Rhodium(III)-Picolinamide Complexes Act as Anticancer and Antimetastasis Agents via Inducing Apoptosis and Autophagy.

{"full_text": " pubs.acs.org/jmc Article\n\n\n\n Rhodium(III)\u2212Picolinamide Complexes Act as Anticancer and\n Antimetastasis Agents via Inducing Apoptosis and Autophagy\n Yun-Qiong Gu, Kun Yang, Qi-Yuan Yang, Huan-Qing Li, Mei-Qi Hu, Meng-Xue Ma, Nan-Feng Chen,\n Yang-Han Liu, Hong Liang,* and Zhen-Feng Chen*\n Cite This: J. Med. Chem. 2023, 66, 9592\u22129606 Read Online\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ABSTRACT: As a continuation of our endeavors in discovering\n metal-based drugs with cytotoxic and antimetastatic activities,\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 12:08:42 (UTC).\n\n\n\n\n herein, we reported the syntheses of 11 new rhodium(III)\u2212\n picolinamide complexes and the exploration of their potential\n anticancer activities. These Rh(III) complexes showed high\n antiproliferative activity against the tested cancer cell lines in\n vitro. The mechanism study indicated that Rh1 ([Rh(3a)-\n (CH3CN)Cl2]) and Rh2 ([Rh(3b)(CH3CN)Cl2]) inhibited cell\n proliferation by multiple modes of action via cell cycle arrest,\n apoptosis, and autophagy and inhibited cell metastasis via FAK-\n regulated integrin \u03b21-mediated suppression of EGFR expression.\n Furthermore, Rh1 and Rh2 significantly inhibited bladder cancer\n growth and breast cancer metastasis in a xenograft model. These\n rhodium(III) complexes could be developed as potential anticancer agents with antitumor growth and antimetastasis activity.\n\n\n \u25a0 INTRODUCTION\n Cancer seriously threatens human health and life, and\n significant downstream effector factors that targets cell\n adhesion to ECM and mediates downstream signal events of\n metastatic spread of cancer cells is the main clinical ECM integrin conjugation.10,11 Thus, FAK plays a vital role in\n complication of cancer that causes more than 90% of cancer- cell migration and invasion.12,13\n Metal complexes are an important class of compounds in\n related deaths.1,2 Chemotherapy is still the most common and\n anticancer drug development owing to their unique physical,\n effective therapeutic method in treating the evolving cancers.3\n chemical, and structural characteristics and biological activities.\n Among the chemotherapy drugs, platinum complexes such as\n Serious side effects of platinum drugs have led to the\n oxaliplatin, carboplatin, and cisplatin are widely applied in the\n investigation of other transition-metal complexes,14 such as\n treatment of solid tumors. However, their dose toxicity, low\n ruthenium,15 rhodium,16 and palladium17 complexes. Com-\n selectivity, drug resistance, and low efficacy for metastatic\n pared with the typical square-planar Pt(II) complexes, Rh(III)\n tumors hindered their wider applications.4 Therefore, the\n complexes show more structural diversity,18 high cellular\n development of new metal complexes that can inhibit both\n uptake,19 strong antiproliferative activity,20 and positive\n tumor growth and metastasis via multiple modes of action is a\n tolerance in normal cells.21,22 The rhodium(III) centers are\n promising chemotherapy strategy for cancer.\n traditionally considered inactive, but recent studies have shown\n Metastasis is a multistep continuous process involving a\n that the reactivity to biological targets could be enhanced by\n complex series of dynamic events, such as epithelial\u2212\n coordinating appropriate ligands.23 These complexes act as\n mesenchymal transition (EMT), angiogenesis, inflammatory\n inhibitors of proteins or regulators of protein\u2212protein\n tumor microenvironment initiation, and apoptotic dysfunc-\n interactions. For example, they are shown to be inhibitors of\n tion.5 Tumor cells metastasize and spread when EMT occurs\n enzymes,24 such as the platelet-activating factor (PAF) and\n in the primary tumor. The early step of EMT is the local\n thrombin,23 isomerase,25 lysine-specific demethylase 5A\n infiltration of the surrounding extracellular matrix (ECM) and\n the stromal cell layer.6 Cancer cells degrade ECMs by\n concentrating the enzyme activity of matrix metalloproteinases Received: February 22, 2023\n (MMPs) using special F-actin-rich projections, which allows Published: July 11, 2023\n cancer cells to migrate and invade outside the microenviron-\n ment.7,8 The EGFR signaling pathway and the integrin\n signaling pathway are involved in regulating many cellular\n functions.9 Focal adhesion kinase (FAK) is one of the most\n\n \u00a9 2023 American Chemical Society https://doi.org/10.1021/acs.jmedchem.3c00318\n 9592 J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n(KDM5A),26 and lysine-specific demethylase 1 (LSD1).27 The (BRAFV600E, Figure 1B) for C-raf inhibitors, BLZ-94536 for\nunique characteristics of these rhodium complexes, especially FMS inhibitor, Klisyri (KX01, Figure 1D) for the dual\ntheir octahedral geometry, allow specific interactions with the microtubulin/Src protein inhibitor,37 and 30m (Figure 1E)\nappropriate regions of chemical space in protein-binding for hypoxia-inducible factor 1 (HIF-1) inhibitor.38 Meanwhile,\npockets that are inaccessible to small organic molecules. pyrazole derivatives occupy a special position in cancer\nRhodium metallo-insertors have high affinity and specific therapy,39 including crizotinib (Figure 1F), ruxolitinib (Figure\nbinding to DNA mismatches in vitro, specifically target nuclear 1G), tartrazine (Figure 1H), pyrazofurin (Figure 1I), and 5-\nDNA, and exhibit cell-selective cytotoxic and antiproliferative anilino-pyrazole analogue I (Figure 1J).40 In addition, some\nactivities,19,28,29 which have attracted our interest in further picolinamide derivatives show high activity in inhibiting tumor\nstudying their biological effects. cell metastasis.41,42 The development of new picolinamide and\n A substantial number of heterocyclic compounds have been pyrazole compounds with low toxicity and high activity has\napproved for cancer treatment,30 and the backbones of their attracted extensive attention in the field of medicinal\nstructures are often nitrogen heterocycles.31 Among them, chemistry.33 The pharmacophore conjugation strategy is an\nmany drugs containing picolinamide or pyrazole have been effective approach to find new chemical entities with improved\nused in clinical practice to treat cancer (Figure 1).32,33 The biological activity.32 Using this strategy, we synthesized a series\n of picolinamide derivatives by conjugating them with pyrazole\n rings with different substitutions (Scheme 1).