pubs.acs.org/IC Article Synthesis and In Vitro Studies of Photoactivatable Semisquaraine- type Pt(II) Complexes Kevin Morales, Sergi Rodríguez-Calado, Jordi Hernando, Julia Lorenzo, Antonio Rodríguez-Diéguez, Carlos Jaime, Pau Nolis, Merce ̀ Capdevila, Ò scar Palacios, Marta Figueredo,* and Pau Bayón* Cite This: Inorg. Chem. 2022, 61, 7729−7745 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: The synthesis, full characterization, photochemical properties, and cytotoxic activity toward cisplatin-resistant cancer cell lines of new semisquaraine-type Pt(II) complexes are presented. The synthesis of eight semisquaraine-type ligands has been carried out by means of an innovative, straightforward methodology. A thorough structural NMR and X-ray diffraction analysis of the new ligands and complexes has been done. Density functional theory calculations have allowed to assign the trans configuration of the platinum center. Through the structural modification of the ligands, it has been possible to synthesize some complexes, which have turned out to be photoactive at wavelengths that allow their activation in cell cultures and, importantly, two of them show remarkable solubility in biological media. Photodegradation processes have been studied in depth, including the structural identification of photoproducts, thus justifying the changes observed after irradiation. From biological assessment, complexes C7 and C8 have been demonstrated to behave as promising photoactivatable compounds in the assayed cancer cell lines. Upon photoactivation, both complexes are capable of inducing a higher cytotoxic effect on the tested cells compared with nonphotoactivated compounds. Among the observed results, it is remarkable to note that C7 showed a PI > 50 in HeLa cells, and C8 showed a PI > 40 in A2780 cells, being also effective over cisplatin-resistant A2780cis cells (PI = 7 and PI = 4, respectively). The mechanism of action of these complexes has been studied, revealing that these photoactivated platinum complexes would actually present a combined mode of action, a therapeutically potential advantage. ■ INTRODUCTION For decades, platinum complexes have focused a great interest However, despite all the efforts being made, there are some aspects that remain unsolved associated with the physical properties of the platinum complexes themselves. One of these because of their particular features.1 In recent years, these limitations is, for example, the stability of the complexes in the complexes have re-emerged as very motivating entities for physiological medium and the dark. This refers not only to the different purposes.2 In particular, photoresponsive complexes photostability of the complex but also to the resistance to have become significant,3 being one of the fields in cancer hydrolysis.8 The solubility of platinum(II) species in aqueous therapies where most efforts have been invested.4 In the last media is another concern. In general, neutral complexes have years, platinum(II) phototherapy has been shown as an low solubility; however, in order to make them work, they opportunity to lessen the side effects associated to cisplatin- must have a certain solubility in physiological media,9 though type drugs.5 Roughly, phototherapy can be classified into two keeping an equilibrium with lipophilicity, as this feature is categories: photodynamic therapy (PDT) and photoactivated believed to be crucial for the passive influx of drug molecules chemotherapy (PACT). Both PDT and PACT are proving to into cells.10 Also, the frequency of irradiation where a complex be good options as new cancer therapies.6 While PDT needs response occurs is critical, as ideally, it should take place in the the presence of molecular oxygen to elicit cell death, PACT acts by means of the species generated by a prodrug as its Received: December 20, 2021 photoresponse.7 In this way, PACT allows controlling where Published: May 6, 2022 and when the active species are generated, selectivity is thus increased, and therefore, the necessary dose is reduced. Such an approach allows minimizing side effects, hence improving the quality of life of the treated patients. © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.inorgchem.1c03957 7729 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article Figure 1. New semisquaraine-type Pt(II) complexes in this study. Scheme 1. Sequential Strategy in the Synthesis of Ligand L1 Scheme 2. Synthesis of Substituted Pyrrole Motives frequency range known as the therapeutic window.5e In this branch as a cooperative cyclobutenedione substituent, article, we want to present a new class of platinum(II) considering its chelation abilities. Also, unlike our former complexes and how we overcome the limitations pointed out squaramide-type ligands, the other substituent of the ring has above. Thus, herein we report the synthesis of new ligands and been used to increase electronic delocalization and, therefore, their corresponding platinum(II) complexes and their modify the absorption range of these new complexes. Finally, complete photochemical characterization. Recently, we have several structural modifications for the improvement of the described the synthesis and photochemical assessment and solubility of the complexes are discussed. activity against HeLa cells of new squaramide-based platinum- (II) complexes.11 Although promising results were obtained, some limitations were noticeable. Those stable complexes ■ RESULTS AND DISCUSSION Synthesis. In view of our previous findings, we planned to showed moderate to low solubility in aqueous media and, synthesize a new series of cyclobutenedione-based platinum- importantly, all of them were photoresponsive in the UV range, (II) complexes fulfilling the following requirements: (i) clearly out of the therapeutic window. In the study herein bearing two 3-thiopropylamino appendixes, since this kind of reported, the aforementioned problems have been alleviated. chelators proved to be efficient for the Pt(II) release from the We believe that these new complexes may have applications complex; (ii) enhanced water solubility, to improve the given their ability of photoresponse. The new ligands here availability in a physiological medium; and (iii) photochemical presented are semisquaraine-type (Figure 1), and based on the response in a biocompatible frequency range. We visualized conclusions of our previous study, they bear a thioalkylamino that the attachment of a pyrrole linker to the cyclobutenedione 7730 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article Scheme 3. Synthesis of Conjugate Pyrrole Ligands L2−8 core could hopefully facilitate the accomplishment of these organic phase, without the need of a previous chromatographic requirements since such a subunit should be amenable to separation. This improved protocol applied to pyrrole 18 subtle structural modifications, oriented to achieve the desired furnished the corresponding ligand L4 as an orange powder in properties in the new photoactive complexes. Thus, an a 57% yield. Eventually, crystallization of L2 from dimethyl- accessory phenyl residue conjugated to the heterocycle formamide (DMF)/H2O provided crystal needles suitable for would extend the π delocalization, providing a chromophore X-ray analysis (Figure 2), which confirmed the 1,2- suitable for a low-frequency photoactivation, while the addition of hydroxyl groups and their PEG derivatives would improve water solubility. Moreover, pyrrole is considered a privileged scaffold for biomedical applications.12 Accordingly, the syn- thesis of complexes C1−8 (Figure 1) was undertaken. The first endeavor was the preparation of the corresponding ligands, L1 to L8. To this aim, we developed a one-pot protocol consisting of the consecutive addition of the pyrrole and the methylthiopropylamino nucleophilic moieties to the cyclobutenedione core. The proper conditions were set up by investigating, in depth, the reaction depicted in Scheme 1. The Figure 2. MERCURY drawing for L2. Thermal ellipsoids are drawn at pyrrole component 10 was prepared by N-alkylation of pyrrole the 30% probability level. H atoms are omitted for clarity. according to a known procedure.13 Successive conjugate addition of 10 (in a molar ratio 9/10 = 1/1) and then a mixture of thioamine 11 and diisopropylethylamine (DIPEA) disubstitution of the cyclobutenedione core, the E config- in a molar ratio 9/11/DIPEA = 1/1/2, to a solution of uration of the alkene, and a preferential fully extended dichlorocyclobutenedione 914 in tetrahydrofuran (THF), conformation of the two substituents, with the NH syn to furnished the expected ligand L1 in a single operation and a the pyrrole moiety. 53% isolated yield after crystallization from MeOH. Treatment of L2 and L4 with tribromoborane in CH2Cl2 Next, we prepared the required pyrrole derivatives for the unveiled the hydroxyl groups, furnishing the corresponding synthesis of the other targeted ligands (Scheme 2). N- phenols L3 and L5 in good yields. Phenol L3 was readily alkylation of 1H-pyrrole-2-carbaldehyde, 12, with (3- converted into the glycol ether derivatives L6 and L8 in 47 and bromopropyl)(methyl)sulfane furnished aldehyde 13, which 92% yield after crystallization from methanol and CH2Cl2/ was subjected to a Horner−Wadsworth−Emmons (H−W−E) Et2O, respectively. However, when the same methodology was alkenylation with the benzylic phosphonates 1415 and 15,16 intended for the synthesis of L7, the O-alkylation of the affording the corresponding pyrrole derivatives 17 and 18, pyrogallol derivative L5 failed. Alternatively, L7 was synthe- respectively, in good yields. It is worth mentioning that alkene sized from pyrrole 19, prepared through an H−W−E reaction 17 showed a limited stability, even at freezing temperatures, between aldehyde 13 and phosphonate 1617 (Scheme 2). As leading to degradation products after several weeks. Hence, it 19 was revealed to be very unstable, for the synthesis of L7, should be soon subjected to the next synthetic step. On the freshly prepared crude 19 was added to cyclobutenedione 9, contrary, its congener 18 showed an unlimited stability. and after a successive addition of thioamine 11, the expected When the conditions developed for the preparation of L1 ligand was obtained in a 62% global yield from 13. Likewise, an from the simpler pyrrole 10 were applied to 17, after alternative synthesis of L6 starting from 13 and an appropriate chromatographic purification followed by crystallization from phosphonate was also developed (see the Supporting CH2Cl2/Et2O, the expected ligand L2 was obtained as a yellow Information, S3 for details), but the overall sequence yield powder in a 60% yield (Scheme 3). Moreover, it was found did not improve the results of the pathway shown in Scheme 3. that the same reaction performed in a two-phase CH2Cl2/H2O All the