Ligand-Controlled Reactivity and Cytotoxicity of Cyclometalated Rhodium(III) Complexes

We report the synthesis, characterisation and cytotoxicity of six cyclometalated rhodium(III) complexes [CpRh(C^N)Z], in which Cp = Cp*, Cp, or Cp, C^N = benzo[h]quinoline, and Z = chloride or pyridine. Three x-ray crystal structures showing the expected “piano-stool” configurations have been determined. The chlorido complexes hydrolysed faster in aqueous solution, also reacted preferentially with 9-ethyl guanine or glutathione compared to their pyridine analogues. The 1-biphenyl-2,3,4,5,-tetramethylcyclopentadienyl complex [CpRh(benzo[h]quinoline)Cl] (3a) was the most efficient catalyst in coenzyme reduced nicotinamide adenine dinucleotide (NADH) oxidation to NAD and induced an elevated level of reactive oxygen species (ROS) in A549 human lung cancer cells. The pyridine complex [CpRh(benzo[h]quinoline)py] (3b) was the most potent against A549 lung and A2780 ovarian cancer cell lines, being 5-fold more active than cisplatin towards A549 cells, and acted as a ROS scavenger. This work highlights a ligand-controlled strategy to modulate the reactivity and cytotoxicity of cyclometalated rhodium anticancer complexes. INTRODUCTION The approval of cisplatin as an anticancer drug in the late 1970s has not only led to new generations of platinum drugs in clinical use (carboplatin and oxaliplatin) or trials, but also to the search for a new era of transition metal-based anticancer agents. Group 9 metals iridium and rhodium have attracted much attention although are less widely studied than platinum. Ir and Rh complexes with low spin d configurations are usually considered to be kinetically inert, however, recent studies have revealed that their reactivity toward biological targets can be adjusted by the rational selection of the surrounding ligands directly coordinated to the metal. Kinetically stable iridium/rhodium pyridocarbazole complexes have been synthesized by Meggers and co-workers as potent enzyme inhibitors, and Sheldrick and co-workers have reported various pentamethyl-cyclopentadienyl (Cp*) iridium/rhodium complexes with chelating polypyridine ligands as potent anticancer agents. More recently, attention has turned to cyclometalated iridium and rhodium complexes in which five-membered chelate rings contain a strong M-C  bond. Nevertheless, research on cyclometalated rhodium complexes has largely focused on non-cyclopentadienyl cyclometalated rhodium complexes that target DNA, enzyme or protein-protein interfaces. Reported cyclopentadienyl rhodium anticancer complexes to date, have mainly contained N^N, N^S, N^O, or O^O chelating ligands. Our recent work has demonstrated that replacing the N^N coordinating ligand 2,2’-bipyridine or 1,10-phenanthroline with C^N chelating 2-phenylpyridne or benzo[h]quinoline in pentamethyl-cyclopentadienyl (Cp*) iridium complexes, not only enhances nucleobase binding and lipophilicity, but also switches on anticancer activity towards human A2780 ovarian cancer cells. Moreover, the chelated iridium(III) biphenyl-tetramethyl-cyclopentadienyl complex [CpIr(phenylpyridine)(pyridine)] can utilize NADH as a hydride source to transfer hydride electrons to oxygen, generating hydrogen peroxide and reactive oxygen species in cancer cells to trigger cell death. Recently, we have described rhodium anticancer complexes [CpRh(N^N’)Cl] (Cp = Cp*, Cp, or Cp) which can be effective transfer hydrogenation catalysts inducing reductive stress when co-administrated with nontoxic does of sodium formate as the hydride donor in cancer cells. Intriguingly,the Cp ring in these catalytic [CpRh(N^N’)Cl] (N^N’ is the bipyridine, dimethylbipyridine, or phenanthroline) complexes can be readily activated by deprotonation and undergo rapid deuteration in aqueous media, so providing a novel activation pathway for halfsandwich Rh(III) complexes. Based on these interesting discoveries, we have extended our studies to include cyclometalated cyclopentadienyl rhodium anticancer complexes with potential catalytic properties. Herein, six cyclometalated rhodium complexes (Scheme 1) bearing different cyclopentadienyl Cp rings with C^N chelating ligand benzo[h]quinoline have been synthesized and fully characterized by H NMR, C NMR, high resolution ESI-MS and elemental analysis. Five novel complexes 2a, 3a, 1b-3b are reported, and the X-ray crystal structures of complexes 2a, 3a and 1b·PF6 have been determined. Complexes 1a-3a contain chloride, while complexes 1b-3b feature pyridine as the monodentate ligand. Their aqueous chemistry including hydrolysis, binding to nucleobase 9ethylguanine (9-EtG) or abundant cellular thiol glutathione (GSH, -L-Glu-L-Cys-Gly), as well as catalytic activity in the oxidation of coenzyme NADH are evaluated and