Synthesis, Characterization, and Anticancer Activity of Rhodium (III) Complexes with Bidentate and Tridentate N‐Donor Ligands

{"full_text": " Research Article\nChemistrySelect doi.org/10.1002/slct.202503583\n\n\n www.chemistryselect.org\n\n\n Synthesis, Characterization, and Anticancer Activity of\n Rhodium (III) Complexes with Bidentate and Tridentate\n N-Donor Ligands\n Haoran Liang,*[a] Huan Hong,[a] Gang Wang,[b] Li Gao,[a] Shui Wang,[a] Yan Shen,*[a]\n and Yuanqiang Wang*[a]\n\n A series of five novel rhodium(III) complexes of the type platin. Molecular docking studies indicated that complexes 3\n [Rh(tpy)(N\u02c6N)Cl](PF6 )2 , incorporating a tridentate 2,2\u0002 :6\u0002 ,2\u0002\u0002 - and 5 effectively insert into the minor groove of calf thymus\n terpyridine (tpy) ligand and various bidentate N-donor ligands, DNA (ctDNA), supported by favorable steric and electrostatic\n were synthesized and fully characterized. The single-crystal X- interactions. Mechanistic studies further confirmed that com-\n ray diffraction analysis of complex 5 [Rh(tpy)(dppz)Cl](PF6 )2 plex 5 induces apoptosis, causes S-phase cell cycle arrest, and\n (dppz = dipyrido[3,2-a:2\u0002 ,3\u0002 -c]phenazine) confirmed its octahe- elevates intracellular ROS levels in AGS cells. Collectively, these\n dral geometry. Cytotoxicity assays revealed potent antiprolifer- results highlight the potential of terpyridine-based Rh(III) com-\n ative activity against four human cancer cell lines (AGS, MCF-7, plexes as promising scaffolds for metal-based anticancer drug\n HeLa, and HepG2). Notably, complex 5 demonstrated enhanced development.\n selectivity and cytotoxicity toward AGS cells compared to cis-\n\n\n\n 1. Introduction with alternative metals, while also exploring a variety of lig-\n and architectures.[5\u20138] For example, palladium, a member of the\n Cancer remains a major global health challenge, imposing platinum-group metals, has emerged as a potential candidate for\n a significant burden on individuals and healthcare systems anticancer drug development, due to its structural and mecha-\n worldwide.[1] Chemotherapy continues to be a cornerstone of nistic similarities to platinum-based compounds.[9,10] Ruthenium\n cancer treatment, with platinum-based drugs such as cisplatin and iridium have been investigated for their potential in photo-\n playing a pivotal role in the management of solid tumors, dynamic therapy, owing to their unique photophysical and redox\n and lobaplatin being crucial in the treatment of certain hema- properties.[11\u201315]\n tologic malignancies.[2] These platinum compounds primarily Rhodium is another member of the platinum-group metals\n exert their therapeutic effects by binding to DNA, resulting in that has traditionally been employed in catalytic processes.[16]\n structural alterations that disrupt cellular integrity and induce In recent years, it has attracted growing attention due to\n apoptotic cell death.[3] However, despite their clinical success, its versatile coordination chemistry and potential biological\n platinum-based chemotherapeutics are associated with signif- activities.[17\u201320] The pharmacological properties of rhodium com-\n icant drawbacks, most notably their high toxicity to normal plexes are heavily influenced by their ligand design, with biden-\n cells.[4] This nonselective cytotoxicity leads to a wide range of tate ligands, such as N\u02c6N,[21\u201324] N\u02c6C,[25\u201327] N\u02c6O[28] or N\u02c6S,[29] as well\n adverse side effects, which can severely affect patients\u2019 quality as N-heterocyclic carbene (NHC) ligands,[30\u201333] being particularly\n of life during treatment. As a result, there has been increas- prevalent in medicinal rhodium chemistry. In our previous stud-\n ing interest in developing alternative therapeutic strategies for ies, we developed a series of cyclometalated octahedral Rh(III)\n cancer treatment. One promising approach is the development complexes that demonstrated DNA-binding properties.[34] More\n of novel metal-based anticancer agents by replacing platinum recently, we have designed novel square-planar Rh(I) complexes\n incorporating tridentate ligands, which exhibit a particularly\n notable impact on breast adenocarcinoma.