Luminescent Cycloplatinated Complexes with Biologically Relevant

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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Luminescent Cycloplatinated Complexes with Biologically Relevant Phosphine Ligands: Optical and Cytotoxic Properties Gonzalo Millań ,† Nora Gimeń ez,† Rebeca Lara,† Jesús R. Berenguer,† M. Teresa Moreno,† Elena Lalinde,*,† Elvira Alfaro-Arnedo,‡ Icíar P. Loṕ ez,‡ Sergio Piñeiro-Hermida,‡ and Jose ́ G. Pichel*,‡ †

Departamento de Química, Centro de Síntesis Química de La Rioja, Universidad de La Rioja, 26006 Logroño, Spain Centro de Investigación Biomédica de La Rioja, Fundación Rioja Salud, 26006 Logroño, Spain

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‡

S Supporting Information *

ABSTRACT: Two series of neutral luminescent pentafluorophenyl cycloplatinated(II) complexes [Pt(C^N)(C6F5)L] [C^N = C-deprotonated 2-phenylpyridine (ppy; a), 2-(2,4difluorophenylpyridine (dfppy; b)] incorporating dimethyl sulfoxide [L = DMSO for 1 (1a reported by us in ref 14)] or biocompatible phosphine [L = PPh2C6H4COOH (dpbH; 2), PPh 2 C 6 H 4 CONHCH 2 COOMe (dpbGlyOMe; 3), P(C6H4SO3Na)3 (TPPTS; 4)] ligands have been prepared and characterized and their optical properties studied. Their cytotoxic activities against tumor A549 (lung carcinoma), HeLa (cervix carcinoma), and nontumor NL-20 (lung epithelium) cell lines, as well as the ability to interact with DNA (plasmid pBR322), were evaluated. Complexes 2 exhibit higher cytotoxicity (IC50 3.89−20.29 μM) than compounds 1 (9.03−20.50 μM), whereas the activities of complexes 3 and 4 are negligible. All cytotoxic complexes show low selective toxicities toward cancer cells. Interestingly, except 1a, these complexes do not show evidence of DNA intercalation. Along the same lines, fluorescence costaining with Hoechst (2,5′-bi-1Hbenzimidazole, 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl), a nuclear DNA stain) reveals that all complexes easily internalize, being mainly localized in the cytoplasm. In order to deepen the mechanism of biological action, the effect of the most cytotoxic complex 2b toward the dynamics of tubulin was explored. This complex displays tubulin depolymerization activity, exhibiting more potent inhibition of microtubule formation in A549 than in HeLa cells, in accordance with its higher antiproliferative activity (IC50 6.98 vs 12.45 μM), placing this complex as a potential antitubulin agent.

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INTRODUCTION Since the discovery of cisplatin [cis-Pt(NH3)2Cl2] antitumor activity,1 research on promising cytotoxic platinum (Pt) complexes is constantly increasing.2 However, only two compounds besides cisplatin, carboplatin and oxaliplatin, are used worldwide in cancer chemotherapy.2g,3 Their anticancer activity relies on the formation of Pt-DNA adducts through covalent bonds of the Pt atoms with the purine nucleobases (intra- and interstrand cross-links), which lead to the inhibition of DNA replication and transcription and ultimately to cell death.2b,c Despite their effectiveness, serious side effects, including acquired intrinsic resistance, a limited spectrum of activity, and high toxicity, are connected with the use of these Pt compounds in clinical application.4 With the aim of finding new and better anticancer drugs, a great variety of metal complexes derived not only from the cisplatin (Pt analogues) but also from different architectures have been reported.2,5 In this context, organometallic compounds with antitumor properties are promising alternatives,6 mainly because of their unique structural features (rich stereochemistry about © XXXX American Chemical Society

metal centers) and the wide versatility of the ligands, which can be chemically modified with specific functionalities and reactivities to improve or create a new pharmacological action. In recent years, studies about applying cycloplatinated(II) complexes in the area of cancer research have attracted increasing attention.2e,7 In these complexes, the presence of strong σ(Pt−C) bonds increases their stability in physiological conditions, avoiding off-target reactions, and, therefore, simplifies the potential therapeutic applications. Concerning the possible mechanisms of action, several studies suggest that the presence of aromatic rings favors their intercalation between base pairs of DNA by noncovalent π···π stacking interactions,8 thus targeting telomeric G-quadruplex9 or key proteins.9a,10 Furthermore, the incorporation of labile molecules as coligands can facilitate a mixed action through more classical covalent interaction with DNA.11 It is also worth mentioning that recent contributions have demonstrated that Received: November 16, 2018