\n Moreover, we are interested in the development of effective\n and selective Rh(III) complexes with both antiproliferation\n and antimetastasis activities, and our group has previously\n reported a mitochondria-accumulating Rh(III) isoquinoline\n complexes with different modes of action from Pt(II)-based\n therapeutics.43 A series of N-(3-bromophenyl)picolinamide\n rhodium(III) complexes exhibited good in vitro cytotoxicity\n and selectivity index (SI) value of Rh(III) trans-diiodide\n complexes, which is more than 25 times higher than that of\n cisplatin.44 Rhodium half-sandwich complexes containing\n (N,N)-bound picolinamide ligands showed good potential as\n anticancer agents.45 Therefore, the rhodium(III) complexes\n with these picolinamide derivative ligands were synthesized via\n N\u2212N\u2212N coordination; the rhodium(III) complexes were\n characterized, and their activity against tumor growth and\n metastasis in vitro and in vivo were investigated.\n\n \u25a0 RESULTS AND DISCUSSION\n Synthesis and Characterization. The synthesis process\n of ligands 3a\u22123k is shown in Scheme 2, and the synthesis of\n their corresponding rhodium complexes Rh1\u2212Rh11 was\n carried out following the reported procedure.46 The complexes\n Rh1\u2212Rh11 were characterized by HRMS, NMR spectroscopy,\n elemental analysis, powder X-ray diffraction (Figures S49\u2212\n S50), and single-crystal X-ray diffraction analysis. The\n coordination mode of each complex is similar. The central\n metal atom Rh(III) adopts a distorted octahedral geometry\n and is bonded to three N atoms from the pyridine ring,\n pyrazole ring, and amide bond,47 two chloride ions, and a\n solvent molecule (methanol for Rh4 and Rh6 and acetonitrile\nFigure 1. Anticancer agents with picolinamide (A\u2212E) or pyrazole for other complexes) (Scheme 3 and Figure 2). 1H NMR data\n(F\u2212J) moiety. of rhodium complexes showed that hydrogen atoms were\n detached from the amide bonds of the ligands after the\nrepresentative anticancer drug sorafenib (Figure 1A), which formation of the complexes. The loss of hydrogen and the\ncontains picolinamide structural units, is an inhibitor of several addition of two chlorine ions make the complex electrically\ntyrosine kinases (rtk) with potent antitumor and antiangio- neutral.48 X-ray powder diffraction results confirmed that the\ngenic activities.34 Many picolinamide derivatives were potent structures of the crystallites of Rh1 and Rh2 obtained by batch\nkinase inhibitors, such as 2-anilinobenzothiazole derivatives35 synthesis are consistent with their single-crystal structures. The\n\nScheme 1. Structure of Picolinamide Derivatives as Potent Antitumor Agents\n\n\n\n\n 9593 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nScheme 2. Reagents and Conditions: (i) SOCl2, DMF, 85 \u00b0C, 3 h; (ii) Aniline, CH3CN, Triethylamine, 80 \u00b0C, 6 h; (iii)\nHydrazine Hydrate, Ethanol, 80 \u00b0C; and (iv) Ethanol, 90 \u00b0C\n\n\n\n\nScheme 3. Synthesis of Complexes Rh1\u2212Rh11; Reagents and Conditions: (i): RhCl3\u00b73H2O, CH3CN/CH3OH (v:v = 4:1, 10\nmL), 100 \u00b0C, 3 Days\n\n\n\n\nFigure 2. Crystal structures of Rh1\u2212Rh11 (thermal ellipsoids at 30% probability level; hydrogen atoms are omitted for clarity).\n\nintensity difference of the diffraction peak may be due to the Stability and Purity of Rh1\u2212Rh11. The stability and\norientation change of the polycrystalline samples. The purity of Rh1\u2212Rh11 were detected by the HPLC method in\nassignments of each of the H and C resonances of the ligands methanol solution (contains no more than 1% DMSO)\n (Figure S51). The results showed that there were no new\nand rhodium complexes are listed in Tables S1 and S4, and the peaks that appeared in complexes after 48 h, and the shift of\ncrystallographic data, selected bond lengths, and angles of the peaks was almost unchanged, which indicated that Rh1\u2212\nRh1\u2212Rh11 are presented in Tables S2 and S3. Rh11 were stable in solution at least for 48 h at room\n 9594 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nTable 1. IC50a (\u03bcM) of Rh1\u2212Rh11 for Five Human Cancer Cell Linesb Determined by the MTT Method for 48 h\n complex T-24 A549 BEL-7404 HeLa SK-OV-3 WI38 SF\n Rh1 0.8 \u00b1 0.1 1.3 \u00b1 0.4 2.8 \u00b1 0.5 2.5 \u00b1 0.7 3.0 \u00b1 0.7 2.7 \u00b1 0.2 3.4\n Rh2 1.6 \u00b1 0.3 7.7 \u00b1 0.3 9.6 \u00b1 0.8 10.0 \u00b1 0.6 13.8 \u00b1 1.4 6.3 \u00b1 0.7 3.9\n Rh3 4.4 \u00b1 1.5 10.3 \u00b1 1.2 >20 15.5 \u00b1 1.7 13.9 \u00b1 2.1 15.7 \u00b1 1.0 3.9\n Rh4 20.7 \u00b1 0.2 >20 >20 >20 >20 >20\n Rh5 2.0 \u00b1 0.2 5.3 \u00b1 0.2 10.5 \u00b1 1.0 8.3 \u00b1 0.3 10.3 \u00b1 0.5 4.3 \u00b1 1.6 2.2\n Rh6 >20 >20 >20 >20 >20 >20\n Rh7 16.9 \u00b1 1.1 >20 >20 16.5 \u00b1 1.7 19.1 \u00b1 1.2 >20\n Rh8 7.2 \u00b1 0.3 9.9 \u00b1 1.2 3.8 \u00b1 0.7 12.1 \u00b1 0.4 7.9 \u00b1 0.4 8.1 \u00b1 0.5 1.1\n Rh9 5.9 \u00b1 1.5 6.4 \u00b1 0.6 3.2 \u00b1 0.5 5.7 \u00b1 0.2 2.8 \u00b1 0.7 3.7 \u00b1 0.6 0.6\n Rh10 11.6 \u00b1 0.2 18.7 \u00b1 0.9 >20 17.4 \u00b1 0.5 >20 13.3 \u00b1 0.8 1.1\n Rh11 2.4 \u00b1 0.3 5.1 \u00b1 0.3 6.5 \u00b1 0.2 4.5 \u00b1 0.3 16.2 \u00b1 0.2 5.3 \u00b1 0.4 2.2\n DDP 11.0 \u00b1 0.5 11.4 \u00b1 0.4 34.2 \u00b1 0.7 16.6 \u00b1 0.4 7.9 \u00b1 1.2 5.4 \u00b1 0.9 0.5\n RhCl3\u00b73H2O >40 >40 >40 >40 >40 >40\na\n IC50 values are expressed as mean \u00b1 standard deviation of three independent experiments. SF (selectivity factor) = IC50(WI38)/IC50(T-24). bT-\n24 (human bladder cell line), A549 (human non-small-cell lung cancer cells), BEL-7404 (human hepatocellular carcinoma cells), HeLa (human\ncervical cell line), SK-OV-3 (human ovarian cancer cells), and WI38 (human embryonic lung cell line).\n\ntemperature. The purity of all complexes was higher than 95%, Rh11 (4-CH3\u2212Ph)> Rh6 (\u2212CH3), and Rh1 > Rh2. (3) To\nas determined by HPLC. The stability of Rh1\u2212Rh11 was also further investigate the effects of substituents R1 and R2 on\ndetected by UV\u2212vis spectroscopy (Figure S52). As shown, the cytotoxicity, we introduced the same substituents at the R1 and\nabsorption peaks near 250 nm showed no changes in peak R2 sites, such as 1-methylethyl (Rh3), trifluoromethyl (Rh4),\nshape and absorbance, indicating that these complexes methyl (Rh6), phenyl (Rh7), and p-methoxyphenyl (Rh10).\nremained stable during the experimental time (48 h). The Complex Rh3 displayed similar cytotoxic activity as Rh2, while\nexperimental results were consistent with that determined by the other complexes Rh4, Rh6, Rh7, and Rh10 revealed poor\nHPLC. cytotoxic activity, which may be related to their poor solubility\n Lipophilicity Property. The uptake of the complexes by or hydrophilicity, leading to low bioavailability in cells.51\ncells is related to their lipophilicity.49 To determine the Notably, when R1=R2=\u2212CH3 and R3=n-butyl (Rh5), the\nlipophilicity of the rhodium complexes, the octanol/water cytotoxicity of Rh5 was significantly enhanced, with an IC50\npartition coefficients (logPo/w) of the complexes were value of 2.0 \u00b1 0.2 \u03bcM, at least 10 times higher than that of Rh6\ndetermined (Figure S53), which suggested the ability of the (R1=R2=\u2212CH3 and R3=H).