through mul\u00ad coordinated square planar geometry. The crystallographic information\ntiple modes of action including cell cycle arrest, apoptosis autophagy, of complexes 1\u20136 is shown in Table S1.\nand metastasis suppression[24]. And the ruthenium(III) complexes of\npyrazopyrimidine derivative showed strong anti-proliferation activity\n 2.2. Stability, purity and log PO/W determination of the complexes\nagainst cancer cells [29]. These results suggested that more bioactive\nmetal complexes could be designed by coordination of bioactive ligands\n The stability of complexes 1\u20136 was measured by HPLC (Fig. S16) and\nwith bioactive metal center. Pyrazopyrimidine scaffolds are one of the\n UV \u2212 Vis spectroscopy (Fig. S17). The complexes showed no new peak\nprivileged heterocycles in drug discovery, which is the main structural\n within 48 h by HPLC, and the peak retention time hardly changed,\ncomponent of many lead molecules. Because pyrazopyrimidine is\n indicating that the complexes were stable for at least 48 h at room\nsimilar to the adenine base in DNA, its fingerprint as a pharmacophore\n temperature in solution. In addition, HPLC also showed that the purity\nhas always attracted attention [30,31]. However, there are few reports\n of the complexes was above 98%, which could be used for further cell\non the metal complexes with pyrazopyrimidine derivative and their\n experiments [32]. Meanwhile, the absorption peaks and shapes of the\nantitumor effects. Herein, we synthesized highly active and low toxic\n UV spectra of these two complexes were unchanged over 72 h, indi\u00ad\nrhodium/palladium metal complexes by coordinating pyrazolopyr\u00ad\n cating that complexes 1\u20136 were stable in PBS solution for at least 72 h,\nimidine derivatives with good antitumor activity with bioactive\n which is consistent with the HPLC results [33]. Furthermore, the com\u00ad\nrhodium/palladium metal centers, and their antitumor mechanisms\n plexes were dissolved in deionized water (1 mg complex/5mL H2O) and\nwere explored.\n LC-MS analysis was performed after 48 h. The mass spectra are shown in\n Fig. S18 \u2212 S22: For complex 1 (Fig. S18) C17H20Cl3N6ORh: HRMS\n2. Results and discussion\n (CH3OH, m/z): calcd for [M\u2212 H2O + Na]+: 536.9611, found, 536.9620;\n calcd for [M + Na]+: 554.0717, found, 554.9723. For complex 2\n2.1. Synthesis and characterization\n (Fig. S19) C26H38Cl3N6ORh: HRMS (CH3OH, m/z): calcd for\n [M\u2212 CH3OH + Na]+: 649.0863, found, 649.0876. For complex 3\n Complexes [Rh(L1)(H2O)Cl3] (1), [Rh(L2)(CH3OH)Cl3] (2), [Rh(L3)\n (Fig. S20) C25H36Cl3N6ORh: HRMS (CH3OH, m/z): calcd for [M\u2212 H2O +\n(H2O)Cl3] (3), [Rh2(L4)Cl6]\u0387CH3OH (4), [Rh(L5)(CH3CN)Cl3]\u03870.5CH3CN\n Na] +: 649.0843, found, 649.0868. For complex 4: The solubility of the\n(5), and [Pd(L5)Cl2] (6) were characterized by single crystal X-ray\n complex in water was too poor to be detected. For complex 5 (Fig. S21)\ndiffraction analysis (Fig. 1). Complexes 1, 2, 3, 5, and 6 had a mono\u00ad\n C29H25Cl3N7Rh: HRMS (CH3OH, m/z): calcd for [M + Na]+: 660.9924,\nnuclear structure with a 1:1 metal to ligand ratio. The central Rh(III)\n found, 660.9927. For complex 6 (Fig. S22) C27H22Cl2N6Pd: HRMS\ncoordinated with two N atoms of the pyridine and pyrazole rings of the\n (CH3OH, m/z): calcd for [M\u2212 Cl]+: 571.0629, found, 571.0626. These\nligands, three chloride ions, and an O or N atom of a solvent molecule\n results indicated that these complexes were stable in aqueous solution\n(H2O molecules for complexes 1 and 3, methanol for 2 and acetonitrile\n for at least 48 h.\nfor 5) to form a distorted octahedral geometry. However, complex 4 was\n Lipophilicity is a physicochemical property of a drug which affects its\na binuclear structure with a metal to ligand ratio of 2:1. The two\n uptake, metabolism and molecular toxicity [34]. To determine the lip\u00ad\nrhodium(III) centers formed a six-coordinated distorted octahedral ge\u00ad\n ophilicity of the complexes, log Po/W values (N-Octanol/water partition\nometry connected by two \u03bc2-Cl. One Rh(III) center had the same coor\u00ad\n constant) of complexes 1\u20136 were measured (Fig. S23). It was found that\ndination pattern as the other rhodium complexes, and the other Rh(III)\n the log Po/W values of complexes 1 and 4 were negative, \u2212 0.50 \u00b1 0.05\ncenter coordinated with the N atom of the pyrazole ring and a C atom of\n and \u2212 0.45 \u00b1 0.06, respectively, indicating that these two complexes are\nthe benzene ring as well as two free chloride ions. The central Pd(II) ion\n hydrophilic. The log Po/W values of the remaining complexes were\nof complex 6 was chelated with two N atoms of the pyridine and pyr\u00ad\n positive, and increased in the order of complex 5 > complex 2 > com\u00ad\nazole rings of the ligands, two chloride anions to forms a four-\n plex 6 > complex 3, indicating that they are lipophilic complexes, and\n\n\n\n\n Fig. 1. Crystal structures of complexes 1\u20136.