A

DOI: 10.1021/acs.inorgchem.8b03211 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry Scheme 1. Preparation of Complexes 1−4

PBT/DMSO derivative showed moderate cytotoxic activity toward the human cell lines A549 and HeLa, which was suggested to be governed by inhibition of the tubulin polymerization. Following previous results, in this work, we decided to evaluate the biological effect of replacing the DMSO molecule by nonleaving substituted triphenylphosphine ligands with different moieties on two luminescent precursors [Pt(C^N)(C6F5)(DMSO)] [C^N = ppy (1a), dfppy (1b)]. Previous studies have shown the cytotoxic activities of several classes of Pt complexes containing functionalized phosphines.7d,15,16 Here, we have chosen the commercially available PPh2C6H4COOH (dpbH) ligand, having a carboxylic acid, which had been previously used as a platform to form peptidic bonds through successful coupling protocols,17 to generate [Pt(C^N)(C6F5)(dpbH)] (2a and 2b). Via the successful coupling between the carboxylic group and glycine methyl ester (dpbGlyOMe) on complexes 2a and 2b, in the presence o f t h e s t a n d a r d co u p l i n g a g e n t N- e t h y l - N′ - [ 3 (dimethylamino)propyl]carbodiimide hydrochloride (EDC· HCl), the peptide-tagged complexes [Pt(C^N)(C 6F5)(dpbGlyOMe)] (3a and 3b) were formed. Finally, for comparative purposes, the water-soluble complexes [Pt(C^N)(C6F5)(TPPTS)] (4a and 4b) were also synthesized. Here we report the synthesis, characterization, optical properties, and theoretical calculations of complexes 1−4. Furthermore, we describe the biological data on their in vitro activity against tumor A549 (lung carcinoma), HeLa (cervix carcinoma), and nontumor NL-20 (lung epithelium) cell lines, and, more importantly, we present a study of the inhibition of the polymerization of microtubules as a plausible mechanism of

some cycloplatinated complexes also have antiangiogenic properties.7d In addition to their usefulness in therapeutic applications, cycloplatinated compounds are interesting for their intrinsic luminescence properties,12 with emissions produced from various excited states including 3MLCT, 3LC, 3ILCT, 3LLCT, 3 LMMCT, 3MMLCT, or 3MLLCT. This behavior allows their application not only in electroluminescent devices12 but also as luminescent labels for intracellular imaging in living cells.9a,13 In this context, by using the appropriate cyclometalated groups and ancillary ligands, cycloplatinated complexes offer the opportunity to generate new metal-based structures capable of displaying therapeutic and/or traceability functions. The choice of the cyclometalated group and ancillary ligands in the coordination sphere of the Pt plays a key role not only in the final emissive behavior but also on the cytotoxic activity, for which small modifications can induce profound changes in the biological effect. In a previous study, our group described the synthesis of the neutral pentafluorophenyl-cycloplatinated solvate complexes [Pt(C^N)(C6F5)(DMSO)] [C^N = 2phenylbenzothiazole (PBT), thienylpyridine (thpy), phenylpyridine (ppy); DMSO = dimethyl sulfoxide], which showed moderate cytotoxicity against the human lung tumor (A549) cell line.14 Fluorescence microscopy experiments confirmed the accumulation of these complexes in the cytoplasmic region, with brighter emission in the perinuclear area. Recently, we also reported complexes based on 2-(4-substituted phenyl)benzothiazoles of the type [Pt(R-PBT)ClL] [R = Br, Me2N; L = DMSO, 1,3,5-triaza-7-phosphaadamantane (PTA), 3,3,3″trisulfonate sodium salt (TPPTS)].15 Among them, complexes containing the nonleaving PTA phosphine and the Me2NB


