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Self-Assembly of Discrete RuII8 Molecular Cages and Their in Vitro Anticancer Activity Aderonke Ajibola Adeyemo,‡,# Abhijith Shettar,†,# Imtiyaz Ahmad Bhat,‡ Paturu Kondaiah,*,† and Partha Sarathi Mukherjee*,‡ ‡

Department of Inorganic and Physical Chemistry and †Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India S Supporting Information *

ABSTRACT: Four new octanuclear Ru(II) cages (OC-1−OC-4) were synthesized from dinuclear p-cymene ruthenium(II) acceptors [Ru2(μ−η4-C2O4)(CH3OH)2(η6-p-cymene)2](O3SCF3)2 (A1), [Ru2(μ−η4-C6H2O4)(CH3OH)2(η6-p-cymene)2](O3SCF3)2 (A2), [Ru2(dhnq)(H2O)2(η6-p-cymene)2](O3SCF3)2 (A3), and [Ru2(dhtq)(H2O)2(η6-p-cymene)2](O3SCF3)2 (A4) separately with a tetradentate pyridyl ligand (L1) in methanol using coordination-driven self-assembly [L1= N,N,N′,N′tetra(pyridin-4-yl)benzene-1,4-diamine]. The octanuclear cages are fully characterized by various spectroscopic techniques including single-crystal X-ray diffraction analysis of OC-4. The self-assembled cages show strong in vitro anticancer activity against human lung adenocarcinoma A549 and human cervical cancer HeLa cell lines as observed from the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Of all the octanuclear cages, OC-3 exhibits remarkable anticancer activity against both cancer cell lines and is more active than that reported for cisplatin. The excellent anticancer activity of OC-3 and OC-4 highlights the importance of the synergistic effects of the spacer component of the dinuclear p-cymene Ru(II) acceptor clips.

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INTRODUCTION Ruthenium chemistry is a rising field in the search for proficient metallodrugs using diverse synthetic methods to symbiotically develop drugs that are not toxic to normal cells and selective for cancer cells.1 Of these synthetic methods, metal−ligand selfassembly stands out as an advanced field of research that has generated large number of intricate structural motifs in a single step from predesigned molecular building units in a facile manner.2 Our group and others have extensively studied and reported varieties of p-cymene ruthenium(II)-based metallacycles and metallacages with diverse shapes, functionalities, and properties using this approach.3 The ambidexterity of the confined space generated in these cage architectures has found applications in encapsulation of compounds, recognition and trapping of guest molecules, protection and stabilization of an otherwise unstable molecule, and as microreactor for specific reactions among others.4 Organometallic anticancer compounds have generated increasing interest since the discovery of the DNA binding property of cisplatin, but the high toxicity © 2016 American Chemical Society

and multifactorial resistance of platinum-based anticancer drugs in clinical applications propelled scientists to design and develop better alternatives.5 Mononuclear and dinuclear ruthenium complexes represent a new class of promising metal-based drugs with low toxicity (fewer side effects) and high activity (broader spectrum) in tumors possessing the potential to overcome platinum resistance.6 Two rutheniumbased anticancer drug candidates: NAMI-A ([ImH]trans[RuIIICl4(DMSO)Im]; Im = imidazole, DMSO = dimethyl sulfoxide) and KP1019 ([HInd][trans-RuIIICl4 (Ind)2]; Ind = indazole) have successfully passed phase I clinical trials;7 however, the need to improve both activity and selectivity is still of utmost importance. Neutral or cationic arene ruthenium complexes provide both hydrophilic and hydrophobic interactions with biomolecules by stabilizing the oxidation states of these complexes due to the robustness of the ruthenium pReceived: October 13, 2016 Published: December 20, 2016 608