synthesized ligands (L1−8) and their previously system allowed the isolation of pure L2 in a 72% yield by unknown precursors (13, 17, and 18) were fully characterized straight crystallization after the addition of Et2O to the dried according to their physical and spectroscopic data. 7731 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article Table 1. Synthesis of Complexes C1−8a entry solvent time (h) precipitate washingb complex (yield) color, mp (from solvent) 1 MeOH/H2O, 1:1 17 MeOH; H2O C1 (56%) brown, 173−178 °C (MeOH/H2O) 2 THF/H2O, 3:2 22 THF; H2O C2 (97%) orange, 184−187 °C (THF/H2O) 3 THF/H2O, 1:1 15 H2O; MeOH; THF C3 (86%) russet, >200 °C (THF/H2O) 4 THF/H2O, 1:1 20 THF; H2O C4 (99%) orange, >200 °C (THF/H2O) 5 THF/H2O, 1:1 16 H2O; MeOH; THF C5 (85%) brown, >200 °C (THF/H2O) 6 THF/H2O, 1:1 16 H2O; MeOH; THF C6 (81%) orange, 135−139 °C (THF/H2O) 7 THF/H2O, 1:1 17 c C7 (65%) brown, 92−95 °C (CH2Cl2/Et2O) 8 H2O 17 c C8 (95%) brown, 50−53 °C (CH2Cl2/Et2O) a All the reactions were performed by the treatment of the starting ligands with 1 M equivalent of K2PtCl4 at room temperature, under an argon atmosphere, except for entry 8, where the molar ratio K2PtCl4/L8 was 1.3. bComplexes precipitated from the reaction mixture were filtered and successively washed with the indicated solvents. cComplexes C7 and C8 were separated by extraction with CH2Cl2. With the ligands in hands, the next challenge was quantum mechanics. As a starting point, the aminothiolated synthesizing their Pt(II) complexes. To this aim, each ligand chains were assumed to coordinate by the sulfur atoms, based was treated with K2PtCl4 in an appropriate solvent at room on XRD analysis for one of our previous analogs.11 temperature under an argon atmosphere. For complexes C1− Considering K2PtCl4 as the source of Pt(II), the trans effect 6, the resulting precipitate was filtered and successively washed seems to point to the trans configuration as the most favorable, with suitable solvents. For C7 and C8, the complexes were as thioether-type ligands are rated as trans-director when separated from the reaction mixture by the addition of brine, compared to chloride ligands. With this in mind, the method of followed by extraction with CH2Cl2. Table 1 summarizes choice was the use of Gaussian program in its 2016 version conditions, yield, and physical description for each particular (abbreviated as Gaussian 16).18 DFT calculations were complex. performed with the LANL2DZ19 base and the M06-2X20 All the synthesized complexes C1−8 were fully characterized function. Structures were built with the help of graphic according to their physical and spectroscopic data. HRMS or program GaussView,21 and their energies were optimized using MS of all the complexes showed the characteristic pattern of the values defined by default in the program. Vibration platinum compounds except for C8, which degraded upon frequencies were calculated for all compounds, always ionization. Since its polydisperse polymeric nature precluded obtaining zero imaginary frequencies, which ensures that elemental analysis, the amount of Pt was determined by ICP- energy minima are considered.22 OES (6.7% Pt), obtaining a value similar to that expected, The complexation of sulfur atoms to the Pt(II) center causes assuming a molecular weight of 2705 Da (7.2% Pt). them to become stereogenic and, therefore, may have the R The cis−trans configuration of Pt(II) complexes usually and/or S configuration. The values of the obtained energies are reveals crucial for the biological response to these species. shown in Table 2, together with the energy differences Unfortunately, all attempts to obtain crystalline complexes for between the cis and trans isomers. The descriptors R and S their structural elucidation by means of X-ray diffraction refer to the absolute configuration of the sulfur atoms (XRD) analysis were unsuccessful. As tert-butyldiphenylsilyl complexed with platinum (see the Supporting Information, (TBDPS) derivatives are usually prone to form crystalline Figure S1). solids, a TBDPS-derivative ligand L20 and its corresponding The results in Table 2 indicate that structures with a trans- dichloride-Pt(II) complex, C20, were prepared (see the configuration at the platinum center, trans-C21, are more Supporting Information, S5). Despite a number of attempts stable than those for cis-C21 at about 10−13 kcal/mol. Thus, at crystallization, C20 systematically turned out as a solid not by extension, it was assumed that all the complexes here suitable for XRD analysis. In view of that, we decided to presented might display a trans configuration. Since, for these investigate this issue on the basis of theoretical calculations. complexes, it seems that both the results of the DFT cis-Pt Versus trans-Pt Configuration Tentative As- calculations and the prediction based on the trans effect (i.e., signment. Density functional theory (DFT) calculations were thermodynamics and kinetics) point in the same direction, it performed to evaluate the relative stability of the two isomers. would be reasonable to tentatively assign these complexes a The relative energies of both isomers were calculated for trans configuration. complex C21 (Figure 3) as a model by methods based on The relative differences of the corresponding trans-C21 isomers are also given. Among trans-2 isomers, the most stable seems to be R″/S‴ (Table 2, entry 1), although a more complete conformational study would be needed to ensure this. The energy differences between the plausible config- urations for trans-C21 are less significant (less than 4.1 kcal/ mol), and given the size of the Pt(II) metallocycle, very likely small conformational changes would alter the stability order. Photochemistry. Once the ligands and complexes were synthesized and characterized, their photochemical behavior was evaluated. First, the UV−vis spectra of both ligands and complexes were recorded (see the Supporting Information, Figure S2), whose absorption was found to be clearly Figure 3. Configurations of the metal center in model complex C21. bathochromically shifted relative to our previously reported 7732 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article Table 2. Energetic Results of DFT Calculations with Gaussian16, M062X/LANL2DZ for cis- and trans-C21 Considering All Possible Absolute Configurations for Sulfur Atoms23 entry isomera cis-C21 E (hartree) trans-C21 E (hartree) Δ(cis−trans)b kcal/mol Δ(trans−trans)b kcal/mol 1 R″/S‴ −1359.7071453 −1359.7283893 13.3 0.0 2 R″/R‴ −1359.7103260 −1359.7218970 11.3 4.1 3 S″/S‴ −1359.7082496 −1359.7247720 12.6 2.3 4 S″/R‴ −1359.7126554 −1359.7253195 9.9 1.9 a The configuration descriptors refer to the sulfur atom of the chain with double prime numbering (′′), and the chain with triple prime (′′′), as shown in Figure 3. bRelative energy values referred to the most stable trans isomer. Figure 4. Time evolution of the UV−vis absorption spectra after irradiation of complexes (a) C1 in water/DMSO, 98:2, (b) C2 in water/DMF, 98:2, (c) C3 in water/DMSO, 98:2, (d) C4 in water/DMF, 98:2, (e) C5 in water/DMSO, 98:2, (f) C6 in water/DMF, 98:2, (g) C7 in water, and (h) C8 in water. For C1 λexc = 365 nm (UV lamp) and for C2−8 λexc = 450 nm (LED). squaramide-type compounds.11 Thus, ligand L1 and the max ∼ 450 redshift, which clearly falls in the visible range (λabs corresponding complex C1 showed spectral maxima at λabs max nm). Therefore, these results validate our strategy for the ∼ 380 nm because of the introduction of a pyrrole substituent design of Pt(II) photocages that are excitable with visible light. to the semisquaraine core. More interestingly, an extension of Illumination of the ligands and complexes prepared resulted the conjugation path in the rest of the compounds by attaching in a negligible fluorescence emission regardless of the solvent an additional phenylvinyl group led to a further absorption used (see the Supporting Information, Table S1). Instead, 7733 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article concomitant irreversible changes were observed in absorption Scheme 4. Synthesis of Simplified Ligand L22 that suggest that photodegradation of these compounds occurs under irradiation. This process was investigated in detail upon photoexcitation at 365 nm for C1 or 450 nm for C2−8 (Figure 4). Interestingly, while for C2−C6 the use of a cosolvent was needed, complexes C7 and C8 bearing PEG chains could be solubilized in pure water without the need for any additional cosolvent. For these and the rest of the compounds, very similar trends in absorption were registered upon continuous irradiation: the characteristic band at λabs max ∼ 380 nm or λmax ∼ abs 450 nm rapidly faded after a few minutes, which in most of the cases resulted in the appearance of a low-intensity hypsochromically shifted signal (e.g., complexes C2−8 in Figure 4). Interestingly, this behavior is reminiscent of that previously observed by us for squaramide-based Pt(II) Supporting Information, Figure S3). After irradiation, a major complexes,11 which could be unambiguously ascribed to the photoproduct was detected by HRMS [443.2 Da (+H+), 465.2 photodegradation of the ligand. Da (+Na+) and 481.2 Da (+K+)]. These peaks were correlated To assess the efficiencies of the photodegradation process with the mass corresponding to L22 + EtOH (see the for all the ligands and complexes, their photoreaction quantum Supporting Information, Figure S4). Also, a carbonyl group yields (Φph) were evaluated (see the Supporting Information, was confirmed by IR analysis (see the Supporting Information, Table S2). In general, the values obtained are rather low (Φph Figure S5). After careful 1H NMR and 13C NMR analyses of a ∼ 10−4 to 10−5) though they are on the same order of pure sample of the major degradation product (see the magnitude as those reported for other visible light-active Supporting Information, Figures S6 and S7 respectively), one photolyzable groups.24 It must be noted that this effect is of the two regioisomeric butenolides, 24 or 25, was assigned as partially counterbalanced by the large molar extinction the most plausible structure (Scheme 5), consisting of a −1 coefficients of our compounds (εabs max > 2.0 × 10 M 4 cm−1, mixture of 3 different conformers. This hypothesis was also Table S2), as the efficacy of photoreactions strictly depends on consistent with a similar process previously described for the product of Φph with the absorptivity of the irradiated bisketenes.25,26 Ultimately, the structure for the major molecule at the excitation wavelength. As a result, they present photoproduct was assigned by additional NOE and HMBC large enough Φph εabs max values for photouncaging applications experiments of the mixture of conformers. NOE Experiments (Table S2). This is especially true for the water-soluble Pt(II) were performed at 250 K (see the Supporting Information, complexes developed herein, which show Φph and Φph εabs max Figure S8), to get a good resolution of the signals values that are about 10-fold larger than for the corresponding corresponding to each conformer at play. For one of the free ligands and, therefore, make them especially suitable for conformers, the spectrum showed an NOE interaction light-induced Pt(II) release. Most probably, this is due to the between methyl 2‴ and the NHMe group. For the other two restriction of conformational mobility upon metal complex- conformers, NOE effects between 2‴ and the SMe group were ation, which must decrease the efficiency of excited state also noticeable. In addition, an HMBC experiment (see the relaxation through intramolecular vibrational and rotational Supporting Information, Figure S9) revealed no cross signal motions. C2′-H5. With all those pieces of evidence, the major At this point, knowing the nature of the photoproducts and photoproduct was assigned as butenolide 25. The formation giving a mechanistic explanation for their formation was of 25 was envisioned as triggered by the nucleophilic attack of considered as required. Initially, photodegradation of the the nucleophilic solvent to a ketene functionality as expected ligands was explored. First, a 60 mM (DMF/H2O, 9:1) (Scheme 5). A careful HRMS analysis of an irradiated sample solution of ligand L2 was irradiated (450 nm LED) until its of complex C2 suggests that the photodegradation pathway in UV−vis spectra revealed complete degradation, and the Scheme 5 is also effective from this Pt(II) complex. Hence, resulting mixture was analyzed by MS and NMR. Three following the Pt(II) traceability, a peak [721.1 Da [C2 + main peaks were detected and correlated with the mass H2O−Cl]+] was detected and assigned to the corresponding corresponding to L2 + H2O {[M + H]+ (489.2 Da), [M + butenolide Pt(II) complex (see the Supporting Information, Na]+ (511.2 Da) and [M + K]+ (527.2 Da)}. Despite several Figure S10). purification attempts, the NMR analysis turned out to be too Biological Assessment. Prior to initiating the evaluation intricate to elucidate its structure. Therefore, as a structurally of the biological activity of the synthesized complexes, a study simpler model, ligand L22 was prepared on purpose through a of their solubility in an aqueous medium was performed. It was synthetic route similar to that of its analogs (Scheme 4). observed that, among all, complexes wearing PEG motives, C7 We hypothesized that the photodegradation products would and C8, presented excellent water solubility, as expected and, likely derive from bisketene intermediates, which are known to therefore, they were finally chosen as the candidates to be formed upon irradiation of cyclobutenediones.25 With this investigate their biological properties. Accordingly, complexes idea in mind, the irradiation of L22 was assayed in DMF/ C7 and C8 were tested against three cancer cell lines of EtOH (6/4) instead of DMF/water, considering that the different origins without illumination and under blue light addition of the nucleophilic solvent to the ketene functionality (light dose of 32 J/cm2) (Table 3). Thus, HeLa, A2780, and would give us more information (particularly for NMR cisplatin-resistant A2780cis cells were incubated for 24 h with analysis) if an ethoxy moiety, instead of a hydroxy, was stock solutions of C7 and C8 at different concentrations, and incorporated to the final degradation product. The irradiation cell viability was evaluated at 72 h. The effect of the compound experiment was monitored by UV spectroscopy (see the cytotoxicity was expressed as a decrease in cell viability (Figure 7734 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article Scheme 5. Mechanistic Proposal for the Formation of Butenolide 24 or 25 as the Major Photoproduct Table 3. IC50 Determination of C7, C8 Complexes and upon illumination. Nevertheless, the effect is lower compared Cisplatin after 72 h (24 h Internalization) in Light and Dark with the corresponding Pt(II) complex C8 (Figure 6). in HeLa, A2780, and A2780cis Cells Cellular Mechanism of Action of Compounds C7 and C8. DNA Interaction. The interaction of these complexes with IC50 (μM) DNA was also investigated. To this aim, 50 μM calf thymus entry complex cell line lighta dark PI DNA (ct-DNA) was incubated with different molar ratios (ri) 1 C7 HeLa 4±3 >200 >50 of Pt(II) complexes C7 and C8 and stock solutions of their 2 C7 A2780 1.4 ± 0.8 18 ± 9 13 corresponding irradiation products C7′ and C8′ (see the 3 C7 A2780cis 2.7 ± 0.7 19 ± 8 7 Supporting Information, Figure S11). Interestingly, the CD 4 C8 HeLa >50 >200 n.d. spectra for both complexes showed an interaction before and 5 C8 A2780 5±3 >200 >40 after irradiation. In their nonirradiated forms, C7 and C8 6 C8 A2780cis 48 ± 22 >200 4 displayed a similar behavior with a decrease in both positive 7 cisplatin HeLa 15.5 ± 3.4 14.7 ± 2.6 and negative bands of ct-DNA (Figure S11a,b, respectively). 8 cisplatin A2780 2.3 ± 0.3 2.8 ± 0.2 Conversely, the irradiation products, C7′ and C8′, showed a 9 cisplatin A2780cis 15.1 ± 1.5 13.8 ± 0.2 differentiated interaction between them. Whereas for C7′, the a λexc = 450 nm and dose ca. 32 J/cm2. PI = photoactive index positive band remains almost unaltered with a considerable (obtained by dividing the baseline IC50 value by the photoactivated decrease in the negative band, for C8′, the positive band IC50 value). decreases, with the negative one being unaffected (Figure S11c,d, respectively). Hence, although the complexes seem to 5),27 and the half inhibitory compound concentration (IC50) change the morphology of the DNA similarly upon incubation values were also calculated (Table 3). in their nonirradiated form, there is a different type of In the absence of light, C7 displayed considerable interaction when the complexes are irradiated: C7′ causing a cytotoxicity toward A2780 and A2780cis cell lines (IC50 of perturbation in the helicity of the DNA and C8′ destabilizing 18 ± 9 and 19 ± 8 μM, respectively) (entries 2 and 3), while in the base stacking. Hypothetically, these different interactions the same conditions, complex C8 revealed no toxicity against should lead to different in vitro activities toward tumor cells. any of the cancer cell lines. To our delight, when the Since the target molecule for most Pt drugs is primarily the complexes were illuminated, the resulting cytotoxicity nuclear DNA, once we assessed binding by CD, a specific increased significantly in all cases, being the most cytotoxic determination of Pt in DNA was conducted by extracting DNA C7 irradiated within A2780 cell line (IC50 = 1.4 ± 0.8 μM) from the exposed A2780 cells. Pt concentration was (entry 2). Remarkably, when comparing the light effect, both normalized to the DNA concentration, which was initially complexes showed noticeable PI (photoactive index) values. In measured spectrophotometrically at 260 nm. Figure S11e particular, C7 in HeLa (PI > 50) and C8 in A2780 (PI > 40) shows the obtained percentage of Pt bound to DNA for the displayed remarkable results (entries 1 and 5), comparable or A2780 cell line exposed to C7 and C8 complexes at their even improving examples for Pt(II) complexes in the PACT respective IC50. In the light of these results, we concluded that literature.5c,28 Pt incorporation into DNA is much more efficient in the case Using 100 μM concentration as a reference, the photo- of compound C7, about 2-fold compared to C8. This result cytotoxicity of the ligands was also evaluated and compared could also explain why complex C7 is capable of exerting a with their corresponding Pt(II) complexes. Although for L7, greater cytotoxicity than C8. this concentration was too high to evaluate the light effect, for Apoptosis Detection. To date, different biological action L8, it was observed how the cytotoxicity was also enhanced mechanisms for Pt(II) PACT-based antitumor agents have 7735 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article Figure 5. Evaluation of cytotoxicity of compounds C7 and C8 in the absence or presence of blue light (dark/light) in HeLa, A2780, and A2780cis human carcinoma cells after 72 h (24 h internalization) using PrestoBlue assay. Results are representative of three independent experiments with a minimum of three replicates per experiment. involved in the final cell cytotoxic effect. Generally, the prevalent cell death type is dependent not only on the structure, intracellular localization, and concentration of the complex but also on the light dose applied and on cell origin. Thus, further biological investigations were warranted to gain insights into the molecular mechanisms of the complexes, which will provide helpful insights into the rational design of photoactivatable platinum-based antitumor complexes. To verify the mechanism of cell death induced by C7 and C8, A2780 cells were assayed for apoptosis or late apoptosis/ necrosis detection. Cells were treated for 24 h with C7 and C8 Figure 6. Cytotoxicity assays after 72 h incubation (24 h internalization) in the light and dark in HeLa, A2780, and A2780cis at their corresponding IC50 after light activation (1.5 and 5 cells with complex C8 and ligand L8, at 100 μM each. μM, respectively) and then photoactivated (light dose of 32 J/ cm2). After a final 72 h of incubation, cells were stained with an apoptosis/necrosis assay kit which senses the phosphatidylser- been proposed.29 It is well-established that the biological ine exposure on cells as a hallmark of apoptosis. Cells were action of Pt(II) is ultimately related with DNA binding examined under a fluorescent microscope to differentiate between the water-activated drug and two adjacent guanine− between apoptotic (red), late apoptotic/necrotic (green), and cytosine base pairs,30 generating a Pt-DNA cross-link adduct healthy (blue) cells (Figure 7). The controls (untreated which impedes cell propagation.31 Nevertheless, proteins can photoactivated and nonphotoactivated cells) displayed a also be platinated, inducing direct cell damage and immune physiological level of ca. 10% of apoptotic cells but exhibited response activation, inducing a mitochondrial-reactive oxygen no late apoptosis/necrosis. Similar results were detected for species (ROS) response.32 The damage in the mitochondrial complexes C7 and C8 without photoactivation. More membrane is enough to hamper apoptosis after cytochrome c interestingly, after photoactivation, the proportion of apoptotic release into the cytosol.33 In addition to apoptosis, Pt(II) cells among the A2780 cells treated with compound C7 rose to PACT complexes could potentially lead to cell death via 42% at its IC50, 1.5 μM (Figure 7). Under the same conditions, necrosis, with a combination of both apoptosis and necrosis compound C8 induced apoptosis in 36% of cells. The