\n\n 2\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\ncomplex 5 is the most lipophilic, with a log Po/W value of 0.50\u00b10.10 the mitochondria and nuclei of cancer cells, and likely causes damage to\n[35,36]. these two organelles.\n\n\n2.3. Cytotoxicity of the complexes 2.5. Effects of complexes 5 and 6 on gene transcription levels in T-24 cells\n\n The cytotoxic activities of two new ligands (L2 and L4), six complexes To investigate the cytotoxicity mechanism of complexes 5 and 6, we\n1\u20136 and cisplatin were investigated in vitro against five human tumor first investigated the transcription levels of genes involved in common\ncell lines (T-24, A549, BEL-7404, HeLa, SK-OV-3 cells) and two normal cell death pathways using high-sensitivity single-cell RNA sequencing\ncell lines (WI38 and HL-7702) by MTT assay (Table 1). The IC50 values (RNA-SEQ). After the complexes were incubated with T-24 cancer cells\nof the complexes against tested cell lines were <20 \u00b5M except for for 24 h, total RNA was extracted and the relevant gene transcript levels\ncomplexes 1 and 4, which may be due to their low solubility. Overall, were analyzed (Fig. 2). The results showed that complexes 5 and 6\ncomplex 5 was the most cytotoxic compound in all cancer cell lines affected the apoptosis and DNA pathway related genes. They activated\ntested, with IC50 values 2\u20136 times lower than that of cisplatin. All the expression of Bax, Bad, Bcl-2 and Caspase 2/3 genes and disrupted\ncomplexes showed the highest anti-proliferation activity against T-24 the expression of other apoptosis-related genes in T-24 cells (Fig. 2A). At\ncell lines, especially complex 5, whose IC50 value was 2.1\u00b10.1 \u00b5M. The the same time, the expression levels of DNA damage-related genes\nactivity of complex 6 with the same ligand as complex 5 against T-24 DRAM2, DDIT4, GADD45A/G, XPA/C, CCI2, DDB1 and MDC1 were also\nwas lower than that of complex 5, and its IC50 value was only 8.8\u00b10.3 changed (Fig. 2B). The results showed that complexes 5 and 6 may\n\u00b5M. For the other cell lines, the same trend was observed, and the induce apoptosis and DNA damage of T-24 cancer cells by regulating the\nselectivity index (SI = IC50 (HL-7702)/IC50 (T-24) of the complexes was expression of related genes, thereby inhibiting the proliferation of T-24\nhigher than that of cisplatin. The cytotoxic activity of rhodium com\u00ad cells.\nplexes with similar coordination pattern is correlated with their ligands\nfor T-24 cancer lines: (1) When R1 and R2 of the pyrazole ring are R1 =\n 2.6. The comet experiment, cell cycle arrest and the expression levels of\nR2 = \u2212 CH3 and the pyrazole ring R3= \u2212 (CH2)3CH3, the anti-\n cycle-related proteins\nproliferation activity of the corresponding complex 3 was significantly\nenhanced than that of complex 1 (R3 = \u2212 H). When the methyl groups of\n DNA has been demonstrated as the main molecular target of various\nR1 = R2 = \u2212 CH(CH3)2 and R3 = H, the anti-proliferation activity of the\n metal complexes [39,40]. Therefore, we evaluated the DNA damage by\ncorresponding complex was also significantly enhanced, e.g. complex 2\n complexes 5 and 6 first. After treatment with complexes 5 and 6 for 24 h,\n\uff1e complex 1, but complex 2\uff1ccomplex 3; (2) When R1 = \u2212 Ph, R2 =\n DNA fragments of T-24 cells were assessed by basic single-cell gel\n\u2212 CH3 and R3 = \u2212 H in complex 5, it was the most cytotoxic complex.\n electrophoresis (comet assay) (Fig. 3A and B). The results showed that\nTherefore, complexes 5 and 6 with different metal centers of the same\n treatment of T-24 cells with 5 and 6 increased the electrophoretic\nligand were selected to further study the mechanism of their anti-\n migration of DNA fragments. The single cell DNA showed the pattern of\nproliferation effects on T-24 cells.