DOI: 10.1021/acs.inorgchem.6b02488 Inorg. Chem. 2017, 56, 608−617

Article

Inorganic Chemistry

Figure 1. Dinuclear p-cymene Ru(II) acceptor clips and tetradentate ligand (L1) units used for the synthesis of OC-1−OC-4. synthesized following the literature procedure.13 The cell lines (lung cancer cell line A549, cervical cancer cell line HeLa, and normal lung epithelial cells HPL1D) were purchased from American Type Culture Collection. MTT and 2′,7′-dichlorofluorescein-diacetate (H2DCFHDA) were purchased from Sigma-Aldrich (USA). Annexin V-FITC kit was purchased from Santacruz Biotechnologies (USA). All cell culture media and components were purchased from Invitrogen (USA). The experimental procedures for the standard assays (cell anticancer activity, annexin V-FITC/PI assay, DCFDA assay for ROS generation) are given in the Supporting Information. NMR spectra were recorded on a Bruker 400 MHz spectrometer. The chemical shifts (δ) in 1H NMR spectra are reported in parts per million relative to tetramethylsilane [(CH3)4Si] as internal standard (0.0 ppm) or proton resonance resulting from incomplete deuteration of the NMR solvents: CD3OD (3.31) and CDCl3 (7.26). The 13C NMR spectra were recorded at 100 MHz, and the chemical shifts are reported in parts per million relative to external CDCl3 (77.84−77.20) and CD3OD (48.84−48.26). The 13C NMR spectrum of OC-4 was not obtained due to poor solubility at high concentration. Electrospray ionization mass spectrometry (ESI-MS) spectra of the octanuclear cages were recorded in positive mode using Agilent 6538 Ultra-High Definition (UHD) Accurate Mass Q-TOF spectrometer in standard spectroscopic grade solvent (CH3OH). Electronic absorption studies were performed on a Shimadzu UV-2600 UV−vis spectro-photometer in spectroscopic-grade solvent using precision cells made of quartz (1 cm). IR spectra were recorded on a Bruker ALPHA FT-IR spectrometer. Detailed NMR spectra of the octanuclear cages OC-1, OC-2, and OC-4 are given in the Supporting Information. General Synthetic Procedure for OC-1−OC-4. A mixture of 1 equiv of the dichloride analogue of A1−A4 and 2.1 equiv of CF3SO3Ag in methanol (20 mL) was stirred at room temperature for 3 h and then filtered to remove AgCl. The tetradentate ligand (L1; 0.5 equiv) was added to the corresponding acceptor solution in a 4 mL glass vial. An immediate visual sharp color change from light yellow to intense yellow for OC-1 or purple to deep red for OC-2 evidenced the progress of the reactions. After the mixture was stirred at room temperature for 24 h in a closed 4 mL glass vial, the reaction mixture was filtered through a cotton plug, and the solvent was removed under reduced pressure to get a clear yellow (for OC-1) or deep red (for OC-2) or dark green (for OC-3 and OC-4) solution. Cold diethyl ether was added to precipitate out the expected octanuclear cages OC1−OC-4, and they were dried under vacuum.

cymene unit, which holds a high potential for the development of metal-based anticancer drugs.8 Very recently, promising in vitro anticancer activity of half-sandwich complexes of osmium, rhodium, and iridium has also been reported.9 In the past few years, in vitro anticancer activity of a small number of discrete dinuclear p-cymene ruthenium(II) cages has been reported.10 As part of our ongoing contribution in this regard, it was our interest to design and develop discrete pcymene Ru(II) cages built from organic ligands and dinuclear p-cymene Ru(II) acceptor clips. Herein, we report the synthesis and characterization of four new Ru8 cages (OC-1−OC-4) via coordination-driven self-assembly of dinuclear p-cymene Ru(II) acceptors [Ru 2 (μ−η 4 -C 2 O 4 )(CH 3 OH) 2 (η 6 -p-cymene) 2 ](O 3 SCF 3 ) 2 (A 1 ), [Ru 2 (μ−η 4 -C 6 H 2 O 4 )(CH 3 OH) 2 (η 6 -pcymene) 2 ](O 3 SCF 3 ) 2 (A 2 ), [Ru 2 (dhnq)(H 2 O) 2 (η 6 -pcymene)2](O3SCF3)2 (A3), and [Ru2(dhtq)(H2O)2(η6-pcymene)2](O3SCF3)2 (A4) separately with tetrapyridyl ligand (L1) in methanol at room temperature (Figure 1). The potential of these octanuclear cages as anticancer agents was tested against human lung adenocarcinoma A549 and human cervical cancer HeLa cell lines using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay for growth inhibition. The two cages (OC-3 and OC-4) with the best IC50 values against the cancer cell lines were further studied by Annexin V-FITC/PI assay and DFCDA analysis to understand how the cell death is progressing.