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Inorganic Chemistry

Figure 1. Views of the molecular structures of (a) 1b, (b) 2a·3.25CH2Cl2, and (c) 3b·2CH2Cl2·H2O.

action for some of these complexes. In addition, fluorescence microscopy studies for their cellular localization are reported.

the coupling of the carboxylic acid on the complexed dpbH ligand of the platinum complexes 2a and 2b and dpbGlyOMe in its hydrochloride form (HGlyOMe·HCl) to form complexes [Pt(C^N)(C6F5)(dpbGlyOMe)] (3a and 3b). As outlined in Scheme 1, a dichloromethane solution of the corresponding complexes 2a and 2b was reacted (5 h) at room temperature with the coupling agents EDC·HCl and NHS (1/1.2/1.2 molar ratio) to generate the active ester before treatment with HGlyOMe·HCl (1.4 equiv) in basic media (NEt3, 4 equiv) for 24 h. By subsequent workup to remove byproducts, complexes 3a and 3b were isolated as yellow solids in good yield (3a, 71%; 3b, 64%). All complexes were characterized by 1H, 31P{1H}, 19F, and 13 C{1H} NMR spectroscopies, mass spectrometry (MS), IR, and elemental analysis (except 4a and 4b). In complexes 4, the presence of TPPTS favors the incorporation of H2O in the isolated solids, as confirmed by IR. MS spectra [MALDITOF(+) or ESI(+)] for complexes 2 and 3 reveal the presence of molecular ions M+ or [M + H]+, whereas the ESI(+) spectra of TPPTS complexes 4a and 4b feature as the most intense peak that corresponding to the positive [M + Na]+ ions. All 19F NMR spectra display the three typical signals (o-F, pF, and m-F, AA′MXX′ spin systems) of the C6F5 ligand and, in the case of 1b−4b, the two expected fluorine resonances of the cyclometalated dfppy ligand (8F and 10F) with long-range Pt satellites (4JF−Pt = 33−60 Hz). The high values of 3Jo‑F−Pt observed (463−505 Hz) are comparable to those found in

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RESULTS AND DISCUSSION Synthesis and Characterization. The synthetic procedures for the preparation of new PtII compounds are summarized in Scheme 1. Complex 1a was recently reported by our group.18 Following a similar procedure, the cyclometalated dfppy complex 1b was obtained as a pale-yellow solid by refluxing the precursor cis-[Pt(C6F5)2(DMSO)2] with 2-(2,4-difluorophenyl)pyridine (Hdfppy) in toluene for 24 h. On the other hand, the phosphine derivatives 2a, 2b, 4a, and 4b were easily prepared in good yield by replacement of the DMSO ligand in 1a and 1b for the appropriate and commercially available 4-(diphenylphosphino)benzoic acid (dpbH) and triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt [P(C6H4SO3Na)3, TPPTS], respectively. Free phosphine ligands bearing pendant amino residues or short peptides have been previously prepared and easily complexed to give well-defined P-coordinated metal complexes,16c,17c,19 which were applied as catalysts or structural models for secondary proteins.17c,19a−e As an alternative, Monkowius et al. recently developed a successful protocol based on the coupling agent EDC·HCl, with N-hydroxysuccinimide (NHS) used to activate the carboxylic function, for the direct peptide coupling of methyl ester amino acids with the carboxylic function of [AuCl(dpbH)].17a We decided to explore a strategy similar to C