\ncomplexes to cross the cell membranes. Complexes Rh7, Rh8, As shown in Table 1, Rh1 (IC50 = 0.8 \u00b1 0.1 \u03bcM) and Rh2\nand Rh10 showed negative logPo/w values, and the other (IC50 = 1.6 \u00b1 0.3 \u03bcM) were the most cytotoxic to T-24 cells,\ncomplexes displayed positive logPo/w values, which indicated wherein the cytotoxicity of Rh1 was 13.5-fold higher than that\nthat the rhodium complexes are lipophilic except for the of cisplatin. Furthermore, the toxicity of Rh1 and Rh2 to\nhydrophilic Rh7, Rh8, and Rh10. The higher lipophilicity of normal WI38 cells was relatively low, and their selectivity\ncomplexes may promote their cellular uptake and enhance factors for T-24 cells were 3.4 and 3.9, respectively (selectivity\ntheir anticancer activity. Particularly, the lipophilicity of Rh1, factor = IC50 (WI38)/IC50 (T-24), indicating that the two\nRh2, Rh9, and Rh11 were greatly improved by introducing a complexes had moderate selectivity for cancer cells. Therefore,\nmethyl or a trifluoromethyl group on the pyrazole ring of the the biological mechanism of the antiproliferative activity of\nligand,50 whose logPo/w values were 0.63 \u00b1 0.10, 0.62 \u00b1 0.11, Rh1 and Rh2 on T-24 cancer cells was studied.\n0.70 \u00b1 0.06, and 0.55 \u00b1 0.06, respectively. The time-dependence of Rh1 and Rh2 on the cytotoxic\n Cytotoxicity of Rhodium Complexes In Vitro. The IC50 activity against T-24 cancer cells was assayed by measuring the\nvalues of Rh1\u2212Rh11 to five cancer cell lines (BEL-7404, IC50 values of the complexes after 24 and 48 h treatments\nHeLa, A549, T-24, and SK-OV-3) and a normal human (Table 2). It showed that the IC50 values of Rh1 against T-24\nembryonic lung cell line WI38 are listed in Table 1. The\nactivity of Rh1\u2212Rh11 against cancer cells (IC50 range of 0.8\u2212 Table 2. IC50 (\u03bcM) of Rh1 and Rh2 after Different Times of\n20 \u03bcM) was significantly higher than that of the metal salt Treatment of T-24 Cells Determined by the MTT Method\nRhCl3\u00b73H2O and the corresponding ligand (both showed IC50 complex IC50 after 24 h treatment IC50 after 48 h treatment\ngreater than 40 \u03bcM), indicating that there was a significant Rh1 1.7 \u00b1 0.3 0.8 \u00b1 0.1\nsynergistic effect after the ligand coordinated with the Rh(III) Rh2 2.5 \u00b1 0.4 1.6 \u00b1 0.3\nion.50 The rhodium complexes showed a similar trend in IC50\nvalues for all tested cancer cells, especially T-24 cells. The\norder of cytotoxic activity was Rh1 > Rh2 > Rh5 > Rh11 > cells were 1.7 \u00b1 0.3 and 0.8 \u00b1 0.1 \u03bcM after 24 and 48 h, while\nRh3 > Rh9 > Rh8 > Rh10 > Rh7 > Rh4 > Rh6. The the values of Rh2 were 2.5 \u00b1 0.4 and 1.6 \u00b1 0.3 \u03bcM,\nanticancer activity of the complexes against T-24 was analyzed respectively, indicating that the cytotoxic activity of the\nbased on the structure: (1) When R2\ufffd\u2212CF3, R3\ufffdH and R1 complexes was treatment time-dependent. For convenience\nsubstituents showed the following order of cytotoxic activity: and consistency, the most subsequent mechanism experiments\nRh1 (\u2212Ph) > Rh8 (4-Cl-Ph)> Rh9 (4-OCH3\u2212Ph) > Rh4 adopted 24 h treatment.\n(\u2212CF3). (2) When R2\ufffd\u2212CH3, R3\ufffdH and R1 substituents Effect on Cell Viability. Cell viability under different\nshowed the following order of cytotoxic activity: Rh2 (\u2212Ph)> treatments was further identified using an image format. Living\n 9595 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 3. T-24 cancer cells incubated with Rh1, Rh2, and DDP (24 h). (A) Comet assay performed by fluorescence microscopy with EB staining\n(with \u00d7400 magnification of the fluorescence microscope). (B) Tail length of the cells in each treated group measured by the comet assay. (C)\nChanges of c-PARP and \u03b3-H2AX in cells treated with Rh1, Rh2, and DDP analyzed by western blot. (D) Histograms displaying the density ratios\nof c-PARP and \u03b3-H2AX. Effects of the cell cycle incubated with Rh1 (0.8, 1.2, and 1.6 \u03bcM) (E) and Rh2 (1.6, 2.4, and 3.2 \u03bcM) (F). (G) Western\nblot assay of the change of the related proteins of the cell cycle. (H) Histograms displaying the density ratios of cell cycle-related proteins.\n\ncells were stained with calcein AM, showing green and Table S5). After 10 h of incubation with Rh1 and Rh2\nfluorescence, while dead cells were stained with propidium (8.0 \u03bcM), the total rhodium accumulation in the cells was\niodide (PI), showing red fluorescence,52 to observe the cell dramatically increased compared with the blank one. Rh1 (Rh\nviability after different treatments in T-24 cells (Figure S54). content increased from 6.300 \u00b1 0.929 to 625.166 \u00b1 78.970\nWith the increasing concentrations of Rh1 and Rh2, the ng/106 cells) showed more dramatic increase than Rh2 (Rh\ndouble staining (merge of calcein and PI) showed a significant content increased from 6.300 \u00b1 0.929 to 379.730 \u00b1 45.860\ndifference in fluorescence staining between living (green ng/106 cells), indicating that Rh1 containing fluorine atoms\nfluorescence) and dead cells (red fluorescence); the number was more readily absorbed by cancer cells.19\nof dead cells marked in red fluorescence increased significantly. The distribution of Rh in T-24 cells including cytoplasm,\nThe colony formation assays were also used to investigate the\n membrane, mitochondria, and nucleus fractions was detected\nantiproliferation activity of the rhodium complexes, which\n after treatment with Rh1 and Rh2. The highest Rh content was\nshowed that cell viability was significantly inhibited by\nincreasing the dose of Rh1 and Rh2 (Figure S55). Especially, found in the cytoplasm, accounting for 73.82 and 44.17% of\nwhen the cells were treated with 0.8 \u03bcM Rh1, almost no living the total uptake of Rh1 and Rh2 by cells, respectively. The\ncells were observed. These results showed that the two contents of Rh in the cell membrane, mitochondria, and\ncomplexes could notably inhibit the activity of T-24 cells, nucleus were similar, which accounted for 10.40, 8.78, and\nwhich was consistent with the MTT assay results. 5.46% of the total Rh1, respectively, while the content of Rh in\n Uptake and Distribution. The cell uptake of rhodium these three organelles accounted for 16.09, 11.04, and 5.03% of\ncomplex was closely related to its antitumor activity and the total Rh2, respectively. These results showed that the two\nmechanisms.53 The uptake and distribution of Rh1 and Rh2 in complexes entered tumor cells after 10 h of incubation, and\nT-24 cells were detected by the ICP-MS method (Figure S56 their contents were different in each organelle fraction.