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EXPERIMENTAL SECTION

General Methods. The acceptor clips [Ru 2 (μ−η4 -C 2 O 4 ) (CH3OH)2(η6-p-cymene)2](O3SCF3)2 (A1), [Ru2(μ−η4-C6H2O4) (CH3OH)2(η6-p-cymene)2](O3SCF3)2 (A2), [Ru2(dhnq)(H2O)2(η6p-cymene)2](O3SCF3)2 (A3), and [Ru2(dhtq)(H2O)2(η6-p-cymene)2](O3SCF3)2 (A4) were synthesized under dry nitrogen atmosphere using standard Schlenk technique following the reported procedures.11 Solvents were dried and distilled according to standard literature procedures. Silver triflate and 1,4-dibromobenzene were purchased from Sigma-Aldrich and used without further purification. 4,4′Dipyridylamine was synthesized using the Graebe−Ullmann reaction;12 N,N,N′,N′-tetra(pyridin-4-yl)benzene-1,4-diamine (L1) was 609

DOI: 10.1021/acs.inorgchem.6b02488 Inorg. Chem. 2017, 56, 608−617

Article

Inorganic Chemistry Octanuclear Cage 1. Acceptor clip A1 (30.0 mg, 0.03 mmol) and L1 (6.78 mg, 0.02 mmol) were stirred in methanol (4 mL) to obtain OC-1. Yield: 90%. 1H NMR (CD3OD + CDCl3, 400 MHz): δ (ppm) 7.90−7.88 (d, J = 6.2, 16H, Ha‑pyridyl), 7.22 (s, 4H, Hc‑phenyl), 7.13−7.12 (d, J = 6.2, 16H, Hb‑pyridyl), 6.98 (s, 4H, HD‑phenyl), 5.86−5.80 (d, J = 6.2, 16H, Hα‑cymene), 5.76−5.72 (d, J = 6.3, 16H, Hβ‑cymene), 2.89−2.85 (m, 8H, H1‑cymene), 2.26 (s, 24H, H3‑cymene), 1.40−1.37 (d, J = 4.4, 48H, H2‑cymene). 13C{1H} NMR (CD3OD + CDCl3, 100 MHz): δ (ppm) 161.64, 152.50, 122.60, 119.40, 116.89, 31.17, 22.44, 17.81. 19F NMR (CD3OD, 376.5 MHz): δ −79.57 ppm. IR (neat): υ/cm−1 3071.26 (w, C−Haromatic), 1256.49 (s, C−F of CF3), 514.84 (s, Ru−O), 445.87 (s, Ru−N). ESI-MS (m/z): Calcd: 562.07 [OC-1(O3SCF3−)2]6+; Found: 562.07 [OC-1(O3SCF3−)2]6+. UV−vis (CH3OH) λmax (ε) [nm (cm−1 M−1)]: 331 (1.23 × 104). Octanuclear Cage 2. Acceptor clip A2 (30.0 mg, 0.03 mmol) and L1 (6.43 mg, 0.02 mmol) were stirred in methanol (4 mL) to obtain OC-2. Yield: 82%. 1H NMR (CD3OD, 400 MHz): δ (ppm) 8.05−8.04 (d, J = 6.4, 16H, Ha‑pyridyl), 7.45 (s, 4H, Hc‑phenyl), 7.41 (s, 4H, HD‑phenyl), 7.18−7.17 (d, J = 6.2, 16H, Hb‑pyridyl), 6.00 (s, 8H, Hq), 5.79−5.78 (d, J = 7.5, 16H, Hα‑cymene), 5.77−5.76 (d, J = 6.8, 16H, Hβ‑cymene), 2.90− 2.87 (m, 8H, H1‑cymene), 2.23 (s, 24H, H3‑cymene), 1.37−1.36 (d, J = 6.9, 48H, H2‑cymene). 13C{1H} NMR (CD3OD, 100 MHz): δ (ppm) 184.50, 153.21, 119.34, 117.21, 103.77, 99.03, 84.00, 83.88, 82.49, 31.66, 21.70, 21.63, 17.32. 19F NMR (CD3OD, 376.5 MHz): δ −79.67 ppm. IR (neat): υ/cm−1 3071.45 (w, C−Haromatic), 1597.97 (s, C− Caromatic) 1514.35 (s, C−Ooxalate), 1251.90 (s, C−F of CF3), 512.28 (s, Ru−O), 445.03 (s, Ru−N). ESI-MS (m/z): Calcd: 2082.58 [OC2(O 3SCF3 −) 6] 2+ , 1339.41 [OC-2(O3SCF3− )5 ]3+, 966.57 [OC2(O3SCF3−)4]4+; Found: 2082.57 [OC-2(O3SCF3−)6]2+, 1339.39 [OC-3(O 3 SCF 3 − ) 5 ] 3+ , 966.56 [OC-2(O 3 SCF 3 − ) 4 ] 4+ . UV−vis (CH3OH) λmax (ε) [nm (cm−1 M−1)]: 495 (4.70 × 103), 331 (1.33 × 104), 204 (2.68 × 104). Octanuclear Cage 3. Acceptor clip A3 (30.0 mg, 0.04 mmol) and L1 (8.21 mg, 0.02 mmol) were stirred in methanol (4 mL) to obtain OC-3. Yield: 94%. 