[35] Tridentate ligand\n tpy is a well-established scaffold in medicinal inorganic chem-\n [a] H. Liang, H. Hong, L. Gao, S. Wang, Y. Shen, Prof. Y. Wang\n Chongqing Key Lab of Medicinal Chemistry & Molecular Pharmacology, istry, known for its diverse biological activities and attractive\n School of Pharmacy and Bioengineering, Chongqing University of features in metal-N\u02c6N\u02c6N complexes.[36] However, terpyridine-\n Technology, Chongqing 400054, P. R. China functionalized rhodium complexes remain largely unexplored in\n E-mail: hrliang@cqut.edu.cn the context of anticancer drug development.[37] In the present\n shenbmy@cqut.edu.cn\n study, we aim to design and synthesize a series of octahedral\n wangyqnn@cqut.edu.cn\n Rh(III) complexes, utilizing terpyridine as a N\u02c6N\u02c6N tridentate lig-\n [b] G. Wang\n School of Life Sciences, Nanjing University, Nanjing 210023, P. R. China\n and in combination with various N\u02c6N-bidentate auxiliary ligands.\n This work will encompass the synthesis, characterization, and\n Supporting information for this article is available on the WWW under\n https://doi.org/10.1002/slct.202503583\n\n\n\n ChemistrySelect 2025, 10, e03583 (1 of 7) \u00a9 2025 Wiley-VCH GmbH\n\f 23656549, 2025, 27, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/slct.202503583 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistrySelect doi.org/10.1002/slct.202503583\n\n\n\n\n Scheme 1. Structures of the five complexes in this work.\n\n\n\n evaluation of the anticancer efficacy of these complexes against\n a panel of human cancer cell lines.\n\n\n\n 2. Results and Discussion\n Figure 1. X-ray crystal structure for complex 5.\n\n 2.1. Synthesis and Characterization\n diffusion of diethyl ether into the acetonitrile solution at ambient\n A series of five rhodium(III) complexes 1\u20135 with the gen- temperature. The crystal structure (Figure 1, Table S1) unequiv-\n eral formula [Rh(tpy)(N\u02c6N)Cl](PF6 )2 were synthesized by reflux- ocally confirms the octahedral coordination geometry around\n ing [Rh(tpy)Cl3 ] with the respective N\u02c6N bidentate ligands in the Rh(III) center, with the tpy adopting its characteristic merid-\n H2 O/EtOH, followed by addition of excess NH4 PF6 to precipitate ional tridentate binding mode (N\u02c6N\u02c6N), while the dppz ligand\n the products (Scheme 1). After purification by recrystallization chelates through its N\u02c6N donor atoms. The chloride ligand com-\n from acetonitrile/diethyl ether, complexes were obtained as air- pletes the coordination sphere, occupying the sixth position.\n stable, crystalline solids in moderate yields ranging from 64% Structural analysis reveals a near-ideal octahedral geometry, evi-\n to 78%. All complexes demonstrated good solubility in polar denced by cis-angles ranging from 80.50(15)\u00b0 to 99.84(15)\u00b0 and\n organic solvents, including acetonitrile, acetone, and DMSO, but trans-angles between 161.57(16)\u00b0 and 179.22(16)\u00b0, with the Rh-N\n were insoluble in nonpolar solvents such as petroleum ether and bond lengths showing remarkable consistency [1.959(4)-2.041(4)\n diethyl ether. This solubility profile is consistent with their ionic \u00c5 (Rh-tpy), 2.046(4)-2.047(4) \u00c5 (Rh-dppz)] that are significantly\n character and suggests potential compatibility with biological shorter than the Rh\u2500Cl bond length of 2.3211(12) \u00c5, reflecting the\n testing conditions. different trans influences of the coordinated ligands. The tpy lig-\n The molecular structures of complexes 1\u20135 were fully char- and imposes considerable geometric constraint, manifested by\n acterized using HR-MS, NMR, IR, and CHN elemental analysis. nearly identical N\u2500Rh\u2500N bite angles of 80.98(17)\u00b0 and 80.62(16)\u00b0,\n Complete spectroscopic data for all complexes are available while the overall structure displays pronounced rigidity due to\n in the Supporting Information (Figures S1\u2013S5). All complexes the planar aromatic systems of both tpy and dppz ligands. The\n showed intense peaks corresponding to the [M + H]+ cation, relatively short Rh\u2500N bonds, particularly to the central pyri-\n with experimental m/z values matching theoretical calculations dine of the tpy ligand (1.959(4) \u00c5), suggest strong metal-ligand\n (Figures S6\u2013S10). Key vibrational bands in the IR spectra of the interactions that may contribute to the complex\u2019s stability. The\n complexes were observed at 1602\u20131606 cm\u22121 (C\u2550N stretch) and crystallographic data have been deposited with the Cambridge\n 830\u2013835 cm\u22121 (PF6 \u2212 counterion), confirming the proposed struc- Crystallographic Data Centre (CCDC no. 2442291).