\n 9596 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 4. Hoechst 33258 staining of T-24 cancer cells incubated with Rh1 (A, B) and Rh2 (C, D) (24 h). Apoptosis of T-24 cancer cells induced\nby Rh1 (E) and Rh2 (G). Representative images and quantitative displays of apoptosis by Rh1 (F) and Rh2 (H).\n\n DNA Damage and Cell Cycle Arrest. Since DNA is the without calf thymus DNA (ct-DNA), the maximum absorption\nmain target by the metal complexes,54 the effects of the peak of Rh1 was observed at 272 nm, and that of Rh2 was 278\ncomplexes on DNA was first studied. Comet assay was used to nm, which exhibited approximately 79.9 and 60.9% hypo-\ndetect DNA damage by Rh1, Rh2, and cisplatin (DDP) chromism (shown by the black arrow) with the increase of ct-\ntreatment in T-24 cancer cells (Figure 3). The cells in the DNA concentration, respectively. According to the fitting\ncontrol one were round in shape, but the cells showed comet- calculation of UV\u2212vis spectral data, the binding constants of\nlike tails after incubation for 24 h with different concentrations the two complexes were similar (Kb = 1.12 \u00d7 105 M\u22121 and Kb =\nof the rhodium(III) complexes (Figure 3A,B). DNA damage 1.09 \u00d7 105 M\u22121, respectively), indicating that Rh1 and Rh2\nwas especially evident after treatment with Rh1 (1.6 \u03bcM), have a high DNA-binding affinity.20,28 The maximum\nwhich indicated that the complexes caused DNA fragmentation absorption peaks of the two complexes did not shift\nand might further induce cell apoptosis. significantly (Figure S57A,B). Moreover, the fluorescence\n PARP-1 is widely recognized as a first-line molecule in DNA intensity of ethidium bromide-DNA (EB-DNA) significantly\ndamage response.55 H2AX (\u03b3-H2AX) is essential for DNA decreased with the increasing concentration of both the\nrepair after checkpoint-mediated cell cycle arrest and DNA rhodium(III) complexes, suggesting that the rhodium(III)\ndouble-strand break.49 To further verify that Rh1 and Rh2 complex effectively competed with EB molecules for insertion\ninduced DNA damage, western blot analysis was employed to sites on ct-DNA by substituting EB molecules (Figure\nassess the expression changes of biomarkers connected with S57C,D). These results indicated that Rh1 and Rh2 had\nDNA damage. After the incubation of T-24 cancer cells with strong interaction with DNA at a lower concentration, and\nthe rhodium(III) complexes for 48 h, the cleavage of PARP-1 they also caused damage to DNA when internalized in cells, as\n(c-PARP) and \u03b3-H2AX protein expressions was significantly verified by the alkaline comet assay,20,56 consistent with those\nincreased (Figure 3C,D), suggesting that the rhodium(III) of the abovementioned cellular experiments.\ncomplexes killed cells by destroying DNA and that Rh1 and Effects of Rh1 and Rh2 on the cell cycle arrest of T-24 cells\nRh2 were potential DNA-damaging agents. Moreover, cleavage were investigated by flow cytometry analysis (Figure 3E,F).\nof PARP (c-PARP) is known as a hallmark of apoptosis, which The results indicated that the G2/M phase distribution of\nwill be further investigated in subsequent studies. tumor cells increased significantly in a concentration-depend-\n Furthermore, to determine whether Rh1 and Rh2 interact ent manner of Rh1 and Rh2, which varied from 18.32 to 43.97\nwith DNA, several methods were used to investigate their and 30.26%, respectively, while the proportion of S and G1\nability to bind to DNA. Relative to the rhodium(III) complex phase cells decreased simultaneously. These results reflected\n 9597 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 5. Detection of mPTP of T-24 cancer cells treated with Rh1 and Rh2 (A\u2212C). ROS determination of cells after incubation with Rh1 (D)\nand Rh2 (E) was analyzed (12 h). Analysis of Ca2+ release in the cells upon treatment with Rh1 (F) and Rh2 (G) after 24 h. Influence of Rh1 (H)\nand Rh2 (I) on the MMP of T-24 cancer cells (24 h).\n\nthat the inhibition of Rh1 and Rh2 on T-24 cells might be tion,15 and the expression level of cyclin B1 was gradually\nthrough blocking the synthesis of cell DNA, causing G2/M downregulated with increasing concentrations of the two\nphase cell cycle arrest, thereby inhibiting cell proliferation.57 complexes compared with the control one, resulting in G2/M\n The changes in G2-related proteins in T-24 cancer cells\n phase arrest. CDK1, a cyclin-dependent kinase, was down-\nincubated with Rh1 and Rh2 for 48 h were studied by western\nblot (Figure 3G,H). Cyclin B, as a G2 phase-related protein, regulated significantly and maintained in a nonactivated state,\nactivates and forms complex with cyclin-dependent protein thereby blocking mitotic initiation, decreasing the expression\nkinases (CDKs) to promote G2/M phase cell transforma- level of CDC25C, and inhibiting the dephosphorylation of\n 9598 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nmitotic kinase CDK1/cyclin B1. These results showed that Effects on the Mitochondrial Membrane Potential.\nRh1 and Rh2 arrested the T-24 cell cycle in the G2/M phase. The effects of Rh1 (0.8, 1.2, and 1.6 \u03bcM) and Rh2 (1.6, 2.4,\n Apoptosis Induction. Apoptosis is a fundamental and and 3.2 \u03bcM) on the mitochondrial membrane potential\ninherent biological process that maintains the balance between (MMP) of T-24 cells were also examined (Figure 5H,I).\ncell proliferation and death.58 Hoechst 33258 staining is Compared with the control one, with the concentration of Rh1\ncommonly used to identify apoptotic cell DNA.59 To observe and Rh2 increasing to 2 \u00d7 IC50, the mitochondrial membrane\nthe effects of Rh1 and Rh2 on cell apoptosis, the treated cells potential decreased from 3.86 to 50.4 and 39.0%, respectively.\nwere stained and detected by fluorescence microscopy (Figure It could be concluded that both complexes significantly\n4A\u2212D), which showed that the apoptosis rates of T-24 cells reduced the membrane potential of T-24 cells and activated\nwere significantly increased after incubation with Rh1 and the caspase cascade, finally leading to cell apoptosis.\nRh2. The cells in the treated group showed typical apoptotic Activation of Caspase-3, Caspase-8, and Caspase-9.