1H NMR (CD3OD, 400 MHz): δ (ppm) 8.16−8.14 (d, J = 6.8, 16H, Ha‑pyridyl), 7.40−7.39 (d, J = 2.4, 4H, Hc‑phenyl), 7.03− 7.01 (d, J = 9.8, 16H, Hb‑pyridyl), 7.00−6.99 (d, J = 7.1, 4H, HD‑phenyl), 7.18−7.15 (d, J = 9.8, 16H, H4), 5.81−5.79 (d, J = 6.2, 16H, Hα‑cymene), 5.59−5.58 (d, J = 5.5, 16H, Hβ‑cymene), 2.85−2.82 (m, 8H, H1‑cymene), 2.13 (s, 24H, H3‑cymene), 1.35−1.33 (d, J = 6.9, 48H, H2‑cymene). 13 C{1H} NMR (CD3OD, 100 MHz): δ (ppm) 171.25, 152.36, 137.60, 116.87, 111.68, 103.73, 99.79, 84.65, 83.06, 82.96, 31.06, 21.59, 16.49. 19 F NMR (CD3OD, 376.5 MHz): δ −79.49 ppm. IR (neat): υ/cm−1 3067.97 (w, C−Haromatic), 1598.41 (s, C−Caromatic) 1531.47 (s, C− Ooxalate), 1258.95 (s, C−F of CF3), 514.96 (s, Ru−O), 448.33 (s, Ru− N). ESI-MS (m/z): Calcd: 2182.61 [OC-3(O3SCF3−)6]2+, 1405.42 [OC-3(O3SCF3−)5]3+; Found: 2182.65 [OC-3(O3SCF3−)6]2+, 1405.43 [OC-3(O3SCF3−)5]3+. UV−vis (CH3OH) λmax (ε) [nm (cm−1 M−1)]: 430 (3.31 × 103), 324 (1.18 × 104). Octanuclear Cage 4. Acceptor clip A4 (30.0 mg, 0.04 mmol) and L1 (7.28 mg, 0.02 mmol) were stirred in methanol (4 mL) to obtain OC-4. Yield: 88%. 1H NMR (CD3OD + CDCl3, 400 MHz): δ (ppm) 8.64−8.56 (d, J = 6.0, 16H, Ha‑pyridyl), 8.16−8.15 (d, J = 7.0, 16H, H4), 7.29 (s, 4H, Hc‑phenyl), 7.91−7.89 (d, J = 6.1, 16H, Hb‑pyridyl), 6.81−6.80 (d, J = 7.2, 16H, H5), 6.59 (s, 4H, HD‑phenyl), 5.86−5.81 (dd, J = 6.2, 16H, Hα‑cymene), 5.66−5.61 (dd, J = 6.0, 16H, Hβ‑cymene), 2.88−2.85 (m, 8H, H1‑cymene), 2.15 (s, 24H, H3‑cymene), 1.30−1.26 (d, J = 16.0, 48H, H2‑cymene). 19F NMR (CD3OD, 376.5 MHz): δ −80.02 ppm. IR (neat): υ/cm−1 3070.82 (w, C−Haromatic), 1593.88 (s, C−Caromatic) 1538.20 (s, C−Ooxalate), 1252.35 (s, C−F of CF3), 513.92 (s, Ru−O). ESI-MS (m/z): Calcd: 2382.18 [OC-4(O 3 SCF 3 − ) 6 ] 2+, 1538.14 [OC4(O3SCF3−)5]3+; Found: 2382.17 [OC-4(O3SCF3−)6]2+, 1538.13 [OC-4(O3SCF3−)5]3+. UV−vis (CH3OH) λmax (ε) [nm (cm−1 M−1)]: 321 (1.10 × 104), 275 (1.65 × 104). X-ray Crystal Data Collection and Structure Solution. Singlecrystal X-ray data of OC-4 was collected on a Bruker D8 QUEST CMOS diffractometer using the SMART/SAINT22 software,14 equipped with a low-temperature device. The intensity data were collected at 110(2) K using graphite-monochromated Mo Kα radiation (0.7107 Å). The structures were solved by direct methods

and refined by the full matrix least-squares method based on F2 with all observed reflections15 employing SHELX-9716 incorporated in WinGX.17 In addition, the structures contain huge void of disordered solvent molecules and anions, so Squeeze program18 was applied to account for embedded solvent molecules that were seriously disordered. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were placed by using the riding models and refined isotropically. The diffraction data quality of OC-3 was not of publishable standard due to poor quality of the crystals. However, gross connectivity of the building units and the formation of an octanuclear cage were evident from the data set. Crystallographic data and refinement parameters are provided in Table 1 and the supporting CIF file.