\n a comet, and the tail length of comet was longer than that of the control\n cell, showing a \u201cbroom\u201d pattern of tail. Moreover, the effect of complex\n2.4. Cellular uptake and distribution of the complexes 5 on DNA damage was concentration-dependent, indicating that com\u00ad\n plexes 5 and 6 induced DNA damage in cells. As a result, cell cycle arrest\n The cellular uptake of metal anticancer drugs is a crucial factor and apoptosis could be induced [41].\naffecting their anti-proliferation activity [37]. To investigate the kinetics The cell cycle is a highly ordered process in cell growth, tissue\nof cellular uptake of complexes 5 and 6, ICP-MS was used to measure regeneration, DNA repair, and apoptosis [42]. Among the cell cycle,\ntheir uptake in T-24 cells. The results showed that the concentration of interphase is mainly composed of three stages: G1, G2 and S [43,44]. At\nrhodium uptake of complex 5 was 859.72\u00b172.97 ng/106 cells after the concentration of 4.0 \u00b5M, both complexes 5 and 6 induced G2/M\ntreatment at 15 \u00b5M for 10 h. The uptake of palladium in complex 6 by phase arrest in T-24 cells, and the arrest effect of complex 5 was more\ncells was 296.84\u00b145.66 ng/106 cells (Fig. S24A and B). The uptake of obvious than that of complex 6. Moreover, the proportion of cells in G2/\ncomplexes by cells affects their anti-proliferative activity against tumor M phase increased from 12.34% (control) to 44.96% and 25.18%,\ncells, and previous studies have shown that the distribution of the respectively, after treatment with complexes 5 and 6 (Fig. 3C). After T-\ncomplexes in subcellular organelles is closely related to different cellular 24 cells were treated with complex 5 (2.0, 3.0 and 4.0 \u03bcM) for 24 h, the\npathways [38]. The distribution of complex 5 rhodium metal in the proportion of G2/M phase cells increased from 20.16% to 44.21% and\nsubcellular compartment was studied (Fig. S24C). Of all the subcellular the proportion of S and G1 phases cells decreased (Fig. 3D).\ncompartments measured, the uptake into cytoplasm was the highest, Phosphorylated cyclin p-CDC25Cs216 (60 KDa) is one of the DNA\nfollowed by mitochondria and nucleus, and cell membrane was the damage-mediated G2 phase checkpoints in cell cycle progression.\nlowest. These experiments indicated that complex 5 was accumulated in Cyclin-dependent protein kinases (CDKs) are a group of serine/\n\nTable 1\nThe cytotoxicity of ligands, metal salts and complexes 1\u20136 against five human tumor cell lines and two normal cell lines by MTT method (IC50a, 48 h).\n Compound T-24 A549 BEL-7404 HeLa SKOV-3 WI38 HL-7702 SI\n\n L4 >20 >20 >20 >20 >20 >20 >20 \u2212\n L5 >20 >20 >20 >20 >20 >20 >20 \u2212\n 1 >20 >20 >20 >20 >20 >20 >20 \u2212\n 2 8.2\u00b10.8 10.9\u00b10.4 15.5\u00b10.9 15.3\u00b10.8 18.0\u00b10.1 9.3\u00b11.6 13.0\u00b10.8 1.6\n 3 4.3\u00b10.5 5.6\u00b10.4 7.4\u00b10.2 8.5\u00b10.8 9.3\u00b10.1 5.1\u00b10.8 7.4\u00b11.0 1.7\n 4 >20 >20 >20 >20 >20 >20 >20 \u2212\n 5 2.1\u00b10.1 4.5\u00b10.4 5.7\u00b10.9 4.2\u00b10.4 5.8\u00b10.2 3.2\u00b10.3 2.5\u00b10.3 1.2\n 6 8. 8\u00b10.3 12.4\u00b10.5 14.4\u00b10.9 12.3\u00b10. 3 12.9\u00b10.8 14.0\u00b10.8 16.9\u00b10.3 1.9\n DDP 11.0\u00b10.5 11.4\u00b10.5 34.2\u00b10.7 16.6\u00b10.5 8.0\u00b11.0 7.4\u00b10.6 9.3\u00b10.50 0.8\n RhCl3\u03873H2O >40 >40 >40 >40 >40 >40 >40 \u2212\n PdCl2 >40 >40 >40 >40 >40 >40 >40 \u2212\n a\n IC50 values are expressed as the mean \u00b1 standard deviation of three independent experiments. SI (Selectivity indices) = IC50(HL-7702)/IC50(T-24).\n\n 3\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\n\n\n Fig. 2. Effects of complexes 5 and 6 on the transcription levels of genes associated with DNA damage (A), apoptosis (B) in T-24 cells.\n\n\nthreonine protein kinases that drive the cell cycle through their concentrations of complex 5 were applied to cells for 24 h (Fig. S25). The\nchemotactic effects on serine/threonine proteins. The cyclin-CDK com\u00ad results clearly confirmed that complexes 5 and 6 effectively induced cell\nplex, through CDK activity, catalyzes the phosphorylation of different apoptosis in a time-and concentration-dependent manner[48], and the\nsubstrates to achieve the advancement and transformation of different apoptosis rate was higher than that of cisplatin at the same\nphases of the cell cycle [45]. Our experimental results demonstrated that concentration.