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Inorganic Chemistry similar complexes14,18,20 and consistent with the relatively low trans influence of the pyridine N atom. Complexation of the phosphines in 2−4 is evidenced in the 31P{1H} NMR spectra, which exhibit a unique resonance in the range of 26.2−29.2 ppm, as a singlet in 2a−4a or a triplet for 2b−4b due to wellresolved long-range 5JP−F (8−10 Hz) to F atoms on the dfppy ligand. The 1JP−Pt (1936−2028 Hz) values are found in the typical range for P trans to C.21 In the 1H and 13C{1H} NMR spectra, the most characteristic signals are those of the cyclometalated groups and the substituents on the phenyl ring of the phosphine ligands. Complete assignments were made on the basis of 2D experiments [1H−1H (COSY and TOCSY) and 1H−13C (HSQC and HMBC)], and as an illustration, a set of spectra for 2b and 3b are given in Figures S1 and S2. Complexation of the phosphines is reflected in distinctive differences in the cyclometalated ring in relation to precursor DMSO complexes (1a and 1b). Thus, the proton H2 is notably shifted from 9.71 ppm in 1a and 1b to 8.00−8.17 ppm in 2a−4a and 2b−4b, a fact attributable to the anisotropic diamagnetic effect of the phenyl rings of the mutually cis phosphine ligand. Similarly, a shift to higher frequencies (ΔδH ≈ 0.4−0.5 ppm from 1 to 2−4) is observed for the H11 resonance, which appears as the most shielded because of its proximity to the diamagnetic current of the C6F5 ring, and displays a more complex appearance due to an additional long-range coupling to the P atom (4JH−P). The OH protons seen at 11.55 ppm in 2a and 11.72 ppm in 2b disappear upon formation of the amide bonds in complexes 3a and 3b, which exhibit the NH proton at ca. 8.2 ppm. In the latter, the H and C atoms of the protected GlyOMe entities appear in the expected region (see the Experimental Section and Figure S2). In the 13C{1H} NMR spectra of 3a and 3b, the two distinct carbonylic C atoms were assigned by the HMBC and HSQC experiments [δCOO/δCONH = 170.77/168.7 (3a) and 171.0/166.8 (3b)]. To elucidate the stability of the complexes toward biological studies, their stability in solution was investigated. For this purpose, complexes 1−3 were dissolved in DMSO-d6 or D2O (4a and 4b), and their NMR (1H, 19F, and 31P{1H}) data were taken after 30 min and 24 h. All complexes remained unaltered within 24 h. After that, D2O was added to solutions of 1−3 in DMSO (9:1 DMSO/D2O). Under these conditions, solutions of complexes 1 and 2 are stable. Complexes 3a and 3b were found to precipitate in part, but no reaction of hydrolysis of the amide groups was observed, indicating that both complexes are sufficiently stable under hydrolysis at ambient conditions. The molecular structures of 1b, 2a, and 3b, established by monocrystal X-ray diffraction (XRD) studies (details in Figures 1 and S3−S5 and Tables S1 and S2), confirm the formulation shown in Scheme 1. The Pt−C, Pt−N, and Pt−P bonding distances around the Pt are similar to those described previously,14,15 whereas the Pt−S bond is slightly larger than the average distance observed in related complexes (see Table S1).22 Photophysical Properties. The electronic absorption spectra of the complexes were recorded in CH2Cl2 and DMSO for 1a−3a and 1b−3b and in H2O and MeOH for 4a and 4b. Data are collected in Tables 1 and S3, and representative spectra (1b−3b) are shown in Figure 2. All complexes displayed intense high-energy absorption bands with shoulders at λ ≈ 220−280 nm and less-intense low-energy absorption bands at λ ≈ 300−400 nm (ε ∼ 0.9 × 103−104 M−1 cm−1). The high-energy absorption bands were assigned as

Table 1. Absorption Data for Compounds 1−4 (1−3, CH2Cl2; 4, H2O; 5 × 10−5 M) λabs, nm (ε × 10−3, M−1 cm−1)

compound 1a 1b 2a 2b 3a 3b 4a 4b a

253.4 (23.6), 312.1sh (6.63), 320 (7.8), 360.7sh (2.5) 270 (52.3), 307sh (8.8), 320 (11.6), 357 (1.9)a 250sh (32.1), 267 (25.4), 274 (23.9), 315sh (6.5), 328 (6.9), 361sh (2.9) 261sh (22.9), 313sh (3.7), 325 (5.6), 360sh (0.9) 266 (22.4), 271 (25.5), 327 (4.2), 361sh (1.9) 258sh (41.0), 312sh (6.9), 325 (10.2), 356sh (2.2) 263 (28), 308sh (5.6), 324 (6.4) 258 (28), 308sh (6.4), 321 (8.6)