Table 1. Crystal Data, Data Collection, and Structure Refinement Parameters for OC-4 octanuclear cage empirical formula formula weight crystal system space group T, K λ (Mo Kα), Å a, Å b, Å c, Å α, deg β, deg γ, deg V, Å3 Z ρcalcd, gcm−3 μ, mm−1 GOFa R1b [I > 2σ(I)] wR2c

OC-4 C216H184F6N12O26Ru8S2 4350.42 triclinic P1̅ 110(2) 0.710 73 15.844(4) 18.858(5) 21.373(6) 102.133(7) 102.954(6) 100.508(6) 5905(3) 2 1.223 0.579 1.015 0.0944(11 473) 0.2767(20 759)

GOF = {∑[w(F02 − Fc2)2]/(n − p)}1/2, where n and p denotes the number of data points and the number of parameters, respectively. bR1 = (∑||F0| − |Fc||)/∑|F0|. cwR2 = {∑[w(F02 − Fc2)2]/∑[w(F02)2]}1/2, where w = 1/[σ2(F02) + (aP)2 + (bP)] and P = [max (0,F02) + 2Fc2]/3. a

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RESULTS AND DISCUSSION The diruthenium(II) acceptor clips A1−A4 were obtained by treating the dichloride analogues with 2.1 equiv of silver triflate in methanol at room temperature for 3 h. Subsequent addition of the methanolic solutions of A1−A4 to the solid form of tetradentate ligand L1 at room temperature immediately consumed the solid followed by visual color change (Scheme 1). The octanuclear ruthenium(II) cages OC-1−OC-4 were isolated as triflate complexes in good yields and are soluble in polar organic solvents such as acetone, methanol, nitromethane, acetonitrile, and dimethyl sulfoxide and partially soluble in chloroform and dichloromethane. These cages were studied by NMR spectroscopy. The appearance of a single peak in the 19F NMR spectra at −79.5 ppm for OC-1, −79.6 ppm for OC-2, −79.4 ppm for OC-3, and −80.0 ppm for OC-4 indicated the presence of free triflate counteranions in the same chemical environment in the cages (Figures 2, S7, S12, and S19). The formation of a single and symmetrical structure was indicated by 1H NMR spectra (Figure 2 and Supporting Information). A significant upfield shift exhibited by the pyridyl protons of OC3 as compared to free tetradentate ligand in the 1H NMR 610

DOI: 10.1021/acs.inorgchem.6b02488 Inorg. Chem. 2017, 56, 608−617

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Inorganic Chemistry Scheme 1. Self-Assembly of the Discrete Octanuclear Cages (OC-1−OC-4)

Figure 2. 1H NMR (left) and 19F NMR (right) spectra of OC-3 recorded in CD3OD at room temperature.

of OC-1 to OC-4 equals 11.62, 10.05, 12.74, and 12.85 Å, respectively (Figures S6, S11, S15, and S18). Notably, the results from the ESI-MS experiment unequivocally establish the formation of the octanuclear cages OC-1− OC-4, where the cages maintain good stability. The ESI-MS analysis of [4 + 2] self-assembled octanuclear cages showed multiply charged fragmented ions for OC-1 at m/z = 562.07 [OC-1(O 3 SCF 3 −) 2 ]6+ ; OC-2 at m/z = 2082.57 [OC2(O3SCF3−)6]2+, 1339.39 [OC-2(O3SCF3−)5]3+, 966.56 [OC2(O 3 SCF 3 − ) 4 ] 4 + ; OC-3 at m/z = 2182.65 [OC3(O3SCF3−)6]2+, 1405.43 [OC-3(O3SCF3−)5]3+; and OC-4 at m/z = 2382.17 [OC-4(O 3 SCF 3 − ) 6 ] 2+ , 1538.13 [OC4(O3SCF3−)5]3+. These peaks (OC-2 to OC-4) were wellresolved isotopically and matched well with the theoretical distributions (Figures S21−S23). Electronic absorption properties of the newly self-assembled octanuclear cages OC-1−OC-4 and the tetradentate ligand L1 were studied by UV−visible spectroscopy recorded in methanol solution (3.5 × 10−5 M) at room temperature (Figure 3). The absorption spectra of OC1−OC-4 show intense bands at λmax = 331 nm for OC-1; λmax = 495, 331, 204 nm for OC-2; λmax = 430, 324 nm for OC-3; and λmax = 321, 275 nm for OC-4. The intense band at 305 nm for L1 corresponds to intraligand π−π* transition, which shifts bathochromically in the spectra of the octanuclear cages. Intraand intermolecular π−π* transitions and metal-to-ligand charge transfer (MLCT) transitions associated with capped p-cymene