\nthe cyclin p-CDC25CS216 was significantly increased, and CDK2 and Bcl-2 family of proteins is the dominant regulator of the normal\nCyclin B1 were significantly decreased in the complexes-treated group apoptotic process, and is also a major player in tumorigenesis, pro\u00ad\ncompared with the control, indicating that the G2/M phase arrest of T- gression and resistance to subsequent drug therapy mediated by\n24 cells by complexes 5 and 6 may be achieved by regulating the apoptotic escape [47], which includes enhancing apoptosis protein (Bad\nexpression of CDK2, Cyclin B1 and p-CDC25C [46], which are in good and Bax) and decreasing antiapoptotic proteins (Bcl-2). To confirm the\nagreement with the flow cytometry results (Fig. 3E and F). apoptosis induced by complexes 5 and 6, the change of Bax and Bcl-2\n were assayed by Western blot. The experimental results showed that\n Bax protein expression was elevated and Bcl-2 protein expression was\n2.7. Cell apoptosis and the expression levels of apoptosis-related proteins decreased compared with the control (Fig. 4C and D). This result indi\u00ad\n cated that complexes 5 and 6 induced T-24 cells apoptosis (Fig. 4C and\n Cell cycle arrest also seriously affects tumor cell apoptosis [47], D).\ntherefore we further evaluated the apoptosis induced by complexes 5\nand 6 with Annexin V and PI staining. In Fig. 4, the apoptosis rates (Q2\n+ Q3) of cells treated with these two complexes for 24 h at the con\u00ad 2.8. Complex 5 induces mitochondrial dysfunction\ncentration of 4.0 \u00b5M were 28.05% and 13.69%, respectively (Fig. 4A).\nWhen treated with the complexes for 48 h, the apoptosis rates (Q2 + Q3) We investigated the effect of complex 5 on mitochondrial function.\nwere 71.1% and 24.54% (Fig. 4B), respectively, indicating that com\u00ad Mitochondrial membrane potential (MMP, \u0394\u03a8m) is a fundamental\nplexes 5 and 6 increased the percentage of apoptosis and obviously feature reflecting mitochondrial integrity [49]. Mitochondrial depolar\u00ad\ninduced apoptosis of T-24 cells. Moreover, the percentage of apoptosis ization induced by complex 5 was detected by JC-1 kit staining by flow\ninduced by complex 5 was concentration-dependent after different cytometry. After treatment with complex 5 (2.0, 3.0 and 4.0 \u00b5M) for 24\n\n 4\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\n\n\nFig. 3. (A) The comet assay was imaged by fluorescence microscopy with EB-staining after incubation with 5 and 6 for 24 h on T-24 cells (400\u00d7). (B) The tail length\nof the cells in each treated group was measured by the comet assay Effects on the cell cycle after treated with complexes 5 (4.0 \u03bcM), 6 (4.0 \u03bcM) (C) and 5 (2.0, 3.0 and\n4.0 \u03bcM) (D). (E) Effect of complexes 5 and 6 on cycle-related proteins in T-24 cells for 48 h. (F) Histograms display the density ratios of cell cycle related proteins\n(***P < 0.001, **P < 0.01, *P < 0.05).\n\n\n\n\n 5\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\n\n\nFig. 4. Effect of complexes 5 and 6 on cell apoptosis of T-24 cells for 24 h (A) or 48 h (B). (C) Effect of complexes 5 and 6 on apoptosis-related proteins in T-24 cells\nfor 48 h (D) Histograms display the density ratios of apoptosis elated proteins (***P < 0.001, **P < 0.01, *P < 0.05).\n\n\n\nh, mitochondrial membrane potential was decreased to 6.94%, 32.2% These findings are consistent with an increased rate of cell apoptosis. In\nand 33.9% compared with the control cells (Fig. 5A), which indicated conclusion, mitochondria dependent apoptosis is the cause of apoptosis\ncell mitochondrial membrane may be damaged by the complex [50]. induced by complex 5 [14]. Without sufficient ATP produced by mito\u00ad\n The production of intracellular reactive oxygen species (ROS) is a chondria, the rapid proliferation of cancer cells is hampered, which is\ncrucial indicator in the induction of apoptosis in multifarious cell lines also consistent with previous conclusions.\n[51]. Since the mitochondria are the main source of ROS, mitochondrial\nfunction damage often leads to increased ROS levels and calcium levels 2.9. Complexes 5 and 6 induce endoplasmic reticulum stress\n[52]. To investigate whether complex 5 would alter the intracellular\nROS levels, we first investigated the intracellular ROS levels treated with Many studies show that ROS production is an important component\ncomplex 5 by 2\u2032,7\u2032-dichlorodihydrofluorescein diacetate (H2DCF-DA) of ER stress responses and may act as an upstream signal [55]. The ef\u00ad\nstaining. After treatment with complex 5 for 10 h, a concentration fects of complexes on ER stress were examined by ER-Tracker dyes. The\ndependent enhancement of green fluorescence intensity was observed in green fluorescence of ER-Tracker was significantly enhanced as shown\ncomplex 5-treated T-24 cells, indicating an increase in intracellular ROS by confocal fluorescence photography after cells were incubated with\n(Fig. 5B). At the same time, we also determined the intracellular calcium complexes 5 and 6 (Fig. 6A), indicating that these two complexes\nand ATP levels, and found that calcium content increased in a induced ER stress in T-24 cancer cells [56].