\ncell morphological changes, including bright blue fluorescence, After cytochrome C (Cytc) is released by mitochondria,\nnuclear chromatin presenting a crescent shape, and shrunken caspase-9 forms a complex with Cytc and Apaf-1 and is\nnuclear membrane, in a concentration-dependent manner. simultaneously activated. Activated caspase-9 can activate\n Quantitative measurement of Rh1 and Rh2 induced caspase-3, the most vital enzyme for cell apoptosis, and then\napoptosis by flow cytometry (Figure 4E\u2212H). With the promote the subsequent apoptotic signaling pathway.\nincrease of the concentration of the rhodium(III) complexes, Activation of caspase-3, caspase-8, and caspase-9 is important\nthe percentage of cell apoptosis (Q2 + Q3) increased. When for the induction of apoptosis.66 Figure S59 shows the\nthe concentration of Rh1 was 0.8 and 1.6 \u03bcM, the proportion activation of caspase-3, caspase-8, and caspase-9 by Rh1 and\nof apoptotic cells was 28.0 and 44.3%, respectively. A similar Rh2 in T-24 cells after incubation for 24 h. Compared with the\neffect was observed for Rh2. When treated with Rh2 (3.2 \u03bcM), blank one, the activity of caspase-3 and caspase-9 was\nthe apoptosis rate of T-24 cancer cells changed from 2.56 to obviously increased in the dose-supplemented group, while\n38.2%. the change of caspase-8 was not obvious. The results indicated\n Mitochondria-Mediated Apoptotic Pathway. Mito- that Rh1 and Rh2 distinctly activated caspase-3 and caspase-9\nchondria are the bioenergetic and metabolic centers of cells, of T-24 cancer cells to induce apoptosis. Therefore, we\nregulating intracellular homeostasis by controlling calcium speculated that the main pathway of the two complexes\nsignaling, energy production, and cellular metabolism, and inducing apoptosis of T-24 cancer cells was through the\nmitochondrial damage may lead to apoptosis,60 which is caspase-dependent pathway.38\ncharacterized by the opening of mitochondrial permeability In the mitochondria-mediated apoptosis pathway, Bcl-2 and\ntransition pore (mPTP), elevated ROS, released Ca2+, caspase family proteins regulate cell apoptosis and promote the\ndecreased MMP, and activation of caspase-3, caspase-8, and release of cytochrome c to activate the caspase cascade and\ncaspase-9.61 induce cell death.67 To further examine the molecular\n Effects on mPTP. The opening of mPTP is a vital event mechanism underlying the action of the compounds against\nthat causes cell death.62 To elucidate the underlying T-24 cancer cells, the change of apoptosis-related proteins was\nmechanism of complex-induced apoptosis, we explored its measured by western blot (Figure 6A,B). The measured\npossible effects on mitochondrial functions. The detection of\nmPTP indicated that Rh1 and Rh2 sharply increased the\nopening of mPTP (Figure 5A\u2212C), which might trigger ROS\nimbalance and calcium homeostasis disruption, resulting in\nmitochondrial dysfunction.62\n Effects on ROS. The release of ROS after the treatment of\nT-24 cancer cells with Rh1 and Rh2 for 12 h was detected by\nfluorescence microscopy after staining with the ROS (H2O2)\nprobe DCFH-DA. The cells, complex-treated, showed\nenhanced green fluorescence with the increase of the\nconcentration of complexes compared with the blank one Figure 6. Change of apoptosis-related proteins after incubation with\n(Figure 5D,E), suggesting that the release of ROS increased Rh1 and Rh2 in T-24 cells (48 h) detected by western blot (A).\nsignificantly. Furthermore, the cellular content of H2O2 was Histograms displaying the density ratios of the related proteins of\n apoptosis (B).\nquantified by a hydrogen peroxide detection kit (Figure S58).\nThe results showed that both Rh1 and Rh2 produced more\nH2O2 at the same concentration (1.6 \u03bcM) than did the control apoptotic proteins included Apaf-1, Bax, Bak, Bcl-2, and Cytc.\ngroup, and the effect of Rh1 was more obvious. These results As shown, compared with the control group, the expression\nindicated that Rh1 and Rh2 effectively caused the increase of level of anti-apoptotic protein Bcl-2 was decreased, while the\nROS in T-24 cells, thus triggering cell apoptosis.63,64 expression level of proteins Apaf-1, Bax, Bak, and Cytc were\n Effects on the Intracellular Ca2+ Levels. Ca2+, as a increased, leading to MMP disruption and the release of Cytc\nsecond messenger or death signal transduction molecule, into the cytoplasm to initiate the mitochondria-mediated\nmediates apoptosis through the activation of apoptosis-related pathway of apoptosis.68 Cytoplasmic Cytc activates the\nprotein kinases and nucleases.65 Figure 5F,G shows that initiator caspase-9 and the subsequent downstream effector\ncompared with the blank one (red peak), the intracellular Ca2+ caspase-3, which further cleaves PARP causing apoptosis.\nlevel was increased after treatment with Rh1 and Rh2. With Cleavage of PARP-1 by caspases is considered a hallmark of\nthe increase of the drug concentration, the intracellular Ca2+ apoptosis.69 It is worth mentioning that the effects of Rh1 and\nlevel increased significantly, indicating that Rh1 and Rh2 Rh2 on related proteins were more obvious than those of\neffectively caused the increase of Ca2+ in T-24 cancer cells. cisplatin.\n 9599 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n Triggering Cell Autophagy. Autophagy degrades and marker of autophagosome.72 Further evidence of autophagy\nrecycles damaged or aged proteins and organelles and is a was manifested by western blot (Figure 7C,D). It was found\ndynamic cellular pathway. It is closely associated with cancer that the expression of autophagy markers LC3-II and Beclin 1\npromotion and prevention. Mitochondrial damage can induce was upregulated. Meanwhile, the level of autophagy substrate\nautophagy or mitochondrial autophagy.70 The autophagy p62/SQSTM1 was downregulated by the treatment with Rh1\ndetection kit was adopted to measure the autophagy of T-24 and Rh2, indicating that the protein was degraded in\ncells induced by Rh1 and Rh2. Brighter blue spots were autophagosomes. These results confirmed that Rh1 and Rh2\nproduced on autophagic vesicles in the complex-treated group significantly triggered autophagy in T-24 cancer cells.\n(Figure 7A), which indicated that Rh1 and Rh2 caused Our study on the anticancer mechanism of Rh1 and Rh2\nautophagy in T-24 cancer cells. demonstrated that cycle arrest caused by DNA damage and\n mitochondrial damage induced by ROS accumulation are the\n main causes of apoptosis and autophagy.\n In Vivo Anticancer Activity in Xenograft Tumor\n Model. The tumor suppression activity by Rh1 and Rh2 in\n vivo was investigated in nude mice (Balb/c) inoculated with T-\n 24 cancer cells. The complexes were dosed via intravenous\n administration in the tail. The results showed that the tumor\n volume after incubation with Rh1 at 7.5 and 15.0 mg/kg\n decreased, compared with the control one on the 15th day and\n 11th day, respectively (Figure 8). The tumor volume after\n\n\n\n\n Figure 8. T-24 tumor growth suppressed by Rh1 and Rh2 in vivo.