spectra was due to the coordination of the pyridyl-N atom to the ruthenium metal center. The aromatic protons corresponding to the capped p-cymene moiety in the cages appeared slightly downfield in the range of δ 6.00−5.58 ppm. The isopropyl and methyl protons of the p-cymene moiety remain almost unchanged as compared to the ruthenium(II) acceptor clips. Similar proton resonances were observed for OC-1, OC2, and OC-4 (Figures S3, S8, and S16). Additionally, the benzoquinone protons of OC-2, the naphthaquinone protons of OC-3, and the naphthacenedione protons of OC-4 showed no significant changes in chemical shift as compared to A2, A3, and A4 acceptors, respectively. Furthermore, the formation of only one product was confirmed by the DOSY NMR, and the hydrodynamic radii (rH) of the cages were calculated from the Stokes−Einstein equation using the diffusion coefficients (D) obtained from the DOSY NMR experiment D=

kBT 6πηrH

where D = diffusion coefficient, kB = Boltzmann constant (1.3806 × 10−23 m2kg−2K−1), T = absolute temperature (298 K), η = viscosity coefficient of CD3 OD (6.02 × 10 −4 Kgm−1s−1), rH = hydrodynamic radius. Inserting the respective values of D = −9.506 (m2 s−1) for OC-1, −9.441 (m2 s−1) for OC-2, −9.554 (m2 s−1) for OC-3, and −9.548 (m2 s−1) for OC-4 in the above equation gave the hydrodynamic radii (rH) 611

DOI: 10.1021/acs.inorgchem.6b02488 Inorg. Chem. 2017, 56, 608−617

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Inorganic Chemistry

summarized in Table 1. Capped stick representations of the structures of OC-4 (Figure 4) show that indeed two tetradentate ligands L1 are coordinated to the four dinuclear Ru(II) acceptor clips A4 with a Ru9−Ru3 height of ∼8.4 Å and a Ru9−Ru4 diagonal distance of 17.3 Å between the edges, respectively. Selected bond lengths, angles, and geometrical parameters of the octanuclear cage structure of OC-4 are given in Table S1. O−Ru−N bond angles around the Ru centers are in the range of 80−85°. The rigid nature of the p-cymene moieties deviates the geometry around Ru centers from the ideal octahedral geometry. The two central phenyl rings in OC-4 are freely rotating (in solution) through the C−N bond attached to pyridyl moiety of L1. Thus, they are almost parallel to each other with a distance of ∼5 Å apart. The molecule of OC-4 in solid-state is centrosymmetric. The solid-state packing diagram of OC-4 through the crystallographic b-axis is presented in Figure S24, which shows the presence of a large rectangular channel. A triflate anion, a methanol molecule, and an assigned diethyl ether molecule all lie parallel to each other on one side of the rectangle. Anticancer Activity Studies. From the MTT assay results, it is conspicuously evident that all the octanuclear cages exhibit good anticancer activity on the cancer cell lines tested in micromolar concentration range after 72 h of exposure to increasing concentrations of OC-1−OC-4. However, among the cages studied, OC-3 and OC-4 possess excellent anticancer activity with the lowest IC50 values against both A549 and HeLa cancer cell lines, which is obviously better than the IC50 value recently reported for cisplatin.19 While OC-1 and OC-2 show very similar IC50 values comparable to that of cisplatin against A549 cancer cell line, OC-1 shows moderate anticancer activity against HeLa cells when compared with the activity of cisplatin against the same cell line (Table 2). Interestingly, the most active of the octanuclear cages (OC-3 and OC-4) contain polyaromatic rings in their Ru(II) acceptor clips, suggesting that the nature and sometimes the number of the aromatic rings in the acceptor unit may improve the anticancer activity of the cages. Additionally, the IC50 value of the Ru(II) acceptor clip A3 (4 equiv) is higher than the IC50 of the octanuclear cage OC-3, indicating there are some synergistic effects coming from the four acceptor clips in OC-3 on the cancer cell lines leading to increasing anticancer activity. However, the IC50 values of the octanuclear cages in normal lung epithelial cells HPL1D