observed 7736 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article both complexes followed by light activation resulted in an elevation in cellular ROS release when compared to control cells (3-fold increase), highlighting the significant ROS production capabilities of these two photoactivatable com- plexes (Figure 8). On the contrary, cells treated with Figure 8. ROS formation measured with the DCFDA assay in A2780 cells for cisplatin (CisPt) and complexes C7 and C8 at the corresponding IC50 values for each complex (Table 1) after treatment for 4 h. Results are represented as the percentage over untreated cells. H2O2 (100 μM) was used as the positive control. complexes but kept in the dark or the control molecules used (cisplatin and H2O2) in the dark or after light activation did not show a significant difference in ROS release. These results are in concordance with the significant cytotoxicity levels previously found for these complexes (Table 3). Thus, upon photoactivation, both complexes C7 and C8 are capable of inducing ROS production and cell death through apoptosis. Having low cytotoxicity in the dark, the present complexes exert a multimechanistic chemotherapeutic effect that may serve for a targeted cancer chemotherapy. Figure 7. (a) Necrosis and apoptosis assays of A2780 cells untreated (control dark), treated with light only (control light), 1.5 μM C7 (C7 dark), light irradiation in the presence of 1.5 μM C7 (C7 light), 5 μM ■ SUMMARY AND CONCLUSIONS Pursuing the improvement of our previous Pt(II) complexes C8 (C8 dark), and light irradiation in the presence of 5 μM C8 (C8 already published11 in reference to the solubility and the light). Healthy viable cells were stained with CytoCalcein Violet 450 photoresponse wavelength, in this study, we have succeeded in (blue), late apoptotic/necrotic cells with DNA nuclear green DCS1 (green), and apoptotic cells with phosphatidylserine (red). (b) synthesizing, characterizing, and studying the photochemical Representative histograms of stained cells. Results are shown as a properties of eight new semisquaraine-type Pt(II) complexes. percentage of total cells. The synthesis of semisquaraine ligands has been carried out by means of a new straightforward approach. Also, XRD analysis of ligand L2 revealed a 1,2-semisquaraine-type constitution results also showed a significantly increased level of late/ which has been consequently extended to all the ligands apoptotic/necrotic cells after photoactivation of the complexes, presented. DFT calculations have allowed us to propose the with a special incidence in the C7 compound (56%). trans configuration of the platinum center. Importantly, by The obtained data evaluating the cellular death mechanism structural modification of the ligands, the corresponding induced by complexes C7 and C8 present promising cancer- complexes are photoactive at wavelengths such that they targeting properties by activation of apoptotic pathways. As allow their activation in cell cultures. Photodegradation shown, the analyzed carcinoma cell line presented apoptosis processes have been studied. Water-soluble complexes C7 only after photoactivation, allowing a better selectivity toward and C8 present quantum yields increased with respect to the irradiated cancer cells, with minimal effect over those kept in corresponding free ligands L7 and L8. By means of NMR the dark. techniques, it has been also possible to determine the structure ROS Generation Study. As previously mentioned, Pt(II) of photoproducts of the presented semisquaraines, concluding PACT-based complexes can be related with an oxidative- that what is generated are butenolide-type derivatives, thus dependent mechanism to trigger cell death. In order to detect justifying the changes observed before and after irradiation. the formation of intracellular ROS in A2780 cells in the Among all the complexes, two have been selected for their presence of the complexes while observing the effect of the biological evaluation, mainly because both have an excellent photoactivation, the 2′,7′-dichlorofluorescein diacetate solubility in physiological media. Thus, complexes C7 and C8 (DCFDA) assay was performed. In brief, DCFDA is cleaved have been demonstrated to behave as promising photo- and oxidized by intracellular esterases and ROS, generating the activatable compounds in the presented cancer cell lines, fluorescent compound dichlorofluorescein. Treatment with showing a potential amelioration over cisplatin-resistance in 7737 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article A2780cis cells. Upon photoactivation, both complexes were 1-(3-{Methylthio}propyl)-1H-pyrrole-2-carbaldehyde, 13. A sol- capable of inducing a higher cytotoxic effect on the tested cells ution of 12 (2.13 g, 22.4 mmol) in anhydrous DMF (10 mL) was compared with nonphotoactivated compounds. Among the added dropwise to a suspension of NaH (60% in mineral oil, 887 mg, observed results, it is remarkable to note that C7 showed a PI 22.2 mmol) in anhydrous DMF (10 mL) previously cooled to 0 °C. The resulting suspension was stirred under argon for 30 min, and (3- > 50 in HeLa cells and C8 showed a PI > 40 in A2780 cells, bromopropyl)(methyl)sulfane35 (3.73 g, 22.1 mmol) was added being also effective over cisplatin-resistant A2780cis cells (PI = dropwise. The suspension was stirred at room temperature for 4 h and 7 and PI = 4, respectively), a finding consistent with the then poured into water (40 mL). The mixture was extracted with previously reported Pt(II)-based compounds. Moreover, after Et2O (3 × 20 mL), and each extract was washed with water (3 × 10 photoactivation, these complexes were also shown to interact mL). The combined organic layers were dried over anhydrous with DNA (PACT-likely) and further cause ROS production Na2SO4 and filtered, and the solvent was removed under reduced (PDT-likely) and ultimately cell death through apoptosis in pressure to give a yellow oil. The product was purified by column the A2780 cancer cell line. We envision this dual effect34 as an chromatography (silica gel, hexane/Et2O, 9:1 to hexane/Et2O, 4:1) to opportunity to overcome one of the current PDT drawbacks, give 13 (3.23 g, 17.6 mmol, 80%) as a yellow oil. IR (ATR): 3105, 2915, 2801, 2721, 1657, 1526, 1480, 1403, 1369, 1321, 1264, 1217, as PDT is based on the conversion of ground-state triplet into 1078, 1031, 957, 887 cm−1. 1H NMR (400 MHz, CDCl3): δ 7.26 (d, J excited-state singlet oxygen. Photo-activated platinum com- = 1.1 Hz, 1H), 6.99−6.97 (ddd, J = 2.5 Hz, 1.7 Hz, 1.1 Hz, 1H), 6.94 plexes would not require the presence of oxygen, a potential (dd, J = 4.0 Hz, 1.7 Hz, 1H), 6.22 (dd, J = 4.0 Hz, 2.5 Hz, 1H), 4.42 advantage since tumor cells are often hypoxic. (t, J = 6.8 Hz, 2H), 2.43 (t, J = 6.8 Hz, 2H), 2.09 (s, 3H), 2.05 (qn, J Having a low cytotoxicity in the dark, complexes C7 and C8 = 6.8 Hz, 2H). 13C NMR (101 MHz, CDCl3): δ 179.4, 131.8, 131.4, may serve as a prodrug that upon photoactivation in a targeted 125.2, 109.7, 47.7, 30.9, 30.1, 15.5. HRMS (ESI+): calcd for cancer tissue, exert a potent multimechanistic chemother- [C9H13NOS]: 184.0791 [M + H]+, 206.0610 [M + Na]+; found, apeutic effect. The controlled photoactivation of these 184.0795 [M + H]+, 206.0616 [M + Na]+. platinum complexes could allow a targeted cell death in (E)-2-(4-Methoxystyryl)-1-(3-{methylthio}propyl)-1H-pyrrole, 17. regions of cancer growth and avoidance of toxic effects on A solution of phosphonate 14 (749 mg, 2.90 mmol) in anhydrous DMF (2.5 mL) was added to a suspension of NaH (60% in mineral normal cells, and thus may be considered a potential lead oil, 273 mg, 6.83 mmol) in anhydrous DMF (2.5 mL) previously molecule for a targeted cancer chemotherapy. cooled to 0 °C under an argon atmosphere. Then, a solution of 13 In all, we have been able to design, synthesize, and (533 mg, 2.91 mmol) in anhydrous DMF (1 mL) was added, and the characterize new platinum complexes that have been shown cooled mixture was heated progressively until 50 °C for 1 h. After this to have a powerful cytotoxic activity, solving problems time, the mixture was cooled to 0 °C, 12 mL of H2O was added concerning their solubility and their photoresponse range. dropwise, and the product was extracted with EtOAc (3 × 12 mL). Ultimately, we hope that this study contributes to the The combined organic extracts were dried over anhydrous Na2SO4 development of new cytotoxic Pt(II) photoactive photocages. and filtered, the solvent was removed under reduced pressure, and the More studies in this line continue to be completed. crude product was purified by column chromatography (silica gel, ■ hexane/Et2O, 9:1) to give 17 (787.7 mg, 2.74 mmol, 94%) as a yellow oil. IR (ATR): 2913, 2834, 1737, 1628, 1604, 1574, 1508, 1474, 1440, EXPERIMENTAL SECTION 1358, 1299, 1278, 1243, 1173, 1125, 1109, 1077, 1031, 951, 847, 814 Materials. K2PtCl4 was purchased from Strem Chemicals. cm−1. 1H NMR (360 MHz, CD3OD): δ 7.43−7.39 (m, 2H), 6.98 (dt, Different organic reagents used for ligands synthesis were purchased J = 16.1 Hz, 0.7 Hz, 1H), 6.90−6.85 (m, 2H), 6.81 (d, Jtrans = 16.1 Hz, from Sigma-Aldrich or Alfa Aesar. Organic solvents were dried before 1H), 6.70 (dd, J = 2.7 Hz, 1.7 Hz, 1H), 6.39 (ddd, J = 3.8 Hz, 1.7 Hz, use when required. Compounds 9,14 10,13 14,15 15,16 and 1617 were 0.7 Hz, 1H), 6.06 (ddd, J = 3.8 Hz, 2.7 Hz, 0.7 Hz, 1H), 4.14 (t, J = synthesized according to previous descriptions. 6.8 Hz, 2H), 3.79 (s, 3H) 2.42 (t, J = 6.8 Hz, 2H), 2.05 (s, 3H), 1.98 Synthesis of Ligands. 3-(1-{3-[Methylthio]propyl}-1H-pyrrol-2- (qn, J = 6.8 Hz, 2H). 13C NMR (91 MHz, CD3OD): δ 160.3, 132.8, yl)-4-({3-[methylthio]propyl}amino)cyclobut-3-ene-1,2-dione, L1. A 132.2, 128.1, 126.3, 123.3, 116.4, 115.1, 109.1, 106.7, 55.7, 45.8, 31.7, solution of 9 (521.3 mg, 3.45 mmol) in dry THF (8 mL) was added 31.7, 15.3. HRMS (ESI+): calcd for [C17H21NOS]: 288.1417 [M + dropwise to a solution of 10 (527.7 mg, 3.40 mmol) in dry THF (12 H]+, 310.1236 [M + Na]+; found, 288.1423 [M + H]+, 310.1239 [M mL). The resulting pink solution was stirred at room temperature + Na]+. under an argon atmosphere overnight. After this time, 11 (374 μL, (E)-1-(3-{Methylthio}propyl)-2-(3,4,5,-trimethoxystyryl)-1H-pyr- 3.41 mmol) and DIPEA (1.2 mL, 6.9 mmol) were added. The role, 18. Phosphonate 15 (774.9 mg, 2.4 mmol) was cooled to 0 °C resulting mixture was stirred at room temperature for 5 h. Then, 120 under an argon atmosphere. Then, anhydrous DMF (5 mL), NaH mL of CH2Cl2 was added, and the organic layer was washed with H2O (60% dispersion in mineral oil, 203.7 mg, 5.1 mmol), and 13 (452.0 (3 × 60 mL), dried over anhydrous Na2SO4, and filtered. The solvent mg, 2.5 mmol) were added. The reaction mixture was stirred at room was removed under reduced pressure, and the crude was purified by temperature for 1 h and then heated to 100 °C for 3 h. After this time, column chromatography (silica gel, hexane to hexane/EtOAc, 2:3) to 25 mL of ice/water were added, and the product was extracted with give L1 (813.0 mg, 2.40 mmol, 71%). Recrystallization in MeOH gave EtOAc (2 × 30 mL). The combined organic extracts were dried over pure L1 (612.3 mg, 1.81 mmol, 53%) as a yellow powder. mp 116− anhydrous Na2SO4 and filtered, the solvent was removed under 119 °C (from MeOH). IR (ATR): 3281, 2955, 2916, 1769, 1613, reduced pressure, and the residue was purified by column 1580, 1530, 1474, 1435, 1420, 1384, 1353, 1257, 1226, 1157, 1126, chromatography (silica gel, hexane/EtOAc, 8:2) to give 18 (696.9 1093, 1063, 1048, 1021, 983, 955, 906, 868, 853, 810 cm−1. 