\n (A) Photographs of tumors isolated from the mice. (B) Weight of\n tumor was monitored. (C) Volume of tumors treated with Rh1 and\nFigure 7. Autophagy determined in the T-24 cancer cells (A and B). Rh2. (D) Body weight of the mice (mean tumor volume (mm3) \u00b1 SD\nRepresentative TEM images of T-24 cancer cells treated with Rh1, (n = 6).\nRh2, and DDP for 48 h (C and D). Changes of the autophagy-related\nproteins after incubation with Rh1 and Rh2 were analyzed by western\nblot (48 h) (E). Histograms displaying the density ratios of treatment with Rh2 at 7.5, 15.0, and 30.0 mg/kg decreased\nautophagy-related proteins (F). significantly on the 15th, 11th, and 9th day compared with the\n control one, with significant statistical differences. The volume\n of tumor in the cisplatin-treated area was significantly\n As an indicator of autophagy, vacuoles of autophagosomes decreased on the 5th day after administration. After 15 days\ncan be observed in TEM images after treatment with Rh1 (0.8 of drug treatment, the inhibitory rates of the different groups\nand 1.6 \u03bcM) and Rh2 (1.6 and 3.2 \u03bcM) for 48 h (Figure 7B), were 27.7, 44.7, 31.2, 49.4, and 54.2%, respectively (Figure\nand the number of vacuoles of autophagosomes rose with the 8A,B). These results suggested that the two complexes\nincrease in the complex concentration. Particularly, more inhibited the growth of the human bladder cancer T-24\nvacuoles were formed after treatment with Rh1 and Rh2 than xenograft model and that Rh2 has superior anticancer activity\nwith cisplatin at the same concentration (1.6 \u03bcM). to Rh1 (Figure 8C). At the same time, no obvious difference\n The effect of autophagy on promoting tumor death is usually was observed in the body weight of nude mice treated with\nthrough autophagy-associated proteins, such as autophagy Rh1 and Rh2 (Figure 8D), compared with the control,\nmarkers p62 and Beclin 1, and conversion of LC3-I to LC3- indicating the in vivo safety of the complexes.\nII.71 During autophagy, LC3-I is modified and processed by a The spleen, liver, heart, kidney, lung, and tumor tissues of\nubiquitin-like system to form LC3-II, which is an important mice were collected and stained with H&E. It was observed\n 9600 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 9. Histological examination of the liver, spleen, and tumor sections in tumor-bearing mice (magnification, 200\u00d7; black stars indicate tumor\ncells; black arrows indicate anomalous cells, which resemble tumor; blue stars in liver and spleen sections indicate normal tissue and those in tumor\nsections indicate tumor necrosis; blue arrows refer to multinucleated megakaryocytes, tumor cells, and necrotic tissue junction or vascular\ncongestion and hemorrhage; and blue pentagrams refer to central veins).\n\nthat the structures of the lung, heart, and kidney of mice wound healing experiment results. Meanwhile, the level of FAK\ntreated with Rh1 and Rh2 were normal, and no drug-related protein expression did not change, but FAK phosphorylation\npathological damage was observed (Figure S60). Complex Rh2 (at the Tyr397 residue) was inhibited effectively. Integrin \u03b21\nwas slightly more toxic to the spleen, and the spleen structure and EGFR levels were obviously decreased after incubation\nwas altered and filled with a large number of tumor cells. It was with Rh1 and Rh2. These results indicated that Rh1 and Rh2\nimportant to note that the control group and the three Rh2- inhibited the metastasis of T-24 cancer cells and could be used\ntreated groups showed different degrees of tumor spread to the as antimetastasis agents.\nliver and spleen; the DDP group showed mild liver metastasis; Inhibition of Lung Metastasis In Vivo. Furthermore, the\nbut, no tumor lesions were found in the spleen and liver tissues antimetastasis activity of Rh1 and Rh2 in vivo was confirmed,\nin the Rh1-treated (15.0 mg/kg) group (Figure 9). This and mouse lung cancer cells 4T1 were injected into the tail\nobservation suggested that Rh1 (15 mg/kg) inhibited liver and vein to establish an experimental lung metastasis model.\nspleen metastasis associated with T-24 tumors, and it had low Significant metastases were observed in the control group, with\ntoxicity for liver and spleen tissues. Overall, Rh1 showed more metastatic nodules on the lung surface. However, the\npromise as a potent anticancer agent in vivo,73 which is mice treated with Rh1 and Rh2 showed a reduced number of\nconsistent with the inhibition of cell-invasive metastasis in metastatic nodules in the lungs due to the combined inhibition\nvitro. Pathological examination of tumor sections showed that of tumor growth and metastasis. Importantly, the Rh1\nthe number of inflammatory cells in each drug administration treatment is more effective (Figure 11A). H&E staining of\ngroup was significantly higher than that in the control group. the lungs further indicated that treatment with Rh1 and Rh2\nRh1 caused the most serious lesions. The different changes in significantly reduced the area of micrometastatic lesions\ncell morphology in each group are indicated by arrows or (Figure 11B,C). These results confirmed the antimetastatic\nasterisks. Pathological examination records of five organs and activity of Rh1 and Rh2 in vivo.\ntumors in T24 tumor-bearing nude are listed in Table S6.\n Inhibition of Metastasis by Rhodium Complexes. The\nmigration of cancer cells is a key step in cancer metastasis.\n \u25a0 CONCLUSIONS\n Eleven rhodium(III)-picolinamide complexes were synthesized\nThus, suppressing the migration of cancer cells is an alternative and characterized, which showed high cytotoxicity to five\nstrategy to treat cancer.74 To determine whether Rh1 and Rh2 cancer cell lines and less toxicity to WI38 normal cells.