\nconcentration-dependent manner (Fig. 5C), while ATP level decreased ER is the major store of intracellular Ca2+ which is an important\nsignificantly (Fig. 5D). marker of cell signaling and survival [57]. When physiological changes\n A decrease in MMP indicates a shift in mitochondrial permeability. of ER occur, Ca2+ is released into the cytoplasm [58]. Several ER stress\nThis phenomenon leads to uncoupling of the respiratory chain, resulting pathways exist in mammalian cells [59]. Elevated expression levels of p-\nin cessation of ATP synthesis, ROS overproduction, and decreased ATP PERK (Phosphorylated RNA-dependent protein kinase-like endoplasmic\n[53]. ROS overproduction and ATP depletion in turn promote mito\u00ad reticulum kinase), p-eIF2\u03b1 (phosphorylated eukaryotic promoter 2\u03b1),\nchondrial dysfunction, leading to rupture of mitochondrial outer mem\u00ad and CHOP (nuclear transcription factor C/EBP homologous protein)\nbrane and release of apoptotic factors [50]. We found cascade changes in indicated ER stress [60]. To verify whether 5 and 6 could induce ER\nmitochondria following complex 5 treatment, such as decreased MMP, stress, the expression levels of endoplasmic reticulum stress signature\nincreased ROS and Ca2+ accumulation, and decreased ATP levels [54]. proteins p-PERK, P-eIF2\u03b1 and CHOP in 5 and 6 treated cells were\n\n 6\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\n\n\nFig. 5. (A) The influence of complex 5 on mitochondrial membrane potential in T-24 cells (24 h). (B) The ROS determination of cells after incubation of complex 5\n(10 h). (C) The measurement of Ca2+ release in the cells upon treatment with complex 5 (24 h). (D) The detection of the intracellular ATP level after incubation of\ncomplex 5 (24 h) (***P < 0.001, **P < 0.01, *P < 0.05).\n\n\ndetected by Western blot. Complexes 5 and 6 were found to significantly 5 (DR5) promoter and is activated by caspase-8 to induce the death\nincrease the expression of p-PERK, P-eIF2\u03b1 and CHOP proteins (Fig. 6B receptor apoptosis pathway [63,64].\nand C), indicating an increased level of ER stress [61]. To explore the complex 5-induced apoptosis pathway, we first\n detected the activation of caspase-3/8/9 by complex 5 in T-24 cells.\n Complex 5-treated cells showed additional peaks in activated caspase-3/\n2.10. Complexes 5 and 6 activate caspase 3/8/9\n 8/9 cells compared with untreated cells (Fig. 7). After treatment with\n complex 5, the proportion of activated caspase-3/8/9 cells in total T-24\n In general, there are two different pathways involved in the\n cells was 36.70%, 23.00% and 32.37%, respectively, which showed\napoptosis of cancer cells, the intrinsic pathway (mitochondria-related\n obvious concentration dependence. These results suggested that com\u00ad\napoptosis pathway) and the extrinsic pathway [50]. The intrinsic\n plex 5 activated caspase-3/8/9 in T-24 cells, thereby inducing apoptosis\npathway is typically dependent on the activation of the initiator caspase-\n mediated by mitochondrial dysfunction [65].\n9 and caspase-3 [62]. Moreover, CHOP interacts with the death receptor\n\n 7\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\n\n\nFig. 6. (A) Confocal micrographs of cells after treatment with ER-Tracker staining (green) and Hoechst 33,258 staining (blue). (B) Effect of complexes 5 and 6 on ER-\nstress-related protein against T-24 cells for 48 h. (C) Histograms display the density ratios of ER-stress-related proteins (***P < 0.001, **P < 0.01, *P < 0.05). (For\ninterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)\n\n\n\n\n Fig. 7. The activation of caspase-3/8/9 (A, B and C) in T-24 cells after incubation with complex 5 (24 h).\n\n\n\n 8\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\n2.11. In vivo anticancer activity in xenograft tumor model obvious. However, the expression level of Bax in the two complex groups\n was increased, indicating that the ratio of Bcl/Bax was significantly\n Acute toxicity test showed that the safe dose of complexes 5 and 6 decreased after the treatment of cells with complexes 5 and 6. These\nwas 30 mg/kg after a single caudal vein injection, and both groups of results indicated that the complexes also induced apoptosis in vivo,\nmice survived. which is consistent with the TUNEL staining results.