Ruthenium-based PACT agents based on bisquinoline chelates: synthesis, photochemistry, and cytotoxicity

The known ruthenium complex [Ru(tpy)(bpy)(Hmte)](PF6)2 ([1](PF6)2, where tpy = 2,2’:6’,2″-terpyridine, bpy = 2,2’-bipyridine, Hmte = 2-(methylthio)ethanol) is photosubstitutionally active but non-toxic to cancer cells even upon light irradiation. In this work, the two analogs complexes [Ru(tpy)(NN)(Hmte)](PF6)2, where NN = 3,3'-biisoquinoline (i-biq, [2](PF6)2) and di(isoquinolin-3-yl)amine (i-Hdiqa, [3](PF6)2), were synthesized and their photochemistry and phototoxicity evaluated to assess their suitability as photoactivated chemotherapy (PACT) agents. The increase of the aromatic surface of [2](PF6)2 and [3](PF6)2, compared to [1](PF6)2, leads to higher lipophilicity and higher cellular uptake for the former complexes. Such improved uptake is directly correlated to the cytotoxicity of these compounds in the dark: while [2](PF6)2 and [3](PF6)2 showed low EC50 values in human cancer cells, [1](PF6)2 is not cytotoxic due to poor cellular uptake. While stable in the dark, all complexes substituted the protecting thioether ligand upon light irradiation (520 nm), with the highest photosubstitution quantum yield found for [3](PF6)2 (Φ[3] = 0.070). Compounds [2](PF6)2 and [3](PF6)2 were found both more cytotoxic after light activation than in the dark, with a photo index of 4. Considering the very low singlet oxygen quantum yields of these compounds, and the lack of cytotoxicity of the photoreleased Hmte thioether ligand, it can be concluded that the toxicity observed after light activation is due to the photoreleased aqua complexes [Ru(tpy)(NN)(OH2)]2+, and thus that [2](PF6)2 and [3](PF6)2 are promising PACT candidates. Graphic abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00775-021-01882-8.


Introduction
In recent years, ruthenium polypyridyl complexes gained attention in the field of phototherapy for their favorable photophysical and photochemical properties [1]. Drug activation by light irradiation at the tumor site provides physical selectivity towards cancerous tissues and minimizes the effect of the drug on the healthy, non-irradiated tissues. Therefore, undesired side effects are expected to be reduced. Two different types of phototherapy are distinguished: photodynamic therapy (PDT) and photoactivated chemotherapy (PACT). In both cases, a molecule is promoted to a singlet metal-to-ligand charge transfer excited state ( 1 MLCT) by photon absorption. From there, the molecule undergoes intersystem crossing (ISC) to a triplet metal-to-ligand charge transfer excited state ( 3 MLCT). This 3 MLCT state can be deactivated via four different pathways: non-radiative deactivation, emission of a photon, energy transfer to molecular oxygen to generate singlet oxygen ( 1 O 2 ), or thermal population of a low-lying triplet metal-centered excited state ( 3 MC), which leads to ligand photosubstitution [1][2][3][4][5][6][7]. In PDT, the production of 1 O 2 leads to serious oxidative damage of the cells, culminating in cell death. In PACT, on the other hand, the prodrug, which is usually poorly toxic in the dark, is activated by ligand photosubstitution [6,[8][9][10][11][12]. The activated drug becomes capable of interacting with biomolecules, causing cell death in an oxygen-independent way [7,10,[13][14][15][16]. Since thermal promotion from the photochemically generated 3 MLCT state into the photosubstitutionally active 3 MC state is a competitive pathway for the quenching of the 3 MLCT state, good PACT agents are usually not emissive and produce only small amounts of 1 O 2 [17].