\n tures (Figure S11). The identity and purity of each complex were\n further confirmed by satisfactory elemental analysis.\n To evaluate the stability of the complexes, a time-dependent 2.2. Antitumor Efficacy of Complexes 1\u20135\n 1\n H NMR spectroscopic stability study was conducted in a mix-\n ture of 85% DMSO-d6 / 15% phosphate-buffered saline (PBS) To evaluate the antitumor therapeutic potential of complexes\n (pH \u2248 7.2, prepared from D2 O) at 37 \u00b0C. No significant changes 1\u20135, their anti-proliferative activity was assessed using an in\n were observed over a 48-h period, indicating that the struc- vitro MTT assay across four tumor cell lines: AGS (human gas-\n tural integrity of the Rh center and its coordinated ligands is tric adenocarcinoma), MCF-7 (human breast adenocarcinoma),\n maintained under conditions that approximate the intracellular HeLa (human cervical adenocarcinoma), and HepG2 (human\n environment (Figure S12). hepatocellular carcinoma). Additionally, a non-tumor cell line,\n Single crystal of complex 5 [Rh(tpy)(dppz)Cl](PF6 )2 suitable GES-1 (human gastric epithelial cells), was included for com-\n for X-ray diffraction analysis was obtained through slow vapor parative purposes. Cisplatin served as the positive control for\n\n\n ChemistrySelect 2025, 10, e03583 (2 of 7) \u00a9 2025 Wiley-VCH GmbH\n\f 23656549, 2025, 27, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/slct.202503583 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistrySelect doi.org/10.1002/slct.202503583\n\n\n\n Table 1. IC50 and selectivity index (SI) of complexes 1\u20135 against different cell lines.a)\n\n Cell Lines AGS MCF-7 HeLa HepG-2 GES-1\n \u2013 IC50 (\u03bcM) SI IC50 (\u03bcM) SI IC50 (\u03bcM) SI IC50 (\u03bcM) SI IC50 (\u03bcM) SI\n\n Complex 1 46.6 \u00b1 1.7 2.1 >100 \u2013 >100 \u2013 >100 \u2013 >100 \u2013\n Complex 2 37.7 \u00b1 2.1 2.7 >100 \u2013 >100 \u2013 >100 \u2013 >100 \u2013\n Complex 3 7.0 \u00b1 0.6 1.0 9.3 \u00b1 1.1 0.7 5.4 \u00b1 1.1 1.3 21.4 \u00b1 4.3 0.3 6.9 \u00b1 0.3 \u2013\n Complex 4 >100 \u2013 >100 \u2013 >100 \u2013 >100 \u2013 >100 \u2013\n Complex 5 8.3 \u00b1 0.8 2.5 13.9 \u00b1 1.5 1.5 23.6 \u00b1 2.3 0.9 34.1 \u00b1 4.0 0.6 20.5 \u00b1 1.3 \u2013\n Cisplatin 38.8 \u00b1 3.8 1.1 14.8 \u00b1 2.5 2.8 24.8 \u00b1 3.8 1.7 13.5 \u00b1 1.5 3.1 42.1 \u00b1 3.1 \u2013\n\n a)\n Data are shown as mean \u00b1 SD (n = 3); SI (Selectivity index) = IC50 (non-tumor cells)/IC50 (tumor cells).\n\n\n\n this experiment. Following a 48-hour incubation of the cell lines\n with varying concentrations of complexes 1\u20135, cell viability was\n measured using the MTT assay, and the results are summa-\n rized in Table 1. Notably, complexes 3 and 5 exhibited higher\n anti-proliferative activity than complexes 1, 2, and 4.\n The selectivity index (SI), reflecting the therapeutic window\n of the complex, was calculated to evaluate their preferential sup-\n pression of malignant cell viability over normal cells. Despite\n showing potent inhibitory effects on cell viability in both tumor\n and non-tumor cells, complex 3 exhibited a narrow therapeu-\n tic window due to insufficient selectivity, which may limit its\n future clinical applications. In contrast, complex 5 demonstrated\n a promising therapeutic profile, with a notably low IC50 and a Figure 2. The binding mode between complexes and ctDNA.\n favorable SI. When compared to cisplatin, complex 5 exhibited\n higher selectivity towards AGS cells and a significantly lower IC50 ,\n suggesting that it may offer superior therapeutic potential in the tion into the minor groove and likely impeding DNA replication.