compared. Cytotoxicity has been screened against human A549 lung and A2780 ovarian cancer cell lines. Cellular Rh accumulation, ROS induction and apoptosis in A549 cancer cells induced by complexes 3a and 3b at equipotent concentrations have been investigated to elucidate their possible mechanism of action. Scheme 1. Structures of the six cyclometalated rhodium(III) benzo[h]quinoline complexes studied in this work. RESULTS AND DISCUSSION Synthesis and Characterization The synthesis of the rhodium dimers [CpRh(μ-Cl)Cl]2 (Cp = Cp, Cp) followed the reported procedure. The rhodium chloride complexes 1a-3a were synthesized via C-H activation of benzo[h[quinoline by the rhodium dimer aided by sodium acetate in dichloromethane at ambient temperature as reported previously. In contrast, pyridine complexes 1b-3b were obtained in good yields through the reaction of the corresponding chloride complexes with silver nitrate and then with excess pyridine (Scheme 2). All complexes were characterized by H and C NMR, elemental analysis and high resolution ESI-MS. Scheme 2. Synthesis route for rhodium complexes containing chloride (1a-3a) and pyridine (1b-3b) ligands. X-ray crystal structures Crystal structures of [CpRh(benzo[h]quinoline)Cl] (2a), [CpRh(benzo[h]quinoline)Cl] (3a) and [CpRh(benzo[h]quinoline)pyridine]PF6 (1b·PF6) with PF6 as the counter anion were determined and are shown in Figure 1 with the atom numbering scheme. Crystallographic data are listed in Table S1, and selected bond lengths and angles in Table S2. All structures adopt the familiar “pianostool” geometry. The distances between Rh and the centroid of the ηcyclopentadienyl ring are within 1.827-1.832 Å. The length of the Rh-C (quinoline) bond in 2a, 3a, 1b·PF6 [2.050(3), 2.0458(15), 2.058(2), respectively] is significantly shorter than the Rh-N (quinoline) bond length [2.082(3), 2.1030(14), 2.1068(18), respectively]. Figure 1. X-ray structures with atom numbering of (a) [CpRh(benzo[h]quinoline)Cl] (2a); (b) [CpRh(benzo[h]quinoline)Cl] (3a); (c) [CpRh(benzo[h]quinoline)pyridine]PF6 (1b·PF6), drawn with thermal ellipsoids at 50% probability. Hydrogen atoms and counterions have been omitted for clarity. Aqueous solution chemistry The hydrolysis of these complexes was monitored by H NMR (Figures 2, S1, S2) as well as UV-vis spectroscopy at 310 K in methanol/water. Methanol was used to ensure the sufficient solubility of the complexes in water. All the chloride complexes showed faster hydrolysis than their pyridine counterparts. Their hydrolysis rates and half-lives of hydrolysis (listed in Table 1) were determined by fitting the UV-vis absorption changes versus time to pseudo-first-order kinetics (Figure S6). With the extension of Cp* to Cp, to Cp, the hydrolysis half-lives of the chloride complexes become shorter, while the hydrolysis half-lives of the pyridine complexes become longer. The hydrolysis extent over 24 h for chloride complexes 1a-3a was 43%-60% at equilibrium based on integration of H NMR peaks. In addition, after 24 h, an additional set of H NMR peaks was visible for complexes 1a-3a which can be attributed to release of the benzo[h]quinoline ligand (ca. 25%), as characterized by H NMR, H-H COSY and confirmed by ESI-MS with m/z 180.13 (calculated m/z 180.08) (Figure S7-S9). In contrast, after 24 h at 310 K, no apparent change was observed for millimolar solutions of the pyridine complexes 1b-3b in d4-MeOD/D2O (1/9-1/2 v/v) by H NMR (Figure S3S5), although the hydrolysis of micromolar solutions of complexes 1b-3b could be observed by UV-vis spectroscopy at 310 K (Figure S10). When 104 mM NaCl (to mimic the chloride concentration in blood plasma), was added to the 24 h equilibrium hydrolysis solution of complexes 1a (Figure 2c) and 2a (Figure S1c), peaks for the aqua adducts disappeared and those for the parent (b) (c) (a) chloride complexes increased in intensity, confirming that hydrolysis of the chloride complexes is reversible. Figure 2. 400 MHz H NMR spectra of [Cp*Rh(benzo[h]quinoline)Cl] (1a) (ca. 0.5 mM) in d4-MeOH/D2O (v/v 4/1) after (a) 15 min; (b) 24 h; and (c) 10 min after addition of NaCl (104 mM). After 15 min, 60% of the parent complex 1a (■) hydrolysed to its aqua adduct [Cp*Rh(benzo[h]quinoline)D2O] (●). After 24 h, one third of the aqua adduct [Cp*Rh(benzo[h]quinoline)D2O] had decomposed into the free chelated ligand and [Cp*Rh(D2O)3] (▲). After additon of NaCl, all the aqua adducts were coverted into 1a, with the released ligand remaining unbound and the [Cp*Rh(D2O)3] reacting with Cl to form [Cp*RhCl2]2 (▲). Table 1. Hydrolysis data for complexes 1a-3a and 1b-3b at 310 K. [a]: Determined by H NMR in d4-MeOH/D2O. [b]: At equilibrium, determined by UVvis spectroscopy in MeOH/H2O. (a) 15 min (b) 24 h (c) Addition of NaCl (104 mM)