1H NMR mg, 2.0 mmol, 83%) as a yellow oil. The oil was cooled in the fridge to (400 MHz, CDCl3): δ 6.98 (dd, J = 2.6 Hz, 1.5 Hz, 1H), 6.58−6.47 give a pale-yellow solid. mp 66−68 °C (from CH2Cl2). IR (ATR): (m, 1H), 6.47−6.42 (m, 1H), 6.32 (dd, J = 4.0 Hz, 2.6 Hz, 1H), 4.63 2921, 2852, 1737, 1575, 1505, 1460, 1444, 1425, 1412, 1354, 1337, (t, J = 7.2 Hz, 2H), 4.03−3.96 (q, J = 6.7 Hz, 2H), 2.66 (t, J = 6.7 Hz, 1315, 1297, 1277, 1232, 1188, 1116, 996, 950, 856, 816 cm−1. 1H 2H), 2.42 (t, J = 7.2 Hz, 2H), 2.14 (s, 3H), 2.07 (s, 3H), 2.03 (qn, J = NMR (400 MHz, CD3OD): δ 7.09 (dt, J = 16.0 Hz, 0.6 Hz, 1H), 6.81 6.7 Hz, 2H), 2.00 (qn, J = 7.2 Hz, 2H). 13C NMR (101 MHz, (d, Jtrans = 16.0 Hz, 1H), 6.80 (s, 2H), 6.73 (dd, J = 2.7 Hz, 1.7 Hz, CDCl3): δ 189.4, 185.7, 177.1, 157.4, 128.7, 123.8, 112.0, 111.0, 48.8, 1H), 6.44 (ddd, J = 3.7 Hz, 2.7 Hz, 0.6 Hz, 1H), 6.08 (ddd, J = 3.7 45.1, 31.9, 31.5, 31.0, 29.4, 15.6, 15.5. HRMS (ESI+): calcd for Hz, 2.7 Hz, 0.6 Hz, 1H), 4.17 (t, J = 6.7 Hz, 2H), 3.87 (s, 6H), 3.76 [C16H22N2O2S2]: 339.1195 [M + H]+, 361.1015 [M + Na]+; found, (s, 3H), 2.44 (t, J = 6.7 Hz, 2H), 2.05 (s, 3H), 1.99 (qn, J = 6.7 Hz, 339.1205 [M + H]+, 361.1025 [M + Na]+. UV (DMSO) λmax, nm (ε, 2H). 13C NMR (101 MHz, CD3OD): δ 154.6, 138.3, 135.7, 132.5, M−1 cm−1): 281 (1.21 × 104), 367 (2.50 × 104). 126.4, 123.7, 118.2, 109.4, 107.3, 104.3, 61.2, 56.6, 45.6, 31.8, 15.3. 7738 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article HRMS (ESI+): calcd for [C19H25NO3S]: 348.1628 [M + H]+, the mixture was heated at 100 °C for 16 h. After this time, the mixture 370.1447 [M + Na]+; found, 348.1636 [M + H]+, 370.1458 [M + was cooled to 0 °C, and H2O (5 mL) was added dropwise. The Na]+. aqueous phase was extracted with EtOAc (3 × 5 mL). Each organic (E)-3-(5-{4-Methoxystyryl}-1-{3-[methylthio]propyl}-1H-pyrrol-2- phase was washed with brine (3 × 5 mL). The combined organic yl)-4-({3-[methylthio]propyl}amino)cyclobut-3-ene-1,2-dione, L2. A extracts were dried over anhydrous Na2SO4 and filtered. The solvent solution of 17 (2.73 g, 9.5 mmol) in CH2Cl2 (90 mL) was added was removed under reduced pressure, and the residue was purified by dropwise to a mixture of a solution of 9 (1.43 g, 9.5 mmol) in CH2Cl2 column chromatography (silica gel, EtOAc to EtOAc/MeOH, 95:5) (200 mL) and H2O (50 mL). The organic layer turned dark red, and to give crude 19 (341.2 mg) as a dark brown oil, which was used in the resulting mixture was stirred at room temperature under an argon the next step without further purification. atmosphere for 1 h. After this time, a solution of thioamine 11 (1.1 A solution of crude 19 (238.8 mg, 0.32 mmol) in CH2Cl2 (3 mL) mL, 9.8 mmol) and DIPEA (3.3 mL, 18.9 mmol) in CH2Cl2 (40 mL) was added dropwise to a mixture of a solution of dichlorocyclobu- was added, and the mixture was stirred for 30 min. Then, the organic tenedione 9 (49.9 mg, 0.33 mmol) in CH2Cl2 (7 mL) and H2O (2 layer was separated, dried over anhydrous Na2SO4, and filtered. The mL). The organic layer turned dark red, and the mixture was stirred at resulting filtrate was cooled to 0 °C, and Et2O was added. A yellow room temperature for 1 h. After this time, a solution of 11 (36 μL, solid precipitated, which was filtered and washed with Et2O to give 0.32 mmol) and DIPEA (113 μL, 0.64 mmol) in CH2Cl2 (1 mL) was pure L2 (3.19 mg, 6.8 mmol, 72%) as a yellow powder. mp 197−200 added. The resulting mixture was stirred at rt for 90 min. Then, the °C (from CH2Cl2/Et2O). IR (ATR): 3247, 2910, 1766, 1702, 1601, organic layer was dried over anhydrous Na2SO4 and filtered. The 1562, 1535, 1509, 1459, 1425, 1390, 1368, 1348, 1294, 1253, 1202, solvent was removed under reduced pressure, and the residue was 1176, 1137, 1110, 1060, 1028, 963, 814, 799, 765 cm−1. 1H NMR purified by column chromatography (silica gel, EtOAc to EtOAc/ (400 MHz, CDCl3): δ 7.48−7.43 (m, 2H), 7.04 (d, Jtrans = 16.0 Hz, MeOH, 95:5) to give pure L7 (245.6 mg, 0.26 mmol, 62% over 2 1H), 6.98 (d, Jtrans = 16.0 Hz, 1H), 6.93−6.88 (m, 2H), 6.67 (d, J = steps) as a red-orange oil. The oil was kept in the freezer turning into 4.3 Hz, 1H), 6.47 (d, J = 4.3 Hz, 1H), 6.44 (t, J = 6.6 Hz, 1H), 4.76 a brown sticky solid. mp 46−49 °C (from CH2Cl2). IR (ATR): 2919, (t, J = 7.3 Hz, 2H), 4.02 (q, J = 6.6 Hz, 2H), 3.84 (s, 3H), 2.68 (t, J = 2876, 1766, 1714, 1596, 1533, 1504, 1466, 1431, 1341, 1295, 1270, 6.6 Hz, 2H), 2.52 (t, J = 7.3 Hz, 2H), 2.15 (s, 3H), 2.07 (s, 3H), 2.04 1109, 952, 854 cm−1. 1H NMR (400 MHz, CDCl3): δ 7.00 (d, Jtrans = (qn, J = 6.6 Hz, 2H), 2.00 (qn, J = 7.3 Hz, 2H). 13C NMR (101 MHz, 16.0 Hz, 1H), 6.92 (d, Jtrans = 16.0 Hz, 1H), 6.75 (s, 2H), 6.66 (d, J = CDCl3): δ 188.5, 185.4, 176.6, 159.9, 156.6, 139.2, 130.9, 129.7, 4.3 Hz, 1H), 6.53 (t, J = 6.5 Hz, 1H), 6.49 (d, J = 4.3 Hz, 1H), 4.75 128.0, 125.0, 114.4, 113.6, 112.5, 109.1, 55.5, 45.2, 45.0, 32.0, 31.3, (t, J = 6.9 Hz, 2H), 4.22−4.13 (m, 6H), 3.99 (q, J = 6.5 Hz, 2H), 31.2, 29.3, 15.7, 15.6. HRMS (ESI+): calcd for [C25H30N2O3S2]: 3.89−3.49 (m, 30H), 3.36 (s, 3H), 3.36 (s, 6H), 2.66 (t, J = 6.5 Hz, 471.1771 [M + H]+, 493.1590 [M + Na]+; found, 471.1781 [M + 2H), 2.51 (t, J = 6.9 Hz, 2H), 2.13 (s, 3H), 2.06 (s, 3H), 2.05−1.95 H]+, 493.1603 [M + Na]+. UV (DMF) λmax, nm (ε, M−1 cm−1): 303 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 188.7, 185.4, 176.8, 156.5, (1.69 × 104), 431 (7.32 × 104), 447 (7.74 × 104). 153.0, 139.1, 138.6, 132.5, 130.8, 125.2, 115.3, 112.6, 109.4, 106.5, (E)-3-(1-{3-[Methylthio]propyl}-5-{3,4,5-trimethoxystyryl}-1H- 72.6, 72.1, 70.9, 70.8, 70.7, 70.6, 69.9, 69.1, 59.1, 45.1, 44.9, 31.9, pyrrol-2-yl)-4-({3-[methylthio]propyl}amino)cyclobut-3-ene-1,2- 31.4, 31.3, 29.5, 15.8, 15.6. HRMS (ESI + ): calcd for dione, L4. A solution of 18 (1.04 g, 3.0 mmol) in CH2Cl2 (40 mL) [C45H70N2O14S2]: 927.4341 [M + H]+, 949.4161 [M + Na]+; was added dropwise to a mixture of a solution of 9 (456 mg, 3.0 found, 927.4326 [M + H]+, 949.4164 [M + Na]+. UV (H2O) λmax, nm mmol) in CH2Cl2 (45 mL) and H2O (30 mL). The organic layer (ε, M−1 cm−1): 212 (2.15 × 104), 235 (1.70 × 104), 308 (1.03 × 104), turned dark red, and the resulting mixture was stirred at room 432 (4.06 × 104). temperature under an argon atmosphere for 1.5 h. After this time, a (E)-3-(5-{4-Hydroxystyryl}-1-{3-[methylthio]propyl}-1H-pyrrol-2- solution of 11 (340 μL, 3.0 mmol) and DIPEA (1.1 mL, 6.3 mmol) in yl)-4-({3-[methylthio]propyl}amino)cyclobut-3-ene-1,2-dione, L3. CH2Cl2 (5 mL) was added, and the mixture was stirred for 2 h. Then, Boron tribromide (1 M in dichloromethane, 10 mL, 10.0 mmol) the organic layer was separated, dried over anhydrous Na2SO4, and was added dropwise to a suspension of L2 (533 mg, 1.1 mmol) in filtered. The solvent of the resulting filtrate was removed partially anhydrous CH2Cl2 (10 mL) at −10 °C (ice/acetone bath). The under reduced pressure, and then the mixture was cooled to 0 °C, and mixture was stirred under an argon atmosphere at 0 °C for 2 h. After Et2O was added. An orange solid precipitated, which was filtered and this time, H2O (20 mL) was added dropwise to the cooled mixture, washed with Et2O to give pure L4 (925 mg, 1.7 mmol, 57%) as an and an orange solid precipitated. The resulting mixture was basified orange powder. mp 149−152 °C (from CH2Cl2/Et2O). IR (ATR): with a NaHCO3 saturated solution (55 mL) and stirred for 1 h. Then, 3337, 2912, 2836, 1760, 1694, 1581, 1526, 1505, 1464, 1444, 1423, the organic phase was removed under reduced pressure, and the 1342, 1295, 1271, 1245, 1230, 1128, 1043, 1003, 951, 805, 769 cm−1. formed precipitate was filtrated and washed with H2O and Et2O. The 1 H NMR (400 MHz, CDCl3): δ 7.08 (d, Jtrans = 16.0 Hz, 1H), 6.99 resulting solid was digested with Et2O and then filtrated to afford pure (d, Jtrans = 16.0 Hz, 1H), 6.76 (s, 2H), 6.69 (d, J = 4.3 Hz, 1H), 6.51 L3 (439.8 mg, 0.96 mmol, 87%) as a brown solid. mp 192−196 °C (t, J = 6.6 Hz, 1H), 6.49 (d, J = 4.3 Hz, 1H), 4.79 (t, J = 6.8 Hz, 2H), (from CH2Cl2). IR (ATR): 3249, 2916, 1766, 1705, 1568, 1536, 4.01 (q, J = 6.6 Hz, 2H), 3.91 (s, 6H), 3.87 (s, 3H), 2.67 (t, J = 6.6 1511, 1459, 1428, 1386, 1368, 1342, 1296, 1270, 1253, 1217, 1173, Hz, 2H), 2.54 (t, J = 6.8 Hz, 2H), 2.15 (s, 3H), 2.07 (s, 3H), 2.07− 1138, 1106, 1061, 1044, 952, 929, 850, 816, 760 cm−1. 1H NMR (400 1.97 (m, 4H). 13C NMR (101 MHz, CDCl3): δ 186.6, 185.4, 176.8, MHz, DMSO-d6): δ 9.65 (s, 1H), 8.57 (t, J = 6.6 Hz, 1H), 7.59−7.43 156.5, 153.6, 138.6, 138.5, 132.7, 131.0, 125.2, 115.3, 112.5, 109.3, (m, 2H), 7.13 (d, J = 16.1 Hz, 1H), 7.05 (d, J = 16.1 Hz, 1H), 6.96 103.7, 61.1, 56.3, 45.2, 44.8, 32.0, 31.4, 31.3, 29.3, 15.8, 15.6. HRMS (d, J = 4.4 Hz, 1H), 6.82 (d, J = 4.4 Hz, 1H), 6.80−6.76 (m, 2H), (ESI+): calcd for [C27H34N2O5S2]: 531.1982 [M + H]+, 553.1801 [M 4.72 (t, J = 7.2 Hz, 2H), 3.77 (q, J = 6.6 Hz, 2H), 2.52 (t, J = 6.6 Hz, + Na]+; found, 531.1996 [M + H]+, 553.1814 [M + Na]+. UV (DMF) 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.05 (s, 3H), 1.99 (s, 3H), 1.90 (qn, J = λmax, nm (ε, M−1 cm−1): 306 (1.29 × 104), 429 (5.93 × 104), 448 6.6 Hz, 2H), 1.82 (qn, J = 7.2 Hz, 2H). 