To be a promising PACT agent, a metal complex has to fulfill three criteria: (1) it should be thermally stable in solution in the dark, (2) it should be photoactivatable with acceptable photosubstitution quantum yields, typically in the order of Φ ~ 0.01-0.05, and (3) it should show an increased cytotoxicity after light activation, compared to the dark. For example, [Ru(tpy)(bpy)(Hmte)](PF 6 ) 2 ([1] (PF 6 ) 2 , where tpy = 2,2':6',2″-terpyridine, bpy = 2,2'-bipyridine, and Hmte = 2-(methylthio)ethanol), is known to undergo photosubstitution of the thioether Hmte ligand under blue light irradiation, to generate an aqua ruthenium-based photoproduct [Ru(tpy)(bpy)(OH 2 )] 2+ [18] that is known to be non-cytotoxic [19]. It is hence a good example for a chemically activated compound, i.e., a compound capable of photosubstitution, that is not expected to be biologically activated because its photoactivated product is not cytotoxic. To obtain high phototoxicity after light activation, ruthenium complexes require efficient cellular uptake, as well as some form of deleterious interaction of the activated photoproducts with biological molecules. Bi-cationic polypyridyl ruthenium complexes such as [1](PF 6 ) 2 often show low cellular uptake [20], can be solved either by lowering the positive charge of the complex, e.g. via cyclometallation [21,22], or by increasing the hydrophobicity of the ligands, e.g. by expanding the aromatic surface of its polypyridyl ligands or adding methyl groups [23,24]. On the other hand, too lipophilic complexes may show too high dark cytotoxicity, which is a problem in phototherapy [25]. For PACT compounds, ligand expansion aimed at increasing steric hindrance and thus photosubstitution efficacy [26,27], may also lead to distorted complex geometries, resulting in uncontrolled ligand release, thus thermal activation in the dark [18,24,28]. Overall, the design of a good PACT compound requires careful balancing of the lipophilicity of the complex and its photoreactivity.

Synthesis and characterization
The bidentate ligand i-biq was obtained following a reported procedure [32]. The ligand i-Hdiqa is also known [33] and was synthesized using a Buchwald-Hartwig coupling as described for the synthesis of other dipyridylamine derivatives in literature [34]. After purification by column chromatography, the ligand was isolated as NMR-pure solid in 48% yield. The two ruthenium-based PACT compounds [2] (PF 6 ) 2 and [3](PF 6 ) 2 were synthesized following the same reaction route as for [1](PF 6 ) 2 (Scheme S1). In short, the bidentate ligand was first coordinated to the ruthenium precursor [Ru(tpy)(Cl) 3 ], before the monodentate chloride ligand was thermally substituted by the protecting thioether ligand Hmte. The desired complexes were obtained in good yield (50 and 60%, respectively), and their purity was confirmed with 1 H NMR, 13 C NMR, and elemental analysis.

Photochemistry
The two complexes have many overlapping 1 MLCT absorption bands extending between 400 and up to 600 nm, with an absorption maximum at 429 nm for [ (Table S3), transferred an electron from an essentially metal-centered 3d xz to the π* orbital centered on terpyridine ( Figure S12a-b). In contrast, for [3] 2+ the metal-based 3d orbital was much more in the xy plane of the terpyridine ligand, and significantly mixed via antibonding orbital overlap with the π system of the bent quinoline moiety of the i-Hdiqa ligand ( Figure S12c), thereby reducing the energy of the 1 MLCT transition into the terpyridine π*-based orbital, which hence appeared at a bathochromically shifted wavelength (476 nm). Overall, in [2] 2+ the extension of the conjugation of the bpy system, compared to [1] 2+ , does not significantly influence the lowest-energy 1 MLCT transition of the complex as this transition involves the terpyridine ligand and not the bidentate chelate, while in [3] 2+ the formation of a 6-membered metallacycle due to the presence of the additional NH bridge, generates a distortion of the planarity of the i-Hdiqa ligand that destabilizes the HOMO, thereby shifting the lowest-energy 1 MLCT transitions towards the red region of the spectrum. Although [2](PF 6 ) 2 and [3](PF 6 ) 2 were perfectly stable in pure water in the dark at 37 °C for 24 h ( Figure S1a and S1b), they were clearly chemically photoactivated. Their photoreactivity was investigated upon green light irradiation (517 nm) in water at 37 °C using UV-vis spectroscopy (Fig. 3). For each complex, upon irradiation a typical bathochromic shift of the absorption maximum was observed, due to the release of the thioether ligand and the formation of the corresponding aqua complex [Ru(tpy)(NN) (OH 2 )] 2+ ([4] 2+ and [5] 2+ for NN = i-biq and i-Hdiqa, respectively, see Scheme 1) [17,41,42]. The formation of the aqua complexes was confirmed with mass spectrometry ( Figure  S4). The UV-vis spectra recorded during irradiation showed isosbestic points (at 369; 375 and 404, respectively), indicating a one-step photosubstitution reaction. The Glotaran software package was used to fit the time evolution of the UV-vis absorption spectra to a single photoreaction, and to obtain the photosubstitution quantum yields Φ 517 (Table 2; Figure S5) [43]. The quantum yields of [1](PF 6 ) 2 and [2] (PF 6 ) 2 were found similar (Φ 517 = 0.022 and 0.023 for [1] 2+ and [2] 2+ , respectively). Thus, changing the bidentate ligand from bpy to i-biq did not alter the photosubstitution efficacy. However, the presence of i-Hdiqa in [3] 2+ increased the quantum yield by a 3.5-fold, to Φ 517 = 0.077, which is quite high.