2.5 × 10−5 M.

ligand-based π−π* transitions. With reference to previous studies14 and calculations on 1b, 2a, 2b, 3a, and 3b, the lowenergy absorption feature in the precursor 1b and in complexes 3a and 3b is mainly assigned to an admixture of 1MLCT/1IL located on the cyclometalated group, with some 1LLCT (C6F5 to C^N) character, having higher contributions in the phosphine complexes 3a and 3b. However, in the dpbH phosphine derivatives (2a and 2b) is ascribed to a more complex mixture of transitions involving intraligand (1IL, C^N), metal to both ligands [1M(L+L′)CT (Pt → C^N, PPh2R)], and ligand-to-ligand (1LLCT, C6F5 → C^N) charge transfer with higher weight in 2b. As shown in Figure 2a, replacement of the DMSO in the precursors by the phosphines causes a slight red shift in the absorption maxima, a feature that correlates with the calculated values [338.6 nm (1b) vs 350.0 nm (2b) and 350.1 nm (3b)]. Also, all dfppy complexes (1b− 4b) display the absorption maximum blue-shifted in relation to the ppy complexes (see Table 1), in line with the lower energy of the aryl-based highest occupied molecular orbital (HOMO). The absorption spectra were also recorded in DMSO to elucidate the stability of the complexes in this solvent used in the biological studies. A negligible variation in the absorption energies was detected in DMSO (see Table S3), suggesting a very low charge-transfer contribution. In order to investigate the occurrence of aggregation phenomena for the less bulky complexes 1, a concentration dependence study in CH2Cl2 was carried out (Figures 2b and S6). The lowest-energy absorption band (361 nm, 1a; 357 nm, 1b), monitoring the absorbance at 375 nm, follows Beer’s law in the concentration range from 2.5 × 10−5 to 5 × 10−3 M, suggesting that no remarkable ground-state aggregation occurs within this concentration range. However, much weaker bands (ε ∼ 20 M−1 cm−1) are barely discernible at lower energy in very concentrated solutions (10−2 M for 1a and 1b; Figures 2b and S6). These bands are ascribed to the direct population of triplet states of mixed (3LC/3MLCT) nature, caused by the high spin−orbit coupling of the PtII center. Complexes 4 are nonemissive in solution (H2O or MeOH) upon photoexcitation, whereas 1−3 are only weakly emissive (see Table 2). In detail, the DMSO complexes 1a and 1b display in a fluid CH2Cl2 solution a weak characteristic phosphorescent emission band at 474 and 462 nm, respectively. The emission energy is similar at low temperature (77 K; 475 nm, 1a; 462 nm, 1b), but the intensity increases notably and residual fluorescence (ca. 430 nm) is observed for 1a. As shown in Figures 3 and S7, the emission spectra of 1a and 1b are not concentration-dependent (range of 1 × 10−2−5 × 10−5 M) at either 298 or 77 K, thus excluding aggregationD


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Inorganic Chemistry

Figure 2. (a) Absorption spectra in CH2Cl2 of 1b (2.5 × 10−5 M) and 2b and 3b (5 × 10−5 M). (b) Low-energy region of the UV−vis absorption spectra of 1b in CH2Cl2 at different concentrations. Inset: Lineal fit of the absorbance at the 375 nm band versus concentration.

and 3b) and a low-energy structured band ascribed to phosphorescence (475 nm, 2a; 478 nm, 3a; 460 nm, 2b; 462 nm, 3b). As expected, both bands present coincident excitation spectra. However, in the glass state at 77 K, all of these 2 and 3 complexes are strongly emissive, exhibiting only vibronic-structured low-energy phosphorescence emission bands. The bands are located at λmax = 473−475 nm for the ppy complexes (Figure 4) and blue-shifted for the dfppy series 1b−4b (458−462 nm; Figures 4b and S9). The emissions show vibrational progressional spacings of about 1410−1565 cm−1, typical of the vibration modes in the cyclometalated ligands.14,15,18,20,21b These observations, together with the long emission lifetimes (τ 38−49 μs) and the support of theoretical calculations (see below), indicate that the emissions predominantly originate from the 3IL excited state, with a minor mixing of the 3MLCT state. The blue shift observed for the dfppy series in relation to the ppy one is in line with the higher-energy gap for the π−π* orbitals of the difluorophenylpyridine (dfppy) ligand. The severe quenching of the emission in a fluid solution for the phosphine complexes could be ascribed to ease nonradiative deactivation through