INTRODUCTION
The approval of cisplatin as an anticancer drug in the late 1970s has not only led to new generations of platinum drugs in clinical use (carboplatin and oxaliplatin) or trials, but also to the search for a new era of transition metal-based anticancer agents. [1][2][3][4] Group 9 metals iridium and rhodium have attracted much attention although are less widely studied than platinum. [5][6][7][8] Ir III and Rh III complexes with low spin d 6 configurations are usually considered to be kinetically inert, however, recent studies have revealed that their reactivity toward biological targets can be adjusted by the rational selection of the surrounding ligands directly coordinated to the metal. [6,9] Kinetically stable iridium/rhodium pyridocarbazole complexes have been synthesized by Meggers and co-workers as potent enzyme inhibitors, [10][11][12][13] and Sheldrick and co-workers have reported various pentamethyl-cyclopentadienyl (Cp*) iridium/rhodium complexes with chelating polypyridine ligands as potent anticancer agents. [14] More recently, attention has turned to cyclometalated iridium and rhodium complexes in which five-membered chelate rings contain a strong M-C  bond. [15,16] Nevertheless, research on cyclometalated rhodium complexes has largely focused on non-cyclopentadienyl cyclometalated rhodium complexes that target DNA, enzyme or protein-protein interfaces. [5,9,[17][18][19][20] Reported cyclopentadienyl rhodium anticancer complexes to date, have mainly contained N^N, [21,22] N^S, [23] N^O, [24][25][26] or O^O [22,27,28] chelating ligands.
Our recent work has demonstrated that replacing the N^N coordinating ligand 2,2'-bipyridine or 1,10-phenanthroline with C^N chelating 2-phenylpyridne or benzo[h]quinoline in pentamethyl-cyclopentadienyl (Cp*) iridium complexes, not only enhances nucleobase binding and lipophilicity, but also switches on anticancer activity towards human A2780 ovarian cancer cells. [29,30] Moreover, the chelated iridium(III) biphenyl-tetramethyl-cyclopentadienyl complex [Cp biph Ir(phenylpyridine)(pyridine)] + can utilize NADH as a hydride source to transfer hydride electrons to oxygen, generating hydrogen peroxide and reactive oxygen species in cancer cells to trigger cell death. [31] Recently, we have described rhodium anticancer complexes [Cp X Rh(N^N')Cl] + (Cp x = Cp*, Cp ph , or Cp biph ) which can be effective transfer hydrogenation catalysts inducing reductive stress when co-administrated with nontoxic does of sodium formate as the hydride donor in cancer cells. [32] Intriguingly，the Cp X ring in these catalytic [Cp X Rh(N^N')Cl] + (N^N' is the bipyridine, dimethylbipyridine, or phenanthroline) complexes can be readily activated by deprotonation and undergo rapid deuteration in aqueous media, so providing a novel activation pathway for halfsandwich Rh(III) complexes. [33] Based on these interesting discoveries, we have extended our studies to include cyclometalated cyclopentadienyl rhodium anticancer complexes with potential catalytic properties.
Herein, six cyclometalated rhodium complexes (Scheme 1) bearing different cyclopentadienyl Cp X rings with C^N chelating ligand benzo[h]quinoline have been synthesized and fully characterized by 1 H NMR, 13