The reason for the increased photosubstitution quantum yield of the Hmte ligand in [3] 2+ remains unclear. Triplet state minimization using DFT afforded, as expected, 2 different triplet states for each complex (Table S7-S12): an 3 MLCT state, characterized by a geometry very similar to the ground state and a highest singly occupied orbital (SOMO) located on the terpyridine ligand; and a 3 MC state, characterized by strongly elongated Ru-S and Ru-N 5 trans bonds (Table 3) and a highest SOMO primarily located on the metal. The difference in energy ΔE between the 3 MC Table 2 Lowest-energy absorption maxima (λ max in nm), molar absorption coefficients at λ max (ε max in M −1 · cm −1 ) in water, singlet oxygen generation quantum yields (Φ Δ ) in aerated methanol-d 4 , phosphorescence quantum yields (Φ P ) in aerated methanol-d 4 , and photosubstitution quantum yields upon irradiation at 517 nm (Φ 517 ) in water for complexes [ 3 MLCT energy level, while the latter shows significantly higher photosubstitution quantum yields; other phenomena, such as interaction with the incoming solvent molecule, may also explain this discrepancy, which should be studied further. To conclude on the photochemistry, the phosphorescence of all three complexes in deuterated methanol was negligible (Φ P < 5 · 10 −4 upon blue light irradiation), while they showed very low singlet oxygen quantum yields Φ Δ , suggesting that their 3 MLCT states might be short-lived, and that these complexes are not suitable for PDT (Table 2; Figure S3). Overall, photochemical generation of the 3

Cytotoxicity and cellular uptake
The thermal stability of PACT complexes is essential not only in pure water, but also in cell-growing conditions, i.e., in OptiMEM complete medium at 37 °C. All three complexes [1](PF 6 ) 2 - [3](PF 6 ) 2 were found stable for at least 24 h under such conditions ( Figure S1c and S1d). In a second step, the cytotoxicity of these complexes was tested under normoxic conditions (21% O 2 ) in 2D monolayers of human lung carcinoma (A549) and human epidermoid carcinoma (A431) cell lines, following a protocol developed in our group [45]. In short, cancer cells were seeded at t = 0 h, treated with six different complex concentrations at t = 24 h, and irradiated after another 24 h with the light of a green LED for 30 min (520 nm, 38 J/cm 2 ). The irradiation time, necessary to fully activate the complexes, was determined in a mock irradiation protocol using UV-vis spectroscopy ( Figure S10). At t = 96 h a Sulforhodamine B (SRB) assay was performed to compare the cell viability in treated vs. untreated cells (Figure S7 and S8). The effective concentrations (EC 50 values), i.e. the concentration at which the cell viability was reduced by 50% compared to untreated cells, are reported in Table 4. The photo index of each compound was calculated as the ratio of the EC 50 values obtained in the dark and upon light irradiation. The bpy-based complex [1](PF 6 ) 2 was found as expected to be non-cytotoxic against A549 cancer cells, whether irradiated or not (EC 50 > 150 µM). The complexes [2](PF 6 ) 2 and [3](PF 6 ) 2 showed low cytotoxicity in the dark (80 vs. 62 µM), but revealed a significant increase in cytotoxicity after light activation characterized by EC 50 values of 21 and 14 µM, respectively. These changes correspond to photo indices of ~ 4 for both complexes, indicating that a more cytotoxic species is released upon light activation. The released thioether ligand Hmte, tested independently, showed neither cytotoxicity in the dark nor upon light irradiation ( Figure S9). In A431 cancer cells, the same trends were observed (Table 4). Therefore, the cytotoxicity observed upon light irradiation of [2] 2+ or [3] 2+ must be based on the metal-containing photoproduct, i.e. the aqua complexes [4] 2+ and [5] 2+ , respectively, and not on the photoreleased Hmte ligand [46,47].