13C NMR (101 MHz, DMSO- (6.20 × 104). d6): δ 188.9, 184.3, 175.6, 157.6, 155.4, 138.7, 130.2, 128.0, 124.5, (E)-3-(1-{3-[Methylthio]propyl}-5-{3,4,5-tris[2-(2-{2- 115.6, 114.5, 112.8, 108.6, 44.2, 43.5, 31.3, 30.2, 30.0, 29.9, 14.6. methoxyethoxy}ethoxy)ethoxy]styryl}-1H-pyrrol-2-yl)-4-({3- HRMS (ESI+): calcd for [C24H28N2O3S2]: 457.1614 [M + H]+, [methylthio]propyl}amino)cyclobut-3-ene-1,2-dione, L7. A suspen- 479.1434 [M + Na]+; found, 457.1604 [M + H ]+, 479.1432 [M + sion of NaH (60% dispersion in mineral oil, 51.7 mg, 1.29 mmol) in Na]+. UV (DMSO) λmax, nm (ε, M−1 cm−1): 306 (1.56 × 104), 436 anhydrous DMF (0.5 mL) was cooled to 0 °C under an argon (5.60 × 104), 452 (5.77 × 104). atmosphere. Then, a solution of diethyl (3,4,5-tris{2-[2-(2- (E)-3-(1-{3-[Methylthio]propyl}-5-{3,4,5-trihydroxystyryl}-1H-pyr- methoxyethoxy)ethoxy]ethoxy}benzyl)phosphonate36 (431.6 mg, rol-2-yl)-4-({3-[methylthio]propyl}amino)cyclobut-3-ene-1,2-dione, 0.60 mmol) in anhydrous DMF (0.7 mL) was added dropwise. The L5. Boron tribromide (1.0 M in dichloromethane, 14 mL, 14.0 mmol) reaction mixture was stirred at 0 °C for 30 min, then a solution of 13 was added dropwise to a suspension of 22 (482 mg, 0.91 mmol) in (112.8 mg, 0.62 mmol) in anhydrous DMF (0.7 mL) was added, and anhydrous CH2Cl2 (14 mL) at −10 °C (ice/acetone bath). The 7739 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article mixture was stirred under an argon atmosphere at 0 °C for 1 h. After 69.4, 58.8, 44.5, 44.4, 31.0, 30.8, 29.7, 15.2. UV (H2O) λmax, nm (ε, this time, 30 mL of H2O was added, and an orange solid precipitated. M−1 cm−1): 192 (3.32 × 104), 234 (1.38 × 104), 308 (1.09 × 104), The resulting mixture was basified with a NaHCO3 saturated solution 435 (3.39 × 104). (40 mL), and the solid was filtrated and washed with H2O and (E)-3-(5-{4-[(tert-Butyldiphenylsilyl)oxy]styryl}-1-{3-[methylthio]- MeOH to give pure L5 (436.6 mg, 0.89 mmol, 98%) as a dark orange propyl}-1H-pyrrol-2-yl)-4-({3-[methylthio]propyl}amino)cyclobut-3- solid. mp 215−218 °C (from CH2Cl2). IR (ATR): 3435, 3269, 2915, ene-1,2-dione, L20. tert-Butyldiphenylsilyl chloride (0.35 mL, 1.32 1765, 1671, 1570, 1524, 1468, 1432, 1366, 1334, 1312, 1270, 1224, mmol) was added dropwise to a solution of L3 (274.4 mg, 0.60 1191, 1174, 1116, 1047, 997, 955, 860, 820, 767 cm−1. 1H NMR (400 mmol) in anhydrous DMF (0.25 mL). The resulting mixture was MHz, DMSO-d6): δ 8.91 (s, 2H), 8.53 (t, J = 7.1 Hz, 1H), 8.50 (s, stirred under an argon atmosphere at room temperature for 18 h. 1H), 6.98 (d, J = 15.9 Hz, 1H), 6.93 (d, J = 4.3 Hz, 1H), 6.89 (d, J = After this time, 5 mL of H2O was added, and the product was 15.9 Hz, 1H), 6.83 (d, J = 4.3 Hz, 1H), 6.56 (s, 2H), 4.72 (t, J = 7.1 extracted with EtOAc (3 × 5 mL). Each organic layer was washed Hz, 2H), 3.77 (q, J = 7.1 Hz, 2H), 2.53 (t, J = 7.1 Hz, 2H), 2.39 (t, J = with H2O (3 × 5 mL). The combined organic extracts were dried over 7.1 Hz, 2H), 2.06 (s, 3H), 2.01 (s, 3H), 1.90 (qn, J = 7.1 Hz, 2H), anhydrous Na2SO4, and the solvent was removed under reduced 1.83 (qn, J = 7.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 188.8, pressure. The residue was purified by column chromatography (silica 184.4, 175.6, 155.4, 146.2, 138.6, 134.0, 131.1, 127.6, 124.5, 114.4, gel, hexane/EtOAc, 1:1) to give pure L20 (225.2 mg, 0.32 mmol, 112.7, 108.7, 105.9, 44.1, 43.5, 31.2, 30.2, 30.0, 29.9, 14.6. HRMS 53%) as a yellow solid. mp 161−163 °C (from CH2Cl2). IR (ATR): (ESI+): calcd for [C24H28N2O5S2]: 489.1512 [M + H]+, 511.1332 [M 3308, 2931, 2857, 1768, 1706, 1601, 1563, 1538, 1507, 1472, 1426, + Na]+; found, 489.1495 [M + H]+, 511.1324 [M + Na]+. UV 1390, 1370, 1254, 1174, 1139, 1108, 1057, 954, 912, 853, 819 cm−1. (DMSO) λmax, nm (ε, M−1 cm−1): 311 (1.33 × 104), 441 (6.93 × 1 H NMR (400 MHz, CDCl3): δ 7.76−7.70 (m, 4H), 7.47−7.41 (m, 104), 455 (7.19 × 104). 2H), 7.41−7.35 (m, 4H), 7.28−7.23 (m, 2H), 6.95 (d, J = 16.0 Hz, (E)-3-(5-{4-[2-(2-{2-Methoxyethoxy}ethoxy)ethoxy]styryl}-1-{3- 1H), 6.90 (d, J = 16.0 Hz, 1H), 6.79−6.74 (m, 2H), 6.62 (d, J = 4.3 [methylthio]propyl}-1H-pyrrol-2-yl)-4-({3-[methylthio]propyl}- Hz, 1H), 6.58 (t, J = 6.6 Hz, 1H), 6.49 (d, J = 4.3 Hz, 1H), 4.72 (t, J = amino)cyclobut-3-ene-1,2-dione, L6. K2CO3 (50.1 mg, 0.36 mmol) 7.1 Hz, 2H), 4.00 (q, J = 6.6 Hz, 2H), 2.66 (t, J = 6.6 Hz, 2H), 2.48 was added to a suspension of L3 (77.0 mg, 0.17 mmol) and 1-bromo- (t, J = 7.1 Hz, 2H), 2.13 (s, 3H), 2.03 (qn, J = 6.6 Hz, 2H), 2.02 (s, 2-(2-(2-methoxyethoxy)ethoxy)ethane37 (53.3 mg, 0.23 mmol) in 3H), 1.97 (qn, J = 7.1 Hz, 2H), 1.11 (s, 9H). 13C NMR (101 MHz, anhydrous MeCN (2 mL). The mixture was refluxed for 24 h under CDCl3): δ 188.5, 185.4, 176.7, 156.6, 156.0, 139.2, 135.6, 132.8, an argon atmosphere. After this time, the mixture was cooled to room 131.0, 130.1, 130.0, 127.9, 127.7, 125.0, 120.3, 113.7, 112.7, 109.1, temperature, K2CO3 was removed by filtration, and the product was 45.1, 45.0, 31.9, 31.3, 31.1, 29.5, 26.6, 19.6, 15.6, 15.6. HRMS (ESI+): recrystallized from hot methanol to give L6 (45.3 mg, 0.08 mmol, calcd for [C40H46N2O3S2Si]: 695.2792 [M + H]+, 717.2611 [M + 47%) as a yellow solid. mp 110−112 °C (from MeOH). IR (ATR): Na]+; found, 695.2794 [M + H ]+, 717.2617 [M + Na]+. UV (CHCl3) 3253, 2911, 2872, 1764, 1705, 1604, 1558, 1508, 1459, 1447, 1390, λmax, nm (ε, M−1 cm−1): 301 (1.21 × 104), 431 (5.59 × 104), 446 1368, 1337, 1292, 1273, 1245, 1203, 1174, 1135, 1057, 1031, 951, (5.75 × 104). 849, 815, 800, 758 cm−1. 1H NMR (400 MHz, CDCl3): δ 7.44−7.40 (E)-3-(5-{4-Methoxystyryl}-1-{3-[methylthio]propyl}-1H-pyrrol-2- (m, 2H), 7.01 (d, J = 16.0 Hz, 1H), 6.95 (d, J = 16.0 Hz, 1H), 6.92− yl)-4-(methylamino)cyclobut-3-ene-1,2-dione, L22. A solution of 17 6.87 (m, 2H), 6.65 (d, J = 4.3 Hz, 1H), 6.63 (t, J = 6.9 Hz, 1H), 6.52 (88.1 mg, 0.31 mmol) in CH2Cl2 (3 mL) was added dropwise to a (d, J = 4.3 Hz, 1H), 4.75 (t, J = 7.0 Hz, 2H), 4.17−4.12 (m, 2H), 3.99 mixture of a solution of dichlorocyclobutenedione 9 (46.5 mg, 0.31 (q, J = 6.9 Hz, 2H), 3.89−3.84 (m, 2H), 3.77−3.71 (m, 2H), 3.71− mmol) in CH2Cl2 (6 mL) and H2O (2 mL). The organic layer turned 3.63 (m, 4H), 3.57−3.52 (m, 2H), 3.37 (s, 3H), 2.66 (t, J = 6.9 Hz, dark red, and the resulting mixture was stirred at room temperature 2H), 2.50 (t, J = 7.0 Hz, 2H), 2.13 (s, 3H), 2.05 (s, 3H), 2.07−1.95 under argon for 1 h. After this time, a solution of methylamine (33% (m, 4H). 13C NMR (101 MHz, CDCl3): δ 188.5, 185.4, 176.6, 159.1, wt in MeOH, 38 μL, 0.31 mmol) and DIPEA (105 μL, 0.60 mmol) in 156.6, 139.2, 130.8, 129.9, 127.9, 125.0, 115.1, 113.7, 112.7, 109.1, CH2Cl2 (2 mL) was added, and the mixture was stirred for 30 min. 72.0, 71.0, 70.8, 70.7, 69.8, 67.6, 59.2, 45.1, 45.0, 31.9, 31.3, 31.2, Then, the organic layer was separated, dried over anhydrous Na2SO4, 29.5, 15.6. HRMS (ESI+): calcd for [C31H42N2O6S2]: 603.2557 [M + and filtered. The resulting filtrate was cooled to 0 °C, and Et2O was H]+, 625.2376 [M + Na]+; found, 603.2564 [M + H]+, 625.2387 [M added. An orange solid precipitated, which was filtered and washed + Na]+. UV (DMF) λmax, nm (ε, M−1 cm−1): 304 (1.43 × 104), 431 with Et2O to give pure L22 (45 mg, 0.11 mmol, 37%) as an orange (6.03 × 104), 448 (6.40 × 104). powder. mp >220 °C (from CH2Cl2/Et2O). IR (ATR): 3293, 2910, α-Methyl-ω-((E)-3-{5-[4-oxystyryl]-1-[3-(methylthio)propyl]-1H- 1766, 1699, 1569, 1507, 1450, 1426, 1386, 1294, 1251, 1172, 1130, pyrrol-2-yl}-4-{[3-(methylthio)propyl]amino}cyclobut-3-ene-1,2- 1110, 1026, 956, 816 cm−1. 1H NMR (400 MHz, DMSO-d6): δ 8.43 dione)poly(oxyethane-1,2-diyl), L8. K2CO3 (421 mg, 3.0 mmol) was (q, J = 4.5 Hz, 1H), 7.61−7.53 (m, 2H), 7.17 (d, J = 16.2 Hz, 1H), added to a suspension of L3 (774 mg, 1.7 mmol) and α-methyl-ω- 7.12 (d, J = 16.2 Hz, 1H), 7.00−6.92 (m, 2H), 6.89 (d, J = 4.3 Hz, (methylsulfonate)poly(oxyethane-1,2-diyl)38 (Mw = 2078, 2.71 g, 1.3 1H), 6.83 (d, J = 4.3 Hz, 1H), 4.75 (t, J = 7.4 Hz, 2H), 3.78 (s, 3H), mmol) in anhydrous MeCN (20 mL). The mixture was refluxed for 3.31 (d, J = 4.5 Hz, 3H), 2.38 (t, J = 7.4 Hz, 2H), 1.99 (s, 3H), 1.82 16 h under an argon atmosphere. After this time, the mixture was (qn, J = 7.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 189.4, cooled to room temperature, K2CO3 was removed by filtration, and 184.3, 175.9, 159.1, 155.1, 138.3, 129.6, 127.9, 124.7, 114.2, 114.0, the solvent was removed under a reduced pressure. Then, CH2Cl2 113.9, 108.8, 55.2, 44.2, 31.3, 29.9, 14.6. HRMS (ESI+): calcd for (100 mL) and brine (60 mL) were added, and the aqueous layer was [C22H24N2O3S]: 397.1580 [M + H]+, 419.1400 [M + Na]+; found, extracted with CH2Cl2 (3 × 20 mL). The combined organic layers 397.1567 [M + H]+, 419.1385 [M + Na]+. UV (DMF) λmax, nm (ε, were dried over anhydrous Na2SO4 and filtered, the solvent was M−1 cm−1): 302 (1.48 × 106), 429 (6.08 × 106), 446 (6.44 × 106). removed under a reduced pressure, and the product was recrystallized Synthesis of Complexes. Complex C1. Ligand L1 (86.7 mg, from CH2Cl2/Et2O to afford L8 (2.89 g, 1.2 mmol, 92%) as a yellow 0.26 mmol) and K2PtCl4 (105.6 mg, 0.25 mmol) were suspended in a solid. mp 51−55 °C (from CH2Cl2/Et2O). IR (ATR): 2880, 2740, mixture of MeOH and water (1:1, 4 mL). The mixture was stirred at 1765, 1708, 1596, 1536, 1511, 1466, 1359, 1341, 1279, 1240, 1146, room temperature under an argon atmosphere for 17 h. After this 1100, 1060, 960, 841 cm−1. 1H NMR (400 MHz, CDCl3): δ 7.44− time, a brown solid precipitated. After filtration, the solid was washed 7.38 (m, 2H), 7.00 (d, J = 16.1 Hz, 1H), 6.94 (d, J = 16.1 Hz, 1H), with hot MeOH and water and then dried to afford C1 as a brown 6.91−6.86 (m, 2H), 6.69−6.61 (m, 3H), 6.54−6.48 (m, 1H), 4.74 (t, powder (82.0 mg, 0.14 mmol, 56%). mp 173−178 °C (from MeOH/ J = 7.0 Hz, 2H′), 4.15−4.09 (m, 2H), 3.96 (q, J = 6.8 Hz, 2H), 3.88− H2O, 1:1). IR (ATR): 3283, 2920, 1772, 1706, 1589, 1537, 1478, 3.39 (m), 3.35 (s, 3H), 2.63 (t, J = 6.8 Hz, 2H), 2.48 (t, J = 7.0 Hz, 1421, 1355, 1264, 1132, 1092, 970 cm−1. 1H NMR (400 MHz, 2H), 2.11 (s, 3H), 2.04 (s, 3H), 2.03−1.91 (m, 4H). 13C NMR (101 DMSO-d6) as a mixture of products due to one Cl exchange: 8.67− MHz, CDCl3): δ 188.4, 184.9, 176.1, 158.6, 156.0, 138.6, 130.1, 8.51 (m), 7.31−7.16 (m), 6.92−6.81 (m), 6.40−6.26 (m), 4.61 (t, J = 129.6, 127.5, 124.7, 115.9, 114.7, 113.4, 108.7, 72.3, 71.7, 70.6, 70.3, 6.9 Hz), 4.54 (t, J = 7.0 Hz), 3.86−3.71 (m), 2.59−2.50 (bs), 2.36− 7740 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article 2.28 (bs), 1.93−1.81 (bs), 2.21−1.96 (bs). 13C NMR (101 MHz, to afford C5 (80.5 mg, 0.11, 85%) as a brown solid. mp >200 °C DMSO-d6) as a mixture of products due to one Cl exchange: δ 190.0 (from THF/H2O). IR (ATR): 3317, 1767, 1699, 1585, 1526, 1462, + 189.8, 184.8 + 184.7, 176.0, 156.4, 156.2, 128.9, 123.3, 114.0− 1426, 1311, 1187, 1139, 1032, 998, 958, 819 cm−1. 1H NMR (400 113.7, 110.6−110.4, 48.0, 47.6, 43.5, 43.1, 35.4, 35.3, 31.4, 30.2, 30.0, MHz, DMSO-d6): δ 8.98−8.81 (m), 8.69−8.35 (m), 7.11−6.75 (m), 29.9, 29.7, 28.4, 21.0, 20.6. HRMS (ESI + ): calcd for 6.65−6.51 (m), 4.89−4.64 (m), 3.93−3.57 (m), 3.44−3.07 (m), [C16H22N2O2S2PtCl2]: 532.0688 [M−2Cl−H]+, 569.0444 [M−Cl]+, 2.65−1.62 (m). MS (ESI+): calcd for [C24H28N2O5S2PtCl2]: 719.1 605.0203 [M + H]+, 610.0827 [M−2Cl−H + DMSO]+, 647.0582 [M−Cl]+, 755.1 [M + H]+, 777.0 [M + Na]+, 797.1 [M−Cl + [M−Cl + DMSO]+; found, 532.0678 [M−2Cl−H]+, 569.0443 [M− DMSO]+; found, 719.1 [M−Cl]+, 755.0 [M + H]+, 777.0 [M + Na]+, Cl]+, 605.0206 [M + H]+, 610.0817 [M−2Cl−H + DMSO]+, 797.1 [M−Cl + DMSO]+. EA calcd for C24H28N2O5S2PtCl2 (%): C, 647.0583 [M−Cl + DMSO]+. EA calcd for C16H22N2O2S2PtCl2 (%): 37.31; H, 3.91; N, 3.63; S, 8.30 [M + H2O]. Found: C, 37.14, H, 3.88, C, 30.87; H, 3.89; N, 4.50; S, 10.30 [M + H2O]. Found: C, 30.76; H, N, 3.31, S, 7.67 [M + H2O]. UV (DMSO) λmax, nm (ε, M−1 cm−1): 3.49; N, 4.22; S, 10.12 [M + H2O]. UV (DMSO) λmax, nm (ε, M−1 306 (1.28 × 104), 439 (5.62 × 104), 455 (5.78 × 104). cm−1): 276 (1.40 × 104), 366 (2.25 × 104), 445 (9.32 × 102), 516 Complex C6. A solution of K2PtCl4 (67.0 mg, 0.16 mmol) in H2O (2.30 × 102). (2 mL) was added to a solution of L6 (96.3 mg, 0.16 mmol) in THF Complex C2. A solution of K2PtCl4 (153.9 mg, 0.37 mmol) in H2O (2 mL). The resulting mixture was stirred at room temperature under (5 mL) was added to a solution of L2 (172.0 mg, 0.37 mmol) in THF argon for 16 h. After this time, a sticky orange solid precipitated, (5 mL). The solution was stirred at room temperature under argon which was filtrated and washed with H2O, MeOH, and THF and for 22 h. After this time, an orange solid precipitated. After filtration, dried. The sticky solid was digested with Et2O to give pure C6 (111.3 the solid was washed with THF and water and dried to afford C2 as mg, 0.13 mmol, 81%) as an orange powder. mp 135−139 °C (from an orange powder (262.2 mg, 0.36 mmol, 97%). mp 184−187 °C THF/H2O). IR (ATR): 2869, 1762, 1704, 1583, 1533, 1509, 1460, (from THF/H2O). IR (ATR): 3302, 2916, 1764, 1701, 1584, 1533, 1424, 1371, 1298, 1236, 1176, 1138, 1093, 1057, 954, 849, 817 cm−1. 