\ncan inhibit the migration of cancer cells, wound healing Mechanism studies revealed that Rh1 and Rh2 suppressed cell\n(Figure 10A\u2212D) and transwell tests (Figure 10E\u2212H) were proliferation and inhibited cancer cell metastasis via multiple\nperformed. The transwell test results showed that the two modes of action such as cell cycle arrest, apoptosis, and\nrhodium complexes significantly inhibited T-24 invasion at an autophagy. They induced DNA damage and arrested the cell\nIC50 concentration. In addition, adhesion experiments (Figure cycle in the G2/M phase and altered the expression levels of\n10I\u2212L) displayed that Rh1 and Rh2 suppressed the adhesion cycle-related proteins, caused apoptosis by opening the\nof T-24 cancer cells dose-dependently. Therefore, Rh1 and mitochondrial permeability transition pore, elevating the levels\nRh2 strongly inhibited the migration, invasion, and adhesion of of intracellular ROS and free calcium, reducing MMP, and\nT-24 cancer cells in vitro.57 activating the caspase cascade. The two complexes exhibited\n MMP-2 and MMP-9 are key biomarkers for the prognosis of efficient in vivo anticancer activity in a T-24 tumor xenograft\ncancer metastasis.74 FAK plays a crucial role in the early stages model. In addition, they showed high anticancer and\nof cell migration and adhesion; integrin \u03b21 and EGFR are antimetastasis effects in vivo. They reduced MMP-2/9-,\nclosely associated with radio resistance, mobility of cancer EGFR-, and EMT-related proteins by inhibiting the integrin-\ncells, and tumor cell growth, migration, and adhesion.57,73 To focal adhesion kinase signaling pathway, thereby inhibiting the\nelucidate the possible mechanisms of metastasis inhibition by tumor cell migration, invasion, and adhesion. Overall, the\nRh1 and Rh2, some related proteins were detected by western picolinamide-based Rh(III) complexes Rh1 and Rh2 are\nblot. As shown in Figure 10M,N, the two complexes reduced promising therapeutic agents due to their high antiproliferative\nthe levels of MMP-9 and MMP-2, indicating that Rh1 and Rh2 and antimetastatic activities against cancer cells, with low\nhave antimetastasis potential, which is consistent with the toxicity on normal cells.\n 9601 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\n Figure 11. In vivo Rh1 and Rh2 inhibited lung metastasis. (A)\n Pictures of representative lesions for 4T1 tumor metastasis on the\n lung surface from complex-treated mice on day 14. (B and C) H&E\n staining images of the lung surface tumor metastases in 14 days\u2019 old\n mice treated with Rh1 and Rh2 (magnification 50\u00d7 and 200\u00d7,\n respectively).\n\n in the presence of CH3CN/CH3OH (v:v = 4:1, 10 mL) under\n solvothermal conditions. The reactions were maintained in a 100 \u00b0C\n oven for 3 days, cooled slowly to room temperature, and filtered, and\n the filtrate was placed in a 50 mL beaker and slowly volatilized for 3\u2212\n 5 days until the crystal was precipitated. Reddish brown crystals of\n Rh1\u2212Rh11 were formed, and the structures were detected by the\n single-crystal X-ray diffraction method, HRMS spectroscopy, NMR\n spectroscopy, elemental analysis, and powder X-ray diffraction\n analysis; the characterization data of Rh1\u2212Rh11 are presented in\n the Supporting Information. The purity of all complexes was higher\n than 95%, as observed by HPLC (Figure S51).\n MMP Measurement. T-24 cancer cells were cultivated and\n incubated with the complexes; after 24 h, the cells were harvested.\nFigure 10. Scratches of T-24 cancer cells were induced with pipette The collected cells were stained with JC-1 (1.0 \u03bcg/mL) for 20 min in\ntips (10 \u03bcL). After 12 or 24 h of incubation with Rh1 (A, C) and Rh2 PBS in the dark, collected by centrifugation, and cleaned with PBS.\n(B, D), images were photographed under a phase contrast Flow cytometry was used to detect the ratio of red to green\nmicroscope. T-24 cancer cells were seeded onto chambers and fluorescence intensity of the resuspension cells in PBS.\nincubated with Rh1 (E, G) and Rh2 (F, H) (24 h). T-24 cells were Autophagy Induced by Complexes. T-24 cancer cells (2 \u00d7 105\ntreated with Rh1 (I and K) and Rh2 (J and L) in six-well plates (24 cells/well) were cultivated in plates overnight; then, Rh1 and Rh2\nh), nonadherent cells were washed off with PBS, and the adhesive were added, respectively, at the specified concentration for 24 h. The\ncells were captured under a phase contrast microscope. (M) Changes cells were collected and cleaned; the detection reagent of the cell and\nof migration-related biomarkers treated with Rh1 and Rh2 were autophagosome (1\u00d7, 500 \u03bcL) were added; incubated in an incubator\nanalyzed by western blot (48 h). (N) Histograms displaying the for 15 min; and washed. Then, the cells were observed and\ndensity ratios of migration-related proteins. photographed with a fluorescence microscope.\n TEM Images. T-24 cells (5 \u00d7 106 cells) were cultivated in a dish\n\n\u25a0 EXPERIMENTAL SECTION\nUnless otherwise indicated, all chemicals were obtained from\n (100 mm) overnight and treated with Rh1 and Rh2 (24 h). After\n removing the medium, 2.5% glutaraldehyde was added to fix the cells\n at 25 \u00b0C for about 5 min. The cells were gently scraped down in one\ncommercial companies. Other biochemical materials are listed in direction with a cell scraper, centrifuged, collected, and fixed with\nthe Supporting Information. All synthesized compounds were dried 2.5% glutaraldehyde overnight. 1% osmium was added to fix the cells\nbefore testing. The purity of all rhodium(III) complexes used in the at 4 \u00b0C for 1 h, followed by gradient dehydration, and the resin was\nexperiments was >95%, which was routinely verified by HPLC. The embedded for 24 h. The samples were cut into 60\u221280 nm thickness\nanimal experiments were approved by the ethics committee for using a Leica EM UC6 ultramicrotome and collected on an elliptical\nlaboratory animals, the Nanjing Han & Zaenker Cancer Institute, copper grid (Hatfield, PA, USA), followed by staining with 2%\nNanjing OG Pharmaceuticals, Co., Ltd. (SYXK(SU)2017-0040). The aqueous uranyl acetate and lead citrate. Subsequently, the samples\nanimal experiments were performed in accordance with the NIH were cleaned and placed under a Hitachi7500 fluoroscopic electron\nGuidelines for the Care and Use of Laboratory Animals. microscope (Philips, Amsterdam, The Netherlands) to observe the\n Synthesis of Ligands. The picolinamide ligands were prepared ultrastructural morphology and status of autophagolysosomes and\naccording to the literature method.46,75 All other reagents are lysosomes in each group of cells after drying.\ncommercially available and were used as received. The character- Acute Toxicity Study. Balb/c nude mice (half male and half\nization data of ligands are presented in the Supporting Information. female) were divided randomly into two groups, with six animals in\n Synthesis of Rhodium Complexes Rh1\u2212Rh11. The reactions each group, namely, the Rh1 (0.5 mg/mL) group and the Rh2 (1.25\nof picolinamide ligands with RhCl3\u00b73H2O (Adamas) were carried out mg/mL) group. After 1 week of adaptive feeding, each group was\n\n 9602 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\ninjected with the correspondingly prepared drug solution (94% Authors\nnormal saline, 5% DMSO, and 1% Tween-80) by a single intravenous Yun-Qiong Gu \u2212 State Key Laboratory for Chemistry and\nadministration through tail. The animals were observed, and the Molecular Engineering of Medicinal Resources, Collaborative\nnumber of deaths was recorded, while the survived animals were\nobserved for a week.