\n treatment of gastric adenocarcinoma. These docking results support the hypothesis that minor groove\n binding contributes significantly to the observed cytotoxicity,\n especially for complexes 3 and 5, and are in line with the\n 2.3. Molecular Docking Studies experimental antiproliferative data (Figure 2).\n\n DNA is a well-established molecular target for many metal-\n based anticancer agents. To investigate the DNA-binding inter- 2.4. Complex 5 Induced Apoptosis in AGS\n actions of the synthesized Rh(III) complexes, molecular docking\n studies were conducted using the Surflex-Dock algorithm with To elucidate the mechanism underlying the anti-proliferative\n ctDNA as the target (PDB ID: 1LU5). Validation of the docking effects of complex 5 on AGS cells, its ability to trigger apopto-\n protocol was confirmed by redocking the co-crystallized lig- sis was assessed via Annexin V-FITC/PI dual staining followed by\n and (cyclohexylamine), yielding a root-mean-square deviation flow cytometry. AGS cells were exposed to increasing concen-\n (RMSD) of 0.676 \u00c5, thereby confirming the reliability of the trations of complex 5 (5, 10, and 15 \u03bcM) for 24 h As shown in\n docking approach (Figure S13). Figure 3, a dose-dependent escalation in apoptotic cell popula-\n All five complexes adopted similar binding poses, primarily tions was observed. At 5 \u03bcM, the early and late-stage apoptosis\n engaging the DNA minor groove through dipole\u2013dipole inter- rates were 17.34% and 15.10%, respectively. When the concentra-\n actions between the electron-deficient Rh(III) center and the tion of complex 5 was increased to 10 \u03bcM, these apoptosis rates\n electron-rich nitrogen atoms on guanine bases, particularly dG- rose to 21.29% and 19.58%, respectively, and at 15 \u03bcM, the rates\n 16. Complexes 3 and 5 exhibited slightly shorter Rh\u00b7\u00b7\u00b7N distances increased further to 26.35% and 34.62%. These data indicate that\n (\u223c4.7 \u00c5) compared to complexes 1, 2, and 4 (\u223c4.9\u20135.0 \u00c5), correlat- apoptosis induction contributes to the reduction of AGS cells\u2019\n ing with their lower IC5n values and enhanced cytotoxic activity. viability by complex 5.\n In contrast, cisplatin displayed stronger interactions (distances\n of \u223c2.6\u20132.7 \u00c5) due to its smaller molecular volume (\u223c89.1 \u00c53 ),\n enabling deeper insertion into the DNA groove. However, its 2.5. Complex 5 Induced S Phase Cell Cycle Arrest in AGS\n greater flexibility may reduce binding specificity. By compari-\n son, complexes 3 (390.6 \u00c53 ) and 5 (413.2 \u00c53 ) demonstrated an The effect of complex 5 on the cell cycle was also examined\n optimal molecular size and rigidity, facilitating effective intercala- (Figure 4). Flow cytometry analysis revealed that complex 5\n\n\n ChemistrySelect 2025, 10, e03583 (3 of 7) \u00a9 2025 Wiley-VCH GmbH\n\f 23656549, 2025, 27, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/slct.202503583 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistrySelect doi.org/10.1002/slct.202503583\n\n\n\n\n Figure 3. Complex 5 induced apoptosis in AGS cells. Apoptotic rates were measured by Annexin V/PI staining after 24 h treatment with 5\u201315 \u03bcM complex 5.\n Data are shown as mean \u00b1 SD (n = 3).\n\n induced a concentration-dependent arrest of the AGS cell cycle 3. Conclusion\n at the S phase. As the concentration of complex 5 increased, the\n proportion of cells in the G0/G1 phase decreased from 58.5% to We have synthesized and structurally characterized five new\n 30.1%, while the proportion in the S phase increased from 31.1% octahedral rhodium(III) complexes bearing terpyridine and\n to 68.4%. Additionally, the G2/M phase population decreased diverse bidentate N-donor ligands. Among them, complex 5\n from 10.4% to 1.5%. At the highest concentration of complex 5 displayed superior cytotoxicity and selectivity toward gastric\n tested, the S-phase population increased by 37.3% compared to adenocarcinoma cells. Mechanistic studies confirmed its abil-\n the control group. These findings strongly suggest that complex ity to bind DNA, arrest the cell cycle in S-phase, elevate ROS\n 5 effectively arrests the AGS cell cycle at the S phase, thereby levels, and trigger apoptosis. The combination of structural\n inhibiting cell proliferation. rigidity, favorable DNA-binding properties, and selective anti-\n cancer activity positions these terpyridine-functionalized Rh(III)\n complexes\u2014particularly complex 5\u2014as attractive candidates for\n 2.6. Complex 5 Increased ROS Levels in AGS further investigation as metal-based chemotherapeutics.