Table 2. Photophysical Data for Compounds 1−4 in the Solid State and Solution (1−3 in CH2Cl2 and 4 in H2O) solid statea

compound

298 K, λem/nm (τ/μs) [ϕ/%]

1a 1b 2a 2b 3a 3b 4a 4b

490 (20.1) [48.0] 473 (40.6) [62.1] 477 (11.4) [23.2] 465 (17.6) [43.4] 481 (9.5) [1.7] 472 (8.0) [1.6] 482 (11.3) [8.1] 484 (9.9) [8.8]

solutionb

77 K, λem(max)/ nm (τ/μs) 500 477 474 460 483 468 482 472

(70.0) (52.2) (12.0) (24.1) (25.6) (34.2) (28.0) (30.0)

298 K, λem(max)/ nm 474c 462c 400, 475c 388, 460c 397, 478c 393, 462c

77 K, λem/ nm (τ/μs) 475 462 475 458 475 459 473 459

(38.8) (49.0) (41.0) (39.0) (41.0) (41.0) (35.0) (46.0)

Neat solid. b5 × 10−4 M. cWeak emission.

a

induced phenomena related with Pt···Pt and/or π···π interactions. As shown in Figure S8, the emission profile of complexes 2 and 3 is composed of a high-energy feature attributed to fluorescence (∼400 nm, 2a and 3a; ∼390 nm, 2b

Figure 3. Emission spectra at 298 K (a) and 77 K (b) of 1a in CH2Cl2 at different concentrations (λexc = 365 nm). E


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Inorganic Chemistry

Figure 4. Normalized emission spectra at 77 K: (a) 1a−3a in CH2Cl2 and 4a in H2O; (b) 3a and 3b in CH2Cl2 (λexc = 365 nm).

Figure 5. Schematic representation of the selected frontier orbitals of 1b, 2a, 2b, 3a, and 3b.

molecular motions and noncovalent intermolecular interactions, enhanced in complexes 2−4 by the substituent nature (−COOH, −GlyOMe, −SO3Na). Besides, the local structural distortion from D4h symmetry, at the PtII center, to D2d symmetry due to thermal access to nonemissive metal-centered d−d excited states and also direct solvent interactions, which lead to low emission efficiencies in fluid solution, should not be ruled out.15,23 At low temperature and in rigid media, all of these deactivation pathways are efficiently restrained. Thus, all complexes are also emissive in the solid state (Table 2), exhibiting broad structured profiles at 298 K and better resolved bands upon cooling (77 K), which are slightly redshifted in relation to those found in glassy solutions. This

bathochromic shift is not uncommon and can be attributed to the contribution of some excimeric character due to the occurrence of π···π intermolecular interactions in the solid state.15,18,21b,24 The presence of F atoms on the cyclometalated ligand leads to higher quantum yields in complexes 1 and 2 (62.1% 1b and 43.4% 2b vs 48.0% 1a and 23.2% 2a). However, lower quantum yields are measured for dpbGlyOMe (1.7% 3a and 1.6% 3b) and TPPTS (8.1% 4a) derivatives, indicating that the presence of amino sulfonate substituents on the aromatics rings favors nonradiative deactivation pathways. Theoretical Studies. To further understand the optical properties, density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations for the precursor 1b and F


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Inorganic Chemistry

Table 3. Cytotoxic IC50 Values (μM)a, Selectivity Indexes,b and Cellular Fluorescence Intensitiesc of Complexes 1a−4a and 1b−4b and Cisplatin in Human Cell Lines A549, HeLa, and NL-20 IC50a

fluorescence intensityc

SIb

complex

A549

HeLa

NL-20

A549

HeLa

A549

.HeLa

1a 1b 2a 2b 3a 3b 4a 4b cisplatin

18.92 ± 0.36d 13.91 ± 1.20 6.07 ± 0.37 6.98 ± 0.99 NT NT NT 93.95 ± 9.30 6.45 ± 0.47d