\n Innovation Centre for Guangxi Ethnic Medicine, School of\n Animal Experiments. A total of 55 Balb/c nude mice (5 \u00d7 106 Chemistry and Pharmaceutical Sciences, Guangxi Normal\ncells per mouse) were inoculated subcutaneously under the right University, Guilin 541004, P. R. China; School of\naxillary with a suspension of T-24 cancer cells at the logarithmic Environment and Life Science, Nanning Normal University,\ngrowth stage. When the tumor grew to about 100 mm3, 42 tumor- Nanning 530001, P. R China\nbearing nude mice with a healthy growth status and almost the same Kun Yang \u2212 State Key Laboratory for Chemistry and\ntumor size were divided into seven groups randomly (six mice per Molecular Engineering of Medicinal Resources, Collaborative\ngroup). Control group, Rh1 (7.5 mg/kg) group, Rh1 (15.0 mg/kg) Innovation Centre for Guangxi Ethnic Medicine, School of\ngroup, Rh2 (7.5 mg/kg) group, Rh2 (15.0 mg/kg) group, Rh2 (30.0 Chemistry and Pharmaceutical Sciences, Guangxi Normal\nmg/kg) group, and DDP (2.0 mg/kg) group were dosed via tail\nintravenous administration according to the dose-setting table (0.2\n University, Guilin 541004, P. R. China\nmL per mouse, once every other day). Nude mice\u2019s body weight and Qi-Yuan Yang \u2212 State Key Laboratory for Chemistry and\ntumor diameter were measured and recorded every other day. After Molecular Engineering of Medicinal Resources, Collaborative\nadministration for 2 weeks, the nude mice were killed by neck Innovation Centre for Guangxi Ethnic Medicine, School of\ndissection. The tissues of tumor and viscera were removed and fixed Chemistry and Pharmaceutical Sciences, Guangxi Normal\nwith 10% neutral formalin. The tumor tissues were weighed, and the University, Guilin 541004, P. R. China\ntumor inhibition rate was calculated. One sample of organ and tumor Huan-Qing Li \u2212 State Key Laboratory for Chemistry and\nwas randomly selected for tissue sections. Histopathological studies Molecular Engineering of Medicinal Resources, Collaborative\nwere conducted after H&E staining. Innovation Centre for Guangxi Ethnic Medicine, School of\n Inhibition of Lung Metastasis. 4T1 cells were resuscitated in\nserum-free medium and were injected with 5 \u00d7 105 cells/0.2 mL\n Chemistry and Pharmaceutical Sciences, Guangxi Normal\nculture medium through the tail vein, and the mice were immediately University, Guilin 541004, P. R. China\ntreated with Rh1 and Rh2 at 15 or 30 mg/kg dosage. Two weeks Mei-Qi Hu \u2212 State Key Laboratory for Chemistry and\nlater, the animals were killed, and lungs were fixed in liquid nitrogen; Molecular Engineering of Medicinal Resources, Collaborative\nthen, the number of metastatic nodules on the lung surface was Innovation Centre for Guangxi Ethnic Medicine, School of\ncalculated. Chemistry and Pharmaceutical Sciences, Guangxi Normal\n Statistical Studies. To ensure reliability of data, every experiment University, Guilin 541004, P. R. China\nwas repeated three times (the control group was treated with blank Meng-Xue Ma \u2212 State Key Laboratory for Chemistry and\nsolution), and the data of the results were expressed as mean \u00b1 Molecular Engineering of Medicinal Resources, Collaborative\nstandard deviation (SD), ***P < 0.001, **P < 0.01, and *P < 0.05.\n Innovation Centre for Guangxi Ethnic Medicine, School of\n\n\u25a0\n*\n ASSOCIATED CONTENT\ns\u0131 Supporting Information\n Chemistry and Pharmaceutical Sciences, Guangxi Normal\n University, Guilin 541004, P. R. China\n Nan-Feng Chen \u2212 State Key Laboratory for Chemistry and\nThe Supporting Information is available free of charge at Molecular Engineering of Medicinal Resources, Collaborative\nhttps://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00318. Innovation Centre for Guangxi Ethnic Medicine, School of\n Chemistry and Pharmaceutical Sciences, Guangxi Normal\n HRMS, 1H NMR, and 13C NMR spectra of ligands 3a\u2212 University, Guilin 541004, P. R. China\n 3k; HRMS spectra, X-ray crystallography, HPLC, and Yang-Han Liu \u2212 State Key Laboratory for Chemistry and\n UV\u2212vis spectra of Rh1\u2212Rh11; cytotoxicity in vitro; cell Molecular Engineering of Medicinal Resources, Collaborative\n viability analysis; lipophilicity measurement; cell apop- Innovation Centre for Guangxi Ethnic Medicine, School of\n tosis; cell cycle studies; comet assay; ROS; Ca2+ of Chemistry and Pharmaceutical Sciences, Guangxi Normal\n intracellular determination; Hoechst staining; caspase 3, University, Guilin 541004, P. R. China\n caspase 8, and caspase 9 assay; migration detection; Complete contact information is available at:\n western blot; and H&E staining (PDF) https://pubs.acs.org/10.1021/acs.jmedchem.3c00318\n Molecular formula strings (CSV)\n Author Contributions\n\n\u25a0 AUTHOR INFORMATION\nCorresponding Authors\n The manuscript was written through contributions of all\n authors. All authors have given approval to the final version of\n the manuscript.\n Hong Liang \u2212 State Key Laboratory for Chemistry and\n Notes\n Molecular Engineering of Medicinal Resources, Collaborative\n Innovation Centre for Guangxi Ethnic Medicine, School of The authors declare no competing financial interest.\n Chemistry and Pharmaceutical Sciences, Guangxi Normal\n University, Guilin 541004, P. R. China; Email: hliang@\n gxnu.edu.cn\n \u25a0 ACKNOWLEDGMENTS\n This work was supported by the National Natural Science\n Zhen-Feng Chen \u2212 State Key Laboratory for Chemistry and\n Molecular Engineering of Medicinal Resources, Collaborative Foundation of China (grant no. 22077022) and Natural\n Innovation Centre for Guangxi Ethnic Medicine, School of Science Foundation of Guangxi Province of China (grant nos.\n Chemistry and Pharmaceutical Sciences, Guangxi Normal\n University, Guilin 541004, P. R. China; orcid.org/0000- GUIKEZY22096015, 2023GXNSFDA026054, and\n 0002-6548-6131; Email: chenzf@gxnu.edu.cn AD17129007).\n 9603 https://doi.org/10.1021/acs.jmedchem.3c00318\n J. Med. Chem. 2023, 66, 9592\u22129606\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\u25a0 ABBREVIATIONS\nApaf-1, apoptotic protease activating factor-1; Bax, Bcl-2-\n IgG that activates integrin-FAK signaling. Cancer Lett. 2018, 430,\n 148\u2212159.\n (13) Shen, M.; Jiang, Y. Z.; Wei, Y.; Ell, B.; Sheng, X.; Esposito, M.;\nassociated X; Bak, Bcl2, antagonist/killer; Bcl-2, B-cell Kang, J.; Hang, X.; Zheng, H.; Rowicki, M.; Zhang, L.; Shih, W. J.;\nlymphoma-2; CDC25C, cell division cycle 25C; CDKs, Celia\u0300-Terrassa, T.; Liu, Y.; Cristea, I.; Shao, Z. M.; Kang, Y. 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