1509, 1461, 1425, 1299, 1247, 1174, 1141, 1029, 957, 850, 816 cm−1. 1 H NMR (400 MHz, DMSO-d6): δ 8.65−8.51 (m), 7.66−7.51 (m), 1 H NMR (400 MHz, DMF-d7): δ 8.75−8.51 (m), 7.75−755 (m), 7.25−7.08 (m), 7.02−6.80 (m), 4.93−4.66 (m), 4.23−4.04 (m), 7.40−7.65 (m), 5.06−4.63 (m), 4.23−3.76 (m), 3.41−2.83 (m), 3.88−3.68 (m), 3.65−3.48 (m), 3.47−3.40 (m), 3.26−3.20 (m), 2.69−2.02 (m). HRMS (ESI+): calcd for [C25H30N2O3S2PtCl2]: 2.92−2.75 (m), 2.61−2.34 (m), 2.18−2.03 (m), 2.01−1.96 (m), 737.0781 [M + H]+, 759.0601 [M + Na]+, 775.0339 [M + K]+; 1.95−1.78 (m). MS (ESI+): calcd for [C31H42N2O6S2PtCl2]: 869.2 found, 737.0791 [M + H]+, 759.0607 [M + Na]+, 775.0353 [M + K]+. [M + H]+, 891.1 [M + Na]+; found, 869.2 [M + H]+, 891.1 [M + EA calcd for C25H30N2O3S2PtCl2 (%): C, 40.76; H, 4.11; N, 3.80; S, Na]+. EA calcd for C31H42N2O6S2PtCl2 (%): C, 41.99, H, 5.00, N, 8.70. Found: C, 40.96; H, 4.33; N, 3.51; S, 8.55. UV (DMF) λmax, nm 3.16, S, 7.23 [M + H2O]. Found: C, 42.07, H, 4.79, N, 3.14, S, 7.15 (ε, M−1 cm−1): 305 (1.61 × 104), 430 (6.18 × 104), 446 (5.95 × 104). [M + H2O]. UV (DMF) λmax, nm (ε, M−1 cm−1): 304 (1.90 × 104), Complex C3. A solution of K2PtCl4 (89.2 mg, 0.22 mmol) in H2O 429 (7.19 × 104), 446 (6.93 × 104). (3 mL) was added to a solution of L3 (95.1 mg, 0.21 mmol) in THF Complex C7. A solution of K2PtCl4 (109.6 mg, 0.26 mmol) in H2O (3 mL). The solution was stirred at room temperature under argon (4 mL) was added to a solution of L7 (250.4 mg, 0.26 mmol) in THF for 15 h. After this time, a brown-red solid precipitated. After (4 mL). The solution was stirred at room temperature under argon filtration, the solid was washed with H2O, MeOH, and THF and dried for 17 h. After this time, 20 mL of brine was added, and the product to afford C3 as a brown-red powder (130.8 mg, 0.18 mmol, 86%). mp was extracted with CH2Cl2 (3 × 20 mL). The combined organic >200 °C (from THF/H2O). IR (ATR): 1736, 1691, 1579, 1532, layers were dried over anhydrous Na2SO4 and filtrated. The product 1510, 1461, 1421, 1267, 1168, 1138, 1044, 955, 850, 810 cm−1. 1H was recrystallized in CH2Cl2/Et2O to give C7 (207.2 mg, 0.17 mmol, NMR (400 MHz, DMSO-d6): δ 9.65 (s), 8.70−8.51 (m), 7.59−7.41 65%) as a brown solid. mp 92−95 °C (from CH2Cl2/Et2O). IR (m), 7.22−6.62 (m), 4.92−4.63 (m), 3.91−3.68 (m), 3.27−3.03 (m), (ATR): 2874, 1764, 1707, 1590, 1530, 1503, 1459, 1429, 1338, 1248, 2.70−2.24 (m), 2.15−1.70 (m). MS (ESI + ): calcd for 1096, 951.92, 851 cm−1. 1H NMR (400 MHz, CDCl3): δ 7.12−6.50 [C24H28N2O3S2PtCl2]: 687.1 [M−Cl]+, 745.0 [M + Na]+, 765.1 (m), 4.44−4.04 (m), 3.97−3.44 (m), 3.39−3.30 (m), 2.62−2.01 (m), [M−Cl + DMSO]+; found, 687.1 [M−Cl]+, 745.0 [M + Na]+, 765.1 1.82 (s). MS (ESI+): calcd for [C45H70N2O14S2PtCl2]: 1215.3 [M + [M−Cl + DMSO]+. EA calcd for C24H28N2O3S2PtCl2 (%): C, 38.45; Na]+; found, 1215.3 [M + Na]+. EA calcd for C45H70N2O14S2PtCl2 H, 4.17; N, 3.74; S, 8.55 [M + 3/2H2O]. Found: C, 38.39; H, 3.82; (%): C, 42.64; H, 5.57; N, 2.21; S, 5.06 [M + KCl]. Found: C, 42.73, N, 3.50; S, 8.27 [M + 3/2H2O]. UV (DMSO) λmax, nm (ε, M−1 H, 5.58, N, 2.11, S, 4.82 [M + KCl]. UV (H2O) λmax, nm (ε, M−1 cm−1): 303 (1.58 × 104), 436 (5.22 × 104), 450 (5.29 × 104). cm−1): 221 (3.19 × 104), 237 (2.83 × 104), 312 (1.11 × 104), 432 Complex C4. A solution of K2PtCl4 (57.2 mg, 0.14 mmol) in water (3.66 × 104), 462 (2.71 × 104). (1.5 mL) was added to a solution of L4 (69.6 mg, 0.13 mmol) in Complex C8. A solution of K2PtCl4 (235.5 mg, 0.57 mmol) in H2O THF (1.5 mL). The solution was stirred at room temperature under (6 mL) was added to a solution of L8 (Mw = 2439, 1.06 g, 0.43 argon for 20 h. After this time, an orange solid precipitated. After mmol) in H2O (7 mL). The solution was stirred at room temperature filtration, the solid was washed with THF and water and dried to under argon for 16 h. After this time, brine (20 mL) was added, and afford C4 as an orange powder (102.9 mg, 99%). mp >200 °C (from the mixture was extracted with CH2Cl2 (3 × 20 mL). The combined THF/H2O). IR (ATR): 3299, 2930, 1762, 1701, 1578, 1529, 1503, organic extracts were dried over anhydrous Na2SO4 and filtered, the 1459, 1416, 1336, 1266, 1241, 1184, 1120, 1041, 997, 951, 808, 760 solvent was removed under reduced pressure, and the residue was cm−1. 1H NMR (400 MHz, DMF-d7): δ 8.74−8.57 (m), 7.52−7.14 recrystallized from CH2Cl2/Et2O to give C8 (1.10 g, 0.41 mmol, (m), 7.13−6.96 (m), 6.95−6.79 (m), 5.07−4.80 (m), 4.07−3.85 (m), 95%) as a brown solid. mp 50−53 °C (from CH2Cl2/Et2O). IR 3.81−3.72 (m), 3.69−3.60 (m), 3.37−2.83 (m), 2.70−2.05 (m), (ATR): 2878, 1765, 1706, 1596, 1510, 1466, 1342, 1279, 1241, 1145, 1.88−1.74 (m). HRMS (ESI+): calcd for [C27H34N2O5S2PtCl2]: 1100, 1060, 961, 842 cm−1. 1H NMR (400 MHz, CDCl3): δ 7.76− 797.0994 [M + H]+, 819.0813 [M + Na]+, 835.0551 [M + K]+; 6.45 (m), 4.99−3.48 (m), 3.46−3.40 (m), 3.36 (s), 2.79−2.00 (m). found, 797.0978 [M + H]+, 819.0798 [M + Na]+, 835.0551 [M + K]+. ICP-OES: 6.7% Pt. UV (H2O) λmax, nm (ε, M−1 cm−1): 235 (2.86 × EA calcd for C27H34N2O5S2PtCl2 (%): C, 39.81; H, 4.45; N, 3.44; S, 104), 308 (1.36 × 104), 434 (2.75 × 104), 462 (2.18 × 104). 7.87 [M + H2O]. Found: C, 39.42, H, 4.15, N, 3.15, S, 7.49 [M + Complex C20. A solution of K2PtCl4 (60.9 mg, 0.15 mmol) in H2O H2O]. UV (DMF) λmax, nm (ε, M−1 cm−1): 307 (1.31 × 104), 429 (2 mL) was added to a solution of L20 (98.1 mg, 0.14 mmol) in THF (6.40 × 104), 448 (6.18 × 104). (2 mL). The solution was stirred at room temperature under argon Complex C5. A solution of K2PtCl4 (54.8 mg, 0.13 mmol) in H2O for 17 h. After this time, 20 mL of brine was added, and the product (2 mL) was added to a brown suspension of L5 (63.8 mg, 0.13 mmol) was extracted with CH2Cl2 (3 × 20 mL). The combined organic in THF (2 mL). The solution was stirred at room temperature under layers were dried over anhydrous Na2SO4 and filtrated. The product argon for 16 h. After this time, a brown solid precipitated. After was recrystallized from CH2Cl2/Et2O to give pure C20 (114.0 mg, filtration, the solid was washed with H2O, MeOH, and THF and dried 0.12 mmol, 86%) as a dark orange solid. mp 190−195 °C (from 7741 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry pubs.acs.org/IC Article CH2Cl2/Et2O). IR (ATR): 2929, 2856, 1764, 1702, 1584, 1533, quartz cuvettes. Measurements were carried out at a constant 1506, 1461, 1425, 1253, 1169, 1141, 1104, 955, 910, 851, 819 cm−1. temperature of 25 °C. CD spectra were measured in 10 mM Tris- 1 H NMR (360 MHz, CDCl3): δ 7.76−7.62 (m), 7.49−7.13 (m), HCl buffer (pH 7.24). The calf thymus DNA concentration was 50 7.01−6.39 (m), 4.82−4.33 (m), 4.24−3.86 (m), 3.33−1.81 (m), μM. Different samples with an increasing amount of the Pt complex to 1.29−0.79 (m). MS (ESI+): calcd for [C40H46N2O3S2SiPtCl2]: 925.2 study (0, 25, and 50 μM) were incubated at 37 °C for 24 h before [M−Cl]+, 961.2 [M + H]+, 983.2 [M + Na]+; found, 925.2 [M−Cl]+, spectra were recorded in the range of 200−350 nm. Complexes were 961.2 [M + H] + , 983.2 [M + Na] + . EA calcd for added from 10−3 M stock solutions in water/DMSO, 98:2, solvent C40H46N2O3S2SiPtCl2 (%): C, 49.99; H, 4.82; N, 2.92; S, 6.67. mixtures. Irradiated stocks were prepared by irradiation with a blue Found: C, 49.72, H, 4.85, N, 2.72, S, 6.25. UV (CHCl3) λmax, nm (ε, LED (λexc ∼ 450 nm, 3 W) until the total disappearance of the M−1 cm−1): 310 (9.15 × 103), 429 (3.83 × 104), 455 (3.02 × 104). absorption maxima band of the initial species (monitored by UV−vis X-ray Structure Determination. Crystals of L2 were mounted measurements of aliquots). on a glass fiber and used for the data collection on a Bruker D8 Cell Culture. Human ovarian cancer cells A2780 and human Venture with a photon detector equipped with graphite-monochro- ovarian cancer cells cisplatin-resistant A2780cis were obtained from mated Cu Kα radiation (λ = 1.54178 Å). The data reduction was the European Collection of Authenticated Cell Cultures (ECACC, performed with APEX239 software and corrected for absorption using UK) and were routinely cultured in Dulbecco’s modified Eagle’s SADABS.40 Crystal structures were solved by direct methods using medium (Invitrogen) containing 10% heat-inactivated fetal bovine SIR97 program41 and refined by full-matrix least-squares on F2, serum (FBS) at 37 °C in a humidified CO2 atmosphere. Human including all reflections using anisotropic displacement parameters by adenocarcinoma cells (HeLa) were obtained from American Type means of WINGX crystallographic package.42 Generally, anisotropic Culture Collection (ATCC, Manassas, VA, USA) and were routinely temperature factors were assigned to all atoms except for hydrogen cultured in modified Eagle’s medium alpha (MEM-α, Invitrogen) atoms, which are riding their parent atoms with an isotropic containing 10% heat-inactivated fetal bovine serum at 37 °C in a temperature factor arbitrarily chosen as 1.2 times that of the humidified CO2 atmosphere. respective parent. Final R(F), wR(F2), the goodness of fit agreement DNA Isolation. To investigate the Pt content associated with the factors, and details on the data collection and analysis can be found in DNA of A2780 cells, DNA was extracted and purified by using the Table S3. Crystallographic data (excluding structure factors) for the phenol/chloroform methodology. In brief, 5 × 106 cells were seeded structure reported in this paper have been deposited with the in 150 mm dishes and allowed to attach overnight. Cells were then Cambridge Crystallographic Data Centre as supplementary publica- incubated with the different compounds for 24 h in a 10% serum- tion Nr. CCDC 2100958 for compound L2. Copies of the data can be containing medium. Following incubation, the supernatant was obtained free of charge on application to the Director, CCDC, 12 discarded, and the cells were washed thoroughly 