20.50 ± 1.61 16.98 ± 0.82 20.29 ± 2.10 12.45 ± 2.50 NT NT NT 150.70 ± 3.69 13.60 ± 0.99e

9.03 ± 0.29d 11.32 ± 0.72 4.36 ± 0.23 3.89 ± 0.09 ND ND ND 56.02 ± 6.16 2.83 ± 0.36d

0.48 0.81 0.72 0.56 − − − 0.60 0.44

0.44 0.67 0.21 0.31 − − − 0.37 0.21

++++ + + + + + − − −

++++ + ++ + ++ +/− − − −

IC50 values are presented as mean ± standard error of the mean of three different experiments. bSelectivity index (SI) = IC50 nontumor cell (NL20)/IC50 cancer cell (A549 or HeLa), as described in refs 30a and 61. cThe intensity was classified on a qualitative relative scale: −, no staining; +, weak staining; ++, medium staining; ++++, very strong staining, as reported in ref 15. dAs determined in ref 14. eAs determined in ref 15. NT = nontoxic. IC50 values could not be determined because a relevant percentage of the cytotoxicity was not achieved at any concentration tested. ND = not determined. a

C^N and dpbH [1M(L+L′)CT], 1IL in 2a, and 1MLCT and LLCT (C6F5 → dfppy) in 2b. For additional support of the photoluminescence behavior of these complexes, the lowest-lying T1 (S0 → T1; Table S6 and Figure S15) and spin-density distribution for the triplet excited states (T1; Figure S16) based on their corresponding optimized S0 and T1 geometries, respectively, have been obtained. Molecular analysis for 1b indicates that, in the ground-state geometry, the S0 → T1 transition mainly results from a mixing of Pt → dfppy and IL(dfppy) excitations, supporting a mixed 3IL/3MLCT assignment. A similar conclusion is obtained by optimization of the lowest T1 state, which reveals that the calculated spin-density distribution in the optimized T1 state is localized on the dfppy and Pt. Differently, in the dpbH complexes 2a and 2b, both the cyclometalated group and C6H4COOH ring of the dpbH ligand contribute to S0 → T1 (HOMO → LUMO/LUMO+1). So, the intraligand (C^N) transition, together with metal charge transfer to both ligands M(L+L′)CT, account for the excitation, thus mimicking the absorption features. However, the spin density in the optimized T1 state as well as the SOMO and SOMO−1 orbitals (Table S7) in both complexes are similar to those of 1b, being also located on the C^N unit and Pt. Consequently, the phosphorescent emission features are also 3IL/3MLCT in nature. In the GlyOMe-tagged complexes 3a and 3b, the calculations show that the T1 state at the S0 ground-state geometry is majority-contributed (70%) by the HOMO → LUMO transition, being therefore of a mixed Pt → dfppy and IL (C^N) character. The spin-density distributions for the optimized T1 states are similar to those of 2a and 2b, indicating a negligible influence of the GlyOMe substituent in the energy of the emission. The calculated energy emission gives a value close to 450 nm for the dfppy (b) complexes and 465 nm for the ppy (2a and 3a; values given in Figure S16) complexes, consistent with the trend observed for the experimental phosphorescence, both in a fluid solution and in rigid media. Cytotoxic Activity (MTS Assays) and Selectiviy Index. Since previous studies had demonstrated antiproliferative properties for different cyclometalated Pt II complexes,2e,7,8c,d,10,14,15,25 including a moderate activity of precursor 1a,14 the cytotoxic activity of 1−4 was determined against human tumor A549 (lung carcinoma), HeLa (cervix