Figure 3. UV−vis spectra of the discrete octanuclear cages and the ligand L1 recorded in methanol (3.5 × 10−5 M) at 298 K.

ruthenium moiety can be assigned to the peaks in the ranges of 204−331 nm and 430−495 nm, respectively. Additionally, intense bands observed at 250−300 nm for OC-2, OC-3, and OC-4 are originating from the aromatic spacers of the dinuclear p-cymene ruthenium(II) acceptor clips A2, A3, and A4, respectively. The infrared spectra of OC-1−OC-4 (Figure S20) are dominated by stretching bands at 1625.10 cm−1 for OC-1, 1514.35 cm−1 for OC-2, 1531.47 cm−1 for OC-3, and 1538.20 cm−1 for OC-4 corresponding to the νC−O symmetrical stretching frequency of the coordinated carbonyl groups of dinuclear Ru(II) acceptor clips A1−A4. The νC−F symmetrical stretching frequency of −CF3 in the triflate counteranion also showed strong stretching bands at 1256.49 cm−1 for OC-1, 1251.90 cm−1 for OC-2, 1258.95 cm−1 for OC-3, and 1252.35 cm−1 for OC-4. Slow vapor diffusion of diethyl ether into methanol solution of OC-4 provided dark green single crystals suitable for X-ray analysis. Although X-ray diffraction of OC-3 clearly evidenced the formation of an octanuclear cage, the crystal data are not good enough for publication due to poor crystal quality, which was not improved even after several attempts. The single-crystal X-ray structure analysis of OC-4 unambiguously provided evidence for the expected octanuclear cage. The crystallographic data and structure refinement parameters of OC-4 is

Figure 4. Capped stick representation of the X-ray crystal structure of OC-4. Solvent molecules, counter-anions, and hydrogen atoms are omitted for clarity. 612

DOI: 10.1021/acs.inorgchem.6b02488 Inorg. Chem. 2017, 56, 608−617

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Inorganic Chemistry

emission maxima at 528 nm. ROS include a number of chemically reactive molecules containing oxygen that damage DNA and RNA and oxidize proteins and lipids. These reactive molecules have paramount functions in many cellular signaling pathways including proliferation, cell activation, and migration. ROS are produced during physiological conditions such as cellular metabolism and under pathological conditions including cancers. High amounts of ROS generated in the intracellular compartments can be harmful; thus, enzymatic antioxidants such as superoxide dismutase and catalase safeguard the cells by converting unsafe free radicals to nontoxic molecules like water.20 However, ROS can also prompt cellular apoptosis and can therefore perform antitumorigenic activities.21 The generation of ROS inside the cell was evident by the detection of DCF inside the cell in the presence of OC-3 and OC-4 as indicated by the shift of the band positions (Figure 7). The larger the shift in the emission band position, the more the quantity of DCF generated, and this can be correlated to the amount of ROS produced inside the cell. OC-3 showed the generation of moderate amount of ROS, while OC-4 showed the formation of a significant quantity of ROS, which is even more than the reference used in this assay (H2O2) indicating that the large amount of ROS generated led to the cell death. The high amount of ROS generated in the presence of OC-4 may be as a result of the increased number of fused polyaromatic rings in OC-4 as compared to number of fused polyaromatic rings in OC-3. A combinatorial effect of the ruthenium(II) metal ion and the fused polyaromatic rings is therefore presumed to result in the ROS generation in OC-3 and OC-4.22

Table 2. Anticancer Activity of OC-1−OC-4, A3, and Cisplatin on Human Lung Adenocarcinoma A549 and Human Cervical Cancer HeLa Cell Lines as Expressed by the IC50 Values IC50 value (μM) octanuclear cages OC-1 OC-2 OC-3 OC-4 A3 cisplatin19