To quantify the effect of the increased hydrophobicity of the complexes on the cellular uptake, uptake experiments were performed. A549 cells were treated with 30 µM of the complex [1](PF 6 ) 2 - [3](PF 6 ) 2 , which is lower than their dark EC 50 values, and the ruthenium uptake was determined after 24 h incubation in the dark ( Table 4). The ruthenium content in nmol Ru per mg cell protein was determined by high-resolution continuum-source atomic absorption spectrometry (HRCS AAS, further details in ESI) under normoxic (21% O 2 ). It should be noted here that in such an assay, we cannot distinguish aggregation of the complexes onto the cell surface, from real internalization of the complex (i.e., by passive or active crossing of the cellular membrane): the "uptake" results actually expressed the sum of both types of molecules. Complex [1](PF 6 ) 2 was less taken up (0.16 nmol per mg cell protein), compared to the other two complexes [2](PF 6 ) 2 and [3](PF 6 ) 2 , for which the ruthenium uptake was 0.32 and 0.69 nmol per mg cell protein, respectively. Probably, the higher lipophilicity of [2](PF 6 ) 2 and [3](PF 6 ) 2 , compared to their bpy analog, is at least partly responsible for their higher uptake. However, the more polar (log P ow = 0.45) i-Hdiqa complex [3] 2+ showed enhanced accumulation, compared to its more hydrophobic i-biq analog [2] 2+ (log P ow = 2.10), so that some active transport may be involved here. Increased uptake with polypyridyl ligands bearing a noncoordinating secondary amine group has been observed for example by Barton et al. with rhodium(III) complexes [29], or in our group by platinum(II) complexes [48]; however, the reason for such phenomenon in ruthenium(II) complexes remain unclear. Next to hypothesizing some form of active transport, we may also speculate that metal complexes bearing non-coordinated NH groups such as [3] 2+ , may partly be deprotonated because of the increased acidity of the NH group upon metal coordination, which may lower the charge of the metal complex and hence improve cellular uptake by passive diffusion. For example, a concentration-dependent pKa value between 4 and 5 was reported in acetonitrile for [Ru(phen) 2 (HDPA)] 2+ (phen = 1,10-phenanthroline, HDPA = 2,2'-dipyridylamine) [49]. We are unaware of similar pKa measurements in aqueous solution for ruthenium(II)-dipyridylamine complexes. We should also mention that for the platinum(II) complex [Pt(H 2 bapbpy)] 2+ (where H 2 bapbpy = is N-(6-(6-(pyridin-2-ylamino)-pyridin-2-yl)pyridin-2-yl)pyridin-2-amine), a pKa of 8.3 was measured in water, which was accompanied by a massive cellular uptake in A549 lung cancer cells (1586 pmol Pt/ million cells), compared to cisplatin (23 pmol Pt/million cells). However, we did not notice during our investigations on [3] 2+ , any sign of deprotonation in aqueous solution near pH = 7.4, so that such arguments remain, at that moment, pure speculation. Another hypothesis is that hydrogen bonding involving the non-coordinated NH bridge and biological anions would lead to better transport of the complex through the cell membrane [50]. All in all, the difference in cellular toxicity between [1](PF 6 ) 2 on the one hand, and [2](PF 6 ) 2 and [3](PF 6 ) 2 on the other hand, probably come from other reasons than differences in cellular uptake. Clear differences of localization and/or toxicity have been observed in other published series of ruthenium polypyridyl complexes containing one or several dipyridylamine ancillary ligands [51,52]. Probably, [Ru(tpy)(bpy)(OH 2 )] 2+ is simply less cytotoxic than its i-biq and i-Hdiqa analogs [4] 2+ and [5] 2+ , because of different cellular localization and/or interaction with biomolecules, which remains to be elucidated.

Conclusions
The chemically photoactivatable ruthenium complex [1] (PF 6 ) 2 is poorly taken up by cells and showed no (photo) cytotoxicity in cancer cells. Therefore, although it is chemically activated by light it is not biologically activated by light in cells, and hence not suitable as a PACT agent. However, two analog ruthenium complexes with more hydrophobic bidentate ligands were shown to be promising PACT compounds. [2](PF 6 ) 2 showed a photosubstitution quantum yield that was comparable with that of [1](PF 6 ) 2 and a higher cellular uptake, overall resulting in increased cytotoxicity upon green light irradiation. [3](PF 6 ) 2 , which has an additional non-coordinated amine bridge, showed enhanced photosubstitution quantum yield compared to [2] 2+ and the highest cellular uptake in the series, but its photoindex was similar, in the tested conditions, to that of [2] 2+ . This work demonstrates that careful considerations on ligand design are necessary to fine-tune light activation of a Ru-based PACT drug. The lipophilicity of the prodrug, which influences cellular uptake and interaction with biomolecules, must be intermediate, and its ligand exchange properties must be slow in the dark and significantly increased upon visible light irradiation.