complexes 2a, 2b, 3a, and 3b at the B3LYP/631G**+LANL2DZ level of theory. The effect of the solvent (CH2Cl2) was taken into account using the polarized continuum model (PCM) approach. The validity of the chosen level of theory is supported by the good agreement between calculated and solid-state structures derived from single-crystal XRD data. The bond lengths and angles of the optimized structures and corresponding values obtained from XRD for 1b, 2a, 2b, 3a, and 3b are collected in Table S4. Figures 5 and S10−S14 display selected molecular orbitals (isosurface plots), while Tables S5 and S6 respectively summarize orbital contributions and some selected low-lying excited states. As displayed in Figure 5, the HOMO is rather similar in all complexes, being located on the aryl ring of the cyclometalated ligand and Pt center, with a higher contribution of the metal in the phosphine complexes (27−30% vs 22% in 1b). The closest occupied orbital, HOMO−1, is mainly contributed from the Pt center, with a higher weight in 1b (82%), and the C6F5 ligand. In complexes 1b, 3a, and 3b, the lowest unoccupied molecular orbital (LUMO) is centered on the π* orbital of the C^N ligand, whereas in complexes 2a and 2b, featuring dpbH, it is spread over both the C^N and dpbH (benzoic ring) ligands, with a lower contribution of dpbH in 2b. In 2a and 2b, the LUMO+1 has a similar composition (C^N/dpbH), whereas in complexes 3a and 3b, it is shifted to the phosphine. For 1b, the two lowest S1,2 states are contributed by HOMO or HOMO−1 to LUMO transitions, supporting mainly a mixed intraligand/metal-to-ligand (1IL/1MLCT) character. Similar transitions are involved in the case of the dppGlyOMe derivatives 3a and 3b, but because of the higher C6F5 contribution to the HOMO−1, the absorptions are assigned to 1IL/1MLCT with 1LLCT character. The slight bathochromic shift observed experimentally by replacement of the DMSO ligand in 1b by the phosphines in 2b and 3b (see Figure 2a and Table 1) is in accordance with in the calculations (calcd: ∼350 nm, 2b and 3b, vs 338.6 nm, 1b) and might be attributed to slightly greater destabilization of the HOMOs in relation to the LUMOs. For dpbH complexes (2a and 2b), the lowest calculated singlet has a mixed configuration (HOMO → LUMO/LUMO+1, 2a; HOMO−1/HOMO → LUMO, 2b), having mainly metal-to-ligand charge transfer to both ligands,

1

G


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Inorganic Chemistry

Figure 6. Fluorescence images of A549 and HeLa cells treated with complexes 1b, 2a, 2b, 3a, and 3b. Living cells were incubated with complexes along with the DNA binder Hoechst 33342 for 1 h. Cells were visualized by microscopy for either fluorescence emission in green of compounds (left panels) and blue of Hoechst bound to DNA (not shown) or Nomarski white-light transmission (not shown). Overlays of green, magenta (pseudocolor for blue emission, red arrowheads), and Nomarski images are shown in the right panels. In all cases, complexes stain the cytoplasm (green) with more efficiency in perinuclear areas (yellow arrowheads). Scale bars: 15 μm.

and 4b, respectively. The lack of cytotoxity of 3a and 3b could be due to the low solubility of the complexes in the aqueous biological medium, as noted in the NMR spectra. For complexes 4a and 4b, the lack of toxicity could be attributed to the strong negative charge associated with these complexes due to coordination of the ionic TPPTS ligand, which might well hinder its cellular uptake.15,27 Interestingly, the addition of the two F atoms to the ppy substituent in type b cytotoxic compounds made them slightly more cytotoxic than their ppy (a) counterparts. These results are consistent with previous findings, which indicate that the fluorination of certain C−H bonds in drugs increases their bioabsorption and, hence, their efficacy and selectivity in pharmaceuticals.28 It is worth mentioning that all cytotoxic compounds (1a, 1b, 2a, 2b, and 4b) showed steeper IC50 curves compared to those displayed by cisplatin in the three cell lines (see Figure S17).14,15 These results indicate a homogeneous response toward these complexes and may suggest different cytotoxic mechanisms of action relative to cisplatin.29 In accordance with the IC50 assessment, low values of the selectivity index (SI;

Luminescent Cycloplatinated Complexes with Biologically Relevant

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