A549 19.73 22.78 0.77 3.86 2.58 21.30

± ± ± ± ± ±

3.65 3.88 0.23 1.79 1.50 3.30

HeLa 9.52 21.27 0.91 5.83 2.15 15.10

± ± ± ± ± ±

2.66 3.77 0.36 2.15 1.39 2.10

(Table S2) show that the anticancer activity of OC-3 is slightly higher in the two cancer cell lines tested. The cell viability plots for OC-3 (Figure 5) revealed very low percentage of viable cells even at high concentration. The cell viability plots for OC-1, OC-2, OC-4, and A3 are included in Figures S25−S28. Moreover, since all the cages showed similar activity and selectivity toward the cancer cell lines tested, the same mode of action is suggested for all of them. Therefore, further studies were performed using OC-3 and OC-4 octanuclear cages. ANNEXIN V-FITC/PI Assay. The annexin V-FITC/PI assay was performed with HeLa cells treated with OC-3 and OC-4. Propidium iodide (PI) emits in the red region, while the annexin V-FITC dye shows green fluorescence. High annexin V-FITC stain was shown by cells that are in early apoptotic stage, while the cells in the late apoptotic stage showed both annexin V-FITC and PI staining. Live cells that gave autofluorescence were neither stained by annexin V-FITC nor PI, whereas dead cells were stained by PI. From the dot plots in Figure 6 we deduce that the apoptotic potential of both cages is evident, as the percentage population of viable cells decreases significantly in the presence of the OC-3 and OC-4. In the presence of OC-3, the third quadrant and fourth quadrant (Q_LR and Q_UR) showed increased percentage of apoptotic cells suggesting that OC-3 triggers both early (77.07%) and late (8.07%) apoptosis in HeLa cells. It is fascinating however that in the presence of OC-4, the fourth quadrant (Q_UR) showed increased percentage of apoptotic cells (99.71%) suggesting that OC-4 triggers late apoptosis in HeLa cells. DFCDA Assay. 2′,7′-dichlorofluorescein-diacetate (H2DCFDA) assay was performed to detect the presence of any reactive oxygen species (ROS) inside the cell using flow cytometry. It is well-known in the literature that some compounds on hydrolysis by intercellular esterases and on oxidation by ROS produce fluorescent 2′,7′-dichlorofluorescein (DCF) having

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CONCLUSION The synthesis and in vitro anticancer activity of four new discrete Ru(II) octanuclear cages built from dinuclear ruthenium(II) acceptor clips and a tetradentate pyridyl ligand have been described. All the octanuclear cages were fully characterized by well-known spectroscopic techniques, and the molecular structure of OC-4 was confirmed by single-crystal XRD analysis. The cages were tested against human lung adenocarcinoma A549 and human cervical cancer HeLa cell lines, with OC-3 and OC-4 showing excellent in vitro anticancer activity as revealed from their very low micromolar IC50 values. It is worth noting that OC-3 and OC-4 were found to possess higher anticancer activity against the tested cancer cell lines than that of cisplatin. Some synergism was also observed to be coming from the dinuclear p-cymene Ru(II) acceptor clip A3, which is evident in the IC50 value of OC-3 against the tested cancer cell lines. Further studies to determine

Figure 5. Cell viability plots showing the anticancer activity of OC-3 in (a) human lung adenocarcinoma A549 cell line and (b) human cervical cancer HeLa cell line. 613

DOI: 10.1021/acs.inorgchem.6b02488 Inorg. Chem. 2017, 56, 608−617

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Figure 6. Annexin V-FITC/PI assay for apoptosis detection of human cervical cancer HeLa cell line with OC-3 and OC-4 using FACS analysis.

Figure 7. DFCDA analysis of OC-3 and OC-4 in human cervical cancer HeLa cell line. 614

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the detailed mechanism happening in the cell revealed that cell death is progressing through the generation of ROS, which is of significant amount in OC-4 and even better than the reference used.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02488. 1 H, 19F, 13C, 1H−1H COSY, DOSY NMR spectra, infrared spectra, ESI-MS data, and cell viability plots of the octanuclear cages (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (P.S.M.) *E-mail: [email protected]. (P.K.) ORCID

Partha Sarathi Mukherjee: 0000-0001-6891-6697 Author Contributions #

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.A.A. acknowledges IISc for research fellowship. A.S. thanks the UGC (India) for Kothari fellowship. P.S.M. thanks the Department of Science and Technology (DST), India, for financial support. P.K. acknowledges financial support from the DBT-IISc partnership program. We sincerely thank Dr. K. Mitra and Dr. S. Narra for useful discussions and Leepika for FACS analysis.

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REFERENCES

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DOI: 10.1021/acs.inorgchem.6b02488 Inorg. Chem. 2017, 56, 608−617

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DOI: 10.1021/acs.inorgchem.6b02488 Inorg. Chem. 2017, 56, 608−617