Synthesis and Characterization
The synthesis of the rhodium dimers [Cp X Rh(µ-Cl)Cl]2 (Cp X = Cp ph , Cp biph ) followed the reported procedure. [32,34] The rhodium chloride complexes 1a-3a were synthesized via C-H activation of benzo[h[quinoline by the rhodium dimer aided by sodium acetate in dichloromethane at ambient temperature as reported previously. [35] In contrast, pyridine complexes 1b-3b were obtained in good yields through the reaction of the corresponding chloride complexes with silver nitrate and then with excess pyridine (Scheme 2). All complexes were characterized by 1 H and 13 C NMR, elemental analysis and high resolution ESI-MS. Scheme 2. Synthesis route for rhodium complexes containing chloride (1a-3a) and pyridine (1b-3b) ligands.

Aqueous solution chemistry
The hydrolysis of these complexes was monitored by 1 H NMR (Figures 2, S1, S2) as well as UV-vis spectroscopy at 310 K in methanol/water. Methanol was used to ensure the sufficient solubility of the complexes in water. All the chloride complexes showed faster hydrolysis than their pyridine counterparts. Their hydrolysis rates and half-lives of hydrolysis (listed in Table 1) were determined by fitting the UV-vis absorption changes versus time to pseudo-first-order kinetics ( Figure S6). With the extension of
The new singlet peak for H8 of the bound 9-EtG appeared at 7.39 ppm, shifted by 0.37 ppm to high field relative to that of free 9-EtG. The ESI-MS of the final NMR solution showed a major peak at m/z 595.3, assignable as the adduct of 1a with 9-EtG [Cp*(Rh-

NADH oxidation
NADH is a crucial coenzyme in numerous biological catalytic processes. Previously, we have found that cyclopentadienyl iridium complexes bearing N^N coordinating ligands can accept the hydride from NADH and induce the reduction of protons to H2, [36] and quinones to semiquinones. [37] The 1-biphenyl-2,3,4,5,tetramethylcyclopentadienyl (Cp biph ) iridium complex with the C^N chelating 2phenylpyridine can also transfer hydride from NADH to oxygen to produce the reactive oxygen species (ROS) hydrogen peroxide. [31] Therefore, the time dependence of reactions between rhodium complexes (0.8 µM) and NADH (75-144 µM) was studied over 24 h in 1.6% MeOH/98.4% phosphate buffer (5 mM, pH 7.4) by UV-vis spectroscopy at 310 K ( Figure S13, S14). The reactions proceeded via first-order kinetics ( Figure S15).
The turnover number (TON) and turnover frequency (TOF) were determined based on the decrease in absorption of NADH at 339 nm due to conversion of NADH to NAD + . In reactions with NADH at a higher concentration (144 µM), pyridine complexes 1b-3b showed a much lower TOF than their respective chloride analogues 1a-3a (Table 2). This might due to the decrease in hydrolysis of pyridine complexes compared to chloride analogues, as hydrolysis is believed to be the first step to interact with NADH. [31] In  Figure S16).  [38] for the same Rh-H species. Meanwhile, a triplet at -19.34 ppm (J = 24 Hz) was also observed in Figure S17. The J value of this triplet is characteristic of hydride bridging two Rh(III) centres, [39] suggesting a possible nucleophilic attack of the Rh-H  Figure S18).

Reactivity with GSH
The From the study of cyclopentadienyl iridium complexes with the C^N chelating ligand 2-phenylpyridine, the extension of Cp X (from Cp* to Cp ph to Cp biph ) greatly enhanced the anticancer activity perhaps due to the increased hydrophobicity facilitating passage through the cell membrane or to the extended phenyl or biphenyl ring which can intercalate between DNA bases. [40] Among the Cp X analogues, Cp biph capped complexes 3a and 3b were the most hydrophobic and exhibited the highest potency as expected. Moreover, pyridine complex 3b was ca. 2 x and ca. 10 x more potent than its chloride analogue 3a towards A2780 and A549 cancer cells, respectively (A2780: IC50 0.88 µM for 3b and 1.60 µM for 3a; A549: IC50 0.74 µM for 3b and 7.69 µM for 3a). Therefore, despite the replacement of the chloride with pyridine decreasing hydrolysis, inhibiting adduct formation with 9-EtG/GSH and lowering the catalytic TOF, the in vitro anticancer activity of pyridine complexes was higher than that of chloride analogues, in line with the behaviour of cyclometalated iridium pyridine complexes which showed slower hydrolysis/less interaction with biomolecules, but higher potency than their chloride analogues. [31]