\n The in vivo anticancer activity of complexes 5 and 6 was studied Histological examination of tumor tissues showed that the tumor\nusing T-24 cell xenograft mouse model. When the tumor volume was cells in complexes 5 and 6 treated groups were closely arranged, round\nsuitable (about 90 mm3), the mice were divided into 4 groups randomly or polymorphic, with abundant cytoplasm, megakaryocytes, large and\n(n = 6) and were treated with vehicle, complex 5 (30 mg/kg), complex 6 hyperchromatic nuclei. Compared with the control, there were more\n(30 mg/kg) and DDP (2 mg/kg), respectively. Intraperitoneal injections pyknotic tumor cells and fewer blood vessels in the tumor tissues of the\nare given every 2 days. Tumor size was measured every 2 days after complexes 5 and 6 treatment groups, indicating that tumor necrosis\ntreatment (Fig. 8A). At the end of treatment on the 15th day, the tumor increased in the complexes 5 and 6 treatment groups. At the same time,\nweight of each group was used to calculate the tumor suppression rate the kidney, lung, spleen, liver and heart tissues of complexes 5 and 6\n(Fig. 8B). Compared with the vehicle treatment group, the tumor growth treated groups were similar to those of the vehicle group, and no obvious\nof the complexes-treated groups was significantly inhibited, and the lesions were observed, indicating no obvious toxicity under the current\ntumor volumes of each complexes-treated groups were significantly treatment mode (Fig. 9D). In conclusion, complexes 5 and 6 exhibited\nreduced (Fig. 8C). The inhibition rates of complexes 5 and 6 on tumor strong inhibitory activity against proliferation in vivo.\ngrowth were 50.8% and 47.6%, respectively, slightly lower than that of\ncisplatin (59%). At the same time, the body weight of mice treated with 3. Conclusion\nthese two complexes did not decrease significantly, and the decreasing\ntrend was smaller than that of cisplatin group, indicating that their Six rhodium(III) or palladium(II) complexes with pyrazolopyr\u00ad\ntoxicity was low (Fig. 8D). imidines derivatives as ligands have been synthesized and characterized.\n Complexes 5 and 6 exhibited strong anticancer activity against several\n2.12. Staining of tissues with TUNEL and H&E cancer cell lines and low toxicity against normal cells. Among the five\n rhodium complexes, complex 5 had the strongest activity when the\n Apoptosis of tumor tissues treated with complexes 5 and 6 was pyrazole ring of the ligand contained phenyl group at the R1 position,\nevaluated by TUNEL staining (Fig. 9A), which suggested that the \u2212 CH3 at the R2 position, and hydrogen atom at the R3 position. Complex\napoptosis of tumor cells in complex 5 and 6 groups was significantly 5 accumulated in mitochondria and nuclei and initiated a series of\nincreased comparing with the control, and complex 5 showed stronger events associated with mitochondrial dysfunction and DNA damage,\neffect than complex 6. Nuclear contraction was not obvious in any such as loss of MMP, ATP depletion, ROS release and elevated calcium\ngroups after DAPI staining. levels, and cell cycle arrest, to induce ER stress response and subse\u00ad\n Changes of Bax and Bcl-2 in tumor tissues of T-24 tumor-bearing quently apoptosis. Complexes 5 and 6 showed effective anticancer ac\u00ad\nmice were detected by Western blot (Fig. 9B and C). The results tivity in a mouse tumor model. These compounds are potential\nshowed that in the T-24 xenograft tumor mouse model treated with promising anticancer metal complexes.\ncomplexes 5 and 6, the expression level of Bcl-2 in group complex 5 was\nobviously decreased, while the change in group complex 6 was not\n\n\n\n\nFig. 8. Complexes 5 and 6 suppressed T-24 tumor growth in vivo. (A) Photographs of tumors separated from the mice. (B) Tumor weight was monitored. (C) Volume\nof tumors affected by 5 and 6 on T-24 tumor growth. (D) The body weight of the mice (n = 6) (***P < 0.001, **P < 0.01, *P < 0.05).\n\n 9\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\n\n\nFig. 9. (A) TUNEL staining of the tumor tissue treated with complexes 5 and 6. (B) and (C) The change of apoptosis-related proteins after incubation of complexes 5\nand 6 in tumor tissue. H&E staining of tumor tissue (D) and the visceral organ (E) of mice after complexes 5 and 6 treatment (200\u00d7) (***P < 0.001, **P < 0.01, *P\n< 0.05).\n\n\n4. Experimental sections yrido[2\u2032,3\u2032:3,4] pyrazolo[1,5-a]pyrimidine).