\n\n To investigate the potential mechanism behind the anticancer\n activity of complex 5, its effect on intracellular reactive oxygen 4. Experimental Section\n species (ROS) levels was evaluated. AGS cells were treated with\n varying concentrations of complex 5 for 24 h, and ROS levels 4.1. Materials and Methods\n were measured using a fluorescence-based assay. As shown in\n The synthesis of dppz followed the reported procedure.[32] All other\n Figure 5, complex 5 induced a concentration-dependent increase\n materials were obtained from commercial sources and were used\n in ROS levels compared to the untreated control group. These without further purification. NMR spectra were recorded on a Bruker\n findings indicate that complex 5 elevates intracellular ROS levels AVANCE III HD-400 MHz. NMR instrument operating at room temper-\n in AGS cells, which may contribute to its anticancer activity by ature (400 MHz for 1 H and 101 MHz for 13 C). Referencing is relative to\n inducing oxidative stress. tetramethylsilane. High-resolution mass spectra were recorded on a\n\n\n ChemistrySelect 2025, 10, e03583 (4 of 7) \u00a9 2025 Wiley-VCH GmbH\n\f 23656549, 2025, 27, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/slct.202503583 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistrySelect doi.org/10.1002/slct.202503583\n\n\n\n\n Figure 4. The distribution of the AGS cell cycle induced by complex 5 at the concentrations of 5 \u03bcM, 10 \u03bcM, and 15 \u03bcM.\n\n\n Shimadzu LCMS-IT-TOF high-resolution liquid chromatograph. Sam- Complex 2. This was prepared following the above procedure for\n ples for microanalysis were dried in vacuo to constant weight (20 \u00b0C, complex 1, except that 5,5\u0002 -dimethyl-2,2\u0002 -dipyridy (dmbpy) (0.047 g,\n ca. 0.1 torr) and analyzed on a Flash EA 111 CHN elemental analyzer. 0.25 mmol) was used in place of 2,2\u0002 -bipyridine. Yield = 0.120 g (yel-\n FT-IR spectra (4000\u2013400 cm\u22121 ) were recorded at room temperature low solid), 71%. HR-MS (ESI+): calcd for [M + H]+ , 846.0059; found,\n using a NEXUS 670 spectrophotometer. 846.5205. Elem. Anal. Calcd for C27 H23 ClF12 N5 P2 Rh: C, 38.34; H, 2.74; N,\n Complex 1. RhCl3 \u00b73H2 O (0.395 g, 1.5 mmol) and tpy (0.350 g, 8.28. Found C, 38.26; H, 2.68; N, 8.37. 1 H NMR (400 MHz, DMSO-d6 ) \u03b4\n 1.5 mmol) in EtOH (10 mL) were refluxed for 2 h. Cooled to room 9.51 (d, J = 1.9 Hz, 1H), 9.11 (d, J = 8.1 Hz, 2H), 8.94 (ddd, J = 8.0, 4.7,\n temperature, the product was filtered off and washed with H2 O, 3.4 Hz, 4H), 8.75 (d, J = 8.4 Hz, 1H), 8.59 (dd, J = 8.4, 1.9 Hz, 1H), 8.42\n EtOH and diethyl ether successively, and then dried in vacuo to (td, J = 7.9, 1.5 Hz, 2H), 8.12 (d, J = 8.3 Hz, 1H), 7.85 (d, J = 5.5 Hz,\n give the Rh(tpy)Cl3 . The product was used in subsequent reactions 2H), 7.67 (ddd, J = 7.4, 5.6, 1.4 Hz, 2H), 7.56 (s, 1H), 2.70 (s, 3H), 2.14\n without further purification. Rh(tpy)Cl3 (0.088 g, 0.2 mmol) and 2,2\u0002 - (s, 3H). 13 C{1 H} NMR (101 MHz, DMSO-d6 ) \u03b4 157.15, 154.71, 153.45, 152.89,\n bipyridine (bpy) (0.039 g, 0.25 mmol) in a mixture of H2 O/EtOH (1:1, 151.26, 150.52, 143.46, 143.09, 142.83, 142.50, 140.56, 130.20, 127.24, 126.96,\n 10 mL) were refluxed for 4 h. NH4 PF6 (1.6 g, 9.8 mmol) and H2 O 125.67, 19.24, 18.62. IR (solid): \u03bd = 1606 cm\u22121 (\u03bd(C\u2550N)), \u03bd = 830 cm\u22121\n (20 mL) were then added into the solution, and the reaction mixture (\u03bd(PF6 \u2212 )).