Complexes [Cp biph Rh(benzo[h]quinoline)Cl] (3a) and [Cp biph Rh(benzo[h]quinoline)-
py]NO3 (3b) bearing the Cp biph ring, were the most potent candidates among the chloride family 1a-3a and pyridine family 1b-3b. However, complex 3b is positively charged and 10 x more potent than neutral complex 3a toward A549 cancer cells ( Figure 5). Thus, to elucidate their different anticancer activities, the cellular accumulation of rhodium in A549 human lung cancer cells after 24 h treatment with complexes 3a and 3b at equipotent concentrations of 0.5 x and 1 x IC50 at 310 K was quantified by ICP-MS as shown in Figure 6 (values in Table S5). From Figure 6, the accumulation of Rh in the cells treated with complex 3a or 3b increased in a

ROS induction in A549 cancer cells
The chloride complexes 1a-3a exhibited higher catalytic efficiency than pyridine analogues in the oxidation of NADH to NAD + with production of hydrogen peroxide. -py]NO3 (3b) at concentrations of IC50 or 2 x IC50 by flow cytometry fluorescence analysis using a total ROS/Superoxide detection kit (Figure 7, details in Table S6).
This assay not only allowed determination of the level of total ROS consisting of H2O2,  standard deviations for three independent samples. The asterisk denotes the p-values obtained by comparing each dataset with the negative control (untreated cells) using a t-test, *p < 0.05, **p < 0.01, a p > 0.05.

Apoptosis assay
Apoptosis is well recognized as a distinct cell death mechanism in tumours responding to anticancer therapies. [41] To investigate whether apoptosis is involved in the  Figure S20).
However, there was a significant increase in necrotic cell population induced by the treatment with complex 3b at 2 x IC50 concentration (Table S7).

CONCLUSIONS
In summary, we have described the synthesis and characterization of novel cyclopentadienyl C^N chelated Rh III anticancer complexes. The reactivity and cytotoxicity of these complexes can be reasonably modulated by selection of the monodentate ligand as chloride or pyridine. The hydrolysis rate of chloride complexes increases in the order Cp*< Cp ph < Cp biph , showing that incorporation of the extended Cp X ring confers more labile kinetics on the monodentate chloride ligand. On the contrary, when the chloride is replaced with pyridine, the rate of hydrolysis is slowed down by orders of magnitude and decreases in the order Cp biph > Cp ph > Cp*. This difference in hydrolysis kinetics for the chloride and pyridine complexes leads to the differences in reactivity, and subsequent differences in cytotoxicity.
The pyridine complex 3b reacts more slowly with glutathione than the chloride analogue, resulting in less deactivation and an order of magnitude greater potency towards lung cancer cells. Meanwhile, complex 3b accumulates to a much lesser extent than the chloride analogue in cancer cells at equipotent IC50 concentrations, indicating that complex 3b requires a lower dose than 3a to achieve the same therapeutic potency. Significantly, the rhodium complexes studied here exhibit a different mechanism of action from cisplatin, which acts through interaction with DNA.
The chloride complex 3a shows the greatest catalytic efficiency in NADH oxidation and induces a remarkable increase in the level of ROS in lung cancer cells. Whereas the pyridine complex 3b can act as a ROS scavenger, which is distinct from the related pyridine iridium complex [Cp biph Ir(2-phenylpyridine)py] + which induces a higher level of ROS in ovarian cancer cells. [31] Although the reason of the ROS scavenging activity is not clear, pyridine complex 3b is the first example of an organometallic rhodium complex with such a behaviour. Cyclometalated rhodium complexes such as these may provide a new generation of transition metal-based chemotherapeutic agents and are worthy of further investigation.

Synthesis of [Cp X Rh(benzo[h]quinoline)Cl] (2a and 3a)
General procedure: [Cp X  After this, supernatants were removed by suction and each well was washed with 100 µL PBS. The cells were allowed a further 72 h recovery in free fresh medium at 310 K. SRB assay was used to determine cell viability. [45] Absorbance Welch's t-tests were carried out to establish statistical significance of the variations.