\n 1\n H NMR (400 MHz, Chloroform-d) \u03b4 7.67 (d, 1H), 6.98 (s, 1H), 6.13\n4.1. Synthesis of ligands (s, 1H), 4.48 (p, 1H), 4.19 (p, 1H), 3.22 (p, 1H), 3.05 (s, 1H), 3.01 (d,\n 3H), 1.50 (d, 6H), 1.42 (d, 6H), 1.33 (d, 6H), 1.26 (d, 6H). 13C NMR\n Pyrazopyrimidine derivatives L1\u00a15 and the complexes 1\u20136 were (151 MHz, Chloroform-d) \u03b4 165.60, 160.13, 159.42, 156.15, 154.48,\nprepared using the previously reported method [24,66]. The structures 153.62, 147.37, 143.87, 111.11, 105.80, 103.97, 102.32, 77.24, 77.03,\nof L1\u20135 and complexes 1\u20136 are shown in Scheme 1 and Scheme 2. 76.82, 36.41, 28.67, 28.13, 26.22, 22.77, 22.63, 22.11, 20.19, 19.00\n L2 (8-(3,5-diisopropyl-1H-pyrazol-1-yl)-2,4-diisopropyl-10-methylp (Fig. S1). HRMS (ESI) calcd for C25H34N6 [M + H]+ (m/z): 419.2923;\n\n\n\n\n Scheme 1. The structures of ligands L1\u00a15.\n\n 10\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\n\n\n Scheme 2. The structures of complexes 1\u20136.\n\n\n\nFound, 419.2918 (Fig. S2). Anal. Calcd for C25H34N6: C, 71.74; H, 8.19; H, 5.68; N, 12.61.\nN, 20.08. Found C, 71.59; H, 8.37; N, 19.84. [Rh(L3)(H2O)Cl3] (3): Yellow block crystal. Yield: 51%. 1H NMR\n L4 (8-(3,5-bis(4-methoxyphenyl)-1H-pyrazol-1-yl)-2,4-bis(4-metho (600 MHz, DMSO\u2011d6) \u03b4 7.63\u20137.62 (m, 1H), 3.12\u20133.10 (m, 3H), 2.96 (s,\nxyphenyl)-10-methylpyrido[2\u2032,3\u2032:3,4]pyrazolo[1,5-a]pyrimidine). 3H), 2.92\u20132.89 (m, 2H), 2.88 (s, 3H), 2.79 (s, 3H), 2.76 (s, 3H), 2.56 (t, J\n 1\n H NMR (600 MHz, DMSO\u2011d6) \u03b4 8.45 (d, 2H), 8.31 (d, 2H), 8.20 (s, = 7.6 Hz, 2H), 1.56 (dq, J = 5.9, 3.2, 2.7 Hz, 2H), 1.50\u20131.44 (m, 4H),\n1H), 7.91 (d, 2H), 7.54 (s, 1H), 7.29 (d, 2H), 7.17 (t, 4H), 7.10 (s, 1H), 1.40 \u2013 1.34 (m, 2H), 0.97 (t, J = 7.3 Hz, 3H), 0.94 (t, J = 7.3 Hz, 3H). 13C\n7.04 (d, 2H), 6.89 (d, 2H), 3.88 (s, 6H), 3.81 (s, 3H), 3.72 (s, 3H), 3.11 (s, NMR (151 MHz, DMSO\u2011d6) \u03b4 159.32, 158.86, 155.37, 152.78, 152.04,\n3H). (Fig. S3) HRMS (ESI) calcd for C41H34N6O4 [M + H]+ (m/z): 144.11, 142.30, 141.31, 125.99, 124.66, 105.42, 105.34, 40.06, 39.94,\n675.2720; Found, 675.2685 (Fig. S4). Anal. Calcd for C41H34N6O4: C, 39.80, 39.66, 39.52, 39.38, 39.24, 39.10, 31.72, 31.03, 27.71, 23.53,\n72.98; H, 5.08; N, 12.46. Found C, 72.75; H, 5.36; N, 12.30. 22.24, 22.09, 21.78, 18.73, 15.04, 13.85, 13.76, 13.31, 12.99 (Fig. S9).\n HRMS (ESI) calcd for C25H36Cl3N6ORh, [M + DMSO-Cl]+ (m/z):\n4.2. Synthesis and characterization of complexes 1\u20136 687.1522, Found, 687.1472\uff1b[M + CH3CN + DMSO-H2O-Cl]+ (m/z):\n 710.1682, Found, 710.1138 (Fig. S10). Anal. Calcd for\n Synthesis of rhodium complexes 1\u20136: Ligands L1\u00a15 (0.1 mmol, 1 C25H36Cl3N6ORh: C, 46.49; H, 5.62; N, 13.01. Found C, 46.68; H, 5.40;\nequiv.) and RhCl3\u22c53H2O] (0.12 mmol, 1.2 equiv.) were added to CHCl3: N, 13.27.\nCH3OH or CHCl3:CH3CN (complex 5) (v:v = 2:1, 9.0 mL) for reflux for [Rh2(L4)Cl6]\u0387\u0387CH3OH (4): Yellow block crystal. Yield: 75%. HRMS\n10 h. After filtration and standing for 5 d, red brown block crystals (ESI) calcd for C42H37Cl6N6O5Rh2, [M + CH3CN + CH3OH + H]+ (m/z):\nsuitable for single crystal X-ray diffraction analysis were collected. 1165.9459, Found, 1165.8799\uff1b[M + CH3CN + CH3OH-Cl]+ (m/z):\nComplex 6 was obtained by replacing RhCl3\u22c53H2O with PdCl2 and 1129.9692, Found, 1129.9768 (Fig. S11). Anal. Calcd for\nreacted with ligand L5 under the same reaction conditions. C42H37Cl6N6O5Rh2: C, 44.87; H, 3.32; N, 7.48. Found C, 44.73; H, 3.48;\n [Rh(L1)(H2O)Cl3] (1): Yellow block crystal. Yield: 62%. 1H NMR N, 7.29.\n(600 MHz, DMSO\u2011d6) \u03b4 7.64 \u2013 7.64 (m, 1H), 7.62 (s, 1H), 6.68 (s, 1H), [Rh(L5)(CH3CN)Cl3]\u0387\u03870.5CH3CN (5): Yellow block crystal. Yield:\n3.17 (s, 1H), 3.12 (s, 3H), 2.96 (s, 3H), 2.93 (s, 3H), 2.78 (s, 3H), 2.75 (s, 83%. 1H NMR (400 MHz, DMSO\u2011d6) \u03b4 8.17\u20138.12 (m, 2H), 7.76\u20137.69 (m,\n3H). 13C NMR (151 MHz, DMSO\u2011d6) \u03b4 160.09, 159.61, 155.49, 152.94, 8H), 7.64 (d, J = 1.8 Hz, 1H), 7.02 (s, 1H), 6.46 (s, 1H), 3.17 (s, 3H),\n152.69, 146.37, 145.84, 143.34, 114.80, 114.78, 105.48, 105.28, 48.63, 2.79 (s, 3H), 2.77 (d, J = 1.8 Hz, 3H), 2.75 (s, 3H). 13C NMR (101 MHz,\n40.06, 39.94, 39.92, 39.80, 39.66, 39.52, 39.38, 39.29, 39.24, 39.10, DMSO\u2011d6) \u03b4 160.66, 160.44, 155.80, 153.10, 151.51, 148.09, 146.07,\n24.42, 18.84, 18.09, 15.17, 14.76 (Fig. S5). HRMS (ESI) calcd for 144.44, 131.39, 131.08, 130.43, 130.02, 129.74, 129.35, 128.99,\nC17H20Cl3N6ORh [M\u2212 Cl\u2212 H2O]+ (m/z): 497.0131; Found, 497.0562 128.90, 128.53, 115.58, 114.80, 106.86, 105.24, 48.61, 24.38, 18.94,\n(Fig. S6). Anal. Calcd for C17H20Cl3N6ORh: C, 38.26; H, 3.78; N, 15.75. 