3 times with 5 mL Union Road, Cambridge, CB2 1EZ, U.K. (Fax: +44-1223-335033; e- ice-cold PBS. Cells were trypsinized and transferred into a 15 mL tube mail: deposit@ccdc.cam.ac.uk). for centrifugation at 500g for 5 min. The obtained pellet was Photochemical Characterization. UV−vis absorption measure- resuspended in lysis buffer (100 mM NaCl, 10 mM Tris-Cl pH 8, 25 ments were recorded on a HP 8453 spectrophotometer. mM EDTA pH 8, 0.5% SDS, 0.1 mg/mL proteinase K), and genomic Fluorescence emission spectra were recorded using a custom-made DNA was purified by the phenol/chloroform extraction. The spectrofluorometer, where the sample was excited with a cw diode extracted DNA was eluted using 1 mL of the TE buffer (10 mM laser (λexc = 405 nm), and emitted photons were detected using an Tris-Cl pH 9, 0.1 mM EDTA). The DNA concentration and purity of Andor ICCD camera coupled to a spectrograph. All the emission the sample were determined by spectral photometry (NanoDrop spectra registered were corrected by the wavelength dependence of 2000c, Thermo Scientific) in triplicate. the spectra response of the detection system. Samples were prepared ICP−MS. For quantification of the metal content, 1 mL of the in spectroscopic grade solvents and adjusted to a response within the DNA solution was wet-digested by adding a freshly prepared mixture linear range. of concentrated HNO3 at 105 °C for 1 h. Samples were subsequently Fluorescence quantum yields were determined using the standard dissolved in dilute HNO3 (1%, v/v) before being analyzed by ICP− method for highly diluted solutions to prevent selfabsorption MS. The platinum content was determined by ICP−MS with an processes (absorption <0.1 at λexc) and relative to 9,10-bis- Agilent 7900 inductively coupled plasma mass spectrometer. (phenylethynyl)anthracene in acetonitrile (Φfl = 0.985).43 Cell Viability Assays. Cell proliferation was evaluated using the Continuous irradiation of the solutions of photoactive compounds PrestoBlue assay. Cells were plated in 96-well sterile plates at a density was performed using a UV lamp (Vilber-Lormat, λ = 365 nm, 4 W) or of 5 × 103 cells per well (100 μL) and allowed to grow for 24 h. Cells a blue LED (λexc ∼ 450 nm,26 3 W). were then incubated with various concentrations of the studied Photodegradation quantum yields were determined using a compounds and cisplatin (0−200 μM for dose−response curves) for reported methodology44 and relative to trans-azobenzene in 24 h at 37 °C. The next day, cells were washed three times with a acetonitrile (Φtrans→cis = 0.14)45 for L1 and C1 [excited with a Nd/ fresh culture medium to remove noninternalized complex excess, YAG (Brilliant, Quantel) pulsed laser emitting at 355 nm] and to 1,2- either untreated or light-activated, for 15 min and left to grow for an bis(5-chloro-2-methyl-3-thienyl-perfluorocyclopentene) in hexane additional 48 h at 37 °C. Following the treatment, 10 μL of the (Φclosed→open = 0.13)46 for L2−8 and C2−8 (excited with a diode PrestoBlue solution was added to each well, and the plates were cw laser with λexc = 445 nm). incubated for an additional 2 h at 37 °C. Afterward, fluorescence was To characterize the photoproducts, 10−2 M solutions of L2 (DMF/ measured using a Victor3 (PerkinElmer) fluorescence multiwell plate H2O, 9:1) and L22 (DMF/EtOH, 6:4) and C2 (DMF-d7/D2O, 9:1) reader with the excitation/emission wavelengths set at 531/572 nm. were irradiated with a blue LED (λexc ∼ 450 nm,26 10 W) until the The cell viability was expressed as percentage values with respect to total disappearance of the absorption maxima band of the initial control cells, and the data are shown as the mean value ± standard species (monitored by UV−vis measurements of aliquots). For L2 error of the mean (SEM) of three independent experiments. Dose− and L22, after total photodegradation, H2O was added, and the crude response curves and the corresponding IC50 values were obtained by products were extracted with CH2Cl2. The combined extracts were means of nonlinear regression (curve fit), calculated with GraphPad dried over anhydrous Na2SO4, filtered, and the solvent was removed Prism 6.0 software. under a reduced pressure. Products were purified by column Apoptosis Detection Assay. Assessment of apoptotic, late chromatography (silica gel, hexane/EtOAc 6:4 to hexane/EtOAc, apoptotic/necrotic, and healthy cells with a fluorescence microscope 3:7). In the case of C2, after total photodegradation, the sample was was performed using the Apoptosis/Necrosis Detection Kit (Abcam freeze-dried to remove the solvent. 176750). A2780 cells (104 cells/well) were plated on 96-well plates CD Spectroscopy. CD spectroscopy was performed using a and allowed to adhere overnight. Cells were either untreated or model J-715 spectropolarimeter (JASCO, Gross-Umstadt, Germany) treated with 1.5 μM of complex C7 and 5 μM of complex C8 for 24 h. equipped with a computer (J-700 software, JASCO) with 1 cm thick Cells were washed with a fresh medium and either untreated or light- 7742 https://doi.org/10.1021/acs.inorgchem.1c03957 Inorg. Chem. 2022, 61, 7729−7745 Inorganic Chemistry ■ pubs.acs.org/IC Article activated for 15 min and incubated for a final time of 72 h. Cells were AUTHOR INFORMATION washed twice with the assay buffer, and then the staining buffer Corresponding Authors (containing 5 μL of apopxin deep red indicator, 5 μL of nuclear green, Marta Figueredo − Departament de Química, Universitat and 5 μL of CytoCalcein 450 to each 1 mL of the assay buffer) was Autònoma de Barcelona, 08193 Cerdanyola del Vallès, added and, finally, incubated for 60 min at room temperature. Afterward, the cells were washed three times with the assay buffer, and Spain; orcid.org/0000-0002-8278-7534; a total of 10 random images were taken using the Cy5 (Ex/Em = 630/ Email: marta.figueredo@uab.cat 660 nm), FITC (Ex/Em = 490/520 nm), and violet (Ex/Em = 405/ Pau Bayón − Departament de Química, Universitat Autònoma 450 nm) channels of a fluorescent microscope at a 20× objective. de Barcelona, 08193 Cerdanyola del Vallès, Spain; Cells from images were counted, and the numbers of each cellular orcid.org/0000-0002-3064-8866; Email: pau.bayon@ state were recorded. uab.cat ROS Production Assay. Measurement of ROS was achieved Authors using the DCFDA reagent ROS detection assay. A2780 cells were Kevin Morales − Departament de Química, Universitat plated in a 96-well plate at 2 × 104 cells/well in a medium supplemented with 10% FBS and allowed to adhere overnight. The Autònoma de Barcelona, 08193 Cerdanyola del Vallès, next day cells were incubated with DCFDA (100 μL/well of a 25 μM Spain; orcid.org/0000-0001-6096-7174 solution) for 30 min in the dark. Cells were then washed, either Sergi Rodríguez-Calado − Institut de Biotecnologia i untreated or treated, with cisplatin or complexes C7 and C8 at their Biomedicina (IBB) and Departament de Bioquímica i IC50 and incubated for an additional 4 h. After this time, cells were Biologia Molecular, 08193 Cerdanyola del Vallès, Spain washed, fresh medium was added, and light-activated for 15 min or Jordi Hernando − Departament de Química, Universitat maintained in the dark to finally be incubated for a 48 h extra time. Autònoma de Barcelona, 08193 Cerdanyola del Vallès, The experiments were run in triplicate. H2O2 was used as a positive Spain; orcid.org/0000-0002-1126-4138 control at 100 μM. The fluorescence of each well was measured in a Julia Lorenzo − Institut de Biotecnologia i Biomedicina (IBB) Microplate Reader Victor3 (PerkinElmer) at 535 nm after an and Departament de Bioquímica i Biologia Molecular, 08193 excitation at 485 nm. Cerdanyola del Vallès, Spain; orcid.org/0000-0001- ■ 5659-6008 ASSOCIATED CONTENT Antonio Rodríguez-Diéguez − Department of Inorganic Chemistry, Faculty of Science, University of Granada, 18071 * sı Supporting Information Granada, Spain; orcid.org/0000-0003-3198-5378 The Supporting Information is available free of charge at Carlos Jaime − Departament de Química, Universitat https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c03957. Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; orcid.org/0000-0002-9690-9053 Synthesis of ligand L6 from 9, Synthesis of complex Pau Nolis − Departament de Química, Universitat Autònoma C20, Gaussview representations of all calculated de Barcelona, 08193 Cerdanyola del Vallès, Spain; conformations, UV−vis absorption spectra of ligands orcid.org/0000-0003-2360-1709 L1−8 and complexes C1−8 in solvent mixtures with Mercè Capdevila − Departament de Química, Universitat water, fluorescence quantum yields of ligands L2−8 and Autònoma de Barcelona, 08193 Cerdanyola del Vallès, complexes C2−8, photochemical properties of ligands Spain; orcid.org/0000-0002-2246-0994 L1−8 and complexes C1−8. UV−vis spectra monitoring Ò scar Palacios − Departament de Química, Universitat irradiation of ligand L22 with 450 nm LED in DMF, MS Autònoma de Barcelona, 08193 Cerdanyola del Vallès, spectrum (positive mode) of the ligand L22 photo- Spain; orcid.org/0000-0002-2987-7303 product, IR spectrum of the ligand L22 photoproduct, 1 H NMR spectrum of the major photoproduct of the Complete contact information is available at: ligand L22, 13C NMR spectrum of the major photo- https://pubs.acs.org/10.1021/acs.inorgchem.1c03957 product of the ligand L22, 1H NMR spectrum and the Notes NOE experiment for the major photoproduct of the The authors declare no competing financial interest. ■ ligand L22, HRMS of the irradiation crude for complex C2: peak [721.1 Da (+H+)] and its assigned structure, ACKNOWLEDGMENTS CD spectra of ct-DNA (50 μM) and ct-DNA incubated with (a) C7, (b) C8, (c) C7′, and (d) C8′ at different The authors acknowledge the Spanish Ministerio de Ciencia e molar ratios (ri), NMR spectra of synthesized Innovación and FEDER for the projects BIO2015-67358-C2- compounds, MS spectra of C1−7 and C20, Micrographs 2-P (M.C. and Ò .P.), CTQ2016-75363-R (K.M., M.F., and of complex-treated (C1−8) A2780 cells with a 10% P.B.). M.C., Ò .P., M.F., and P.B. are members of the “Grup de FBS-containing cell culture medium, crystal data for L2, Recerca de la Generalitat de Catalunya”, ref. 2017SGR-864. CIF file for compound L2, final structures obtained from J.L. and S.R.-C. want to thank the Spanish Ministerio de the DFT calculations in the PDB format (PDF) Ciencia e Innovación for the project (RTI2018-098027-B- C22). The authors also thank the Ministerio de Educación, Cultura y Deporte for a FPU Grant to K.M. ■ Accession Codes CCDC 2100958 contains the supplementary crystallographic REFERENCES data for this paper. These data can be obtained free of charge (1) Chemistry of the Platinum Group Metals: Recent Developments; via www.ccdc.cam.ac.uk/data_request/cif, or by emailing Hartley, F. R., Ed.; Elsevier, 1991. ISBN-13: 978-0444881892. 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