\n was refluxed for 2 h. Cooled to room temperature, the product was Complex 3. This was prepared following the above procedure\n filtered off. The crude product was dissolved in acetonitrile (5 mL) for complex 1, except that 3,4,7,8-tetramethyl-1,10-phenanthroline\n and recrystallized by the slow addition of diethyl ether (10 mL) to (TMPhen) (0.060 g, 0.25 mmol) was used in place of 2,2\u0002 -bipyridine.\n yield the desired compound as a yellow solid, which was dried in Yield = 0.141 g (pale yellow solid), 78%. HR-MS (ESI+): calcd\n vacuo. Yield = 0.120 g, 74%. HR-MS (ESI+): calcd for [M + H]+ , for [M + H]+ , 898.0372; found, 898.3321. Elem. Anal. Calcd for\n 817.9746; found, 817.9872. Elem. Anal. Calcd for C25 H19 ClF12 N5 P2 Rh: C31 H27 ClF12 N5 P2 Rh: C, 41.47; H, 3.03; N, 7.80. Found C, 41.38; H, 2.95; N,\n C, 36.72; H, 2.34; N, 8.56. Found C, 36.63; H, 2.23; N, 8.68. 1 H NMR 7.88. 1 H NMR (400 MHz, DMSO-d6 ) \u03b4 9.70 (s, 1H), 9.14 (d, J = 8.2 Hz,\n (400 MHz, DMSO-d6 ) \u03b4 9.76 (d, J = 1.7 Hz, 1H), 9.12 (d, J = 8.1 Hz, 3H), 2H), 8.95 (t, J = 7.9 Hz, 3H), 8.70 (d, J = 9.4 Hz, 1H), 8.51 (d, J = 9.5 Hz,\n 8.96\u20138.89 (m, 4H), 8.78 (td, J = 7.9, 1.5 Hz, 1H), 8.42 (td, J = 7.9, 1.4 Hz, 1H), 8.36 (td, J = 7.9, 1.4 Hz, 2H), 7.91 (s, 1H), 7.68 (d, J = 5.6 Hz, 2H),\n 2H), 8.33 \u2013 8.27 (m, 2H), 7.85 (d, J = 5.6 Hz, 3H), 7.67 (ddd, J = 7.4, 5.6, 7.50 (ddd, J = 7.3, 5.6, 1.3 Hz, 2H), 3.08 (s, 3H), 2.83 (s, 3H), 2.75 (s, 3H),\n 1.4 Hz, 2H), 7.50 (ddd, J = 7.4, 5.8, 1.4 Hz, 1H). 13 C{1 H} NMR (101 MHz, 2.27 (s, 3H). 13 C{1 H} NMR (101 MHz, DMSO-d6 ) \u03b4 157.05, 154.93, 153.50,\n DMSO-d6 ) \u03b4 156.97, 155.92, 154.58, 152.99, 151.75, 151.26, 143.69, 142.94, 149.50, 143.83, 142.60, 140.19, 138.07, 137.50, 133.62, 132.05, 131.40, 130.04,\n 142.61, 142.39, 130.29, 129.73, 127.24, 126.96, 126.73, 126.19. IR (solid): \u03bd = 127.21, 25.96, 22.57. IR (solid): \u03bd = 1602 cm\u22121 (\u03bd(C\u2550N)), \u03bd = 832 cm\u22121\n 1605 cm\u22121 (\u03bd(C\u2550N)), \u03bd = 833 cm\u22121 (\u03bd(PF6 \u2212 )). (\u03bd(PF6 \u2212 )).\n\n\n ChemistrySelect 2025, 10, e03583 (5 of 7) \u00a9 2025 Wiley-VCH GmbH\n\f 23656549, 2025, 27, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/slct.202503583 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistrySelect doi.org/10.1002/slct.202503583\n\n\n\n 4.3. Cell Culture\n Tumor cell lines AGS, MCF-7, HeLa, HepG2, and non-tumor cell\n line GES-1 were purchased from the China Center for Type Cul-\n ture Collection. AGS and GES cells were cultured in Roswell Park\n Memorial Institute 1640 medium (RPMI 1640), while MCF-7, HeLa, and\n HepG2 cells were maintained in Dulbecco\u2019s modified Eagle\u2019s medium\n (DMEM). All media were supplemented with 10% fetal bovine\n serum (FBS), 100 U/mL penicillin, and 100 \u03bcg/mL streptomycin,\n and cells were incubated in a humidified atmosphere with 5%\n CO2 at 37 \u00b0C.\n\n\n\n\n 4.4. Anti-proliferation Assay\n The anti-proliferation effect of complexes 1\u20135 was evaluated by the\n 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)\n colorimetric assay as described previously. AGS, MCF-7, HeLa, HepG-\n 2, and GES-1 cells (7000 cells/well) were seeded into 96-well plates\n and incubated overnight. The cells were incubated with complexes\n (1\u2013100 \u03bcM) or cisplatin, with 0.5% DMSO as vehicle control. MTT solu-\n tion (0.5 mg/mL final) was added for 4 h at 37 \u00b0C. The medium\n Figure 5. The levels of ROS with AGS cells induced by complex 5 at the was aspirated, and formazan crystals were dissolved in DMSO.\n concentrations of 5 \u03bcM, 10 \u03bcM, and 15 \u03bcM. Absorbance at 490 nm was measured, and viability was normalized\n to controls.\n\n\n Complex 4. This was prepared following the above proce-\n dure for complex 1, except that 2,2\u0002 -bipyridine-4,4\u0002 -dicarboxylic acid\n 4.5. Molecular Docking Method\n (dcbpy) (0.061 g, 0.25 mmol) was used in place of 2,2\u0002 -bipyridine.\n Yield = 0.120 g (pale yellow solid), 66%. HR-MS (ESI+): calcd The method used to explore the binding mode between design\n for [M + H]+ , 905.9542; found, 906.2628. Elem. Anal. Calcd for compounds and ctDNA could be referenced from our previous\n C27 H19 ClF12 N5 O4 P2 Rh: C, 35.80; H, 2.11; N, 7.73. Found C, 35.73; H, 2.03; study.