14.38 (Fig. S12). HRMS (ESI) calcd for C30H26.5Cl3N7.5Rh,\nFound C, 38.09; H, 3.66; N, 15.93. [M\u2212 1.5CH3CN + H]+ (m/z): 641.0075, Found, 641.1432\uff1b[M + DMSO\n [Rh(L2)(CH3OH)Cl3] (2): Yellow block crystal. Yield: 59%. 1H NMR + H2O-0.5CH3CN-Cl]+ (m/z): 740.0849, Found, 740.0044 (Fig. S13).\n(600 MHz, DMSO\u2011d6) \u03b4 7.65\u20137.64 (m, 1H), 7.62 (s, 1H), 6.88 (s, 1H), Anal. Calcd for C30H26.5Cl3N7.5Rh: C, 51.38; H, 3.81; N, 14.98. Found C,\n4.39\u20134.36 (m, 1H), 3.97 (dt, J = 33.0, 6.7 Hz, 3H), 3.17\u20133.14 (m, 3H), 51.23; H, 3.69; N, 15.16.\n2.08 (s, 3H), 1.53 (d, J = 6.9 Hz, 6H), 1.41 (d, J = 3.0 Hz, 6H), 1.40 (d, J [Pd(L5)Cl2] (6): Yellow block crystal. Yield: 78%. 1H NMR (600\n= 3.3 Hz, 6H), 1.29 (d, J = 6.9 Hz, 6H). 13C NMR (151 MHz, DMSO\u2011d6) \u03b4 MHz, DMSO\u2011d6) \u03b4 8.12\u20138.09 (m, 2H), 7.67 (s, 1H), 7.62\u20137.58 (m, 3H),\n170.01, 168.53, 156.95, 155.56, 155.03, 153.05, 152.84, 143.39, 7.49 (s, 1H), 7.28\u20137.24 (m, 5H), 6.53 (s, 1H), 3.00 (s, 3H), 2.76 (s, 8H),\n118.14, 108.98, 108.06, 106.02, 105.58, 40.06, 39.94, 39.80, 39.66, 2.32 (s, 3H) (Fig. S14). HRMS (ESI) calcd for C27H22Cl2N6Pd, [M\u2212 Cl]+\n39.52, 39.38, 39.24, 39.10, 35.68, 28.78, 26.93, 26.20, 22.62, 22.61, (m/z): 571.0629; Found, 571.0613; M\u2212 Cl + DMSO]+ (m/z): 651.0773;\n21.94, 21.75, 21.73, 19.86, 18.69 (Fig. S7). HRMS (ESI) calcd for Found, 651.0740 (Fig. S15). Anal. Calcd for C27H22Cl2N6Pd: C, 53.35; H,\nC26H38Cl3N6ORh, [M + DMSO + H]+ (m/z): 737.1445, Found, 3.65; N, 13.83. Found C, 53.09; H, 3.86; N, 13.65.\n737.0673\uff1b[M + H]+ (m/z): 659.1306, Found, 659.1990 (Fig. S8). Anal.\nCalcd for C26H38Cl3N6ORh: C, 47.32; H, 5.80; N, 12.74. Found C, 47.06;\n\n 11\n\fY.-Q. Gu et al. Bioorganic Chemistry 141 (2023) 106838\n\n\n4.3. Cellular uptake of complexes 4.9. Statistical studies\n\n The cells were incubated in 100 mm cell culture dishes for overnight, All experiments were repeated three times and the data were\nand then treated with complexes 5 and 6 (10 \u00b5M) for 24 h. After the cells expressed as mean \u00b1 standard deviation (SD).\nwere collected, washed, harvested, and digested with HNO3, the metal\ncontent in the diluent was detected by ICP-MS. The cytoplasm, mito\u00ad\nchondria, and cell membrane were isolated, digested with concentrated Declaration of Competing Interest\nHNO3, and their metal contents were determined by ICP-MS.\n The authors declare that they have no known competing financial\n interests or personal relationships that could have appeared to influence\n4.4. Detection of cell cycle the work reported in this paper.\n\n T-24 cells (1\u00d7106 cells/dish) in 70 mm dish were treated with Data availability\ncomplexes 5 (2.0, 3.0, 4.0 \u00b5M) and 6 (4.0 \u00b5M) for 24 h. The cells were\ncollected and washed with PBS. Ice-cold ethanol and PBS were added Data will be made available on request.\ndrop by drop while shaking, and then fixed in 70% ethanol in \u2212 20 \u25e6 C\nrefrigerator for 24 h. The cells were centrifuged, washed with PBS, and\n Acknowledgments\nincubated with dye in 5% CO2 humidified environment at 37 \u25e6 C for 30\nmin, and then tested by flow cytometry.\n This work was supported by the National Natural Science Foundation\n of China (Grant: 22077022) and Natural Science Foundation of Guangxi\n4.5. Assessment of apoptosis Province of China (Grants: GUIKEZY22096015,\n 2023GXNSFDA026054).\n T-24 cells (2\u00d7105 cells/well) were cultured in 6-well plates over\u00ad\nnight. After treatment with complexes 5 (2.0, 3.0, 4.0 \u00b5M) and 6 (4.0 Appendix A. Supplementary data\n\u00b5M) for 24 h and 48 h, respectively, the cells were harvested, washed\nand incubated with PI (5.0 \u03bcL) and annexin V (5.0 \u03bcL) in a 5% CO2 Supplementary data to this article can be found online at https://doi.\nhumidified environment for 30 min. Cell apoptosis was determined by org/10.1016/j.bioorg.2023.106838.\nflow cytometry.\n References\n4.6. Detection of intracellular ATP level\n [1] F. Bray, M. Laversanne, E. Weiderpass, I. Soerjomataram, The ever-increasing\n importance of cancer as a leading cause of premature death worldwide, Cancer 127\n After T-24 tumor cells were treated with complex 5 for 24 h, the cells (2021) 3029\u20133030.\nwere fully lysed and centrifuged at 12,000 g for 5 min. Subsequently, [2] T. Yan, X. Zheng, S. Liu, Y. Zou, J. 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