[35] The binding site of ctDNA (ww.rcsb.org, ID: 1LU5) was\n N, 7.82. 1 H NMR (400 MHz, DMSO-d6 ) \u03b4 9.89 (d, J = 5.8 Hz, 1H), 9.56 defined using SYBYL-X based on the co-crystal ligand, and the vol-\n (s, 1H), 9.33 (s, 1H), 9.13 (d, J = 8.0 Hz, 2H), 8.95 (dd, J = 8.1, 3.4 Hz, ume of the pocket and compounds were determined by MOLCAD\n 3H), 8.65 (dd, J = 5.8, 1.7 Hz, 1H), 8.41 (t, J = 7.9, 1.4 Hz, 2H), 7.94 (dd, with the Fast Connolly method.[26] The structure of all complexes\n J = 15.0, 5.7 Hz, 3H), 7.74 (dd, J = 6.0, 1.7 Hz, 1H), 7.68\u20137.63 (m, 2H). was optimized with the Tripos force field and MMFF95 charge.\n C{ H} NMR (101 MHz, DMSO-d6 ) \u03b4 165.31, 164.61, 156.89, 156.46, 154.62,\n 13 1\n The docking program Surflex-Dock GeomX (SFXC) in SYBYL-X was\n 153.29, 152.07, 143.83, 142.61, 130.24, 129.53, 127.26, 126.97, 126.34, 25.96. used to dock the complexes into the pocket. The main proto-\n IR (solid): \u03bd = 1602 cm\u22121 (\u03bd(C\u2550N)), \u03bd = 832 cm\u22121 (\u03bd(PF6 \u2212 )). cols or parameters of docking were addressed in our previous\n Complex 5. This was prepared following the above procedure publications.[40] Briefly, the docking parameters were as follows: (a)\n for complex 1, except that dppz (0.070 g, 0.25 mmol) was used in the \u201cnumber of starting conformations per ligand\u201d was set to 10, and\n place of 2,2\u0002 -bipyridine. Yield = 0.120 g (pale yellow solid), 64%. HR- the \u201cnumber of max conformations per fragment\u201d was set to 20; (b)\n MS (ESI+): calcd for [M + H]+ , 943.9964; found, 944.1374. Elem. Anal. the \u201cmaximum number of rotatable bonds per molecule\u201d was set to\n Calcd for C33 H21 ClF12 N7 P2 Rh: C, 41.99; H, 2.24; N, 10.39. Found C, 41.92; 100; (c) flags were turned on at \u201cpre-dock minimization\u201d, \u201cpost-dock\n H, 2.17; N, 10.46. 1 H NMR (400 MHz, DMSO-d6 ) \u03b4 10.20 (d, J = 7.9 Hz, minimization\u201d, \u201cmolecule fragmentation\u201d, and \u201csoft grid treatment\u201d;\n 1H), 10.13 (d, J = 5.4 Hz, 1H), 9.74 (d, J = 7.9 Hz, 1H), 9.18 (d, J = 8.2 Hz, (d) \u201cactivate spin alignment method with density of search\u201d was set\n 2H), 8.99 (dd, J = 10.5, 8.1 Hz, 3H), 8.79 (dd, J = 8.3, 5.4 Hz, 1H), 8.64 to 9.0; and (e) the \u201cnumber of spins per alignment\u201d was set to 12.\n (dd, J = 7.7, 2.1 Hz, 1H), 8.55 (dd, J = 8.2, 1.8 Hz, 1H), 8.42 \u2013 8.35 (m, To validate the docking program, we redocked the co-crystal ligand\n 3H), 8.27 (ddd, J = 8.0, 5.8, 1.8 Hz, 2H), 7.99 (dd, J = 8.2, 5.5 Hz, 1H), and calculated the RMSD based on their superimposition.\n 7.85 (d, J = 5.6 Hz, 2H), 7.59 (ddd, J = 7.4, 5.6, 1.3 Hz, 2H). 13 C{1 H} NMR\n (101 MHz, DMSO-d6 ) \u03b4 157.05, 154.93, 153.49, 149.84, 149.50, 142.60,\n 140.19, 139.86, 138.08, 137.50, 133.69, 132.05, 131.40, 130.04, 129.20, 127.21,\n 127.04. IR (solid): \u03bd = 1606 cm\u22121 (\u03bd(C\u2550N)), \u03bd = 835 cm\u22121 (\u03bd(PF6 \u2212 )).\n 4.6. Apoptosis Analysis by Annexin V/PI Staining\n AGS cells (2 \u00d7 105 /well) were seeded in 6-well plates and cultured\n overnight. Cells were treated with complex 5 (5, 10, and 15 \u03bcM) for\n 4.2. X-ray Crystallography\n 24 h, harvested, washed, and resuspended in pre-cooling PBS. Apop-\n Diffraction data of complex [Rh(tpy)(dppz)Cl](PF6 )2 was measured at tosis was quantified using an Annexin V-FITC/PI Apoptosis Detection\n 196.0 K using MoK\u03b1 radiation (\u03bb = 0.71,073 \u00c5) on a Bruker APEX- Kit (Beyotime, C1062m) per the manufacturer\u2019s protocol. Briefly, cells\n II CCD diffractometer. Using Olex2, the structure was solved with were stained with FITC-conjugated Annexin V and PI for 15 min in\n the ShelXT structure solution program using Intrinsci Phasing and the dark, followed by immediate analysis on a FACSCalibur flow\n refined with the ShelXL refinement package using Least Squares cytometer (BD FACSAria SORP). Data were processed with FlowJo\n minimisation.[38,39] software (FlowJo 10.8.1).\n\n\n ChemistrySelect 2025, 10, e03583 (6 of 7) \u00a9 2025 Wiley-VCH GmbH\n\f 23656549, 2025, 27, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/slct.202503583 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemistrySelect doi.org/10.1002/slct.202503583\n\n\n\n 4.7. Cell Cycle Analysis [6] S. De, S. Kazi, S. Banerjee, S. Banerjee, N. Sarkar, S. Kumar Shah, Y.-C.\n Kuo, S. K. Ashok Kumar, Coord. Chem. Rev. 2024, 498, 215462.\n AGS cells (2 \u00d7 10 /well) were seeded in 6-well plates and allowed to\n 5\n [7] Z.-F. Wang, X.-F. Zhou, Q.-C. Wei, Q.-P. Qin, J.-X. Li, M.-X. Tan, S.-H.\n adhere overnight. Cells were treated with complex 5 (5, 10, or 15 \u03bcM) Zhang, Eur. J. Med. Chem. 2022, 238, 114418.\n for 24 h, harvested, and fixed in 70% ice-cold ethanol. After fixation, [8] Y. Chen, X. Hu, L. Yuan, S. Zhang, H. Ma, J. Mol. Struct. 2025, 1340, 142499.\n cells were incubated with 100 \u03bcg/mL RNase A at 37 \u00b0C for 30 min, fol- [9] M. Vojtek, M. P. M. Marques, I. M. P. L. V. O. Ferreira, H. Mota-Filipe, C.\n lowed by staining with 50 \u03bcg/mL propidium iodide (PI) for 30 min on Diniz, Drug Discov. Today 2019, 24, 1044\u20131058.\n ice in the dark. Cell cycle distribution was analyzed using a FACSCal- [10] R. Czarnomysy, D. Radomska, O. K. Szewczyk, P. Roszczenko, K.\n ibur flow cytometer (BD FACSAria SORP), and data were processed Bielawski, Int. J. Mol. Sci. 2021, 22, 8271.\n [11] R. Guan, L. Xie, L. Ji, H. Chao, Eur. J. Inorg. Chem. 2020, 3978\u20133986.\n with FlowJo software (FlowJo 10.8.1).\n [12] J. Shen, T. W. Rees, L. Ji, H. Chao, Coord. Chem. Rev. 2021, 443, 214016.\n [13] Y. Wu, S. Li, Y. Chen, W. He, Z. Guo, Chem. Sci. 2022, 13, 5085\u20135106.\n [14] R. Das, U. Das, N. Roy, C. Mukherjee, S. U, P. Paira, Dyes Pigm. 2024, 226,\n 4.8. ROS Determination 112134.\n [15] N. Manav, A. Janaagal, I. Gupta, Coord. Chem. Rev. 2024, 511, 215798.\n Following treatment with complex 5 (5\u201315 \u03bcM, 24 h), cells were [16] H. Fang, Z. Sun, M. Xu, W. Xue, Z. Qian, Z. Li, C. Zhang, Q. Wu, J. Xu, R.\n harvested and incubated with 10 \u03bcM DCFH-DA in PBS for 20 min Li, H. Fu, X. Zheng, H. Chen, Ind. Eng. Chem. Res. 2025, 64, 1488\u20131496.\n at room temperature in the dark. After washing three times with [17] I. Audzeyenka, A. Piwkowska, D. Rogacka, M. Makowski, M. Kowalik, J.\n PBS, cell suspensions were filtered through a 70-\u03bcm nylon mesh to Med. Chem. 2024, 67, 21364\u201321379.\n [18] C. G. L. Nongpiur, C. Soh, D. F. Diengdoh, A. K. Verma, R. Gogoi, V.\n remove aggregates. Intracellular ROS levels were analyzed using a\n Banothu, W. Kaminsky, M. R. Kollipara, J. Organomet. Chem. 2023, 998,\n flow cytometer with excitation/emission settings of 488/530 nm (FL1\n 122788.\n channel). Data were processed with FlowJo software (FlowJo 10.8.1). [19] E. Skelton, U. Erasquin, A. Sukul, A. Zuercher, J. White, B. J. Bythell, K.\n L. A. Cimatu, Inorg. Chem. 2023, 62, 3368\u20133380.\n [20] N. Toupin, M. K. Herroon, R. P. Thummel, C. Turro, I. Podgorski, H.\n Gibson, J. J. Kodanko, Chem. -Eur. J. 2022, 28, e202104430.\n Supporting Information [21] W. Li, S. Li, M. Zhu, G. Xu, X. Man, Z. Zhang, H. Liang, F. Yang, J. Med.\n Chem. 2024, 67, 17243\u201317258.\n 1\n H and 13 C{1H} NMR spectra, HRMS spectra, FT-IR spectra, crystal- [22] M. Kowalik, J. Masternak, M. Olszewski, N. Maciejewska, K. Kazimierczuk,\n lographic data, and cell apoptosis data. J. Sitkowski, A. M. Dabrowska,\n \u02db A. Chylewska, M. Makowski, Inorg. Chem.\n 2024, 63, 1296\u20131316.\n [23] W. K. Chu, C. K. Rono, B. C. E. Makhubela, Eur. J. Inorg. Chem. 2024, 27,\n e202300541.\n Acknowldgements [24] L. Guo, P. Li, J. Li, Y. Gong, X. Li, Y. Liu, K. Yu, Z. Liu, Inorg. Chem. 2023,\n 62, 15118\u201315137.\n [25] Y. Zheng, X.-X. Chen, D.-Y. Zhang, W.-J. Wang, K. Peng, Z.-Y. Li, Z.-W.\n This work was supported by the Science and Technology Mao, C.-P. Tan, Chem. Sci. 2023, 14, 6890\u20136903.\n Research Program of Chongqing Municipal Education Commis- [26] A. Nahaei, Z. Mandegani, S. Chamyani, M. Fereidoonnezhad, H. R.\n sion (Grant No. KJQN202201167). Shahsavari, N. Y. Kuznetsov, S. M. Nabavizadeh, Inorg. 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Manuscript received: June 24, 2025\n\n\n\n\n ChemistrySelect 2025, 10, e03583 (7 of 7) \u00a9 2025 Wiley-VCH GmbH\n\f", "pages_extracted": 7, "text_length": 49528}